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“Selective Enumeration, Isolation and Identification of Streptococcus

thermophilus from Indian traditional curd to produce starter culture along with
elevation of nutrient media fo...
Research · June 2015

DOI: 10.13140/RG.2.1.1494.0324
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CONTENTS
Chapter 1 Introduction Page

No.
1.1) SIGNIFICANCE OF THE STUDY 1
1.2) RESEARCH OBJECTIVES 2
1.3) RESEARCH HYPOTHESIS 2
1.4) SCOPE AND LIMITATION OF THE STUDY 2
1.5) EXPECTED RESULTS 3
Chapter 2 Literature Review

2.1) MICROBES AND FERMENTATION 4
2.1.1) Industrial microbiology 4
2.1.2) Fermentation Technology & Microbes 6
2.2) LACTIC ACID BACTERIA 13
2.2.1) Classification of Lactic acid bacteria 15
2.2.2) Biochemistry and genetics of lactic acid bacteria 18
2.2.3) Natural Habitat of Lactic acid bacteria 25
2.2.4) Lactic acid bacteria in food products 26
2.2.5) Lactic acid bacteria in agricultural products 31
2.2.6) Lactic acid bacteria as Probiotics 32
2.3) STREPTOCOCCUS THERMOPHILUS 42
2.3.1) Physiology of S.thermophilus 43
2.3.2) Characterization of S. thermophilus 45
2.3.3) Factors affecting the growth of lactic acid bacterial fermentations 57
2.4) Media formulations and nutrient requirement of S.thermophilus 59
2.5) Starter Culture and its role in Dairy Industry 61
2.5.1) Functions of Starter Culture 64
2.5.2) Types of Starter Culture 65
2.5.3) Fermented Milk Products 66
2.5.4) Bacteriophage 69
2.5.5) Fermented milk products in India 69
Chapter 3 Material & Methods
3.1) CHEMICAL AGENTS, NUTRIENTS AND CULTURES 78
3.2) SCREENING AND IDENTIFICATION 80
3.2.1) Sample Collection 80
3.2.2) Selective Enumeration and isolation methods 80
3.2.3) Morphological Characterization 81
3.2.4) Biochemical Characterization 82
3.2.5) Molecular Characterization 86
3.3) Media Formulations and effects of different sources of nutrition 88
3.3.1) Effect of carbon sources on mass production of S.thermophilus 88
3.3.2) Effect of nitrogen sources on mass production of S.thermophilus 89
3.4) EFFECT OF OTHER FACTORS AFFECTING THE GROWTH 89
3.4.1) Temperature Optimization 90
3.4.2) pH optimization 90
3.5) UP-SCALING PROCESS STANDARDIZATION 90
3.5.1) Inocolum Preparation 90
3.5.2) Process standardization for incubating conditions 91
3.5.3) Commercial viability of the standardized process 92
3.6) VIABILITY AND CFU STANDARDIZATION 92
3.7) DOWN-STREAM PROCESS STANDARDIZATION 93
3.7.1) Centrifugation and micro-filtration 93
3.7.2) Cryoprotectant preparation and standardization 94
3.7.3) Freezing temperature and incubation 95
3.7.4) Lyophilization standardization 95
3.8) APPLICATION TESTING OF ISOLATED STARTER CULTURE 96
3.8.1) Curd Preparation 96
3.8.2) Comparative testing 98
3.9) COMMERCIAL UP-SCALING TRIALS 99
3.10) PROTOCOL FOLLOWED AT TBI 100
Chapter 4 Results & Discussion
4.1) SCREENING AND ISOLATION OF LACTIC ACID BACTERIA 101
4.1.1) Isolation of Lactic acid bacteria 101
4.1.2) Selection of LAB for curd trials and lactic acid production 101
4.1.3) Morphological and Phenotypic characterization of Lactic
acid bacteria ` 105
4.2) Biochemical Characterization of Isolates 113
Chapter 5 Molecular Characterization

5.1) Molecular Characterization 123
5.1) 16s rDNA sequencing 125
5.1.1) 16s rDNA sequencing results for ST-100 126
5.1.8) 16s rDNA sequencing results for LB-100 135
5.1.9) 16s rDNA sequencing results for LB-200 136
5.2) Whole Genome Sequencing of ST-500 140
Chapter 6 Media Standardization

6.1) Nutritional Requirements of Bacteria 147
6.2) Effect of different sources of nutrition on ST-500 148
6.2.1) Effect of Carbon Sources 148
6.2.2) Effect of Nitrogen Sources 152
6.2.3) Effect of Vegetable protein and Whey protein sources 155
6.2.4) Effect of Yeast Extract, Buffers and other salts used 157
6.3) Influence of factors on lactic acid bacteria fermentation 159
6.3.1) Effect of various temperatures on cell mass production 160
6.3.2) Effect of various initial pH on cell mass production 161
6.3.3) Effect of various agitation speeds on cell mass production 163
6.4) Media Costing 165
6.5) Batch Fermentation of ST-500 and process standardization 171
6.6) Down Stream processing of ST-500 181
6.6.1) Lyophilization process standardization 181
Chapter 7 Dairy Applications

7.0) Dairy Applications 189
7.1) Selected Parameters for application testing 190
7.1.1) Texture of Curd 190
7.1.2) Sensory Evaluation 191
7.1.3) Syneresis 191
7.1.4) Acidity 191
7.1.5) pH 191
7.1.6) Rheological 191
7.2) Application testing for cheese production from ST-500 197
7.3) Chemical & Physiological Parameters for Fermented Milk (set curd) 202
7.4) Testing with market Cultures 202
Chapter 8 Summary & Conclusion 205

References 208
Appendix 227
FIGURE CONTENT
FIGURE
S.N. LIST & TITLE OF FIGURES
NUMBER
Consensus tree, based on comparative sequence analysis
1 Figure 2.1 of 16S rRNA, showing major phylogenetic groups of
lactic acid bacteria
2 Figure 2.2 Characterization of Lactic acid bacteria

Homo-fermentative pathway illustrating the production
3 Figure 2.3
of lactic acid from glucose
Hetero-fermentative pathway showing the production of

4 Figure 2.4 lactic acid, carbon-dioxide and either ethanol or acetic
acid
Phylogenetic tree and important commercial importance

5 Figure 2.5
of Lactic acid bacteria
6 Figure 2.6 Silent features of lactic acid bacteria
Various applications of Lactic acid bacteria modulating

7 Figure 2.7
human health
Electron microscopic image of S. thermophilus (Source:

8 Figure 2.8
Durso and Hotkins 2003)
Phylogenetic tree of Lactobacillales based on

9 Figure 2.9
concatenated alignments of ribosomal proteins
Flow diagram of the main steps involved in the

10 Figure: 2.10
production of Stirred yogurt
11 Figure: 2.11 Flow sheet for the preparation of acidophilus milk
12 Figure: 2.12 Flow sheet for the preparation of Curd
13 Figure: 3.1 Step elaboration of Inocolum preparation from 1st. 2nd

and 3rd generation
14 Figure: 3.2 Chart for preparation of Indain traditional curd from
starter culture
15 Figure: 4.1 Sample colonies of ST-500(A) and ST-600(B) growing
on Streptococcus thermophilus isolation agar
16 Figure: 4.2 Sample colonies of LB-100(C) and LB-200(D) growing
on MRS agar
17 Figure: 4.3 Microscopic view of ST-200 (A), ST-300(B), ST-400(C),
ST-500(D), LB-100(E) and LB-200(F)
18 Figure: 4.4 Analysis of biochemical results of all selected 10 strains
by Kit menthod
Results of MR test with occurrence of a Ruby pink color
19 Figure: 4.5 in all tubes except BLANK which indicates that the
organism produces a large amount of organic acid.
Change in color indicates MR positive
Results of VP test with no change in color in all tubes
20 Figure: 4.6 except BLANK which indicates that the organism is not
capable of converting pyruvate into acetonin. No
acetonin in broth indicates negative test.
Digest results of the 16s rDNA genes of representative
isolates along with the reference. Sample IDs of the
21 Figure: 5.1 different lanes are mentioned on the top of the lane along
with the last lane of Ladder (L) Hind III digested lambda
ladder.
22 Figure 5.2 Results of phylogenetic analysis of ST-100, ST-200 and
ST-300 in the form of Phylogenetic tree
23 Figure 5.3 Results of phylogenetic analysis of ST-400, ST-500 and
ST-700 in the form of Phylogenetic tree
24 Figure 5.4 Results of phylogenetic analysis of LB-100 and LB-200
in the form of Phylogenetic tree
Figure presents the library preparation report with peak
25 Figure 5.5
indicating the ST-500 library optimal of sequencing
Circular genomic map of ST-500 (Streptococcus

26 Figure: 5.6 thermophilus) in comparison with Streptococcus
thermophilus MN-ZLW-002
Influence of carbon sources on lactic acid fermentation

for mass cell production supplied with modified MRS
27 Figure 6.1 (A) broth and 1% of the selected carbon sources. All the
carbon sources were tested indiviually with their 10gm/L
content in media.

28 Figure: 6.1 (B) broth and 2% of the selected carbon sources. All the
content in media.
29 Figure: 6.1 (C) broth and 3% of the selected carbon sources. All the
content in media

30 Figure: 6.1 (D) for mass cell production supplied with modified MRS
broth and 4% of the selected carbon sources. All the
content in media
Effect of nitrogen sources on lactic acid fermentation for
mass cell production supplied with modified MRS broth
31 Figure: 6.2 (A) and 1% of the selected nitrogen sources. All the nitrogen
sources were tested indiviually with their 10gm/L content
in media

mass cell production of supplied with modified MRS
32 Figure: 6.2 (B) broth and 2% of the selected nitrogen sources. All the
nitrogen sources were tested indiviually with their
20gm/L content in media

33 Figure: 6.2 (C) and 3% of the selected nitrogen sources. All the nitrogen
in media

34 Figure: 6.2 (D) and 4% of the selected nitrogen sources. All the nitrogen
in media
Effect of vegetable protein and milk protein sources on

lactic acid fermentation for mass cell production supplied
35 Figure: 6.3 (A) with modified MRS broth and 2% of the selected sources.
All the sources were tested indiviually with their 20gm/L
content in media
Effect of vegetable protein and milk protein sources on

lactic acid fermentation for mass cell production supplied
36 Figure: 6.3 (B) with modified MRS broth and 4% of the selected sources.
All the sources were tested indiviually with their 40gm/L
content in media
Influence of sodium, potassium, ammonium and

magnesium salts on lactic acid fermentation for mass cell
37 Figure: 6.4
production supplied with modified MRS broth and were
tested indiviually with their mentioned content in media.
Influence of yeast extract (amino acid source) on lactic

acid fermentation for mass cell production supplied with
38 Figure: 6.5
modified MRS broth and were tested indiviually with
1gm/L to 5gm/L content in media.
The influence of temperature on cell mass yield on self
39 Figure: 6.6 designed Media 5 by Streptococcus thermophilus NCIM
5539
The influence of initial set point of pH on cell mass yield
40 Figure: 6.7 on self designed Media 5 by Streptococcus thermophilus
NCIM 5539
41 Figure: 6.8 Reducing sugar concentrations (Sucrose) during batch
fermentation of Streptococcus thermophilus NCIM 5539
Effect of agitation speed on the cell growth, reducing
42 Figure: 6.9 sugar concentration and mass production by
Streptococcus thermophilus NCIM 5539: 100 rpm (A),
200 rpm (B), and 300 rpm (C).
43 Figure 6.10 Price & Fresh cell mass weight gm/L comparison of
media from different vendors
44 Figure 6.11 Price & Fresh cell mass weight gm/L comparison of
media M17, MRS and Media 5 from same vendor
45 Figure: 6.12 Prepared media poured in 2L Fermenter
46 Figure: 6.13 Media after autoclaving for 15 minutes
47 Figure: 6.14 Assembling of fermenter for inoculation
48 Figure: 6.15 Completely assembled fermenter ready for inoculation
49 Figure: 6.16 Turbidity observed after 16 hours of incubation

50 Figure: 6.17 Batch closed after 24 hours of incubation
51 Figure: 6.18 Collection of turbid broth in sterile flasks for further

processing
52 Figure: 6.19 Collected material with cell mass for further processing
History plot of the 5 L batch of Streptococcus
53 Figure: 6.20 thermophilus showing the change in pH and consumption
of acid and base for maintaining the set pH balance.
54 Figure: 6.21 Fermentation profile of ST-500 recorded on the basis of
OD at 660nm
Batch fermentation profile of ST-500 in 5L fermenter
55 Figure: 6.22 showing cell mass concentration, lactic acid percentage
and reducing sugar concentration
Media preparation and mixing in sterile buckets [Fig.
6.23 (A, B)], Media addition in 300 L fermenter (Fig.
56 Figure: 6.23 6.23 (C), Seed inoculation in fermenter with heat sterile
conditions (Fig. 6.23 (C), Adjustment of standardized
parameters through manual mode of operation (Fig. 6.23
(D).
Technology Based incubator facility at Delhi University.
(A) Visible turbidity, (B) Peristatic pumps and control
57 Figure: 6.24 panel showing the flow of base to maintain pH, (C, D)
Overall view of the facility with 50 L fermenter on
extreme left, 200 L in middle and 300 L fermenter on the
right
58 Figure: 6.25 Fermentation profile of ST-500 recorded on 300L
fermenter on the basis of OD at 660nm
Effect of different lyoprotectants on the viability after
59 Figure: 6.26 lyophilization. If not other indicated 10% (w/w) solution
were used. The error bars show the standard deviation
Front view of BioSata A plus lyophilizer, (B, C, D)

Lyophilization of cell mass mixed with lyophilization
60 Figure: 6.27 media in plates and vials at -47° C and vacuum pressure
of 0.04mbar, (E) Final lyophilized product (starter
culture) after complete drying
(A) presents the commercial model lyophilizer for large

Figure: 6.28 scale lyophilization. (B, C) presents the lyophilized
61
culture in trays after complete lyophilization cycle of 16
hours
Effect of different Lyophilization media on the viability

62 Figure: 6.29 after lyophilization. The error bars show the standard
deviation
A, B, C presenting the texture, cut ability, thickness,

63 Figure: 7.1 water binding and robustness of the curd prepared from
ST-500 starter culture
A and C presenting the texture, cut ability, thickness,

water binding and robustness of the flavored yogurt
64 Figure: 7.2
prepared by mixture of ST-500 and LB-200. Images 7.2
B and D presents stirred yogurt with different flavors.
Graphical presentation of pH drop rate after inoculation

65 Figure 7.3 of ST-500 with different fruit and sugar percentages on
incubation at 42°C
Application testing results of ST-500 (Streptococcus

66 Figure: 7.4 thermophilus NCIM 5539 for cheese preparation after 30
days of aging and processing incubation period
Graphical presentation of pH drop rate after inoculation

67 Figure 7.5 of ST-500 with different percentage of milk fat on
incubation at 42°
TABLE CONTENT
TABLE
S.N. LIST & TITLE OF TABLES
NUMBER
Examples of industrial fermentation products and their
1 Table 2.1
producer microorganisms
Examples of microorganisms classified as GRAS
2 Table 2.2
(Generally regarded as safe)
3 Table 2.3 Examples of aseptic and non-aseptic fermentations
Differentiating Lactic acid bacteria and a comparison
4 Table 2.4
with current taxonomic classification
Some of the major fermented food products along with
5 Table 2.5
the microbe involved around the world
Differences between Probiotics, Prebiotics and
6 Table 2.6
Synbiotics
Health benefits along with the proposed mechanism of
7 Table 2.7
action of lactic acid bacteria
8 Table 2.8 Classification of cocci lactic acid bacteria
9 Table 2.9 Characterizing features of S. thermophilus
Genomic details of Streptococcus thermophilus strains
10 Table 2.10 with whole genome sequencing available data till Feb
2014
Taxonomy of dairy Starter Cultures with old and new
11 Table 2.11
names and their products
Major starter culture producer companies all over the
12 Table 2.12
world
13 Table 2.13 Characteristics of sweet and sour Dahi
14 Table 3.1 pH indicators for carbohydrate fermentation media
15 Table 3.2 Details of the analytical tools and reference genome used
Percentage lactic acid yield and pH drop results of the
16 Table 4.1
128 isolates after first selective enumeration.
Phenotypic characterization, colony observation and gas
17 Table 4.2
production results of the selected 32 isolates.
Results of complete morphological and phenotypical
18 Table 4.3
analysis of 10 selected strains
Fermentation results of selected isolates from different
19 Table 4.4
sugars
20 Table 4.5 Results of enzyme assays conducted on selected isolates
Biochemical characterization results on the basis of
21 Table 4.6
different biochemical test analysis
Quality Control reports of all of sample isolates. DNA
22 Table 5.1 Concentration and purity of samples estimated using
nanodrop spectrophotometer
16s rDNA sequencing results of selected 10 isolates with
23 Table 5.2
best hit similarities at data in NCBI
24 Table 5.3 Qubit values of prepared library
Summary of the platforms and alignments of WGS of
25 Table 5.4
ST-500
Details of alignments and Raw data sequences obtained
26 Table 5.5
from WGS
Alignment statics of ST-500 in comparison with
27 Table 5.6 S.thermophilus MN-ZLW-002 and S. thermophilus
MTCC_5461
Results of different media compositions concluded after
28 Table 6.1 individual studies of media ingredients for mass cell
production of Streptococcus thermophilus NCIM 5539
Media per litre cost comparison between MRS and M17
29 Table 6.2 on individual ingredient basis from Himedia, India
catalogue.
Costing of the designed media on the basis of ingredient
30 Table 6.3
prices available with Himedia
Costing of the designed media on the basis of
31 Table 6.4
commercial prices provided by Himedia
Comparison of Batch results with complete up-stream
32 Table 6.5
analysis of ST-500
33 Table 6.6 Cell growth rate with respect to incubation hours
Combinations of protectants along with the ratio of
34 Table 6.7
addition
35 Table 6.8 Viability loss during up-downstream bio-processing
Results of application testing done on the basis of certain
36 Table 7.1 parameters
Selected Parameters and standards for comparative

37 Table 7.2 analysis
Results of comparative testing done on selected

38 Table 7.3 parameters
List of abbreviations
AAD : Antibiotic associated diarrhea

ASA : Associated chronic gastritis
ATCC : American Type Culture Collection
BIS : Bureau of Indian Standards
BLAST : Basic local alignment search tool
bp : Base pair
CFU : Colony Forming Unit
DGGE : Denaturing gradient gel electrophoresis
DNA : Deoxyribonucleic acid
EDTA : Ethylenediaminetetraacetic acid
EMP : Embden–Meyerhof–Parnas pathway
EPS : Extracellular polysaccharides
g : Gram
GC : Guanine Cytosine
GRAS : Generally recognized as safe
h : hours
H2SO4 : Sulphuric acid
HCl : Hydrochloric acid
HGT : Horizontal gene transfer
IBD : Inflammatory bowel disease
IL : Interleukin
K2HPO4 : dipotassium hydrogen phosphate
Kg : Kilogram
KH2PO4 : monopotassium phosphate
L : Liter
LAB : Lactic acid bacteria
LDH : Lactate dehydrogenases
LPS : Lipopolysaccharides
mbar : milli bar
mL : Milliliter
MRS : De Man, Rogosa and Sharpe
MR-VP : Methyl red and Voges-Proskauer
MTCC : Microbial type culture collection
N : Normality (Normal)
NaCl : Sodium Chloride
NaOH : Sodium Hydroxide
NCBI : National center for Biotechnology information
NCIM : National Collection of Industrial Microorganisms
NSLAB : Non-starter lactic acid bacteria
ºC : Degree Centigrade
OD : Optical Density
PCR : Polymerase chain reaction
PFGE : Pulse-field gel electrophoresis
psi : Pound per square inch
PTS : Phosphotransferase
PYR : Pyrrolidonyl Arylamidase
RFLP : Restriction fragment length polymorphism
RNA : Ribonucleic acid
Rpm : Revolution per minute
SMP : Skim Milk powder
SNF : Solid not Fat
SWP : Sweet whey powder
TGGE : Temperature gradient gel electrophoresis
TNF : Tumor necrosis factor
WGS : Whole Genome sequencing
WPC : Whey protein concentrate
Chapter 1
Introduction
1.1 SIGNIFICANCE OF THE STUDY
Milk and milk products provide wealth of nutrition benefits with their healthy
contents along with the micro-flora these products carry. The major group present in milk
and dairy products with tremendous potential and most desirable in domestic and
commercial food fermentation is lactic acid bacteria (LAB) group. Some bacteria in this
group are described as psychrophilic, which means that they grow best at cold
temperatures, while others are severely retarded by being in the refrigerator and grow
rapidly only at warmer temperatures. Fermentation was invented long before
microorganisms were discovered, and therefore the process seemed mysterious. The need
for an inocolum was understood and usually satisfied by keeping a sample from the
previous production. But with advancement in science and technology over years, the
capability of microorganisms to perform fermentation leading to various useful end
products is well documented now. LAB are widely used in the production of fermented
food, and they constitute the majority of the volume and the value of the commercial
starter cultures. The demand of a cost economic starter culture is also swelling with
modernization in fermented food industries. It is; therefore, very attractive to develop a
low cost single strain starter culture for Indian food fermentation industries for
manufacturing of Indian traditional curd, as in India, food industries are still using
imported starter cultures due to the unavailability of any domestic starter culture
producer.
1.2 RESEARCH OBJECTIVES
This research work focuses on isolation of Streptococcus thermophilus and

development of a complete up-stream down-stream fermentation process for the isolated
strain including a cost efficient media development with complete process optimization.
This involves bacterial enumeration, morphological, biochemical and molecular
characterization of the isolated strain followed by standardization of the lyophilization
process and finally making sure the commercial viability of the end product. The
objectives are:
• To isolate and identify potential Streptococcus thermophilus strain capable of fast

acid production.
• Complete morphological and biochemical analysis of the isolated strain.
• To study the molecular behavior of the strain including 16s rDNA sequencing
followed by whole genomic sequencing on Ion torrent platform.
• To optimize fermentation conditions best suitable for high yield basis cell mass
production.
• Low cost high yield media designing and development for economic production.
• Commercial trails of the isolated strain on large scale fermenters (200-300L).
• Standardization of the commercial viability of the strain by comparing with the
existing imported cultures available in the market
1.3 RESEARCH HYPOTHESIS
Isolated lactic acid bacteria could produce lactic acid as end product leading to
production of mild flavored Indian traditional curd and capable of multiplying fast on low
cost medium with high yield. The behavior of the strain could be analyzed on complete
characterization and positive approach will lead this type of study to the development of
an economical starter culture with commercial viability.
1.4 SCOPE AND LIMITATION OF THE STUDY
Present work involves investigation about production of starter culture by

isolating a potential strain of lactic acid bacteria with special interest on Streptococcus
thermophilus for production of Indian traditional curd. The ultimate objective is to
develop a cost efficient starter culture especially for Indian market by economically
standardizing the required media along with process development. Designing of media
includes changes in the nutritional requirements of the isolated strain by using
inexpensive sources of carbon and nitrogen to obtain optimum yield of viable cell mass.
In addition, fermentations were carried out from laboratory scale fermenters to pilot scale
commercial bioreactor facilities. Only a few strains (18-20) of Streptococcus
thermophilus are openly available with their complete genomic study by sequencing the
whole genome with NCBI (National Center for Biotechnology Information). The isolated
strain in current study has been used as starter but its whole genome sequence has also
been studied on Ion torrent platform. Commercial trails of the product for its use as
starter culture were also conducted with several big dairies in the country to check the
performance of strain against the cultures already available in the market.
1.5 EXPECTED RESULTS
• Low cost high yield viable cell mass from the fermentation process under the
optimum conditions.
• Standardized nutritional and lyophilization parameters with minimum cell loss
during agitation and freezing shocks.
• Successful continuous recovery of cell mass from both laboratory scale and
commercial scale pilot plant bioreactors.
• A final lyophilized product with all potentials of a good starter culture, to resist
competition with the available imported cultures in the market.
Chapter 2
Literature Review
2.1 MICROBES AND FERMENTATION
The microorganisms are the most successful group of all living species occupying
each habitat in water, soil, plants and animals including humans with enormous success.
This leads to a fundamental impact on all research areas in modern biology and medicine.
Microorganisms are capable of growing on a wide range of substrates and can produce a
remarkable spectrum of products. The advent of in vitro genetic manipulation has
extended the range of products that may be produced by microorganisms and has
provided new methods for increasing the yields of existing ones. The commercial
exploitation of the biochemical diversity of microorganisms has resulted in the
development of the fermentation industry and the techniques of genetic manipulation
have given this well-established industry the opportunity to develop new processes and to
improve existing ones. The term fermentation is derived from the Latin verb fervere, to
boil, which describes the appearance of the action of yeast on extracts of fruit or malted
grain during the production of alcoholic beverages. However, fermentation is interpreted
differently by microbiologists and bio-chemists. To a microbiologist the word means any
process for the production of a product by the mass culture of microorganisms. To a
biochemist, however, the word means an energy-generating process in which organic
compounds act as both electron donors and acceptors, that is, an anaerobic process
where energy is produced without the participation of oxygen or other inorganic
electron acceptors.
2.1.1 Industrial microbiology
Surprisingly enough an estimated 99% of all living microorganisms have not even
been discovered although (or maybe because) they colonies any habitat with great
success. From an evolutionary point of view the microbes show a large degree of
biodiversity, commonly being unified by the feature of their small size.
Biotechnologically designed and employed microorganisms significantly increase the
importance of microbes for applications in food industry, chemistry and pharmacy.
Industrial microbiology is primarily associated with the commercial exploitation of
microorganisms, and involves processes and products that are of major economic,
environmental and social importance throughout the world. There are two key aspects of
industrial microbiology, the first relating to production of valuable microbial products via
fermentation processes. These include traditional fermented foods and beverages, such as
bread, beer, cheese and wine, which have been produced for thousands of years. In
addition, over the last hundred years or so, microorganisms have been further employed
in the production of numerous chemical feed stocks, energy sources, enzymes, food
ingredients and pharmaceuticals. The second aspect is the role of microorganisms in
providing services, particularly for waste treatment and pollution control, which utilizes
their abilities to degrade virtually all natural and man-made products. However, such
activities must be controlled while these materials are in use, otherwise consequent
biodeterioration leads to major economic losses. Over the last twenty years, many
traditional and established industrial fermentation processes have been advanced through
the contribution of genetic engineering (in vitro recombinant DNA technology) which
has also facilitated the development of many novel processes and products. It not only
accelerates strain development, leading to improvement in the production of
microorganisms, but can aid in downstream processing and other elements of the process.
It involved the manipulation of bacteria, cloning in yeasts, filamentous fungi, and plant
and animal cells. Microbial biosurfactants with high ability to reduce surface and
interfacial surface tension and conferring important properties such as emulsification,
detergency, solubilization, lubrication and phase dispersion have a wide range of
potential applications in many industries. Significant interest in these compounds has
been demonstrated by environment, bioremediation, oil, petroleum, food, beverage,
cosmetic and pharmaceutical industries with their low toxicity, biodegradability and
sustainable production technologies. Despite having significant potentials associated with
emulsion formation, stabilization, antiadhesive and antimicrobial activities, significantly
less output and applications have been reported in food industry (Campos et al., 2013).
Enzymes are considered as a potential biocatalyst for a large number of reactions.
Particularly, the microbial enzymes have widespread uses in industries and medicine. In
addition, the microorganisms represent an alternative source of enzymes because they can
be cultured in large quantities in a short time by fermentation and owing to their
biochemical diversity and susceptibility to gene manipulation. Industries are looking for
new microbial strains in order to produce different enzymes to fulfill the current enzyme
requirements (Anbu et al., 2013).
2.1.1.1 Industrial Microbes

The development of industrial microbial processes is gaining unprecedented
momentum. Increased concern for environmental issues and the prospect of declining
petroleum resources has shifted the industrial focus increasingly to microorganisms as
biocatalysts. At the same time systems biology and synthetic biology supply industry and
academia with new tools to design optimal microbial cell factories (Sauer and
Mattanovich, 2012). Microorganisms are used extensively to provide a vast range of
products and services (Table 2.1). They have proved to be particularly useful because of
the ease of their mass cultivation, speed of growth, use of cheap substrates (which in
many cases are wastes) and the diversity of potential products. Their ability to readily
undergo genetic manipulation has also opened up almost limitless further possibilities for
new products and services from the fermentation industries. Most of these
microorganisms, irrespective of their origins, were subsequently modified by
conventional strain improvement strategies, using mutagenesis or breeding programmes,
to improve their properties for industrial use. Several processes developed in the last 20
years have involved recombinant microorganisms and genetic engineering technology to
improve established industrial strains. In most cases, regulatory considerations are of
major importance when choosing microorganisms for industrial use. Fermentation
industries often prefer to use established GRAS (generally regarded as safe)
microorganisms (Table 2.2), particularly for the manufacture of food products and
ingredients.
Table: 2.1 Examples of industrial fermentation products and their producer
microorganisms
Products Bacteria Yeast and Filamentous

fungi
Traditional Products
Bread, beer, wine and Lactic acid bacteria Mainly Saccharomyces
spirits Cheeses, other cerevisiae
dairy products Ripening
of blue and
Camembert-type cheeses Agaricus bisporus,

Fermented meats and Mostly lactic acid Lentinula edodes,
vegetables, Mushrooms, bacteria Aspergillus oryzae,
Soy sauce Zygosaccharomyces rouxii
Sufu (soya bean curd) Acetobacter species Mucor sp.

Vinegar
Agricultural Products
Gibberellins, Fungicides Bacillus thuringiensis, Fusarium moniliforme,
Insecticides, Silage Lactic acid bacteria Coniothyrium minitans
Corynebacterium
Amino acids glutamicum,
l-Glutamine, l-Lysine Brevibacterium
l-Tryptophan lactofermentum,
Klebsiella aerogenes
Enzymes Aspergillus niger
Carbohydrates Kluyveromyces species
a-amylase, b-amylase Bacillus subtilis Kluyveromyces lactis
amyloglucosidase, glucose Streptomyces olivaceus Trichoderma viride
isomerase, invertase, Candida cylindraceae
lactase (b-galactosidase) Aspergillus wentii
Cellulases
Lipases, Pectinases
Clostridium species
Fuels and chemical Clostridium Saccharomyces cerevisiae
feedstock acetobutylicum Zygosaccharomyces rouxii
Acetone, Butanol, Ethanol Zymomonas mobilis
Glycerol, Methane Methanogenic archaeans
Nucleotides Bacillus subtilis,

5¢-Inosine Brevibacterium
monophosphate ammoniagenes
5¢-Guanosine
monophosphate
Organic acids
Acetic Acetobacter xylinum Aspergillus niger
Citric Yarrowia lipolytica
Fumaric, Gluconic , Acetobacter suboxydans, Rhizopus species,

Itaconic
Lactobacillus delbrueckii Aspergillus itaconicus,
Kojic , Lactic
Aspergillus flavus
Pharmaceuticals and Claviceps purpurea,

related compounds Claviceps fusiformis,
Alkaloids Claviceps paspali
Ergotamine, ergometrine,
d-lysergic acid
Antibiotics Streptomyces griseus, Penicillium chrysogenum

Aminoglycosides Streptomyces Acremonium chrysogenum
streptomycin b-Lactams clavuligerus
penicillins, cephalosporins
clavulanic acid
Lantibiotics
Nisin, Macrolides Lactococcus lactis
Erythromycin, Peptides Saccharapolyopora
bacitracin gramicidin erythraea
Tetracyclines, Bacillus licheniformis
chlortetracycline Bacillus brevis
Streptomyces
aureofasciens
Hormones Recombinant
Human growth hormone Recombinant Saccharomyces cerevisiae
Insulin Escherichia coli
Vitamins
B12 (cyanocobalamin) Pseudomonas Blakeslea trispora
b-Carotene (provitamin A) denitrificans Ashbya gossypii
Ascorbic acid (vitamin C) Acetobacter suboxydans
Riboflavin Recombinant Bacillus subtilis
Polymers
Alginates Cellulose Azotobacter vinelandii Aureobasidium pullulans
Dextran, Gellan Acetobacter xylinum Sclerotium rolfsii
Polyhydroxybutyrate Leuconostoc Xanthomonas campestris
Pullulan Scleroglucan mesenteroides
Xanthan Sphingomonas
paucimobilis
Ralstonia eutropha
Single cell protein Methylococcus Candida utilis

capsulatus Fusarium venenatum
Methylophilus Kluyveromyces marxianus
methylotrophus Paecilomyces variotii
Saccharomyces cerevisiae
Immunosuppressants Recombinant Trichoderma polysporum
Cyclosporin Interferon Escherichia coli Recombinant
Steroids Arthrobacter species Saccharomyces cerevisiae
Rhizopus species
Table: 2.2 Examples of microorganisms classified as GRAS (Generally regarded as

safe)
Streptococcus thermophilus
Bacillus subtilis
BACTERIA Lactobacillus bulgaricus
Lactococcus lactis
Leuconostoc oenos
Candida utilis
Kluyveromyces marxianus
YEASTS Kluyveromyces lactis
Saccharomyces cerevisiae
Aspergillus niger
Aspergillus oryzae
FILAMENTOUS FUNGI Mucor javanicus (Mucor circinelloides f.
circinelloides)
Penicillium roqueforti
2.1.2 Fermentation Technology and Microbes
Industrial microbiology came into existence, primarily, based on a naturally

occurring microbiological process called fermentation. Fermentation is a metabolic
process that converts sugar to acids, gases and/or alcohol. Commercially, fermentation is
the process of controlling bacteria, yeast, and moulds to modify food, producing a desired
product. Microbiologists use the term fermentation in two different contexts. First, in
metabolism, fermentation refers to energy-generating processes where organic
compounds act as both electron donor and acceptor. Second, in the context of industrial
microbiology, the term also refers to the growth of large quantities of cells under aerobic
or anaerobic conditions, within a vessel referred to as a fermenter or bioreactor. Apart
from their use for cell cultivation, and for live vegetative cell and spore
biotransformations, similar vessels are used in processes involving cell-free and
immobilized enzyme transformations.
Lactic acid bacteria are the most important bacteria in desirable food fermentations,
being responsible for the fermentation of sour dough bread, sorghum beer, all fermented
milks, and most "pickled" (fermented) vegetables. L. acidophilus, L. bulgaricus, L.
plantarum, L. pentoaceticus, L. brevis and S. thermophilus are examples of lactic acid-
producing bacteria involved in food fermentations. Some of the species are
homofermentative, because they produce lactic acid only, while others are
heterofermentative and produce lactic acid plus other volatile compounds and small
amounts of alcohol. Leuconostoc mesenteroides is a bacterium associated with the
sauerkraut and pickle fermentations. This organism initiates the desirable lactic acid
fermentation in these products. L. mesenteroides produces carbon dioxide and acids
which rapidly lower the pH and inhibits the development of undesirable microorganisms.
The carbon dioxide produced replaces the oxygen, making the environment anaerobic
and suitable for the growth of subsequent species of lactobacillus. Removal of oxygen
also helps to preserve the colour of vegetables and stabilises any ascorbic acid that is
present. Organisms from the Gram-positive Propionibacteriaceae family are responsible
for the flavor and texture of some fermented foods, especially Swiss cheese, where they
are responsible for the formation of 'eyes' or holes in the cheese. These bacteria break
down lactic acid into acetic, propionic acids and carbon dioxide. Several other bacteria,
for instance Leuconostoc citrovorum, Streptococcus lactis and Brevibacterium species are
important in the fermentation of dairy products (Table 2.3). Microorganisms vary in their
optimal pH requirements for growth. Most bacteria favour conditions with a near neutral
pH. The varying pH requirements of different groups of microorganisms are beneficial
for the fermented foods where successions of microorganisms take over from each other
as the pH of the environment changes. Certain bacteria are acid tolerant and will survive
at reduced pH levels. Notable acid-tolerant bacteria include the Lactobacillus and
Streptococcus species playing a key role in the fermentation of dairy and vegetable
products. Most lactic acid bacteria work best at temperatures of 18 to 22 ºC and tolerate
high salt concentrations.
The salt tolerance gives them an advantage over other less tolerant species and
allows the lactic acid fermenters to begin metabolism, which produces acid that further
inhibits the growth of non-desirable organisms. In general, bacteria require a fairly high
water activity (0.9 or higher) to survive. There are a few species which can tolerate water
activities lower than this, but usually the yeasts and fungi will predominate on foods with
a lower water activity. Nearly all food fermentations are the result of more than one
microorganism, either working together or in a sequence. Bacteria from different species
and the various microorganisms – yeast and moulds - all have their own preferences for
growing conditions, which are set within narrow limits. There are very few pure culture
fermentations. An organism that initiates fermentation will grow there until its by-
products inhibit further growth and activity. During this initial growth period, other
organisms develop which are ready to take over when the conditions become intolerable
for the former ones. In general, growth will be initiated by bacteria, followed by yeasts
and then moulds.
The advantages of the use of starter cultures against spontaneous fermentation are
well known and widely spread especially for dairy and meat products, but are not often
used in the vegetable fermentations. The spontaneous fermentation of sauerkraut can
result in the formation of biogenic amines. The utilization of starter culture enables
producers to make food products with a standard quality in a shorter time. Selection of
starter culture, however, should not be only done considering the lactic acid production of
the strains but also their activity for biogenic amine synthesis. Although numerous
studies have been carried out on cabbage, olive and pickle fermentation, little is known
on the lactic fermentation of other vegetables. Usually, lactofermented vegetables are
pasteurized and there is no information on the behavior of lactobacilli during storage of
unpasteurized fermented vegetables. With fermentation of beetroot by appropriately
selected lactobacilli a juice could be produced which combines benefits of betalains and
lactobacilli (Halász et al., 1999).
Non-starter lactic acid bacteria (NSLAB) can be commonly isolated from Cheddar
cheese. The majority of NSLAB are Lactobacillus sp., although Pediococcus and
Leuconostoc sp. also can be present (Peterson and Marshall 1990). According to their
proteolytic activity lactobacilli can contribute to the development of desirable flavours in
cheese but can also cause the accumulation of bitter peptides that result in off-flavours. In
addition NSLAB can cause defects such as gas formation and calcium lactate
crystallization on the surface of the cheese, which forms a white haze. These crystals are
the end result of lactate racemization by some NSLAB that convert L(+) -lactic acid to
the less soluble D(-) isomer. The source of NSLAB contamination is believed to be
originated from post-pasteurization, usually through contact with equipment surfaces or
from the air.
Table: 2.3 Examples of aseptic and non-aseptic fermentations
ASEPTIC NON ASEPTIC
AEROBIC ANAEROBIC AEROBIC ANAEROBIC
Animal and plant Acetone Acetification of Alcoholic

cell cultures,
ethanol in vinegar beverages; beer,
Alkaloids, Amino Butanol
acids, Most production, wine etc, Primary
antibiotics, Most
Ethanol Ripening of some dairy fermentations,
biomass (SCP)
production, Most cheeses, Mushroom Silage production,
enzymes, Most Glycerol
production, Aerobic Anaerobic waste-
organic acids
rDNA, proteins, Lactic acid waste-water water treatment
Steroid
treatment
biotransformations Some toxins
Some toxins
Most vaccines
Most vitamins
Xanthan gum.
2.2 LACTIC ACID BACTERIA (LAB)
LAB are heterogeneous group of bacteria contributing to various sectors of food,

beverage, health tonics and pharmaceutical industries worldwide. All the member of the
group shares a common property which is their gram-positive appearance and can be
differentiated on their physiology and the mode of metabolic pathway they choose; either
homo-fermentative or hetero-fermentative. This group on fermentation produces various
anti-microbial substances that promote their activity of health modulation and many
organic compounds producing flavors and aromas in the fermented food products.
Lactic acid bacteria (LAB) are regarded as a major group of probiotics (Sharma et
al., 2012; Schrezenmeir and de Vrese 2001). These lactic acid bacteria are industrially
important organisms recognised for their fermentative ability as their health and
nutritional benefits. They are comprised of an ecologically diverse group of
microorganisms united by formation of lactic acid as the primary metabolite of sugar
metabolism (Carr et al. 2002). These bacteria are basically Gram-positive non-spore
forming cocci, cocci-bacilli or rods, non-rispiring, catalase-negative bacteria that are
devoid of cytochromes and are of non-aerobic habit but are aero-tolerant, fastidious, acid
tolerant and strictly fermentative; lactic acid is the major end-product of sugar
fermentation. They are chemo-organotrophic and grow in complex media and generally
are non pathogenic to man and animals. LAB consist of several genera, which include
Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc,
Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus
and Weissella (Ercolini et al. 2001; Holzapfel et al. 2001). Based on similarities in
physiology, metabolism and nutritional needs, these genera are grouped together. A
primary similarity is that all members produce lactic acid as a major or virtually sole end
product of the fermentation of sugars.
LAB were first isolated from milk (Carr et al. 2002) and have since been found in
such foods and fermented products as meat, milk products, vegetables, beverages and
bakery products (Liu 2003; O’Sullivan et al. 2002). These bacteria occur naturally in
fermented food and have been detected in soil, water, manure and sewage (Holzapfel et
al. 2001). LAB exist in human (Martin et al. 2003; Schrezenmeir and de Vrese 2001) and
in animal. However, some lactic acid bacteria are part of the oral flora which can cause
dental caries (Sbordone and Bortolaia 2003). LAB can work as spoilage organisms in
foods such as meat, fish and beverages (Liu 2003). Several lactobacilli, lactococci and
bifidobacteria are held to be health-benefiting bacteria (Rolfe 2000; Tuohy et al. 2003),
but little is known about the probiotic mechanisms of gut microbiota (Gibson and Fuller
2000). LAB constitute an integral part of the healthy gastrointestinal (GI) microecology
and are involved in the host metabolism and Streptococcus thermophilus, inhibit food
spoilage and pathogenic bacteria and preserve the nutritive qualities of raw food material
for an extended shelf life (O’Sullivan et al. 2002; Heller 2001). The taxonomy of LAB
based on comperative 16S ribosomal RNA (rRNA) sequencing analysis has revealed that
some taxa generated on the basis on phenotypic features do not correspond with the
phylogenetic relations. Molecular techniques, especially polymerase chain reaction
(PCR) based methods, such as rep-PCR fingerprinting and restriction fragment length
polymorphism (RFLP) as well as pulse-field gel electrophoresis (PFGE), are regarded
important for specific characterization and detection of LAB strains (Gevers et al., 2001;
Holzapfel et al., 2001). Recently, culture-independent approaches have been applied for
the detection of intestinal microbiota (Zoetendal et al., 2002). Denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) analysis of
faecal 16S rDNA gene and its rRNA amplicons have shown to be powerful approaches in
determining and monitoring the bacterial community.
2.2.1 Classification of Lactic acid bacteria
The term Lactic Acid Bacteria (LAB) was gradually accepted in the beginning of
the 20th century (Carol et. al., 2010). In earlier times some other terms were used for
lactic acid bacteria like milk sourcing and lactic acid producing which created confusion.
All the confusion came to an end in 1919 when Orla-Jensen proposed a monograph about
lactic acid bacteria having great impact over systematic lactic acid bacteria (Axelsson,
1989). Classification of LAB genera was based on morphology, mode of glucose
fermentation, growth at certain temperatures, and range of sugar utilization and
configuration of the lactic acid produced, ability to grow at high salt concentrations, and
acid or alkaline tolerance. Even though the taxonomy has been revised since then,
characters used by Orla-Jensen are still very important in current classification of LAB.
Lactic acid bacteria constitute a group of bacteria that have morphological, metabolic and
physiological similarities with relatively closely related phylogeny. For some of the
newly described genera (Pilar et. al., 2008), additional characteristics such as fatty acid
composition and motility are used in classification.
The measurements of true phylogenetic relationship with rRNA sequencing have

aided the classification of lactic acid bacteria and clarified the phylogeny of the group
(Figure 2.1). Most genera in the group form phylogenetically distinct group, but some, in
particular Lactobacillus and Leuconostoc are heterogeneous and the phylogenetic cluster
do not correlate with the current classification based on phenotypic characters. New tools
for classification and identification of lactic acid bacteria are underway (Sascha and
Magdalena 2010). The most promising for routine used are nucleic acid probing
techniques, partial rRNA gene sequencing using the polymerase chain reaction, and
soluble protein patterns. The growth is optimum at pH 5.5-5.8 and the organisms have
complex nutritional requirements for amino acids, peptides, nucleotide bases, vitamins,
minerals, fatty acids and carbohydrates. Orla-Jensen used morphology (cocci or rods,
tetrad formation), mode of glucose fermentation (homo- or heterofermentation), growth
at certain “cardinal” temperatures (e.g., 10ºC and 45ºC), and form of lactic acid produced
(D, L, or both) (Kenji et al., 2009), as the basis for classifying them.
Table: 2.4 Differentiating Lactic acid bacteria and a comparison with current
taxonomic classification
These characters are still very important in current lactic acid bacterial
classification (Figure 2.2). The general description of the bacteria within the group is
gram-positive, nonsporulating, non-respiring cocci or rods, which do, through
fermentation of carbohydrates, produce lactic acid as their major end product. The
common agreement is that there is a core group consisting of four genera; Lactobacillus,
Leuconostoc, Pediococcus and Streptococcus (Table 2.4). The taxonomic revisions have
proposed several new genera and the remaining group now comprises the following:
Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Enterococcus,
Globicatella, Lactococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weissella.
Lactobacilli, Carnobacteria and some Weissella are rods while the remaining genera are
cocci (Jin et al., 2009).
Figure 2.1: Consensus tree, based on comparative sequence analysis of 16S rRNA,
showing major phylogenetic groups of lactic acid bacteria with low
mol% guanine plus cytosine in the DNA and the nonrelated gram-
positive genera Bifidobacterium and Propionibacterium. (Schleifer and
Ludwig, 1995).
Lactic acid bacteria lack the ability to synthesize cytochromes and porphyrins
(components of respiratory chains) and therefore cannot generate ATP by creation of a
proton gradient. The LAB can only obtain ATP by fermentation, usually of sugars. Since
they do not use oxygen in their energy production, lactic acid bacteria happily grow
under anaerobic conditions.
Figure 2.2: Characterization of Lactic acid bacteria
2.2.2 Biochemistry and genetics of lactic acid bacteria
Lactic acid bacteria are chemotrophic; they find the energy required for their
entire metabolism from the oxidation of chemical compounds. The oxidation of sugars
constitutes the principle energy producing pathway. Lactic acid bacteria of the genera
Lactobacillus, Leuconostoc and Pediococcus, the important bacteria to winemaking,
assimilate sugars by either a homofermentative or heterofermentative pathway.
2.2.2.1 Homo-fermentative metabolism
Homo-fermentative bacteria ferment glucose with lactic acid as the primary by-
product. Homofermentative LAB includes Lactococcus sp., used in dairy starter culture
applications where the rapid development of lactic acid and reduced pH is desirable.
Other homofermentative LAB include yogurt strains consisting of rods (Lactobacillus
delbruckii subsp. bulgaricus, L. acidophilus) and cocci (Streptococcus salivarius subsp.
thermophilus) and thermophilic strains that might be used in cheese (L. helveticus).
Other homofermentative cocci that might be found in milk and dairy products, but are
rarely used as starter cultures include other Streptococcus sp., Enterococcus, Pediococcus
and Aerococcus.Lactic acid bacteria utilize sugars (glucose) to form lactic acid by either
the homo- or hetero-fermentative pathway. The homo-fermentative pathway (Figure 2.3)
results in the transformation of glucose to pyruvate through the Embden–Meyerhof–
Parnas pathway (EMP, or glycolysis), eventually yielding lactic acid. NADH produced
by the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate is reoxidized
to NAD+ in the formation of lactate from pyruvate through the action of lactate
dehydrogenases (LDHs). The LDH enzymes vary in their stereospecificity and can yield
d- or l-lactic acid or the racemic mixture.
2.2.2.2 Hetero-fermentative metabolism
Hetero-fermentors, ferment glucose with lactic acid, ethanol/acetic acid and
carbon dioxide (CO2) as by-products. Testing for heterofermentative fermentation
generally involves the detection of gas (CO2). With the exception of certain fermented
milk products, hetero-fermentative LAB are rarely used as dairy starter cultures, although
they are not uncommon in milk and dairy products. If allowed to grow to significant
numbers, they can cause defects related to their acid and CO2 production, such as slits in
hard cheeses or bloated packaging in other dairy products. Hetero-fermentative LAB
includes Leuconostoc sp. (Gram-positive cocci) and Gram-positive rods such as
Lactobacillus brevis, L. fermentum, and L. reuteri. Other Lactobacillus species are
considered “facultatively” hetero-fermentative, meaning they will produce CO2 and other
by-products only under certain conditions or from specific substrates. These strains
would include L. plantarum, L. casei and L. curvatus. Other hetero-fermentors like
Oenococcus oeni (known as Leuconostoc oeni until 1995) L. brevis, L. hilgardii,
L.fructivorans, and L. kunkeei) lack aldolase and must divert the flow of carbon through a
different series of reactions, the pentose phosphate, or phosphoketolase, pathway (Figure
2.4).
Figure 2.3: Homo-fermentative pathway illustrating the production of lactic acid
from glucose.
Figure 2.4: Hetero-fermentative pathway showing the production of lactic acid,
carbon-dioxide and either ethanol or acetic acid.
From 1 mole of glucose, hetero-fermentative bacteria produce 1 mole each of

lactate, CO2, and either acetic acid or ethanol. In reality, these bacteria produce 0.8 mole
lactate from glucose. Unlike homofermentative microorganisms, these bacteria do not
have aldolase but possess phosphoketolase, the enzyme responsible for the cleavage of
xylulose-5-phosphate to form glyceraldehyde-3-phosphate and acetyl phosphate. Due to
the biosynthesis of five-carbon sugars in this pathway (ribulose-5-phosphate and
xylulose-5-phosphate), some strains can utilize the pentose present in wine such as
ribose, xylose, and arabinose. An important consequence of only half of the carbon from
glucose going to glyceraldehyde-3-phosphate is formation of only 1 mole of ATP per
mole glucose. However, hetero-fermentative bacteria can gain additional energy though
conversion of acetyl-phosphate to acetate.
2.2.2.3 Genetics
Currently, more than 19 complete genomes of streptococci are available, covering
different strains of five species. A program aimed at extensive sequencing of the genomes
of non-pathogenic LAB was announced in 2002 by the Lactic Acid Bacteria Genome
Sequencing Consortium (Klaenhammer et al., 2002), but the actual breakthrough
occurred only in the last 6 years (2005 and 2006). The Lactobacillales have relatively
small genomes for non-obligatory bacterial parasites or symbionts (characteristic genome
size, ~2 megabases, with ~ 2,000 genes), with the number of genes in different species
spanning the range from ~1,600 to ~3,000. This variation in the number of genes suggests
that the evolution of LAB involved active processes of gene loss, duplication, and
acquisition. The current collection of LAB genomes is a unique data set that includes
multiple related genomes with a gradient of divergence in sequences and genome
organizations. This set of related genomes is amenable to detailed reconstruction of
genome evolution, which is not yet attainable with other groups of bacteria.
Divergence of Lactobacillales from their ancestor in the Bacilli was marked by

the loss of 600–1200 genes, including many genes encoding biosynthetic enzymes. Other
losses include genes related to sporulation, a function that is seemingly unnecessary in
nutrient-rich food environments (Makarova and Koonin, 2007). Besides gene losses
occurring early in the lineage of the LAB, more recent events have contributed to shaping
these species, including parallel losses in genes involved in various metabolic processes.
The most notable example of gene loss occurred in Streptococcus thermophilus, which
diverged from pathogenic Streptococcus species through the loss and decay of virulence-
associated genes, such as those involved in antibiotic resistance and adhesion. This
genomic record has thus far provided solid evidence supporting the ‘generally recognized
as safe’ status for use of S. thermophilus in foods.
Gene gains in the LAB also reflected a shift toward a nutrient-rich lifestyle during
specific niche adaptations. Soon after the divergence of the Lactobacillales, there
occurred duplication of genes involved in the transport and metabolism of carbohydrates,
including genes for enolases and phosphotransferase (PTS) systems. Genes involved in
amino acid transport and peptidases were also duplicated, further enhancing the ability of
these species to exploit protein-rich environments (Makarova and Koonin, 2007).
Horizontal gene transfer (HGT) has also shaped these genomes. For example, many sugar
transport and metabolism genes in Lactobacillus plantarum are clustered in a lower GC
content area of the genome, and it is possible that many of these genes were acquired as a
result of HGT (Kleerebezem et al. 2003). HGT has also shaped the genome of S.
thermophilus, which possesses a 17-kb region that contains extensive identity with genes
in L. lactis and L. bulgaricus subsp. delbrueckii, two species that are also associated with
growth in milk. The genes from L. bulgaricus enable S. thermophilus to synthesize
methionine, which is rare in milk.
Genome sequencing has shown that the metabolic capabilities of these two
organisms make them reliant on each other for maximum growth. For example, L.
bulgaricus encodes a complete folate biosynthesis pathway, but lacks the ability to
produce p-aminobenzoic acid (PABA), a key intermediate that is supplied by S.
thermophilus (van de Guchte et al. 2006). In addition, an exchange of polyamines might
occur between these organisms that could have a role in their oxidative stress tolerance.
A major difference in the genomes of L. bulgaricus and S. thermophilus is reflected in
their biosynthetic capabilities. The presence of an extracellular protease in L. bulgaricus,
but the absence of many amino acid biosynthesis pathways, reflects the adaptation of this
species to the protein-rich milk environment. By contrast, S. thermophilus has retained
the pathways to synthesize all amino acids except histidine. It is unclear if S.
thermophilus exploits the proteolytic capabilities of L. bulgaricus or retains some
advantage in synthesizing its own amino acids (van de Guchte et al. 2006).
Application of molecular genetic techniques to determine the relatedness of food-

associated lactic acid bacteria has resulted in significant changes in their taxonomic
classification. During the 1980s the genus Streptococcus was separated into the three
genera Enterococcus, Lactococcus and Streptococcus.
Figure 2.5: Phylogenetic tree and important commercial importance of Lactic
acid bacteria
The lactic acid bacteria associated with foods now include species of the genera
Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus,
Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus
Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging
from 33 to 55), of which about one-third are strictly heterofermentative. However, many
changes have been made and reorganization of the genus along lines that do not follow
previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus
is being studied (Figure 2.5). Phylogenetically belonging to the Actinomyces branch of
the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on
account of its greater than 50 mol% G+C content. It is therefore no longer considered one
of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the
bacteria.
The new genus Weissella has been established to include one member of the
genus Leuconostoc (L. paramesenteroides) and heterofermentative lactobacilli with
unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of
the streptococci, morphological and physiological features of Weissella do not directly
support this grouping which now incorporates species that produce D(-)- as well as DL-
lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but
it shares some physiological similarities (growth at pH 9.5) and a common phylogenetic
branch with the genus Enterococcus (Stiles and Holzapfel, 1997).
2.2.3 Natural Habitat of Lactic acid bacteria
LAB can be isolated from various natural sources. For example, Lactococci,
Streptococci, Lactobacillus can be found in milk and milk products. Studies revealed the
presence of lactic acid bacteria in goat, cow, sheep, buffalo and camel milk (Sharma et
al., 2013). Recent studies suggest that composite bio-waste is a preferred habitat of lactic
acid bacteria, suggesting that the unsterilized bio-waste and its natural flora could be used
in a fermentation process for lactic acid production (Probst et al., 2013). The sequencing
of multiple complete genomes has created unprecedented opportunities for evolutionary
genomics of LAB. Some of the genes acquired by Lactobacillales are clearly adaptations
to existence in the nutrient-rich habitats of these bacteria (Makarova and Koonin, 2007).
Barley is also a natural habitat for lactic acid bacteria. The numbers of LAB on barley
have been shown to increase as a result of the malting process during brewing, possibly
because the steeping conditions create a favorable environment for their growth.
However, LAB counts start to decrease during barley germination and continue to decline
throughout the kilning process (Rouse et al., 2008). Diversity and density of lactic acid
isolated from Algerian raw goats' milk in arid zones were studied by determination of
morphological, cultural, physiological and biochemical characteristics. Two hundred and
six lactic acid bacterial strains were isolated, 115 of them belonging to lactic acid cocci
and others to the genus Lactobacillus. The representative species of the total cocci were
Lactococcus sp. (76.16%), S. thermophilus (14.78%) and Leuconostoc sp. (8.6%),
respectively. The dominating species is L. lactis subsp. lactis. Lactobacilli species found
in local raw goats' milk and their proportion were: L. curvatus (25.25%), L. helviticus
(10.98%), L. plantarum (9.89%), L. reuteri (9.89%), L. casei (7.69%), L. brevis (5.49%),
L. bulgaricus (5.49%), L. paracasei (4.39%) and L. acidophilus (2.19%) (Badis et al.,
2004). It has also been reported that some lactic acid bacteria isolated from the
gastrointestinal tract of fish can act as probiotic (Olympia et al., 1995). The variation of
the ecological parameters acting on the microbial association such as the nature of cereal,
temperature, size of inoculum, and length of propagation intervals leads in each case to a
characteristic species association. Cereals are suitable fermentable substrates for the
growth of potentially probiotic microorganisms. Previous studies showed that four
potentially probiotic strains (Lactobacillus fermentum, L. reuteri, L. acidophilus and L.
plantarum) were cultured in malt, barley and wheat media. All strains attained high cell
log10
populations (8.1-10.1 cfu/g). The malt medium supported the growth of all strains
more than barley and wheat media due to its chemical composition, while L. plantarum
and L. fermentum appeared to be less fastidious and more resistant to acidic conditions
than L. acidophilus and L. reuteri (Talamond et al., 2002). Another study showed that the
natural sour cassava starch fermentation was mainly due to the action of lactic acid
bacteria. Fermentation temperature and duration as well as the composition of the
microflora influenced the expansion properties of the final cassava sour starch. However,
some LAB strains (such as L. lactis, Streptococcus sp., Enterococcus saccharolyticu, L.
plantarum, and L. mesenteroides) involved in the natural sour cassava starch
fermentation were isolated, identified and characterized using classical microbiological
techniques (Ampe et al., 2001).
2.2.4 Lactic acid bacteria in food products
LAB has a long tradition of use in the food industry, and the number and diversity
of their applications has increased considerably over the years. These are the most
important group of microorganisms used in the food industry for the production of
various fermented products, such as yogurts, cheese, and pickled vegetables (Figure 2.6).
In addition, LAB can inhibit the growth of spoilage microbes and/or pathogens in their
environment by lowering the pH and/or through the production of antimicrobial peptides,
called bacteriocins. Both LAB and Bifidobacteria are also thought to have health-
promoting abilities and many are used as probiotics for the prevention, alleviation, and
treatment of intestinal disorders in humans and animals. LAB is very important in the
food and dairy industries because lactic acid and other organic acids produced by these
bacteria act as natural preservatives as well as flavor enhancers. LAB find increasing
acceptance as probiotic which aid in stimulating immune responses, preventing infection
by enteropathogenic bacteria, and treating and preventing diarrhea. Fermented foods
constitute a substantial part of the diet in many African countries are considered as an
important means of preserving and introducing variety into the diet, which often consists
of staple foods such as milk, cassava, fish and cereals. For example, Ben saalga is a
traditional Burkinabè gruel obtained by cooking a diluted fermented paste of pearl millet
(Pennisetum glaucum). This fermented food is widely accepted and consumed by the
population, particularly by young children.
The processing of pearl millet into ben saalga comprises the following successive
main steps: soaking the grains (first fermentation), grinding and 13 filtration of humid
flour, decanting (second fermentation) and cooking (Sanni et al., 2002). The production
of this fermented food is still largely a traditional art associated with poor hygiene,
inconsistent quality presentation and short shelf life. The preparation of this indigenous
food generally depends on a spontaneous or chance inoculation by naturally occurring
lactic acid bacteria and the use of starter cultures is still at very early development stages.
LAB play an essential role in the majority of food fermentations, and a wide variety of
strains are routinely employed as starter cultures in the manufacture of dairy, meat, and
vegetable and bakery products. One of the most important contributions of these
microorganisms is the extended shelf life of the fermented product by comparison to the
raw substrate.
Figure: 2.6 Silent features of lactic acid bacteria
Growth of spoilage and pathogenic bacteria in these foods is inhibited due to

competition for nutrients and the presence of starter-derived inhibitors such as lactic acid,
hydrogen peroxide and bacteriocins. Bacteriocins are heterogeneous group of
antibacterial proteins that vary in spectrum of activity, mode of action, molecular weight,
genetic origin and biochemical properties (Lee, 2005). Currently, artificial chemical
preservatives are employed to limit the number of microorganisms capable of growing
within foods, but increasing consumer awareness of potential health risks associated with
some of these substances has led researchers to examine the possibility of using
bacteriocins produced by LAB as biopreservatives as well as the application of
bacteriocinogenic LAB in starter cultures. According to the generally poor sanitary
conditions of ben saalga and other traditional fermented foods, the use of selected
bacteriocinogenic LAB with antimicrobial activity against the most frequent foodborne
pathogenic bacteria could be an affordable way to improve the safety of these fermented
foods. For examples, a total of 14,020 lactic acid bacteria (LAB) are isolated from Nham
and two traditional Indonesian fermented foods “Tapai” (fermented tapioca), and
“Tempoyak” (fermented durian flesh). Chilli puree and fresh goat’s milk are used as
sources 14 for the isolation of lactic acid bacteria (LAB), and the total amount of 126
isolates are obtained (Visessanguan et al., 2006).
In many societies including our own where yogurt has been heralded as a health
food since the 19th century, fermented food has gained a reputation for its beneficial
effects on immunity, intestinal health and general well-being. Modern researchers are just
beginning to understand what the sages of old were tuned in to: fermented food conveys
clear and calculable health benefits to the human diet. Lactic acid fermentation in and of
itself enhances the micronutrient profile of several foods (Table 2.5). For example, milk
that undergoes lactic acid fermentation either in the wild as in the case of clabbered milk
or inoculated by a starter culture as in the case of yogurt, piima, matsoni and other
fermented dairy products conveys more vitamins to the eater in comparison to raw milk
and, particularly, pasteurized and ultra-high-temperature pasteurized milk.
Table: 2.5 Some of the major fermented food products along with the microbe
involved around the world.
NAME OF INGREDIENT MICROBE INVOLVED COUNTRY

FOOD
Beer Barley Saccharomyces cerevisiae, Worldwide
Leuconostoc, Lactobacillus,
Lactococcus etc
Cheese Milk Lactic acid bacteria Worldwide
Dahi Milk Lactic acid bacteria India
Dawadawa Locust beans Bacillus, Staphylococcus Africa
Kefir Milk Streptococcus, Lactobacillus Eastern
and Leuconostoc sp, Candida Europe
kefyr, Kluyveromyces fragilis.
Koko Maize, Shorgum Lactic acid bacteria Ghana
Mahewu Maize L lactis, Lactobacillus sp South Africa
Gari Cassava Leuconostoc, Alcaligenes, Nigeria
Corynebacterium, Lactobacillus
I-sushi Fish Lactic acid Bacteria Japan
Kaanga piro Maize Lactic acid bacteria New Zealand
Kimchi Vegetables L. mesenteroides, L brevis, L Korea
plantarum
Idli/Doas Rice & Black gram L. mesenteroides, E. faecalis, India
yeast
Salami Meat Lactic acid bacteria Worldwide
Yogurt Milk L.bulgaricus and S. Worldwide
thermophilus
Soy sauce Soy beans Lactic acid bacteria South Asia
Sauerkraut Cabbage Lactic acid bacteria Europe,
North
America
Trahanas Milk & wheat Lactic acid Bacteria Greece
Tempeh Soy Bean Lactic acid Bacteria Indonesia
Palm Wine Palm Sap Lactic acid Bacteria Worldwide
Nono Milk Lactic acid Bacteria Nigeria
Ogi Maize L. plantarum, Corynebacterium Nigeria
sp,Yeast
Nam Pork, Garlic, rice P. cerevisiae, L plantarum, L Thailand
brevis
Lambic Beer Barley Yeast and Lactic acid bacteria Belgium
Poi Taro Lactic acid bacteria Hawaii
Injera Tef L. mesenteroides, P. cerevisiae, Euthopia
L. plantarum, S. cerevisiae
2.2.5 Lactic acid bacteria in agricultural products

The major genera found in the microflora of fermented or sour cassava-starch
were Streptococcus, Bacillus, Lactobacillus and Saccharomyces with amylase activity
(Lacerda et al., 2005). Lactic acid bacteria predominated whereas the presence of moulds
was not significant. Traditional fermentation of cassava is dominated by a lactic acid
bacteria population. A total of 139 predominant strains isolated from fermenting cassava
were identified using phenotypic tests and genotypic methods. Moreover, fermented
tapioca was used as sources for the isolation of lactic acid bacteria. A total of 126 isolates
were obtained. In addition, by sequential screening for catalase activity and Gram-
staining, 55 were determined to be LAB, out of which 16 were established to be homo-
fermentative. Moreover, Thailand is the world’s largest exporter of tapioca starch and
starch derivatives with annual production of over 2 million tons of starch.
Development of lactic acid production using cassava as the main substrate is very
attractive because it is a cheap source, contains high starch content with low quantity of
impurities, and also abundant in Nakhon Ratchasima province (Tonukari, 2004).
2.2.6 Lactic acid bacteria as Probiotics
Probiotics can be defined as a food (feed) or drug containing live microbes, when
ingested, is expected to confer beneficial physiologic effects to the host animal through
microbial actions. Microbial components and metabolites are essentially excluded from
the definition of probiotics. Microbes used in probiotics should be able to express their
activities in the host body. The first consideration is the bacteria that normally inhabit the
intestinal tract, and ingestion of these bacteria may affect the intestinal microbial balance.
The human digestive tract is inhabited by numerous microbes. For bacteria to exert any
probiotic effect, they have to be able to survive both the stomach acids (pH as low as 1.5)
and the bile acids (pH as low as 2). This is true of most lactobacilli. Secondly, the
bacteria must arrive in the intestines in sufficient quantities to have an effect. The amount
required depends on the strain and the health benefit being studied. The minimum
effective level for individual bacteria and specific health benefits is not yet established.
The bacteria may need to adhere to the wall of the intestine (“implant”) and colonize in
order for there to be an effect. Sherwood Gorbach, one of the discoverers of
Lactobacillus GG, states, “Our research over the previous 20 years had established
beyond doubt that implantation in the gut was the critical feature that a strain must
possess to influence the intestinal mili” (Gorbach, 1996). However, others contend that
continuous transit (continually eating a prebiotic food) is an alternative to the organism
implanting and colonizing (Saloff-Coste, 1997). Finally, the bacteria must show some
beneficial effects on human health. Some examples of beneficial effects under
investigation include alleviation of lactose intolerance, prevention and treatment of
diarrhea, maintenance of normal intestinal flora, antagonism against pathogens, and
stimulation of the immune system, anti-carcinogenic activity, and reduction of serum
cholesterol levels.
Table 2.6: Differences between Probiotics, Prebiotics and Synbiotics
PROBIOTICS PREBIOTICS SYNBIOTICS

Live microbial food A non-digestible food A combination a probiotic and
supplement having ingredient stimulating the probiotic again with beneficial
beneficial effects on available microbes in colon effects
host through microbial resulting beneficial effects
actions
It should be non- It should neither be This combination should have
pathogenic, non-toxic, hydrolyzed nor absorbed in the capability to increase the
capable of surviving in the upper part of survival chances of probiotic,
the gut, and should gastrointestinal tract.
contain a large number
of viable cells.
Should confer various Prebiotics should be able Synbiotics show a synergistic
postulated health as a consequence to alter effect, as probiotics acts in
advantages like the colonic micro-flora small intestine and prebiotics
improved digestion, towards a healthier acts in large intestine, so
anti-cancerous, lactose composition. synbiotics acts symbiosis.
intolerance and various
nutritional benefits.
Example: Lactic acid Example: FOS Example: Bifidobacteria +
bacteria (oligofructose and FOS, Lactobacilli + Lactitol,
neosugar), Inulin, Bifidobacteria + GOS.
Lactulose, Lactitol.
The balance of this microbial flora greatly influences the intestinal environment.
Among the numerous intestinal microbes, those that are expected to beneficially affect
the host by improving the intestinal microbial balance, and hence are selected as
probiotics, include species of the genera Lactobacillus, Bifidobacterium, and
Enterococcus. The representative species include Lactobacillus acidophilus,
Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus
rhamnosus, Lactobacillus plantarum, Bifidobacterium longum, Bifidobacterium breve,
Bifidobacterium bifidum, Bifidobacterium infantis, Enterococcus faecalis, and
Enterocuccus faecium. Bifidobacterium species that specifically inhabit the intestinal
tracts of animals, such as Bifidobacterium thermophilum and Bifidobacterium
pseudolongum, are used in animal probiotics. Some bacteria that do not normally inhabit
the intestinal tract may also come under the category of probiotics. They are used as
starters in dairy products and include mainly Lactobacillus bulgaricus, Streptococcus
thermophilus, Leuconostoc and Lactococcus species (Sharma et al., 2012). Lactic Acid
bacteria are beneficial for both human and animals because of various properties which
directly or indirectly enhance the immune system and help us to fight against various
infectious diseases. This group of bacteria have been placed among the best human
friendly microbe which not only reduce the effect of infections and kill pathogens; they
even modulate our immune system and reduce hypertension.
In some countries the use of Enterococcus sp. as a probiotic has been questioned
because of safety aspects with regard to transfer of genes conferring antibiotic resistance.
Most scientists agree that probiotic strains shall be able to survive transit through the
gastric acid environment as well as exposure to bile and pancreatic juice in the upper
small intestine to be able to exert beneficial effects in the lower small intestine and the
colon, although there are convincing data on beneficial immunological effects also from
dead cells (Mottet and Michetti, 2005). Best effect is achieved if the microorganisms
colonise the intestinal surface mucus layer since they then can affect the intestinal
immune system, displace enteric pathogens, provide antioxidants and antimutagens, and
possibly other effects by cell signalling. The intake of LAB influences multiple systems
was elegantly shown for Lactobacillus GG using microarray analysis (Di Caro et al.,
2005). One month treatment resulted in up-regulation of 334 genes and down-regulation
of 92 genes involved in inflammation, apoptosis, cell-cell signalling, cell adhesion and
differentiation and signal transcription and transduction. In recent years, multiple reports
have described beneficial effects from various aspects on important diseases, like
intestinal infections, inflammatory bowel disease (IBD), and allergy by addition of
selected strains to food products, often together with fiber or a prebiotic substance. In
many countries, there are now several probiotic products on the market but the
documentation is often based upon case reports, animal studies or uncontrolled small
clinical trials.
Furthermore, there is no general acceptance on how to characterize prebiotic
microorganisms, and few products declare the actual content of live microorganisms.
There are various health modulating properties of the bacteria some most
effective and commonly known are:-
• Immunomodulation
• Anti-cancerous
• Bio-therapeutic
• Anti-inflammatory
• Prevention from diarrhoea
• Hypocholesterolemic effects
• As Live Vaccine
• Anti-viral properties.
An impressive number of reviews and books have reported the constantly
evolving knowledge and state of the art in lactic acid bacteria. The various health benefits
are summarized in short are as under: -
2.2.6.1 Immunomodulation
The lactic acid bacteria produces some extracellular polysaccharides
(EPS) associated with rheology, mouthfeel and texture of fermented milk products. These
extracellular polysaccharides from Lactobacillus bulgaricus purified from the supernatant
have certain immuomodulatory activities shown in case of mice. The yoghurt containing
immunostimulative EPS would have an immunomodulatory effect on human body
(Makino et al. 2006). A halophilic lactic acid bacterium, Tetragenococcus halophilus,
was found to possess an immunomodulatory activity that promotes T helper type 1 (Th1)
immunity in addition to its important roles in soy sauce brewing (Masuda et al. 2008).
Oncococcus oeni and Pediococcus parvulus also found to have immune-stimulatory
activities as the strains found to stimulate cytokines released by peripheral blood
mononuclear cells (Foligne et al. 2010). Studies on the relationship between nutritional
food and immune-modulation have been increased based on the hypothesis that
consumption of some foods create a barrier for immunological diseases (Sandre et al.
2001). Gut microflora participates in immune exclusion. It prevents other bacteria from
adhering by competition for nutrients and places of adhesion, it produces anti-bacterial
agents, and it stimulates the production of specific antibodies (Premalatha and
Dhasarathan 2011). There are many reports on the immunomodulatory activities of lactic
acid bacteria (Wells 2011; Izumo et al. 2011; Foligne et al. 2010). Probiotics can
influence the microflora composition by increasing the number of Lactobacilli and other
anaerobes (Salminen et al. 1998). Dietary supply of probiotic bacteria stimulates IgA
immune response (Kaila et al. 1992) and the transport of target antigens through Peyer’s
patches (Isolauri et al. 1993).
2.2.6.2 Anti-Cancerous
Lactic acid bacteria play an important role in retarding colon
carcinogenesis by possibly influencing metabolic, immunologic, and protective functions
in the colon (Roberfroid et al. 1995). Concentrations of LAB may increase in the colon
after the consumption of foods containing probiotics; however, probiotic ingestion also
increases the number and metabolic activity of LAB in the colon of humans and animals
(Salminen and Salminen, 1997). In animals, LAB ingestion was shown to prevent
carcinogen-induced preneoplastic lesions and tumors (Rowland et al. 1998). A reduced
activity of pro-carcinogenic enzymes in humans also was shown as a consequence of
prebiotic intake. However, in humans, there is no evidence available on whether
probiotics and prebiotics can prevent the initiation of colon cancer. Epidemiologic studies
are contradictory; some studies could not find an association between the consumption of
fermented-milk products and the risk of colon cancer, whereas other studies showed a
lower incidence of colon cancer in persons consuming fermented- milk products or
yogurt. In spite of various controversies, lactic acid bacteria have been shown to affect
intestinal barrier interfering the metabolic activity of tumor cells preventing and treating a
variety of cancers (Qi-Wei et al. 2011).
2.2.6.3 Bio therapeutic

Lactic acid bacteria supplements are becoming more and more popular all
over the world in which a live food microbial food ingredient is ingested found to be very
beneficial to human health. Streptococcus thermophilus is found to be potentially
therapeutic against the associated chronic gastritis (ASA) (Rodriguez et al. 2010).
Gastritis is a common disorder in which discontinuity of the gastric mucosa is observed.
It is caused by various factors like excess alcohol, infection, intensive consumption of
anti-inflammatory drugs with Helicobacter pylori or may be stress. Also, ASA affects
various mucosal defense lines such as bicarbonate secretion, mucus synthesis, and
decrease of mucosal blood flow. The first therapeutic effect of the fermented milk with
the polymer producing strain of S. thermophilus on chronic gastritis induced by ASA was
experimented in mice. It was able to generate immune response in mice and increased the
thickness of the gastric mucus gel layer. Studies suggest that recombinant lactic acid
bacteria are the excellent candidates for the production of various bio therapeutic proteins
and also their delivery to specific places of requirement within the gastrointestinal tract
(Daniel et al. 2011).
Figure 2.7: Various applications of Lactic acid bacteria modulating human

health
2.2.6.4 Anti-inflammatory
The genus Lactobacillus includes a restricted set of intestine-indigenous

species from a pool of more than 100 Lactobacillus species and studies suggest that some
strains of the species have potent anti-inflammatory effects (Versalovic et al. 2008). The
strains have the capability to down regulate the production of TNF-• by macrophage cell
lines and successfully suppressed inflammation in mouse colitis models. Human-derived
Lactobacillus reuteri strains have been identified with potent human TNF-• -inhibitory
effects on lipopolysaccharide (LPS)-activated human myeloid cell lines and primary
monocyte-derived macrophages from children with Crohn's disease (Versalovic et al.
2008). Some Lactic acid bacteria have shown to regulate mucosal immune response by
modulating the production and liberation of regulatory agents such as cytokines by the
host.Some of these cytokines, such as the anti-inflammatory interleukin-10 (IL-10),
modulate the inflammatory immune response, thus immunomodulation is a mechanism
by which LAB can prevent certain inflammatory bowel diseases (IBD). Extracts from
soymilk fermented with lactic acid bacteria and Bifidobacteria showed the inhibitory
effect on LPS-induced pro- inflammatory cytokines including tumor necrosis factor
(TNF)-• , interleukin (IL)-6, IL-1β and prostaglandin E2 (PGE2) produced by RAW 264.7
macrophages (Liao et al. 2010).
2.2.6.5 Prevention from diarrhoea
Intestinal microflora maintains a barrier against the development of
pathogenic bacteria in the digestive tract and is mandatory to the establishment of oral
tolerance to food antigens. Lactic acid bacteria may be useful in preventing and
shortening the duration of several types of diarrhea. It is believed that lactic acid
bacterium competes with the pathogenic bacteria in terms of nutrition and space in the
intestines shortening its chances of survivability. Undergoing a strong metabolism
process, the lactic acid bacteria produce various metabolites like acidophilin and
bulgarican inhibiting the growth of other bacteria preventing and shortening the duration
of diarrhoea. Recent studies suggest that children in India who receive probiotic
demonstrated protective efficacy of 14% prevention from diarrhoea (Hajela et al. 2010).
Approximately 20% of the patients treated with antibiotics will develop AAD because
their intestinal flora, responsible for the natural colonization resistance, is disturbed or
reduced. The intestinal flora modification (in particular in the LAB population) could be
the cause of diarrhea, dehydration and electrolytic imbalance. Also, the fermentation in
the colon can be reduced. Many preparations have been tested for their preventive
efficacy against AAD (Contardi, 1991). However, more studies need to be performed
using well controlled conditions and strains, before one can finally understand which
prophylactic probiotics should be taken against secondary effects of specific antibiotics,
applied at a specific dose in a specific type of patient.
2.2.6.6 Hypocholesterolemic effects
Hyperlipidemia is a leading death cause in many countries of the world
with the risk of cardiovascular disease. A number of scientific studies suggest that lactic
acid bacterial fermented food shows hypolipidimic effects by reducing the cholesterol
biosynthesis and decreasing low density lipoproteins (Gao et al. 2011). The
hypocholesterolemic effects of lactic acid bacteria (LAB) have now become an area of
great interest and controversy for many scientists. Some strains of L. acidophilus can take
up cholesterol in the presence of bile, suggest that lactic acid bacteria can reduce
cholesterol level up to 50% in presence of bile salt (Lavanya et al. 2011). Some lactic
acid bacteria could adjust blood lipid and lower cholesterol which can also prevent some
of the diseases by stimulating antioxidant enzymes (Koiche and Dilmi 2010). These
strains have the ability to tolerate both acid and bile concentrations typically found in the
upper gastrointestinal tract of humans. In a study milk fermented with lactic acid bacteria
was feeded to rats and it was observed that it reduces the serum total cholesterol and LDL
cholesterol levels after 12 days of feeding (Pato et al. 2004). Hypocholesterolemic effect
of Lb. lactis subsp. lactis was attributed to its ability to suppress the reabsorption of bile
acids into the enterohepatic circulation and to modulate the excretion of bile acids in
feces of hypercholesterolemic rats (Pato et al. 2004).
Table: 2.7 Health benefits along with the proposed mechanism of action of lactic
acid bacteria
Health Benefit by Lactic acid Proposed Mechanism

Bacteria
 Increase in IgA production.
Immuno-modulation  Non-specific defence against infection.
 Increased phagocytic activity of WBC.
 Proliferation of intra-epithelial lymphocyte.
Intestinal Tract Infections  Alteration of toxic binding sites.
 Stimulation of the systemic or secretory immune
response.
 Adjuvant effect increasing antibody production.
 Adherence to intestinal mucosa, preventing
pathogen.
 Competition for nutrients.
Lactose Intolerance  Bacterial β -galactosidase acts on lactose.
Anti-inflammatory and  Restoration of the homeostasis of the immune
Anti-allergic. system
 Regulation of cytokine synthesis
 Prevention of antigen translocation into blood
stream
Anti- Colon cancer  Mutagen binding
 Carcinogen deactivation
 Alteration of activity of colonic microbes
 Immune response
 Influence on secondary bile salt concentration
Urogenital infections  Adhesion to urinary and vaginal tract cells
 Competitive exclusion
 Inhibitor production (H2O2, biosurfactants)
2.2.6.7 Lactic acid bacteria as live vaccine
The most promising new application for LAB is their use as live delivery
vectors for antigenic or therapeutic protein delivery to mucosal surfaces. Such engineered
lactic acid bacteria are able to elicit both mucosal and systemic immune responses. The
delivery of vaccine in the body through mucosal routes is much beneficial compared to
direct systemic inoculation. But human mucosal surface is a site in which the host
encounters a large variety of micro organisms initiating infections. Lactic acid bacteria
have proved to be effective delivery vehicles for functional proteins to mucosal tissues.
Oral administration of Lactococcus lactis has shown to induce antigen-specific oral
tolerance (OT) to secreted recombinant antigens (Wells 2011). The food manufacturing
sector has developed the so called functional food containing ingredients for promoting
health. Non pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being
tested for their efficacy as live antigen carriers (Mercenier et al. 2000). The advantages of
using live bacterial vaccines include their mimicry of a natural infection, intrinsic
adjuvant properties and their possibility to be administered orally. Derivatives of
pathogenic and non-pathogenic food related bacteria are currently being evaluated as live
vaccines (Detmer and Glenting, 2006).
The first evidence that recombinant commensal bacterium can be used as a live
vaccine vector was obtained with S. gordonii. There are two major approaches being
followed to achieve efficient mucosal delivery of antigens. A variety of synthetic (non-
living) delivery systems, in which purified antigens are entrapped in microspheres,
liposomes, nanoparticules, or ISCOMS, are presently being investigated. An attractive
alternative consists in the use of live viral or bacterial vectors for the production of
replicative particulate antigens in vivo. This technology, which alleviates the drawbacks
of subunit vaccine development, was first described in the early 1980s. Most of the lactic
acid bacteria are quite acid resistant and various strains are able to effectively survive
passage through stomach. The bacterial group has today created a vaccine vehicle system
depending upon their immunization routes and models of antigens they carry. It is found
that the lactic acid bacteria have a low innate antigenicity even though several strains
clearly exhibit immunoadjuvant properties (Pouwels et al. 1998). The ideal GMM for use
in humans should therefore contain the minimal amount of foreign DNA and must not
include an antibiotic resistance marker (Lee 2010).
2.2.6.8 Anti-viral activities

Lactic acid bacteria were found to be effective against the ‘Transmissible
Gastroenteritis Coronavirus (TGEV) and Rotavirus RF strain (RV)’. A selected number
of strains of lactic acid bacteria were used in the study along with the CLAB cell line
from pig. However, the co-incubation of the CLAB cell line with specific LAB strains
resulted in increased survival percentages, from 40% up to 80%. Various literature and
clinical studies have confirmed the beneficial and alleviating effects of probiotic bacteria,
such as Lactobacillus rhamnosus GG, on the infection and symptoms of rotavirus
diarrhea. Also probiotics have been reported on their antiviral activities on animals
(Zhang et al. 2008). L. acidophilus NCFM has a significant immune-stimulating effect on
the intestinal and systemic HRV-specific T and B cell immune responses induced by the
AttHRV vaccine and is safe in neonates; therefore it may have the potential to be used as
an adjuvant for rotavirus vaccines (Versalovic et al. 2008).
2.3 STREPTOCOCCUS THERMOPHILUS
Streptococcus thermophilus was of major importance for the food industry since it
was massively used for the manufacture of dairy products and it was considered as the
second most important industrial dairy starter after Lactococcus lactis. Nevertheless, over
1021 live cells of Streptococcus thermophilus ingested annually by the human population
(Trevan et al., 2004). Streptoccocus thermophilus (S. thermophilus) belongs to the group
of the thermophilic lactic acid bacteria and is traditionally and widely used as a starter in
manufacturing dairy products (Emmental, Gruyere, Parmigiano, Mozarella, Yoghurt etc).
Yoghurt results from the fermentation of milk by S. thermophilus and Lactobacillus
delbrueckii sp. bulgaricus (L. bulgaricus), fulfils the current specifications required to be
recognized as a probiotic product (Guarner et al., 2005). The health beneficial effect of
yoghurt consumption is linked to the metabolic properties of S. thermophilus and L.
bulgaricus. Streptococcus thermophilus is an important bacterium that is extensively used
in starter cultures in the dairy industry. It is also found growing spontaneously in
traditional products around the world, and is believed to persist in the farm environment.
As such, it improves lactose digestion in the gastro-intestinal tract (GIT) through their
lactose hydrolyzing activity present in yoghurt and in the GIT, thus reducing symptoms
of lactose intolerance (Lomer et al., 2008; Rabot et al., 2010). Yoghurt cultures were
shown to induce other health benefits such as reduction of diarrhoea or allergic disorders
as well as modulation of the immune system (Guarner et al., 2005; Higashikawa et al.,
2010). S. thermophilus is also present at high concentration in VSL#3, a probiotic
mixture of eight different bacterial strains that possesses beneficial effects in several
intestinal conditions (Pagnini et al., 2010; Preidis et al., 2009). Recent data indicate that
strains related to S. thermophilus LMD-9 are among the 57 bacteria species found in 90%
of 124 European individuals intestinal microbiota (Qin et al., 2010). In comparison with
the overall human intestinal microbiota, S. thermophilus is numerically non dominant
species with variable levels (Mater et al., 2005; Elli et al., 2006; Firmesse et al., 2008;
Qin et al., 2010). At birth, Streptococcus genus -with in some studies a precision at the
level of S. thermophilus species- is among the first coloniser of the GIT, since it has been
detected in infant faeces and breast milk (Palmer et al., 2007; Perez et al., 2007). Thus,
Streptococcus, as pioneer bacteria colonising a yet immature GIT, may impact the
maturation and homeostasis of intestinal epithelium after birth.
2.3.1 Physiology of S. thermophilus
S. thermophilus is closely related to Lactococcus lactis, but it is even more closely

related to other streptococcal species including several pathogens (Mitchell, 2003). S.
thermophilus is highly adapted to grow on lactose, the main carbon source in milk and
rapidly converts it into lactate during growth. Lactose is transported into the cell by a
lactose permease (LacS), which operates as a galactosideproton symport system or as a
lactose-galactose antiporter. Lactose is efficiently transported into the cell and
subsequently hydrolyzed by an intracellular β -galactosidase. The vast majority of S.
thermophilus strains only metabolized the glucose moiety of lactose, while galactose is
excreted into the medium. The milk is poor in free amino acids (AA) and short peptides,
therefore for optimal growth; S. thermophilus requires either hydrolysis of caseins
followed by the internalization and the degradation of the resulting peptides or de novo
AA biosynthesis (Hols et al., 2005). For many LAB including S. thermophilus, the
hydrolysis of milk caseins (AA supply) mostly depends on the activity of a cell-wall-
anchored proteinase (Herve-Jimenez et al., 2008).
Fortunately, nature has provided us with different kind of nucleic acids for
different kind of taxonomic studies. Close relations (at species and subspecies level) can
be determined with DNA-DNA homology studies. For determining phylogenetic
positions of species and genera, ribosomal RNA (rRNA) is more suitable, since the
sequence contains both well-conserved and lessconserved regions. It is now possible to
determine the sequence of long stretch of rRNA (~1500 bases of 16S rRNA) from
bacteria (Huili et al., 2011) comparisons of these sequences are currently the most
powerful and accurate technique for determining phylogenetic relationships of
microorganisms (Philippe et. al., 2009).
With this technique, a clearer picture of phylogeny of lactic acid bacteria is

emerging, and the ideas of Orla-Jensen can be examined with some accuracy. The genera
that, in most respects, fit the general description of the typical lactic acid bacteria are (as
they appear in the latest edition of Bergey’s Manual from 1986) Aerococcus,
Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. Major revision of
taxonomy of lactic acid bacteria, in particular of streptococci, was anticipated in Bergey’s
Manual of 1986 (Barry 2009) and to some extent already realized by the year of that
issue. Thus, the former genus Streptococcus was first divided into three: Enterococcus,
Lactococcus, and Streptococcus sensu stricto (Alexander et al., 2001; Zongzhi et. al.,
2008). Later, some motile lactic acid bacteria, otherwise resembling lactococci, were
suggested to form separate genus, Vagococcus (Aly et al., 2004). The genera
Lactobacillus, Leuconostoc, and Pediococcus have largely remained unchanged, but
some rod-shaped lactic acid bacteria, previously included in Lactobacills, are now
forming the genus Carnobacterium (Elliot et al., 1991), and the former species
Pediococcus halophilus has been raised to genus level, forming the genus
Tetragenococcus (Facklam et al., 1995). Revisions after 1986 are supported by extensive
chemotaxonomic and genetic data (Khalid, 2011).
2.3.2 Characterization of Streptococcus thermophilus
2.3.2.1 Phenotypic Characterization
Streptococcus thermophilus is the only starter dairy streptococci in Streptococcus

genus. According to DNA-DNA homology and membrane fatty acid profile studies,
researchers have thought that S. thermophilus should be reclassified as S. salivarius sub
sp. thermophilus. However, S. thermophilus and S. salivarius are not found in the same
ecological niche possessing large number of physiological differences. In 1987, S.
thermophilus has been restored to species level again as a result of more sophisticated
homology studies with huge phenotypic differences (Zirnstein and Hutkins 1999).
Figure: 2.8 Electron microscopic image of S. thermophilus (Source: Durso and
Hotkins 2003)
S. thermophilus prefers the disaccharides lactose and sucrose, and its growth on
the constituent monosaccharides, glucose, fructose and galactose is slower than the
disaccharides suggesting that the transport systems required to accumulate
monosaccharides might be absent or has low activity. It appears that it is dependent on
the availability of the necessary transport system. (Hutkins and Ponne 1991). This
phenomenon has been explained with the findings that the enzymes of the Leloir pathway
for galactose metabolism are present in S. thermophilus, however their activities are very
low and the activity of the first enzyme galactokinase is undetectable under normal
growth conditions (Grossiord et al. 1998). General characters are:
 Gram positive, non-motile coccus
 Spherical/ovoid cells of 0.7-0.9µm diameter
 Occurs in pairs or in long chains of 10-20 cells
 Homo-fermentative, L(+) lactic acid as the major end product
 Facultative anaerobe
 Catalase negative
 Lacks cytochromes
 Optimum growth temperature is between 40-45°C, a minimum at 20-25°C and
maximum at 50-52°C.
 Thermo tolerant, survives at 60°C for 30 min
 Weak or no growth at 2 % NaCl
 Does not utilize arginine
 Lacks group specific antigen
 G-C mol ratio is 37-40%
 Above 10g of lactic acid/kg of yoghurt, the growth reaches exp8-9 and, the
metabolism of bacterium ceases. The final pH in broth culture is 4-4.5
S. thermophilus has limited proteolytic activity and requires free amino acids for
its growth. These are glutamic acid, histidine, cysteine, methionine, valine, leucine,
isoleucine, tryptophan, arginine and tyrosine. However, free aminoacids naturally found
in milk are not sufficient. The free amino acids are supplemented during heat treatment in
milk or the absorption of short-chain peptides released by the breakdown of milk proteins
by Lactobacillus delbrueckii sub sp. bulgaricus (Pearce and Flint 1999).
2.3.2.2 Biochemical Characterization
In traditional identification, most commonly used characteristics are morphology,

staining reactions, nutritional requirements, cell wall chemistry, ability to use different
energy sources, fermentation byproducts, gas requirements, temperature and pH
tolerance, antibiotic sensitivity, pathogenicity, immunological characteristics and habitat
(Morata et al. 1999). The genus Streptococcus includes Gram positive bacteria with
similar metabolic properties but they live in different habitats and have many
physiological differences. In the past two decades, several important Streptococcus
species have been reclassified as members of recently named genera Enterococcus and
Lactococcus. The only dairy Streptococcus remained is S. thermophilus. Streptococci
grouped as “oral”, “pyogenic” and “other streptococci”. “Oral” streptococci are also
subdivided into four groups; S. mutans, S. mitis, S. anginosus and S. thermophilus groups
(Gobbetti and Corsetti 1999). Although S. thermophilus is a member of “S. thermophilus
group” phylogenetically, it is the only bacterium in Streptococci with dairy origin. The
Gram positive and cocci genera sharing the same habitat with S. thermophilus includes
enterococci, lactococci, pediococci and leuconostocs (Table 2.8; 2.9). The pediococci is
readily distinguished from other genera by the tetrad morphology in broth media. Some
of the physiological differences which are helpful for the first grouping at the genus level
are given in the table below:
Table: 2.8 Classification of cocci lactic acid bacteria

Argin
Microrganism Growth Growth Growth Type Lactate Gas from Growth in ine
at 10°C at 45°C in 6.5% formed Glucose broth at Hydr
NaCl pH 9.6 olysis
Enterococcus + + + ND - + +
Lactococcus + - - L - - V
Streptococcus - + - L - - V
Leuconostoc + + - D + ND -
* ND indicates no data available, V indicates variable: some produce (+) results and
some (-), L indicates levo-lactic acid and D indicates dextro-lactic acid
S. thermophilus is highly adapted to the dairy environment, and in the wild. It can
only be isolated from dairy products. S. waius is a recently identified thermophilic
Streptococcus isolated from stainless still pasteurization machinery of milk. It shares
many phenotypic characteristics with S. thermophilus but can be distinguished by the
fermentation of galactose, salicin, cellobiose, maltose, melibiose and D-raffinose. S.waius
is also tolerant up to 7% NaCl (Pearce and Flint 1999). Unlike other streptococci, S.
thermophilus does not possess a group- specific antigen. The peptidoglycan structure is
identical to Enterococcus faecalis; however it is distinguished from enterococci and
lactococci by its sensitivity to salt. It does not grow in the presence of 4% salt, and some
strains will not grow in as little as 2% salt (Zirnstein and Hutkins 1999). The mechanism
of lactose transport in S thermophilus differs from lactococci, which possess a specific
system for lactose transport, the phosphoenolpyruvate (PEP)-dependent
phosphotransferase system (PTS). Lactose-6-phosphate formed during transport is
hydrolyzed by an intracellular phospho-β -galactosidase into glucose and galactose-6-
phosphate which are then metabolized into lactic acid. S. thermophilus does not possess
lactose PTS or phospho-û- galactosidase, but has a lactose permease system including a
proton dependent membrane-located permease.
Table: 2.9 Characterizing features of S. thermophilus

Characteristics of Streptococcus thermophilus B4 isolated from Goat Milk (Sharma et al
2013)
Gram Cell Spore Catalase Fermentation Glucose Nitrate

Staining Morphology Formation Activity Type Fermentation Reduction
Cocci in
Positive chains Negative Negative Homo Positive Negative
Sugar Fermentation Streptococcus thermophilus B4 isolated from Goat Milk (Sharma et al
2013)
Sucrose Lactose Maltose Dextrose Ribose Sorbitol Mannose
Positive Positive Positive Positive Negative Negative Positive
2.3.2.3 Genotypic Characterization
DNA hybridization assays are not without their shortcomings, however, being
time-consuming, labor-intensive, and expensive to perform. Today, fewer and fewer
laboratories worldwide perform such assays, and many studies describing new species are
solely based upon small subunit (SSU) sequences or other polyphasic data. In the early
1990s the availability DNA sequencers in terms of cost, methodologies, and technology
improved dramatically, such that many centers can now afford such instrumentation. In
1994, Stackebrandt and Goebel summarized the emergence of SSU sequence technology
and its potential usefulness in the definition of a species. Although it has been
demonstrated that 16S rRNA gene sequence data on an individual strain with a nearest
neighbor exhibiting a similarity score of <97% represents a new species, the meaning of
similarity scores of >97% is not as clear (Petti, 2007). This latter value can represent a
new species or, alternatively, indicate clustering within a previously defined taxon. DNA-
DNA hybridization studies have traditionally been required to provide definitive answers
for such questions. Whereas 16S rRNA gene sequence data can be used for a multiplicity
of purposes, unlike DNA hybridization (>70% reassociation) there are no defined
“threshold values” (e.g., 98.5% similarity) above which there is universal agreement of
what constitutes definitive and conclusive identification to the rank of species.
One of the most attractive potential uses of 16S rRNA gene sequence informatics
is to provide genus and species identification for isolates that do not fit any recognized
biochemical profiles, for strains generating only a “low likelihood” or “acceptable”
identification according to commercial systems, or for taxa that are rarely associated with
human infectious diseases. The cumulative results from a limited number of studies to
date suggest that 16S rRNA gene sequencing provides genus identification in most cases
(>90%) but less so with regard to species (65 to 83%), with from 1 to 14% of the isolates
remaining unidentified after testing (Drancourt et al, 2000).
Bioinformatics is now an established and vital resource for molecular biology

research and is also a mainstay of routine analysis of DNA. The use of bioinformatics has
been driven by the increase in genetic sequence information and the need to store,
analyse and manipulate the data. There are now a huge number of sequences stored in
genetic databases from a variety of organisms, including the human genome. Indeed the
genetic information from various organisms is now an indispensable starting point for
molecular biology research. The main primary databases include GenBank at the
National Institutes of Health (NIH) in the USA, EMBL at the European Bioinformatics
Institute (EBI) at Cambridge, UK and the DNA Database of Japan (DDBJ) at Mishima in
Japan. These databases contain the nucleotide sequences which are annotated to allow
easy identification. There are also many other databases such as secondary databases that
contain information relating to sequence motifs, such as core sequences found in
cytochrome P450 domains, or DNA-binding domains. Importantly all of the databases
may be freely accessed over the internet. Consequently the new expanding and exciting
areas of bioscience research are those that analyse genome and cDNA sequence databases
(genomics) and also their protein counterparts (protomics).
This is sometimes referred to as in silico research. Sequencing the genomes of

many species in a class of bacteria enables the examination of their evolution and
divergence. Although the phenotypic techniques have proven to be useful, there is a
general awareness that strains with similar phenotypes do not necessarily have closely
related genotypes. Phenotypic methods have also poor reproducibility, ambiguous, and
poor discriminatory power. Wild type strains isolated from natural habitats show
phenotypic variability and are often classified as “atypical”. Genotypic techniques have
different levels of discrimination, from species level to individual strain level. Most of
them are based on Polymerase Chain Reaction (PCR), which enables the amplification of
targeted DNA fragments by the use of designed primers under controlled reaction
conditions. The most powerful and most extensively used phylogenetic marker is 16S
ribosomal RNA and the genes code for it. The advantages result from the fact that the
ribosomes must have been present in the earliest prokaryotic cells, the components of the
ribosomes have not changed their function and the presence of multiple genes coding for
rRNA makes horizontal gene transfer unlikely. That is why there is a high degree of
conservation within the tRNA genes sequences. More than 12.000 sequences of 16S
rDNA are available for prokaryotic strains in gene banks (Morata et al. 1999). PCR-
RFLP (Restriction Fragment Length Polymorphism) is the most commonly used method
to identify isolates at intra-species level. Method is based on the digestion of amplicons
resulting from PCR with restriction enzymes. When RFLP is applied to ribosomal genes,
the method is called amplified ribosomal DNA restriction analysis (ARDRA). PCR-
ARDRA with EcoR I has been to be a reliable and rapid method for identifying L.
delbrueckii isolates at the subspecies level and for differentiating this species from L.
helveticus and L. acidophilus (Bouton et al. 2002, Coeuret et al. 2003). This was also
justified by another study, which differentiated the subspecies of L. delbrueckii and also
reclassified some of the ATCC type strains known to be L. delbrueckii sub sp. bulgaricus
as L. delbrueckii sub sp. lactis (Delley and Germond 2002).
The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy
has been by far the most common housekeeping genetic marker used for a number of
reasons. These reasons include (i) its presence in almost all bacteria, often existing as a
multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not
changed, suggesting that random sequence changes are a more accurate measure of time
(evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics
purposes. In 1980 in the Approved Lists, 1,791 valid names were recognized at the rank
of species. Today, this number has ballooned to 8,168 species, a 456% increase. In the
early 1990s the availability DNA sequencers in terms of cost, methodologies, and
technology improved dramatically, such that many centers can now afford such
instrumentation.
In 1994, Stackebrandt and Goebel summarized the emergence of SSU sequence

technology and its potential usefulness in the definition of a species. Although it has been
demonstrated that 16S rRNA gene sequence data on an individual strain with a nearest
neighbor exhibiting a similarity score of <97% represents a new species, the meaning of
similarity scores of >97% is not as clear. This latter value can represent a new species or,
alternatively, indicate clustering within a previously defined taxon. DNA-DNA
hybridization studies have traditionally been required to provide definitive answers for
such questions. Whereas 16S rRNA gene sequence data can be used for a multiplicity of
purposes, unlike DNA hybridization (>70% reassociation) there are no defined “threshold
values” (e.g., 98.5% similarity) above which there is universal agreement of what
constitutes definitive and conclusive identification to the rank of species. DNA
fingerprinting methods that solely relie on PCR include Randomly Amplified
Polymorphic DNA (RAPD). This analysis makes use of short arbitrary primers and low-
stringency conditions to amplify DNA randomly. Fragments obtained are then separated
electrophoretically to produce a fingerprint. The great flexibility in primer choice enables
it to be used to differentiate LAB at different taxonomic levels, from genus to intra-
specific level.
However, since the primers are random and not directed to a specific region,
reproducibility is major problem. RAPD-PCR was shown to be superior in distinguishing
individual L. delbrueckii strains. On the other hand, S. thermophilus strains showing
phenotypic anomalisms were not easy to locate within S. thermophilus clusters using this
method (Moschetti et al. 1998). Species-specific PCR is another method which relie on
PCR for rapid and accurate identification for S. thermophilus isolates. Differentiation of
S. thermophilus strains from other Streptococcus species such as S. mutans, S. salivarius,
and other bacteria such as L. delbrueckii subspecies, Lactococcus lactis subspecies,
L.acidophilus, L. brevis, L. fermentum, L. helveticus, L. plantarum, Enterococcus
subspecies and E. coli was accomplished clearly using primers homologous within the
lacZ gene. Phylogenetic tree of Lactobacillales based on concatenated alignments of
ribosomal proteins of Lactobacillaceae, Leuconostocaceae and Streptococcaceae is
presented in Figure 2.9. This method was also justified by a study which makes use of the
polyphasic approach to show the genotypic and phenotypic heterogeneity of S.
thermophilus strains (Giraffa et al. 2001) and also with the study, which investigates the
species composition of commercial dairy starters (Giraffa and Rossetti 2004). Pulsed
Field Gel Electrophoresis (PFGE) is another molecular typing method. It has an
alternating field of electrophoresis to separate large DNA fragments resulting from
restriction with rare cutting enzymes. The crucial point in PFGE is the extraction of intact
chromosomal DNA, which is more time consuming than other fingerprinting techniques.
Since large DNA fragments, representing the whole genome, are analyzed with PFGE, it
has a superior discriminatory power at subspecies and strain levels. Ribotyping combines
an enzymatic restriction digestion and the detection of the restriction fragments by means
of rDNA probes. Fluorescent or radioactively labeled probes can be used for
hybridization. Discriminatory power of this method is dependent on the number and type
of endonucleases and probes. In a study, where ribotyping has been applied to 30
different L. delbrueckii strains, it has been found that only ribotyping with EcoR I
allowed the differentiation of three subspecies on the basis of a typical hybridization
pattern (Miteva 2001). Differentiation at strain level has also been achieved for S.
thermophilus strains both by restriction endonucleases digestion combined with pulsed
field gel electrophoresis and by ribotyping (Salzano et al 1994). 16S or 23S rDNA
sequencing is another useful method, when unknown isolates are to be identified.
Obtained sequences are compared with the sequences previously deposited in a database.
Stackobrandt and Goobel stated that strains that are more than 3% divergent in 16S rRNA
are nearly always members of different species, as determined by DNA-DNA
hybridization studies. Whereas, the strains with less than 3% divergency are generally
members of the same species. A cutoff of 3% is a recommended limit as a conservative
criterion (Cohan 2002). DNA-DNA hybridization has a higher discrimination power than
16S sequencing. Various approaches such as nitrocellulose filter methods, free-solution
methods, and recently the microarray technology have been used. As a general rule,
strains, which have a DNA-DNA relatedness of more than 70% and 5°C or less
difference in melting point, belong to the same species. However, use of isotopes and
lack of database affect the popularity of method negatively. As was observed in Archaea
and Proteobacteria (Snel et al., 2002), genome reduction was an overall trend during the
evolution of the LAB. Divergence of Lactobacillales from their ancestor in the Bacilli
was marked by the loss of 600–1200 genes, including many genes encoding biosynthetic
enzymes (Makarova and Koonin, 2007). Other losses include genes related to
sporulation, a function that is seemingly unnecessary in nutrient-rich food environments
(Hufner et al., 2007). Besides gene losses occurring early in the lineage of the LAB, more
recent events have contributed to shaping these species, including parallel losses in genes
involved in various metabolic processes. The most notable example of gene loss occurred
in Streptococcus thermophilus, which diverged from pathogenic Streptococcus species
through the loss and decay of virulence-associated genes, such as those involved in
antibiotic resistance and adhesion. This genomic record has thus far provided solid
evidence supporting the ‘generally recognized as safe’ status for use of S. thermophilus in
foods.
Figure 2.9: Phylogenetic tree of Lactobacillales based on concatenated alignments
of ribosomal proteins. Colors represent current taxonomy, with
Lactobacillaceae in blue, Leuconostocaceae in red and
Streptococcaceae in green
Table: 2.10 Genomic details of Streptococcus thermophilus strains with whole
genome sequencing available data till Feb 2014
Primary NCBI / Gene Genomic

Strain application bank accession Size Protein Reference
s
Streptococcus
thermophilus Starter culture NC_006449 1.8 Mb 1915 Bolotin et
CNRZ1066 al., 2004
Streptococcus Makarova
thermophilus Starter culture NC_008532 1.8 Mb 1710 et al., 2006
LMD-9
Streptococcus Bolotin et
thermophilus Starter culture NC_006448 1.8 Mb 1889 al., 2004
LMG 18311
Streptococcus Starter culture CP002340. N/A 3840 Zhihong

thermophilus Sun et al.,
ND03 2011
Streptococcus Non Starter- Delorme
thermophilus Multi locus FR875178. N/A 4305 et al., 2011
JIM8232
Streptococcus
thermophilus Starter culture CP003499 N/A 3822 Kang et
MN-ZLW-002 al., 2012
Streptococcus Starter culture AYSG0000000 N/A 1724 Treu et al.,

thermophilus 0 2014
TH1435
Streptococcus Treu et al.,
thermophilus Starter culture AYTT0000000 N/A 1698 2014
TH1436 0
To date, the complete genomes at the chromosome level of S. thermophilus strains

CNRZ1066, JIM8232, LMD-9, LMG18311, MN-ZLW-002, and ND03 have been
published. However, S. thermophilus TH1435 and TH1436 represent the first cases of S.
thermophilus strains isolated in Italy and the only ones from goat milk (Table 2.10).
S. thermophilus TH1435 and TH1436, collected from two alpine huts (malghe)
from raw goat milk used for the artisanal production of Italian cheese, are capable of
rapidly lowering the pH of milk, which represents a key technological feature for the
dairy industry. Moreover, strain TH1436 is capable of utilizing galactose, while TH1435
lacks this feature. Whole-genome sequencing of strains TH1435 and TH1436 was
performed with an Illumina MiSeq sequencer at the Ramaciotti Centre, Sydney,
Australia. Genomic libraries were prepared using the Nextera XT kit Illumina (Illumina,
Inc., San Diego, CA), which produced a mean insert size between 800 and 1,200 bp. A
total of 776,373 and 1,527,857 paired-end reads (2 × 250 bp) were generated and gave
134- and 159-fold coverages of the TH1435 and TH1436 genomes, respectively.
Approximately 85% of these reads were assembled into 36 and 28 large scaffolds,
respectively, using a manually curated consensus of assemblies obtained using version
1.2.10 of the Velvet software (Zerbino and Birney, 2008) and version 2.8 of the 454
Newbler Assembler (454 Life Sciences, Branford, CT). The draft genome of S.
thermophilusTH1435 is a single circular chromosome of 1,750,348 bases in length, with
a mean G+C content of 38.9% and two putative plasmids individuated by BLAST match
(scaffolds 33 and 35). The draft genome of strain TH1436 is a single circular
chromosome of 1,780,473 bases, with a mean G+C content of 39.0%; no plasmid
sequences were detected for this strain.
S. thermophilus MN-ZLW-002 was originally isolated from a traditional

fermented dairy food called Yogurt Block, originating from the Gannan region of Gansu
province, China by Kang et al., 2012. MN-ZLW-002 has many good fermentation
characteristics, but the most prominent is the ability to produce exopolysaccharides
(EPSs). EPSs produced by LAB in fermented milk or yogurt improve the viscosity, body,
texture, and taste of thefinal product (De Vuyst et al., 2001). In addition, LAB -derived
EPSs contribute to human health through their potential antitumor, antiulcer,
immunomodulating, and cholesterol-lowering properties (Duboc et al., 2001; Ruas-
Madiedo et al., 2002). Whole-genome sequencing of S. thermophilus strain MN-ZLW-
002 was performed with a combined strategy of 454 sequencing (Margulies et al., 2005)
and Solexa paired-end sequencing technologies (Bentley et al., 2008). Genomic libraries
containing 8-kb inserts were constructed, and 188,861 paired-end reads and 64,276
single-end reads were generated using the GS FLX system, giving 45-fold coverage of
the genome. The majority (97.4%) of reads were assembled into three large scaffolds,
including 50 nonredundant contigs, using the 454 Newbler assembler (454 Life Sciences,
Branford, CT).
A total of 339,700,400 reads (500 bp library) were generated to reach a depth of
180-fold coverage with an Illumina Solexa GA IIx (Illumina, San Diego, CA) and
mapped to the scaffolds using Burrows-Wheeler alignment (BWA) (Li and Durbin,
2009). The gaps between scaffolds were filled by sequencing PCR products using an ABI
3730 capillary sequencer. With decreasing costs for sequencing and annotation, it is
likely that most industrial strains will be sequenced, which could aid in strain selection
and performance in an industrial setting. Genome sequences alone, however, do not
provide a full understanding of a microorganism. In the post-sequencing era, scientists
are taking the first steps to integrating sequence data with transcriptional and functional
studies so as to better define complex traits.
New methods of analysis, such as metabolomics and metagenomics, can also aid
in characterization and should be added to the repertoire of tools for investigation of
complex microbial ecosystems. Humans have relied on the LAB for thousands of years
for food preservation. Our understanding of LAB has increased exponentially with the
applications of genomics and biotechnology, opening up new horizons in bioprocessing,
human health and food production.
2.3.3 Factors affecting the growth of lactic acid bacterial fermentations
However, there are still several researches that need to be addressed in order to
produce lactic acid within the targeted cost, development of high performance lactic acid
producing microorganisms and lowering the cost of the raw material. Many factors
affected in lactic acid fermentation have been investigated. The optimization of
fermentation processes requires profound knowledge of the factors determining microbial
metabolism, and the influence of process parameters
2.3.3.1 Temperature
Temperature and pH are the key environmental parameters that affect the
fermentation process (Yuwono and Kokugan, 2008). Low temperature has been reported
to positively influence the outgrowth of contaminating microorganism, thereby
influencing the performance of the lactic acid production (Neysens and Vuyst, 2005;
Hujanen and Linko, 1996). For Lactobacillus amylophilus, which is known to grow at 15
°C but not at 45 °C, the optimal temperatures were 25 °C and 35 °C for maximum
productivity and yield, respectively (Yumoto and Ikeda, 1995). The results from
measuring the residual starch and reducing sugar in 4 h and 8 h indicated that there was
increased in starch hydrolysis and reducing sugar accumulation as the temperature
increased from 22-30 °C, and a further increase from 30-40 °C resulted in a slight
improvement for the saccharification in both Rhizopus oryzae 2062 and Rhizopus oryzae
36017 cultures (Huang et al., 2005). Naturally occurring lactic acid bacteria (LAB) load
was found to vary between 1.97×105 cfu/g to 4×105cfu/g at10˚C. The yeast and mold
counts decrease from 1.04 ×105cfu/g to 0cfu/gr at 10˚C. Lactic acid bacteria load was
found to vary between1.97×105cfu/g to 4.3×105cfu/g at 20˚C. The yeast and mold counts
decrease from 1.04 ×105cfu/g to 3×104 cfu/g at 20˚C and salt content 0.5%. Lactic acid
bacteria load was found to vary between1.97×105cfu/g to 1.1×106cfu/g at 37˚C. The yeast
and mold counts decrease from 1.04 ×105cfu/g to 3×104 cfu/g at 37˚C and salt content 0.5%.
The largest increase in the numbers of LAB was noted during the first 24 h of
fermentation and further incubation led to decrease (Tabatabaei-Yazdi et al., 2013).
2.3.3.2 Incubation temperature
An increase in lactose utilization and subsequent lactic acid production was found
up to 36 h of incubation and thereafter no improvement in both the functions was
observed (Panesar et al., 2010). This could be attributed to the growth of the culture
reached to the stationary phase and as a consequence of metabolism, microorganisms
continuously change the characteristics of the medium and the environment. The
incubation period of 48 h has been generally used for lactic acid production using
different lactobacilli cultures (Gandhi et al., 2000). In addition, different optimal
conditions reported by various workers for maximum lactic acid production could be
explained by the differences in the nature of the strains and medium composition used in
their studies.
2.3.3.3 pH
The fermentation pH is either set at the beginning and then left to decrease due to
acid production or it is controlled by an addition of alkaline solutions. The optimal pH for
lactic acid production varies between 5.0 and 7.0. A pH below 5.7 was optimal for
Lactobacillus strains, which are known to tolerate lower pH than lactococci. The
optimum pH for cell growth of Enterococcus faecalis RKY1 was seen to be 8.0, the lactic
acid fermentation at pH 7.0 was completed faster than that at pH 8.0. The cell growth at
pH 5.0 almost ceased after 10 h of fermentation (Wee et al., 2004). At initial pH 6.5, cell
started to utilize glucose earlier and at a faster rate than at other initial pH. Maximum
lactic acid concentration was attained at initial pH 6.5. Further increase in initial pH
beyond 6.5 does not improve the lactic acid production (Idris and Suzana, 2006). It is
possible that the higher initial pH brought too much stress on the microorganism
metabolic abilities (Vijayakumar et al., 2008).
2.3.3.4 Agitation
Different lactic acid bacterial strains differed in their requirement for growth
conditions. The maximum lactic acid concentrations from Lactobacillus rhamnosus
strain, could be achieved when fermentation was carried out at pH 6, temperature of 40°C
and agitation speed of 150 rpm, in accordance with a previous report (Hofvendahl and
Hagerdal, 2000) the optimal condition for lactic acid is pH 5.0-6.8, temperature 30- 45°C
with continuously agitating at 100-200 rpm (Timbuntam et al., 2008).
2.4 MEDIA FORMULATIONS AND NUTRIENT REQUIREMENT OF S.

THERMOPHILUS
Lactic Acid Bacteria (LAB) are characterized by fastidious nutritional

requirements leading to an important biosynthesis deficiency (Djeghri-Hocine et al.,
2010; Cogan et al., 1997; Loubiere et al., 1996; Monnet and Grippon, 1994). LAB
growth required complex and rich media, containing complex nitrogen sources
(peptides), carbon sources, vitamins and minerals (to supply for trace elements)
(Amouzou et al.,1985; Juillard et al., 1995). These nutrients should be supplied at
optimal concentrations (Benthin and Villadsen, 1996; Cocaign-Bousquet et al., 1995;
Desmazeaud, 1994). Several specific and selective culture media are available for the
isolation and selection of LAB (De Mann et al., 1960; Rogosa et al., 1951; Talwalkar and
Kailasapathy, 2004; Terzaghi and Sandine, 1975; Vinderola and Reinheimer, 1999;
Chamba et al., 1994). The variability in the nutritional requirements varied significantly
with the strain used (Chamba et al., 1994). Consequently, a selective media cannot allow
the numeration of all LAB genera and species. The available selective media are based on
the tolerance of LAB to acidity, to inhibitory compounds or the replacement of glucose
by other sugars (Coeuret et al., 2003; Dave and Shah, 1996; Roy, 2001).
M17 media is found to be critical for the growth of Streptococcus thermophilus.

Therefore, a cheaper media is necessary and the development of economical medium
requires selection of carbon, nitrogen, phosphorous, potassium and trace element sources
(Naveena et al., 2005). Prebiotics are non-digestible carbon sources that stimulate the
growth of beneficial microbial populations (Pinheiro et al., 2012; Mishra and Mishra,
2013), and the growth rates of Streptococcus strains increased when an amino acid
mixture was added. Carbon sources and amino acids have a huge influence on viable
bacteria of S. thermophilus. Xylooligosaccharides, lactose, isomaltooligosaccharide,
glutamate, lysine and valine out of selected substances affect the growth of S.
thermophilus significantly. In addition, xylooligosaccharides, lactose,
isomaltooligosaccharide, glutamate and lysine showed positive effect, but valine had
negative trend. Whatever, these positive effectors can be used in the cultivation of S.
thermophilus and are also conducive to the accumulation of bacterial cell. In addition, the
negative trend of valine for growth of S. thermophilus suggested that this kind of amino
acid must be limited in the medium (Chen et al., 2013).
A culture medium for LAB, based on deproteinated whey supplemented with

yeast autolysis and de-lapidated egg yolk was developed (Djeghri-Hocine et al., 2007).
Egg yolk, an economically attractive local resource (Algeria), contains about 160 g kg-1
proteins, mainly lipoproteins along with high number of vitamins and was found to be
efficient for LAB supplementation (Djeghri-Hocine et al., 2007). The protein efficiency
ratio, namely the ratio of body weight gain to net protein intake, was higher than that of
milk casein for rat nutrition (Sakanaka et al., 2000). Pure cultures of some LAB strains
were considered to evaluate its potential for LAB growth.
Deproteinated whey supplemented with yeast autolysate and de-lipidated egg yolk
showed an interesting potential for growth of the bacterial flora of the various tested dairy
products (Djeghri-Hocine et al., 2007). The addition of sodium azide, to inhibit the
contamination flora, including Gram negative flora, as well as purple bromocresol
allowed a direct selection of the Gram + and lactose + flora, characteristics of the
majority of the LAB species. An acidic pH (5.0) appeared is also helpful, but should be
considered after protein hydrolysis to avoid protein denaturation (or flocculation) and to
favor the release of amino acids and especially small peptides, essential metabolites for
LAB growth.
2.5 STARTER CULTURE AND ITS ROLE IN DAIRY INDUSTRY
Starter cultures are preparations to assist the beginning of

the fermentation process in preparation of various foods and fermented drinks. A starter
culture is a microbiological culture which actually performs fermentation. These starters
usually consist of a cultivation medium, such as grains, seeds, or nutrient liquids that
have been well colonized by the microorganisms used for the fermentation. Dairy starter
cultures are carefully selected microorganisms, which are deliberately added to milk to
initiate and carry out desired fermentation under controlled conditions in the production
of fermented milk products. Most of them belong to lactic acid bacteria (Lactococcus,
Lactobacillus, Streptococcus and Leuconostocs). The taxanomy of the wide range of
starters is explained in Table 2.11. In some cases, few non-lactic starters (bacteria, yeast
and mold) are also used along with lactic acid bacteria during manufacturing of specific
fermented milk products, such as kefir, kumiss and mold ripened cheeses. The
preservation of food by fermentation is one of the oldest methods known to mankind. A
typical example is lactic acid fermentation, which is widely used for the preparation of
several fermented milk products, such as dahi (curd), yoghurt, acidophilus milk, shrikhand
and various varieties of cheeses. In the modern dairy industry, dairy starter cultures are pre-
requisite for the production of safe products of uniform quality. Lactic acid bacteria are often
called dairy starter cultures, which are used for the production of various fermented milk
products. There are 13 major starter producing industries in the world covering the whole
dairy sector across the globe for production of fermented food products (Table 2.12)
Dairy starter cultures are microorganisms that are intentionally added to milk in
order to create a desired outcome in the final product, most often through their growth
and “fermentation” processes. The most common use of starter cultures is for the
production of lactic acid from lactose (milk sugar), which in most cases causes or assists
in the coagulation of milk protein by lowering its pH value. Cultures that produce lactic
acid are generally referred to as “lactic acid bacteria” (LAB). Certain starter organisms
are added specifically for their ability to produce flavor compounds such as diacetyl,
although lactic acid and other culture created compounds contribute to flavor as well.
Starter organisms can also influence flavor and texture of cultured and/or aged products
through the breakdown of proteins, fats and other milk constituents in addition to the pH
effect. The lower pH of cultured products can be inhibitory to certain spoilage organisms,
although inhibition is also associated with other by-products of growth with some
starters. More recently, probiotic cultures are finding their way into cultured milk
products. These are organisms that have some claimed health benefit for those that
consume them, e.g., better digestion, anti-cancer compounds, and prevention of heart
disease. Probiotic cultures may be added as adjuncts or they may be directly involved in
the fermentation process. Slow acid development by cultures can result in an inferior
product or the loss of a vat full of milk. Starter activity can be influenced by a number of
factors including the age of the culture, handling and storage practices, incubation
temperature, the quality of the raw milk, bacteriophage, and the presence of inhibitors
such as drugs or sanitizers. Penicillin and related antibiotics can inhibit cultures at levels
as low as 1-2 parts per billion. Sanitizers can cause inhibition, especially those that leave
residues, such as quaternary ammonia compounds. Natural inhibitors associated with
high somatic cells and late lactation can also slow growth.
Table: 2.11 Taxonomy of dairy Starter Cultures with old and new names and
their products
OLD NAME NEW NAME MAJOR PRODUCT USE

FUNCTION
Mesophilic Lactococcus lactis Acid Production Buttermilk, sour
Starter sub-sp. lactis cream, many types
Streptococcus of cheese
lactis
Streptococcus Lactococcus lactis Acid Production Buttermilk, sour
cremoris sub-sp. cremoris cream, many types
of cheese
Streptococcus Lactococcus lactis Flavor & Acid Sour cream, ripened
diacetylactis sub-sp. lactis biovar butter,cheese,
diacetylactis buttermilk
Leuconostoc Leuconostoc Flavor Buttermilk, sour
cremoris mesenteroides sub- cream cottage
sp. cremoris cheese, ripened
butter
Leuconostoc lactis Unchanged Flavor Buttermilk, sour
cream cottage
cheese, ripened
butter
Thermophilic Unchanged Acid (& Flavor) Yogurts, fermented
Starter milks, Italian
Streptococcus cheese, emmenta
thermophilus
Lactobacillus Lactobacillus Acid & Flavor Yogurts, fermented
bulgaricus delbrueckii sub-sp. milks, Italian
bulgaricus cheese, emmental
Lactobacillus Lactobacillus Acid & Flavor Yogurts, fermented
lactis delbrueckii sub-sp. milks, Italian
lactis cheese, emmental
Lactobacillus Unchanged Acid & Flavor Yogurts, fermented
helveticus milks, Italian
cheese, emmental
Table: 2.12 Major starter culture producer companies all over the world
S.N. Company Country
1 Alce Italy
2 ASCRC Australia
3 Centro Sperimentale del Latte Italy
4 Chr. Hansen Denmark
5 CSK The Nederlands
6 Danisco Denmark
7 Degussa Germany
8 DSM The Nederlands
9 Gewu¨rzmu¨ller Germany
10 Lallemand Canada
11 NZDRI New Zealand
Quest International
12 The Nederlands
13 Rhodia France
2.5.1 Functions of Starter Culture
Starter cultures can be used as single strain, mixed strain and multiple strains
depending upon the type of products to be prepared. The ability of starter culture to
perform its functions efficiently during manufacture of fermented dairy foods depends
primarily on purity and activity of starter cultures. The major roles of starter culture
during fermentation of milk are:
a) Production of primarily lactic acid and few other organic acids, such as formic acid
and acetic acid.
b) Coagulation of milk and changes in body and texture in final products.
c) Production of flavouring compounds, e.g., diacetyl, acetoin and acetaldehyde.
d) Help in ripening of cheeses by their enzymatic activities.
e) Produce antibacterial substances in the finished product.
f) In addition, they may possess functional properties.
Thus, an ideal starter culture should be selected for the preparation of various fermented
milks with the following characteristics.
• It should be quick and steady in acid production.
• It should produce product with fine and clean lactic flavour.
• It should not produce any pigments, gas, off-flavour and bitterness in the finished
products.
• Should be associative in nature in product development.
2.5.2 Types of Starter Culture
There are two major groups of starter cultures which are used in the preparation of
fermented milk products classified on the basis of their physiological, biochemical and
growth characteristics:
2.5.2.1 Mesophillic starter culture
These cultures have optimum temperature for growth between 20 to 30°C and
include Lactococcus and Leuconostoc. These mesophillic lactic cultures are used in the
production of many cheese varieties where important characteristics are:
• Acid producing activity
• Gas production, and
• Production of enzymatic activity for cheese ripening, e.g., proteases and

peptidases enzymes.
2.5.2.2 Thermohillic starter culture
These cultures have optimum temperature for growth between 37 to 45°C.

Thermophilic cultures are generally employed in the production of yoghurt, acidophilus
milk, swiss type cheese. Thermophilic cultures include species of Streptococcus and
Lactobacillus. These cultures grow in association with milk and form the typical yoghurt
starter culture. This growth is considered symbiotic because the rate of acid development
is greater when two bacteria are grown together as compared to single strains.
Thermophilic starter cultures are microaerophillic and fresh heated milk should be used
to achieve a better growth of the culture since heat treatment reduces amount of oxygen
in the product. The important metabolic activities of thermophilic cultures in
development of fermented milk products are:
• Acid production, e.g. lactic acid
• Flavour compounds, e.g., acetaldehyde
• Ropiness and consistency, e.g., polysaccharides
• Proteolytic and lipolytic activities, e.g., peptides, amino acids, fatty acids
• Possesses therapeutic significance, such as Improvement of intestinal organisms,
Produce antibacterial substances, and immunity improvement.
2.5.2.3 Homo-fermentative lactic starter
These lactic acid bacteria are characterized for their ability to ferment lactose
almost exclusively to lactic acid while pentoses and gluconate are not fermented. The
examples of these cultures are: L. acidophilus, L. bulgaricus.
2.5.2.4 Hetero-fermentative lactic starter
Main characteristics of these bacteria are the ability to ferment hexoses and
pentoses to lactic acid, acetic acid, alcohol and CO . The examples of these cultures are
2
Lb. brevis, Lb. fermentum.

2.5.3 Fermented Milk Products
Fermented milks are sour milk products prepared from milk, whole, partially or
fully skimmed concentrated milk or milk substituted from partially or fully skimmed
dried milk, homogenized or pasteurized or sterilized and fermented by means of specific
dairy starter cultures. The origin of cultured dairy product is obscure and it is difficult to
be precise about the date when they were first made. In the early part of the century,
Metchnikoff (1845-1916) claimed that owing to lactic acid and other products present in
sour milks, fermented by lactic acid bacteria, the growth and toxicity of anaerobic, spore-
forming bacteria in the large intestine are inhibited. Lactic acid is biologically active and
capable of suppressing harmful microorganisms, especially putrefactive ones and so has a
favorable effect on human vital activities. Metchnikoff’s theory of longevity considerably
influenced the spread of fermented milk products to many countries, particularly in
Europe. He also promoted extensive studies concerning biochemical and physiological
properties of fermented milks. Milk fermentation for processing of milk into fermented
milk products for increasing the shelf-life and having different flavour and texture
characteristics have been practiced in different parts of the world. Milk has been
processed into cheese, yoghurt, acidophilus milk, kefir, dahi, kumiss and various other
fermented products. In the preparation of various fermented milk products, lactic starters
occupy the key position as the success or failure of such products is directly related to the
types of starter used.
Spoilage of fermented milk products on storage also takes place due to non-lactic
contaminants, such as sporeformers, micrococci, coliform, yeast and molds. These
undesirable organisms rapidly increase in number when the starters are weak and the
ratio of non-lactic to lactic organisms is high. Containers having a large surface of air in
contact with the fermented milk accelerate the process of spoilage. Fermented milk
products are generally spoiled by yeasts and molds and also by lactic acid bacteria which
may cause sour, bitter and cheesy flavor. From dietary point of view, sour milk products,
such as yoghurt, dahi, acidophilus milk, kumiss and other fermented milks are far more
valuable than milk. During fermentation of milk, the composition of the minerals remains
unchanged, while those of proteins, carbohydrates, and vitamins and to some extent fat
constituents change which produce special physiological effects. Dietary and therapeutic
qualities of sour milk products are determined by microorganisms and substances formed
as a result of biochemical process accompanying milk souring. These substances are
lactic acid, alcohol, carbon dioxide, antibiotics and vitamins. Following biochemical
processes make fermented milk products more nutritive than milk:
• Milk Proteolysis
Proteolysis in milk takes place by exo- or endo-peptides of lactic acid bacteria. The
biological value of protein increases significantly from a value of 85.4 to 90 per cent.
This increase is due to breakdown of protein into peptones, peptides and amino acids.
The contents of essential amino acids such as leucine, isoleucine, methionine,
phenylalanine, tyrosine, threonine, tryptophane and valine increase considerably to
offer special advantages not only to healthy people but also particularly to the
physically weak persons. Fermented milks (yoghurt, kefir, dahi) are having higher
protein digestibility due to precipitating into fine curd particle by lactic acid that
contributes to its higher nutritional value and capacity to regenerate liver tissue.
During fermentation and storage, the amount of free amino acids increases,
particularly lysine, proline, cystine, isoleucine, phenylalanine, and arginine. Due to
these biochemical changes in milk protein during fermentation make these products
dietetic in nature.
• Lactose Hydrolysis
Lactose in milk is hydrolysed by metabolic activity of bacteria. Approximately 45-

50% lactose; 16–20% galactose and 0.6-0.8% glucose are obtained from lactose
hydrolysis on the basis of on average 5% lactose in milk. Lactose hydrolysis takes
place due to β -galactosidase production by lactic acid bacteria. The importance of
lactose is due to the lactic acid produced from the hydrolysis of lactose leading to a
pH range in the bowel inhibiting the growth of putrefactants. In addition to this, lactic
acid is important for organoleptic properties and calcium absorption.
• Lipolysis
The homogenization process reduces the size of fat globules, which become
digestible. The production of free fatty acids as a consequence of lipolytic activity
increases due to lactic acid bacteria as compared to milk. This leads to some
physiological effects.
• Changes in vitamins
There is more than two fold increase in vitamins of B-group especially thiamine (B ),
1
riboflavin (B ) and nicotinamide as a result of biosynthetic process during milk

2
fermentation. Subsequently, vitamin B ascorbic acid and vitamin B decrease by

2 1
approximately one half as they are utilized by the bacteria present in milk. However,
the increase or decrease in vitamin content depends on the type of culture.
• Antibacterial activities
The bactericidal properties of fermented milk products are determined by antibiotic

activity of bacteria growing in the product. The antibiotic properties are generally
associated with lactobacilli in yoghurt and materials responsible for such antibacterial
actions are described as lactic acid, hydrogen peroxide and other substances such as
antibiotics and bacteriocins.
• Changes in Minerals
Infact there is not any significant changes in minerals in milk after or during
fermentation process by lactic acid bacteria and the nutritional values of fermented
milk products remain intact.
2.5.4 Bacteriophage
Bacteriophages (phage) are viruses that attack and destroy bacteria. They are very
small and cannot be seen with an ordinary microscope. Phage requires a host cell to
reproduce; one phage per bacterial infection can result in up to 200 phage being released,
each of which can infect a new bacterial cell. Phages are very strain specific, which is
why culture rotation and resistance are used as control mechanisms. Phage can enter the
dairy plant through the raw milk supply although some culture strains are “carriers.”
Problems with “dead vats” due to phage can often be linked to phage in the plant
environment (poor plant hygiene, residual culture). Stringent culture handling and plant
sanitation programs are essential in preventing phage problems.
2.5.5 Fermented milk products in India
2.5.5.1 Yogurt
Yogurt (also spelled jugurt or yoghurt) is a semisolid fermented milk product,
which originated centuries ago in Bulgaria. Its popularity has grown and is now
consumed in almost all parts of the world. Although the consistency, flavour and aroma
may vary from one region to another, the basic ingredients and manufacturing processes
are consistent. Yogurt is strictly defined as a milk product produced by the action of two
bacteria – Streptococcus thermophilus and Lactobacillus delbrueckii sub sp. bulgaricus.
In addition, yogurt may contain bifidobacteria and supplementary flora like Lactobacillus
acidophilus for improving its therapeutic significance. Although milk of various animals
has been used for yogurt production in various parts of the world, most of the
industrialized yogurt production uses cow’s milk, whole milk, partially skimmed milk,
skim milk or cream. Good quality milk is clarified and then standardized to achieve the
desired fat content. The various ingredients are then blended together in a mix tank
equipped with a powder funnel and an agitation system. The mixture is then pasteurized
for 30 min at 85°C or 10 min at 95°C. These heat treatments, which are much more
severe than fluid milk pasteurization, are necessary to:
• Produce a relatively sterile and conducive environment for the starter culture
• Denature and coagulate whey proteins to enhance the viscosity and texture
The mix is homogenized using pressures of 2000 to 2500 psi before final heat treatment.
Besides thoroughly mixing the ingredients, homogenization also prevents creaming and
wheying off during incubation and storage (Figure 2.10). Stability, consistency, body and
texture are enhanced by homogenization. After the final heat treatment, the mix is cooled
to an optimum growth temperature and inoculated with the yoghurt starter culture. A ratio
of 1:1 of Streptococcus thermophilus and Lb. bulgaricus inoculation is added to the
jacketed fermentation tank. A temperature of 42°C is maintained for about 4 h without
agitation, till the milk sets. This temperature is suitable for the two microorganisms. The
titratable acidity is carefully monitored until the titrable acidity is 0.85 to 0.90 per cent.
At this time, chilled water is circulated in the jacket and agitation begins, both of which
slow down the fermentation. The coagulated product is cooled to 5-22°C, depending on
the product. Fruit and flavour may be incorporated at this time, and then packaged. The
product is now cooled and stored at refrigeration temperatures (5°C) to slow down the
physical, chemical and microbiological degradation. There are two types of plain yogurt:
stirred yogurt and set yogurt. In set style, the yogurt is packaged immediately after
inoculation with the starter and is incubated in the packages. Other yogurt products
include fruit and flavored yoghurt, frozen yoghurt and liquid yoghurt.
Figure 2.10: Flow diagram of the main steps involved in the production of Stirred
yogurt
2.5.5.2 Acidophilus Milk
Acidophilus milks are sour milk products in which milk is allowed to ferment
under conditions that favour the growth and development of larger number of
Lactobacillus acidophilus alone or in combination with other lactic acid bacteria or
lactose fermenting yeasts.
• Types of acidophilus milks Organisms

Acidophilus sour milk Lb. acidophilus
Acidophilus yoghurt Lb. acidophilus + S. thermophilus
Bioghurt Lb. acidophilus, L. bulgaricus, S. thermophilus
Acidophilus yeast milk Lb. acidophilus + Lactose fermenting yeast
Acidophilin Lb. acidophilus, Lc. lactis, Kefir fungi
• Acidophilus concentrates
Acidophilus paste Lb. acidophilus
Dried acidophilus Lb. acidophilus
Lyophilized form of milk Lb. acidophilus
Therapeutic importance of acidophilus milk products
• Possesses significant nutritional and prophylactic properties.

• Control gastrointestinal disorders, such as diarrhoea, constipation, dyspepsia,
flatulence and colitis.
• Acidophilus yeast milk, which is rich in alcohol and CO2, excite respiratory and
central nervous system.
• Induction of L. acidophilus into the intestine return to normalcy in the intestinal
microflora and body comforts.
• Possible lowering of blood cholesterol.
• Possible improvement in immune status.
• Lower proliferation of cancerous cells.
 Preparation of acidophilus milk
The milk for this product can be skimmed from full cream milk but because L.
acidophilus does not grow well in milk and would be easily overgrown by usual
microflora, the base milk has to be virtually sterile when the culture is added. The milk is
then left to incubate at 37°C for 12-16 h or till the acidity of the product reaches around
0.8 to 0.9 per cent (as lactic acid). Consequently, the optimum acidity is achieved by
cooling the milk to 5°C or less and halting any further activity by the culture (Figure
2.11). The culture could generate up to 1.0 to 2.0 per cent lactic acid, but the impact of
such levels on cell viability over 2-3 weeks can be devasting in a low solid product. After
cooling, the acidophilus milk is bottled and consumed under chilled conditions.
Acidophilus milk has shelf life of two weeks under refrigeration.
Figure: 2.11 Flow sheet for the preparation of acidophilus milk

2.5.5.3 Curd
Dahi or curd is an Indian fermented milk product which is equally known for its
palatability, refreshing taste and therapeutic importance as claimed in the ayurvedic
literature. Some of its characteristics are similar to other fermented milk products such as
yoghurt and acidophilus milk but it differs with regard to heat treatment of milk, starter
culture, chemical composition and taste. In addition, dahi also has antibacterial properties
against pathogenic and non-pathogenic organisms.
Types of Curd
Some of the fermented milks and different types of dahi consumed throughout India have
been categorized as follows:
• North Zone : Dahi, Lassi
• South Zone : Dahi, Buttermilk (Mattha)
• East Zone : Payodhi or Lal dahi or Mishti dahi
• West Zone : Shrikhand, Chakka, Chhash, Dahi
Based on the acidity level (% lactic acid), dahi has been classified into categories
such as sweet dahi with a maximum acidity of 0.7 per cent and sour dahi with 1.0 per
cent acidity. Starter culture used in the preparation of dahi is normally dahi left over from
previous day. The composition of microflora varies from one household to another and
from one place to another. In general, it has been found that dahi culture is dominated by
streptococci and lactobacilli. In sour dahi, however, lactobacilli predominate. For
commercial manufacture by organized dairy, single starter culture (Lactococcus lactis
subsp. diacetylactis) or mixed culture is used. The raw materials used are cow and/or
buffalo milk, standardized milk, skim milk and reconstituted skim milk powder. The
traditional method for preparation of dahi invariably involves a small scale, either in
consumers’ household or in the sweet makers shop in urban areas. In the household, milk
is boiled, cooled to about 37°C and inoculated with 0.5 – 1 per cent of starter (previous
day’s dahi or butter milk) and allowed to set overnight (Figure 2.12). It is then stored
under refrigeration and consumed
Figure: 2.12 Flow sheet for the preparation of Curd
According to Bureau of Indian Standards (1978), specifications for fermented

milk, dahi, should have a pleasing flavour and a clean acid taste, devoid of undesirable
flavour, should have firm, solid body and texture and be uniform with negligible whey
separation. Some important characteristics have been defined in Table 2.13.
Table 2.13: Characteristics of sweet and sour Dahi
Characteristics Sweet dahi Sour dahi
Acidity (% lactic acid) 0.7 1.0

Yeast and molds (per gram) Max. 100 100
Coliforms (per gram) Max. 10 10
Phosphatase test Negative Negative
A good quality dahi made from whole milk has a cream layer on the top, the rest
being made up of a homogenous body of curd and the surface being smooth and glossy,
while the cut surface should be firm and free from cracks of gas bubbles and it should
have a pleasant acid taste with sweetish aroma. Composition and quality of dahi vary
widely from one locality to another as it is being prepared under different domestic
conditions as well as milk, with variable chemical and bacteriological quality used for the
preparation. However, the chemical composition of dahi has been reported as fat ranging
from 5 to 8 per cent, protein 3.3 to 3.4 per cent, ash 0.75 to 0.79 per cent and lactic acid
0.5 to 1.1 per cent. Quality of dahi can be improved with regard to increase in riboflavin
and folic acid by incorporating propionic acid bacteria such as Propionibacterium
shermani along with dahi starter culture. Regarding palatability and therapeutic
importance of dahi, it has been known to create relish for food, promote the appetite,
increases strength and leads to longevity.
2.5.5.4 Cultured Butter Milk
Buttermilk is really the liquid left from butter making. However, cultured
buttermilk is a fermented milk product made from pasteurized skim milk low fat milk in
which mesophilic lactic acid bacteria is added as starter.
• Starter culture for cultured buttermilk
Starter cultures are typically mixtures of flavour and acid producers Leuconostoc sp.
and Lactococcus lactis sub sp. diacetylactis produces diacetyl, the flavour most
commonly associated with flavored butter and Lactococcus lactis is used to produce
lactic acid which contributes to the acidic flavour typically associated with cultured butter
milk.
• Preparation of cultured butter milk
The starting ingredient for buttermilk is skim or low-fat milk. The milk is pasteurized at
82° to 88°C for 10 - 30 minutes. This heating process is done to destroy all naturally
occurring bacteria and to denature the protein in order to minimize wheying off
(separation of liquid from solids). The milk is then cooled to 22°C and starter cultures of
desirable bacteria, such as Lactococcus lactis, L. cremoris, L. citrovorum and L.
dextranicum are added to develop buttermilk’s acidity and unique flavour. These
organisms are used in proper combination to obtain the desired flavour. The ripening
process takes about 12 to 14 hours (overnight). At the correct stage of acid and flavour,
the product is gently stirred to break the curd, and it is cooled to 7.2°C (45°F) in order to
stop fermentation. It is then packaged and stored under refrigeration.
2.5.5.5 Cheese
A dairy product prepared from cow, buffalo, goat or sheep’s milk that is set aside
to thicken until it separates into liquid, called whey, and semisolids, called curd. The
whey is drained off and the curd is formed into the shape as per specification of cheese. It
is packaged immediately; making it a fresh cheese like Ricotta cheese or cottage cheese,
or it is aged using various curing methods.
 Types of cheeses
There are 400 varieties of cheeses, of which 18 are distinct. Important varieties of cheeses
are given below:
• Cheddar
Cheddar is a hard variety with about 40% moisture and has a diverse selection of tastes
that range from mild to sharp. This is dependent upon the age of the cheese. Mild
Cheddar is perfect for sandwiches because it has a mellow balance of flavors. Sharp
Cheddar is good for cooking because its flavour is released when heated and it shreds
well with other cheeses.
• Mozzarella
Mozzarella has a mild, milky taste and is more of a cooking cheese due to its good
binding properties, moist texture and ability to melt. It is a “stretched-cured” cheese
meaning that during the manufacturing process the curd is pulled, kneaded and shaped
while it is still pliable. Therefore, it absorbs the flavors and juices of the ingredients
surrounding it and is perfectly designed for cooking. Mozzarella is also low in fat;
therefore, it is ideal to use even when dieting. Mozzarella is an ideal cheese for Pizza
making.
• Swiss
Swiss cheese, which is also known as Emmental or Schweizer, is a firm cheese with a
sweet, mildly nutty flavour. This cheese is known for the holes or eye formation that
develops as it ripens. These holes or eyes range in diameter from ½ inch to 1 inch and
begin forming when the cheese is about 3 weeks old.
• Camembert
Camembert has a soft texture with a buttery taste and mushroom smell. It tastes best
when it is at room temperature and the center becomes soft and it is a mold-ripened
cheese.
• Processed Cheeses
It is prepared by melting one or more pressed cooked or uncooked cheeses, and adding
milk, cream, butter and sometimes flavouring agents. One or several ripened cheeses are
heated and mixed, then pasteurized at high temperature (130-140°C) after other dairy
products, such as liquid or powdered milk, cream, butter, casein, whey, and seasoning
have been added.
Chapter 3
Material & Methods
3.1 CHEMICAL AGENTS, NUTRIENTS AND CULTURES.
All chemicals used for chemical reaction were of analytical grades and media
ingredients like yeast extract, casein enzyme hydrolysate, casein peptone, soya peptone,
skim milk powder, beef extract, tryptone, tryptose, protease peptone were of extra pure
grade purchased from Himedia, India. Other chemicals like sodium acetate, tri-
ammonium citrate, di-potassium hydrogen sulphate, magnesium sulphate, manganous
sulphate, di-sodium glycerol phosphate, ascorbic acid, sucrose, lactose, maltose, dextrose,
arabinose, malto dextrin, sodium chloride, potassium chloride, sodium hydroxide,
hydrochloric acid, sulphuric acid were obtained from Fisher Scientific, India; Himedia,
India; Rankem, India; Fluka, UK and Merck, Germany. Some pre-prepared agar medium
were also obtained from Himedia, India like Streptococcus thermophilus isolation agar
(M948), M-16 (M600), Lactobacillus selection agar (M1180), Lactobacillus bulgaricus
agar (M927), Lactobacillus MRS agar (M369), M-17 agar (M929), Brain Heart infusion
agar (M211A), Tomato Juice agar (M048) and L.S differential media (M582). Reference
bacterial strains were purchased from American Type Culture Collection (ATCC), three
lactic acid bacterial strains for reference were purchased from Microbial Type Culture
Collection and Gene Bank (MTCC) Chandigarh, India and five reference strains were
obtained from National Collection of Industrial Microrganism, Pune, India.
 Lactococcus lactis (ATCC 19435)

 Lactobacillus acidophilus (ATCC 4356)
 Streptococcus lactis (MTCC 460)
 Lactobacillus acidophilus (MTCC 10307)
 Lactococcus lactis (MTCC 440; ATCC 11454)
 Streptococcus thermophilus (NCIM 2904)
 Streptococcus thermophilus (NCIM 2412)
 Lactobacillus acidophilus (NCIM 2903)
 Lactobacillus bulgaricus (NCIM 2057)
 Lactobacillus lactis (NCIM 2368)
3.2 SCREENING AND IDENTIFICATION OF SELECTED ISOLATES OF

LACTIC ACID BACTERIA
3.2.1 Sample Collection
Lactic acid bacteria (LAB) in this study were isolated from different sources
which includes cow milk, buffalo milk, goat milk, sheep milk, camel milk, Indian
traditional curd and grapes collected from the surrounding area of Gwalior district of
Madhya Pradesh, India. The sources of cow, buffalo and goat milk were urban dairy
farms while as sheep and camel milk samples were obtained from local milk suppliers.
Curd samples were collected from both dairy farms and local curd suppliers. Grape
samples were randomly picked from different fruit markets and grape farms in the city.
Grape sample was taken aseptically and packaged into clean bag, then stored at 4°C
during their transport. These fruit samples were analyzed within 24 hours of acquisition
at the grape farms. On the other hand raw milk samples were collected in sterile tubes
and maintained in chilled condition (8-10o C) during their transport to Microbiology
Biotechnology Laboratory, Tropilite Foods Pvt. Ltd. Gwalior, for further analysis and
research.
3.2.2 Selective Enumeration and isolation methods
Serial dilutions of all raw milk samples in 0.1% peptone saline were used for
microbial isolation separately. All the milk samples were serially diluted one by one and
were pour plated on MRS (DE MAN, ROGOSA and SHARPE), M17 agar,
Streptococcus thermophilus isolation agar and Lactobacillus selection agar. Plates were
incubated for 24-48 hours at 15, 32, 37 and 45°C in both anaerobic and aerobic
conditions. Same protocol was followed every time for different raw milk samples of
cow, buffalo, goat, sheep and camel (Sharma et al., 2013). In case of grapes, 10 g of
grape sample was homogenized with 90 mL of peptone water (mother solution), 1 mL of
mother solution was transferred into 9 mL of slain solution (8.5 g NaCl, 1000 mL
distilled water, pH 7.0) and serial dilutions up to 10 were made. Then, 1 mL form each
dilution was cultivated in the following selective media: M17 (Himedia, India) and
Streptococcus thermophilus isolation agar to count Streptococcus, incubation at 45°C/48h
(Terzaghi and Sandine, 1975), MRS (Himedia, India) to count Lactobacillus and
Pediococcus, incubation at 30°C/48h (De Man et al., 1960) And Elliker (Himedia, India)
to count Lactococcus, incubation at 30°C/48h (Elliker et al., 1956). Randomly picked
colonies were transferred to suitable media and purification of colonies was made by
repeated streaking on suitable media. Randomly picked colonies were transferred to
suitable media and purification of colonies was made by repeated streaking on suitable
media (Patil et al., 2010). The colonies were randomly (different colony types) picked
from plates with 30-300 colonies. Several representative strains displaying the general
characteristics of lactic acid bacteria were chosen from each plate for further studies.
Each of the isolates were repeatedly streaked in order to purify the isolates, which were
maintained on MRS agar slants for immediate use and in 15% glycerol for storage at -20
°C. Strains of LAB were identified according to their microscopical, morphological,
physiological and biochemical properties (Samelis et al., 1994; Bisen 2014).
3.2.3 Morphological Characterization
Cell morphology was observed by Gram’s staining under bright field

microscopy (CX 21i, Olympus). Selected isolate was heat fixed on a glass slide followed
by flooding the sample with crystal violet for 30 seconds. Crystal violet dissociates in
aqueous solution CV+ and chloride (Cl–) ions. These ions penetrate through the cell wall
and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts
with negatively charged components of bacterial cells to stain the cells purple. Gentle
rinsing was required to wash the violet layer followed by flooding with iodine. Iodine, in
the form of negatively charged ions, interacts with CV+ to form large complexes of
crystal violet and iodine (CV–I complexes) within the inner and outer layers of the
cell. Iodine acts as a trapping agent to retain the purple crystal violet color in the cell.
This step was followed by decolourization in which the decolorizing agent will remove
the crystal violet stain from both gram-positive and negative cells. Counter stain is
applied last to stain the decolorised gram-negative bacteria a pink or red shade. Basic
fuchsin, which stains anaerobic bacteria more intensely, may be substituted for safranin,
but it is less commonly used. Gram-positive bacteria were stained dark blue or violet by
crystal violet and safranin. Gram positive bacteria stain violet due to the presence of a
thick layer of peptidoglycan in their cell walls, which retains the crystal violet.
Catalase activity was initially tested by placing a drop of 3% hydrogen peroxide

solution on the cells (Guyot et al., 1998). Immediate formation of bubbles indicated the
presence of catalase in the cells. Catalase test differentiates between two or more similar
looking unknown coccus bacteria. Streptococcus bacteria can be differentiated from
staphylococcus, planococcus and micrococcus as streptococcus are always catalase
negative while as the other three mentioned are catalase positive. A catalase positive will
produce bubbles of oxygen within one minute after addition of hydrogen peroxide, while
as no bubble release will determines catalase negative. For gas production determination,
bacterial cells were added in MRS test tubes containing the Duram tube (Nomura et al.,
2006) to determine whether the isolate strains produces carbon dioxide during
fermentation (CO2 from glucose or gluconate). The identification procedures were
determined according to the criteria established in Bergey’s Manual of Determinative
Bacteriology. An isolate was deemed to be a homo-fermentative lactic acid producer if no
gas was produced (no CO2 from glucose or gluconate). Plates were incubated at 30 °C for
24-48 h (Holt et al., 1994). All tests were performed in duplicate.
3.2.4 Biochemical Characterization
The previous procedure was repeated in order to purify the isolates, and to be
able to select the high ability in term of growth and good characterization. According to
these tests, the isolates were selected as potential lactic acid bacteria. Biochemical
characterization of the selected strains was carried out with following biochemical tests:
3.2.4.1 MR-VP tests (Methyl red and Voges-Proskauer test)
MR-VP test differentiates bacteria upon the fermentative end product they
produce. Some bacterial strains produce large amount of acids and some produce a
neutral acetoin as the end product. MR-VP are basically two different tests but the reason
behind performing the two tests together is the results they produce as if methyl red tests
gives positive results then Voges-Proskauer must be negative of the same and vice-versa.
Material requirements:
• MRVP broth tubes (Peptone 7g, Dextrose 5g, and Potassium phosphate 5g)
• Methyl Red pH indicator
• Napthol solution
• 40% potassium hydroxide
MR-VP broth was prepared and selected strains were tested against the standard.
Eight tubes were prepared for each bacterial strain separately. Six were inoculated and
incubated at 35°C for 48 hours while as two were kept un-inoculated. On completion of
incubation, five drops of methyl red were added to any three tubes with clear turbidity,
while as 12 drops of napthol solution and 2-3 drops of 40% potassium hydroxide were
added to the remaining three. No color change in the tubes added with methyl red
indicates MR positive and color changing to yellow indicates MR negative. Ruby pink
color on the tubes with napthol indicates VP positive and no color change shows VP
negative. If the MR test is positive it means that the organism produces a large amount of
organic acids like lactic acid, formic acid, succinic acid by fermenting glucose but a
negative MR test shows that the organic acids produced by the organism faced some
enzymatic conversion and get converted into non acidic products like ethanol or acetoin.
On the other hand VP test also determines the presence of acetoin in the under testing
broth. The organisms capable of converting pyruvate into acetoin shows VP positive
(showing red colour) while as no acetoin in broth shows negative test (no colour change).
3.2.4.2 Nitrate reduction test
Bacterial species can also be differentiated on the basis of their ability to reduce
nitrate to nitrite or nitrogenous gases. The reduction of nitrate may be coupled to
anaerobic respiration in some species. Selected isolated strains were also tested against
their nitrate reducing capacity. Material requirements:
• Nutrient broth and KNO3 (Peptone: 5g, Beef Extract: 3g, Nacl: 5g, KNO3: 5g)
• Solution A (Sulfanilic acid 8 g + acetic acid 5N 1000 mL)
• Solution B (Alpha-naphthylamine 5 g + acetic acid 5N 1000 mL)
Inoculate nitrate broth with an isolate and incubate for 48 hours. Add 10-15 drops each
of sulfanilic acid and followed by addition of Alpha-naphthylamine. If the bacterium
produces nitrate reductase, the broth will turn a deep red within 5 minutes at this step. If
no color change is observed, then the result is inconclusive. Add a small amount
of zinc to the broth. If the solution remains colorless, then both nitrate reductase
and nitrite reductase are present. If the solution turns red, nitrate reductase is not present.
3.2.4.3 Casein Hydrolysis
Caseinase is an exoenzyme that is produced by some bacteria in order to degrade

casein. Casein is a large protein that is responsible for the white color of milk. This test is
conducted on milk agar which is a complex media containing casien, peptone and beef
extract. If an organism can produce caseinase, then there will be a zone of clearing
around the bacterial growth. Material requirements are:
• SMP 100 g, peptone 5 g, agar 15 g pH 7.2
• Actively growing culture
Inoculate the organism on the plate either in a straight line or a zig-zag and incubate at 25
or 37 0C. Hold the plate up to the light to see the zones. Positive reactions may be
recorded as strong + or weak + reactions. There is no reagent or indicator in the agar. A
zone of clearing around the growth area identifies the presence of the enzyme caseinase
3.2.4.4 Carbohydrate Fermentation

Carbohydrate fermentation tests detect the ability of microorganisms to ferment a
specific carbohydrate. Fermentation patterns can be used to differentiate different
bacterial groups or species (Forbes et al., 2007). During the fermentation process,
an organic substrate serves as the final electron acceptor. The end-product of
carbohydrate fermentation is an acid or acid with gas production (Mahon et al., 2011).
The end-product depends on the organisms involved in the fermentation reaction, the
substrate being fermented, the enzymes involved, and environmental factors such as pH
and temperature. Common end-products of bacterial fermentation include lactic acid,
formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon
dioxide, and hydrogen (MacFaddin, 2000). Fermentation reactions are detected by the
color change of a pH indicator when acid products are formed. This is accomplished by
adding a single carbohydrate to a basal medium containing a pH indicator. Because
bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH
changes only when excess acid is produced as a result of carbohydrate fermentation.
Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests
because most of the end-products of carbohydrate utilization are organic acids. However,
other pH indicators such as bromocresol/bromcresol purple, bromothymol/bromthymol
blue, and Andrade’s can be used (Table 3.1).
Table: 3.1 pH indicators for carbohydrate fermentation media
pH Uninoculated Acid Alkaline

indicator media (fermentation) (negative)
pH Color pH Color pH Color
Andrade’s 7.1- Light pink 5.0 Pink-red 12.0- Yellow, colorless
7.2 14.0
Bromocresol 7.4 Deep purple 5.2 Yellow 6.8 Purple
purple
Bromothymol 7.0 Green 6.0 Yellow 7.6 Deep Prussian blue
blue
Phenol red 7.4 Reddish- 6.8 Yellow 8.4 Pink-red
orange
When using phenol red as the pH indicator, a yellow color indicates that enough acid
products have been produced by fermentation of the sugar to lower the pH to 6.8 or less.
A delayed fermentation reaction may produce an orange color. In such cases, it is best to
re-incubate the tube. Bubbles trapped within the Durham tube indicate the production of
gas. Even a single bubble is significant and denotes evidence of gas production. No
bubbles within the Durham tube indicate a non-gas-producing or anaerogenic organism.
A reddish or pink color indicates a negative reaction. In negative tubes, the presence of
turbidity serves as control for growth. A reddish or pink color in a clear tube could
indicate a false negative.
3.2.5 Molecular Characterization
Genomic DNA from nearly all prokaryotic and eukaryotic organisms is also
complexed with protein and termed chromosomal DNA. Each gene is located at a
particular position along the chromosome, termed the locus, whilst the particular form of
the gene is termed the allele. In mammalian DNA each gene is present in two allelic
forms which may be identical (homozygous) or which may vary (heterozygous). The use
of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far
the most common house keeping genetic marker used for a number of reasons.
3.2.5.1 16s rDNA gene sequencing
16S rDNA sequencing has played a pivotal role in the accurate identification of
bacterial isolates and the discovery of novel bacteria in clinical microbiology
laboratories. For bacterial identification, 16S rDNA sequencing is particularly important
in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing
bacteria, uncultivable bacteria and culture-negative infections. The hyper variable regions
of 16S rRNA gene sequences provide species-specific signature sequences useful for
bacterial identification. In medical microbiology, 16S rRNA sequencing serves as a rapid
and cheap alternative to phenotypic methods of bacterial identification.
The genetic interrelationships of members of the lactic acid bacteria have been
studied extensively in 16S rDNA sequence, and DNA-DNA hybridization experiments.
Total genomic DNA of all isolated strains was prepared by using the following procedure
(Cardinal et al., 1997). DNA (214 ng/µl) was subjected to PCR utilizing the primer 1
(AGA GTT TGA TCC TGG CTC AG) and primer 2 BOX A1R (CTA CGG CAA GGC
GAC GCT GAC G) as Versalovic et al described. Each 27 µl PCR reaction contained 5
µl 5× Gitschier buffer (1 M (NH4)2SO4, 1 M Tris-HCl (pH 8.8), 1 M MgCl2, 0.5 M
EDTA (pH 8.8) and 14.4 M β -mercapto-ethanol add double distilled water till 200 mL),
0.6 mg/ mL BSA (Sigma, A-7906), 100% DMSO (Sigma, D-8418), 0.2 mM dNTP
(Sigma, D7295), 0.5 µM oligonucleotide primer, 1 units of Taq DNA polymerase
(Sigma, D1806) and distilled water. PCR amplifications were performed in a DNA
thermal cycler with an initial denaturation step (95°C, 7 min), followed by 30 cycles of
denaturation (94°C, 1 min), annealing (53°C, 1 min) and extension (65°C, 8 min), and a
single final extension step (65°C, 16 min). The amplified fragments were fractionated on
a 1.5% w/v agarose gel during 200 min at a constant voltage of 40 V in 0.5×TAE (Tris-
Acetat EDTA) at 4°C. A 10-kb reference marker (Sigma, D7058) was used to allow
standardization, followed by staining with ethidium bromide and visualization.
3.2.5.2 Whole Genome Sequencing
De-novo sequencing and assembly of novel strain of ST-500 (Streptococcus

thermophilus) on Ion torrent platform. 1.6 ug of Qubit quantified DNA was sonicated for
300 sec on Covaris. 950 ng of DNA was taken for library preparation (Table 3.2). End-
repair and adapter ligation was done according to the protocol. The sample was bar coded
at this step. The sample was cleaned using Ampure XP beads. Sample was size selected
using 2% Low melting agarose gel. The gel purified sample was amplified as per the
protocol. The amplified product was cleaned up using Ampure XP beads. The sample
was run on Bioanalyzer HS chip to check the distribution and was quantified using Qubit
Fluorometer.
Table: 3.2 Details of the analytical tools and reference genome used
[1] Sequencing Platform Ion torrent
SeqQC, MIRA-3.4.0, MUMmer, T-MAP, BLAST,

[2] Data Analysis Tools
RAST
Streptococcus thermophilus MTCC_5461.Only
Reference genome
[3] scaffolds are available. (gi|444751733|ALIL01000001-
(1)
ALIL01000144)
a. Link:
http://www.ncbi.nlm.nih.gov/nuccore/NC_003888.3
b. Genome size: 1619896 bp (1.61MB)
c. GC content: 39.33 %
[4] Reference genome Streptococcus thermophilus MN-ZLW-002
(2) (gi|387908808|ref|NC_017927.1|)
a. Link:
http://www.ncbi.nlm.nih.gov/nuccore/NC_017927.1
b. Genome size: 1848520 bp (1.84MB)
c. GC content: 39.01 %
The experimental process of whole genome sequencing was conducted at Genotypic
Technology Pvt. Ltd, Bangalore, India, on paid basis as analysis of the whole genome
sequences and genome mapping requires a number of latest bioinformatics tools and
techniques.
3.3 MEDIA FORMULATIONS AND EFFECTS OF DIFFERENT SOURCES OF

NUTRITION ON MASS PRODUCTION OF STREPTOCOCCUS
THERMOPHILUS
Since a potential lactic acid bacterium was isolated, it was essential to test the
growth parameters of the strain on the minimal media and other selective media
prescribed for the strain to check the final bacterial mass. Selective media were studied
for a brief idea on the nutrition requirements of the strain on specific source basis. Effect
of carbon, nitrogen, amino acids, buffers, surfactants were studied.
3.3.1 Effect of carbon sources on mass production of S. thermophilus
The optimum conditions for growth and fermentation were investigated by using
the selected lactic acid bacterium. To investigate the requirements for complex carbon
sources, the experiments were performed either omitting one of the organic carbon
sources from the modified MRS medium. Effects of different carbon sources like sucrose,
dextrose, lactose, maltose, glucose, arabinose, malodextrine, and fructose were studied.
The initial concentrations of all the selected carbon sources varied from 20-100 g/L (2-
10%). For cultivation, a modified MRS and M948 (Himedia, India) was used: carbon
source (2- 10%), nitrogen source (5%), sodium acetate (0.5%), tri-ammonuim citrate
(0.2%), di-hydrogen potassium phosphate (0.2%). All the compositions were uniformly
mixed sterilized under total controlled conditions and later inoculated with 5% inocolum
size. The concentrations were in triplicates prepared in 500 mL conical flasks with
working volume of 200 mL. Experiments were carried out with incubating temperature of
40°C in orbital shaking incubator (Remi, India) with agitation speed of 150 RPM. Media
formulations resulting in high cell mass were selected for further analysis by cultivation
on 2L fermenter (BIOSTAT A PLUS, Sartorius Stedim, Germany) with working volume
of 1.5 L. Formulations were selected on the basis of their spectra-photometric results
(UV-Visible spectrophotometer 119, Systronics, India) and differentiated on the basis of
optical density (OD) taken at 660 nm. Sampling was done every 5 hour interval for first
15 hours and then every 2 hour for total 24 hours. The optimized condition and medium
were further used in process development.
3.3.2 Effect of nitrogen sources on mass production of S. thermophilus
To investigate the requirements for organic nitrogen sources, the experiments

were performed omitting one of the organic nitrogen sources from the modified MRS
medium. The highly effective lactic acid bacterium which possesses desirable
characteristics such as homo-fermentative ability, good fermentation performance was
selected. Different nitrogen sources were investigated such as casein peptone, meat
peptone, tryptone, soya peptone, beef extract, tryptose, whey protein concentrate, corn
steep liquor, sweet whey powder, yeast extract, respectively. The initial concentrations of
all the selected nitrogen sources varied from 20-80 g/L (2-8%). For cultivation, a
modified MRS and M948 (Himedia, India) was used: carbon source (4%), nitrogen
source (2-8%), sodium acetate (0.5%), tri-ammonium citrate (0.2%), di-hydrogen
potassium phosphate (0.2%). However, it was reported that spent yeast extract lacks
growth factors especially iron. Therefore, 2 mg/L of ferrous sulfate (FeSO4) was added.
Effect of polysorbate 60 (0.1-0.2%), polysorbate 80 (0.1-0.2%), KH2PO4 (0.2-0.4%),
K2HPO4 (0.2-0.4%), MgSO4 (0.05-0.1%), MnSO4 (0.005-0.01%) NaCl (0.2-0.4%),
sodium acetate (0.3-0.5%), tri-ammonium citrate (0.2-0.4%), sodium glycerophosphate
(1.5-2.5%) ammonium nitrate (0.1-0.2%) was also studied. Formulations were selected
on the basis of their spectra-photometric results along with the morphological
observations of the cells to check cell rupturing and viability staining for further analysis.
Experiments were carried out with the same protocol followed for the standardization of
carbon sources.
3.4 EFFECT OF OTHER FACTORS AFFECTING THE GROWTH OF S.
THERMOPHILUS
Other than nutrition sources, there were several other factors and conditions
which needs to be standardized for successful process optimization like incubation
temperature and pH standardization.
3.4.1 Temperature Optimization
The temperature giving the highest productivity was in some cases lower than the
temperature resulting in highest lactic acid concentration and yield, whereas in others the
same temperature gave the best results in all categories (Hofvendahl and Hagerdal, 2000).
The effect of temperature between 30 °C, 37 °C, 40 °C, 45 °C, 50 °C, or 55 °C were
investigated for lactic acid fermentation (Busairi, 2002). All of determinations were
analyzed and the optimum temperature was selected for further study
3.4.2 pH optimization
Some enzymes have ionic groups on their active site, and these ionic groups must
be in the correct form (acid or base) to function. Variation in the pH of the medium
results in changes in the ionic form of the active site. Therefore, the activity of the
enzymes were significantly affected the reaction rate for cell growth and lactic acid
production. The effects of initial pH (4.0, 5.0, 6.0, 7.0, or 8.0) were investigated for lactic
acid fermentation (Yuwono and Kokugan, 2008). The optimum pH was selected for
further study.
3.5 UP-SCALING PROCESS STANDARDIZATION
The up-scaling process for bacterial mass production is one of the critical
standardization points in order to obtain maximum cell mass during the fermentation
process for which a number of parameters were standardized staring from inocolum
preparation to viable cell mass as end product.
3.5.1 Inocolum Preparation
Pure viable colonies of the isolated strain were selected after complete
morphological and biochemical observations and were cultured on agar media (M948
Himedia) followed by incubation at 37°C for 24 hours. Sub-cultured colonies were
checked again for any traces of other bacterial contaminants and were cultured on slants
followed by storage at 8°C. Each experiment was performed using a preserved slant by
inoculating loop-full culture in 80 mL of broth medium. After incubation and visible
turbid growth, broth to broth sub-culturing was done for four to five bacterial generations
and further used as inocolum for different generations (Figure 3.1).
Figure 3.1: Step elabaration of Inocolum preparation from 1st. 2nd and 3rd
generation
3.5.2 Process standardization for incubating conditions
Different incubation conditions were provided to the isolated strains in order to

check the effect of the same on viability of the cells. Both anaerobic and aerobic
conditions were provided and results for oxygen and carbon dioxide demand were
concluded. Temperature was controlled with an attached heating jacket on the fermenter
along with a chilling rod supplied with cold water, while as amount of dissolved oxygen
in the fermenter was calculated with an attached probe. Air supply inside the fermenter
vessel was managed externally through a 0.20 µm air filter (Midisart 2000). Extra
pressure was managed with the help of an attached exhaust. The acid-base ratio inside the
vessel was maintained by an external supply of 1N hydrochloric acid and 5N sodium
hydroxide. The effects of pH, air pressure, dissolved oxygen, temperature, working
volume were studied. The external supply of sodium hydroxide and hydrochloric acid on
the basis of its normality was studied from 1N to 5N and 0.1N to 1N respectively on the
basis of the pH drop due to lactic acid secretion and managed according to the working
volume of the vessel.
3.5.3 Commercial viability of the standardized process
In order to check the commercial viability of the developed process, it was

essential to calculate the final CFU (colony forming units) of the end product of the up-
streaming process, as cell mass with low viability is unsuitable for commercial
production. Final number of CFU was calculated by taking 1 mL sample of the turbid
broth followed by serial dilution of the same. Plates were incubated for 24 hours and
colonies were counted on colony counter (Remi, India). The effect of various parameters
on CFU was also studied and process with maximum CFU was selected for further
experiments.
3.6 VIABILITY AND CFU STANDARDIZATION
Cell viability is defined as the number of living cells present based on a total cell
sample. Quantifying bacteria can be a difficult task to achieve using direct methods of
enumeration. Cell-counting instruments exist that can be used to count numbers of
organisms in a sample using electrical or light impedance, but these tools are often not
found in every lab setting. The viable cell count is an estimate of bacterial population in
an original sample being tested. To perform viable cell counts on agar plates, it is often
necessary to dilute the original sample to make counting easier. Countable plates are
typically considered to hold 30-300 colonies on the surface of the agar. Serial dilution
was performed as per following protocol:
One milliliter of the original sample was transferred to a tube containing 9 mL of sterile
water which makes it a total of 10 mL of solution, 1 part sample and 9 parts water. One
milliliter of solution was moved from this tube to another tube with 9 mL of sterile water
creating a 1:100 dilution. This process is carried out until desired dilution factors were
met. After the proper dilutions the contents of each tube were used to create plates.
• Using a sample of milk, transfer 1 mL into the first sterile water blank. Label this
tube 1:10.
• Mix the tube contents using the vortex mixer on the end of each table.
• Using another transfer pipette, transfer 1 mL of the 1:10 tube to the next sterile
blank tube. Label this tube 1:100.
• Continue making transfers until 1:100 and 1:10000 dilutions have been made.
• After all dilution have been completed, transfer ½ mL from the 1:10 dilution and
place it on a plate labeled 1:20.
• Spread the liquid across the surface of the plate with a clean spreading rod.
• Continue making plates in this fashion from each of the dilution tubes until
created four plates: 1:20, 1:200, 1:2000, 1:20000.
• Place the plates in inverted position in an incubator at fixed temperature.
3.7 DOWN-STREAM PROCESS STANDARDIZATION
Down stream process for production of starter culture includes a lot of bio-
instrumentation and a number of processes like centrifugation, ultra micro-filtration,
cryoprotectant standardization, freezing and lyophilization. It also includes the
application testing of the final product on different parameters. The effects of all the
processes were studied in order to obtain a remarkable commercially viable product.
3.7.1 Centrifugation and micro-filtration
Centrifugation of the turbid broth was standardized to overcome the issue of

viability loss during the process. Specific number of revolutions per minute (rpm) were
standardized by studying the effect of revolutions on cells starting from 4000 to 6000
(4000, 4500, 5000, 5500, 6000 rpm). It was observed during the process that, excessive
centrifugal time was leading to cell rupturing, so the effect of centrifugal time was tested
from 5 minutes to 20 minutes. On the other hand, the effect of micro-filtration on cells
was also studied with both single and double membrane system and it was observed that
the process was less viable compared to centrifugation. The cell loss during
centrifugation was also studied by taking 0.1 g of the fresh pellet sample followed by
serial dilution and colony counting after incubation. 5000 rpm for 15 minutes was
standardized with maximum CFU or lowest cell loss and was selected for performing
further experiments. The 15 minute centrifugal time was again distributed into two
phases with 10 minutes of centrifugation followed by discarding the supernatant and 5
minute washing of pellet at the same 5000 rpm with saline water.
3.7.2 Cryoprotectant preparation and standardization
The turbid broth samples were then centrifuged at 5000 RPM for 10 minutes
followed by pellet washing with physiological NaCl (0.9%) solution. The pellet were
resuspended in 10% sterile solution of defatted skim milk powder (Himedia, India) and
then distributed in sterile vials of 1 mL capacity followed by immediate freezing at -80°
C for further use. The following compounds were used to check the viability
improvement.
Lactose, monohydrate (min 99.5%, milk sugar, Himedia, India), Sucrose, Extra
pure (min 99.5%, Himedia, India), D-(-)-Fructose, Extra pure (min 99%, Himedia, India),
D-(+)-Glucose monohydrate (min 99.5%, Himedia, India), D-(-)-Mannitol, Extra pure
(min 99%, Himedia, India), D-(-)-Sorbitol (min 99%, Himedia, India), D-(+)-Maltose
monohydrate (min 95%, Himedia, India), Maltodextrine (Himedia, India), D-(-)-Ribose
(Min 99%, Himedia, India), D-(-)-Arabinose (Min 99%, Himedia, India). Polymers,
inorganic compounds and other media: Distilled water, Monosodium-L-glutamate
monohydrate (min 98%, Himedia, India), meso-Inositol (min 98%, Himedia, India),
glycerol, purified (min 99%, Himedia, India), sodium chloride, extra pure (min 99%,
Himedia, India), Skim Milk powder, defatted (Himedia, India), di-Potassium hydrogen
phosphate (min 99%, Himedia, India), Potassium dihydrogen orthophosphate, purified
(min 99%, Himedia, India), tri-ammonium citrate, extra pure (min 97%, Himedia, India),
whey protein concentrate (Fonterra, New Zealand), sweet whey powder (Fonterra, New
Zealand), sodium caseinate (Arla Foods, Denmark). Different lyoprotectant combinations
were prepared to check the cell viability after lyophilization. A total of 18 lyoprotectants
were used to develop 40 combinations for evaluating viability results and escalation. All
the lyoprotectants (10% w/w) were prepared and sterilized except monosodium glutamate
(1% w/w), meso-inositol (0.5% w/w), KH2PO4 (1% w/w), K2HPO4 (1% w/w),
ammonium citrate (1% w/w) which were sterilized with mentioned w/w. These
protectants in single and in combinations were mixed with freezed vials in 1:5 ratios (1
mL inocolum vial and 5 mL lyoprotectant). Lyophilization of the samples was done at
0.04mbar vacuum at -50° C (Alpha 1-2, LO plus, Martin Christ).
3.7.3 Freezing temperature and incubation
Freezing is a critical step in lyophilization process as randomness of ice

nucleation and growth is not easy to control. Impact of freezing on lyophilization
includes inter vial and intra vial uniformity, cake appearance, reconstitution time, cake
integrity, container-closure system, in-process stability and long term shelf life stability.
Two phase freezing system was tested in current study. Primary freezing temperature
range from -20°C to -60°C and secondary freezing from -60°C to -80°C were studied.
Freezing cycle time and high degree of intra-vial heterogeneity was also studied.
3.7.4 Lyophilization standardization
Freeze drying technique is a dehydrating method in which biological materials are

first frozen followed by sublimation (primary drying) and desorption (secondary drying),
generally used for long term preservation of lactic acid bacteria starters, but freeze drying
also brings changes in physical state of membrane lipids and structure of sensitive
proteins leading to viability issues (Leslie et al., 1995). However, demand of starter in
modern era is continuously expanding the interest to create ready to use lyophilized
starter with improved stress and shock tolerance (Broadbent and Lin, 1999).
Consequently, some compounds such as polyols, polysaccharides, disaccharides, amino
acids, proteins, vitamins, and various salts have been examined for their potential role to
improve the survival of LAB throughout freeze drying process (Champagne et al., 1991).
Use of S. thermophilus as starter culture for curd, depends on the concentration and
preservation technologies employed, which are required to guarantee long-term delivery
of stable cultures in terms of viability and functional activity. Lyophilization process was
standardized by studying the effect of vacuum pressure from 0.02 mbar to 0.1 mbar and
results were compared and finalized with different freezing period and temperature. The
results were evaluated on the basis of CFU present on different vacuum pressures
analyzed by serial dilution.
3.8 APPLICATION TESTING OF ISOLATED STARTER CULTURE
Monitoring and screening starter culture is of pivotal importance in modern dairy

technology to guarantee quality of products like e. g. curd, yoghurt, cheese, or butter
milk. During milk fermentation organic acids, mainly lactic acid, are produced by the
microorganisms lowering the pH value. This acid production can be used to characterize
milk fermentation and is therefore an important method to test the activity of starter
cultures.
3.8.1 Curd Preparation
Curd is an important part of Indian diet. In most Indian homes curd is prepared
almost every day. Homemade curd is not only very simple to prepare but is also
delicious. Moreover, it has no preservatives and is also economical.
Figure 3.2: Chart for preparation of Indain traditional curd from starter culture
The milk was slowly stirred to distribute the culture organisms uniformly and
the inoculated milk was poured into 250 mL capacity beakers which were then incubated
at 42°C for about 6 h. After fermentation, the beakers were shifted to refrigerator to cool
the curd for about 24 h so that it was cooled to about 5°C. The curd was then analysed for
sensory quality, rheological attributes and physico-chemical attributes. Following
parameters are checked on curd formation:
 Texture of Curd: The texture of curd depends mainly upon the heat treatment
given to milk. Cow milk (3.5% fat and 8.5% SNF) was subjected to two separate
treatments: (1) heating at 63oC for 30 min and (2) boiling treatment without
holding period. The milk was cooled to about 40oC and inoculated with S.
thermophilus culture and incubated at 42oC for about 6 hours. The curd formed
was chilled to 5oC and evaluated for quality. Firmness, consistency and index of
viscosity as measured by Texture Analyser increased with increased heat
treatment and the highest values were observed in curd prepared from boiled
milk. Boiling treatment of milk resulted in least syneresis of whey in the curd.
Based on the results, it was recommended that milk be subjected to boiling
treatment to produce best quality curd.
 Sensory: The chilled curd (5°C) was served to a panel of seven judges and its
colour and appearance, flavor, body and texture and overall acceptance were
evaluated on 9- point Hedonic scale (Amerine et al., 1965). The sensory
evaluation was conducted in Sensory Evaluation Laboratory of the Institute under
fluorescent lights. The sensory acceptance data were tabulated by taking average
of scores awarded by all the judges for different treatments in three replications.
 Syneresis: The set curd at 5°C was slowly transferred to 15 mL capacity

centrifuge tubes causing minimum disturbance to the coagulum. The centrifuge
tubes were balanced by adjusting their weights and centrifuged at 2000 rpm in a
Remi centrifuge for 5 min. The quantity of whey separated at the top of the
coagulum inside centrifuge tubes was recorded as mL. The higher the volume of
whey separated, the higher will be the syneresis and vice versa.
 pH: pH was determined by potentiometric method i.e. by potential difference

between the sample and electrolyte solution present inside the electrode of pH
meter, using digital pH meter (Systronic Co., Bangalore). The electrode of the pH
meter was directly dipped in the set curd and the pH was recorded (5°C). pH was
recorded to check the lactic acid secreting capability of the culture in specific
duration with favorable temperature. Curd was prepared in cups with 100 mL
volume and was incubated. pH was recorded after 3, 3.5, 4, 4.5, 5, 5.5, 6 hours on
incubation from different cups of same experiment, so that the coagulation will
not get disturbed.
 Acidity: The acidity of the curd samples was analyzed by BIS method and
expressed as per cent lactic acid (Bureau of Indian Standards, 1981).
 Rheological: Firmness, consistency and viscosity of curd are important
rheological or textural parameters that govern the quality of curd. These attributes
can be measured objectively by Texture Analyzer or cone penetrometer. The
following method was employed for measuring firmness, consistency and index
of viscosity by Texture Analyzer (Stable Microsystems, UK). The working of
Texture Analyser is based on the principle that a cylindrical steel probe penetrates
into the curd samples and experiences resistance during the penetration. The
resistance offered by the curd sample (5ºC) during the penetration of the probe up
to a specified distance is recorded as firmness of curd.
3.8.2 Comparative testing between prepared starter and starters available in

market
Starter cultures are available in Indian market for curd preparation. Starter
culture JAMA and Yoflex (CHR Hansen, Denmark) and FlavoGard (Danisco Dupont,
Copenhagen) collected from sources and local distributors for comparative studies on
performance for curd preparation. Culture packs were equally diluted in milk and were
inoculated in conditions which were maintained similar for both self isolated and market
starters.
The conditions provided were: Milk FAT 6%, Milk SNF 8.5%, Inoculation temperature
42° C, Incubation temperature 42° C, pH check 3-6 hours.
Parameters observed
• Gel formation
• Cut ability
• Creaminess
• Texture
• Taste
• Acidity
• Mouth feel
• Water separation
• Shelf life
3.9 COMMERCIAL UP-SCALING TRIALS
Commercial up-scaling trials were conducted on 300 L fermenter at a rented

facility with TBI (Technology Based Incubator) Center, Delhi University South campus,
New Delhi. The facilities used were – Shaking incubator, seed fermenter (14, 30 L), pilot
fermenter (300L), Micro and Ultra filtrate, pilot centrifuge and lyophilizer. At
commercial facility inocolum was first prepared in 500 mL flasks (10) with 180 mL each
and lyophilized vials were used as inocolum multiplication. 7 L media was prepared for
14 L fermenter and sterilized at 15psi. After 8 hours of incubation the total volume in the
14 L fermenter was transferred to 250 L media (working volume) in a 300 L fermenter as
its inocolum.
3.10 PROTOCOL FOLLOWED AT TECHNOLOGY BASED INCUBATOR
CENTER (TBI) DELHI UNIVERSITY FOR FERMENTATION TRIALS ON
PILOT PLANT
MEDIA PREPARATION IN 500 mL FLASKS WITH WORKING VOLUME 180 mL
INOCULATION IN FLASKS WITH PREPARED LYOPHILIZED VIAL INOCOLUM
INCUBATION IN ORBITAL SHAKER INCUBATOR FOR 12-16 HOURS TILL
MORPHOLOGICAL OBSERVATION OF THE STRAIN AFTER MULTIPLICATION

ST
PREPARATION OF 10L MEDIA FOR 14L SEED FERMENTER FOR INOCOLUM
1.44 L BROTH WAS TRANSFERRED TO 14L FERMENTER AS SEED INOCOLUM
FERMENTATION WAS CARRIED OUT AT 200RPM & 40°C FOR 10-12 HOURS
MORPHOLOGICAL OBSERVATION AFTER SEED PREPARATION (2ND gen)
10.5 L BROTH WAS TRANSFERRED TO 250L MEDIA IN 300L FERMENTER
FERMENTATION WAS CARRIED OUT AT 200rpm & 40°C FOR 10-12 HOURS
1N HCL AND 5N SODIUN HYDROXIDE WERE PREPARED AS BUFFERS
CENTRIFUGATION WAS CARRIED OUT AT 5000rpm FOR 10 MINUTES
LYOPHILIZATION MEDIA WAS PREPARED AND MIXED WITH PELLET
PELLET AND LYOPHILIZATION MEDIA WERE MIXED IN RATIO OF 1:1
FREEZING WAS DONE AT -60°C FOR 12 HOURS (UNTILL CAKE FORMATION)
LYOPHILIZATION WAS CARRIED OUT AT 0.04MBAR VACUUM AT -40°C FOR 12
CREAMISH WHITE POWDER WAS RECOVERED AS END PRODUCT

Chapter 4
Results & Discussion
4.1 SCREENING AND ISOLATION OF LACTIC ACID BACTERIA
4.1.1 Isolation of Lactic acid bacteria
Streptococcus thermophilus Isolation (STI) agar, M 17 agar and MRS agar were
employed as selective media for isolation of lactic acid bacteria. 10 colonies were
randomly picked from different agar plates containing 20 to 200 lactic acid bacteria in
each isolation experiment and more than 500 isolation experiments were conducted by
serial dilution of different samples from different region. Plates were examined by eye,
and the different colony types were individually picked. They were propagated twice and
streaked on the selective media to obtain cultures. Gram positive and catalase negative
cultures were maintained on selective media slants for further studies while others were
discarded. A total of 128 isolates of LAB were isolated from curd, ripen grapes, cow
milk, buffalo milk, camel milk, sheep milk and goat milk. Initially, the isolated strains
were named according to their origin. For example; thirty seven isolates were isolated
from curd were named as C (n), twenty two from goat milk were named as G (n), twenty
eight from buffalo milk were named B (n), Twelve from cow milk were named CW (n),
seven from camel milk were named CAM (n), six from grapes were named GP (n),
sixteen from sheep milk were named S (n), respectively.
4.1.2 Selection of LAB for curd trials and lactic acid production
All the isolated LAB strains were subjected to study their ability to coagulate milk
to produce curd. Isolated 128 isolates were maintained in MRS, M17 and STI broth
depending upon their original selective media of isolation at 32, 37 and 45°C temperature
in orbital shaker incubator at 150 rpm for 24 hours. Pellets of the isolates were collected
by centrifugation at 4°C in cooling centrifuge at 5000 rpm for 10 minutes followed by
washing with saline water and supernatant was discarded. 300 mg of fresh cell mass was
inoculated in 300 mL of pasteurized milk (6% FAT, 8.5% SNF) which was allowed to
cool up to 42°C before inoculation. The experiment was carried out in triplicates. After
six hours of incubation, pH was recorded (Table 4.1) for each isolate and potent strains
were selected for further experiments. The concentrations of lactic acid were determined
from the inoculated broth medium after 24 hours of incubation. A 10 mL sample was
collected during fermentation aseptically produced from 100 mg of sugar. Centrifugation
of fermented broth samples was carried out at several intervals at 12000 RPM for 10
minutes and supernatant was analyzed by HPLC. The cultured medium was filtered
through 0.45 µm membrane filter. Organic acids were analyzed by HPLC (Merck–
Hitachi). Five microliters of the sample was injected into the HPLC system equipped
with an Aminex HPX-87 H column and RI detector. The column temperature was
maintained at 65ºC. The mobile phase was 10 mM H2SO4 at flow rate of 0.6 mL/min
(Sharma et al., 2013; Thang and Novalin, 2008). Isolates with tendency to ferment sugar
into lactic acid efficiently were further allowed to multiply individually and
concentrations of lactic acid productivity was determined (Table 4.1) by using HPLC and
the yield was calculated by folowing equation.
Percentage yield = x 100
It was observed that 32 isolates could produce high yields of lactic acid (more
than 75%) with pH drop to 4.22 - 4.47 after six hours of incubation (Table 4.1). These 32
isolates were also identified for their better milk coagulating properties, fine texture and
taste of the final curd, whereas other 96 isolates may be classified as hetero-fermentative
LAB because of their low yield of lactic acid production. As a result, 32 isolates showing
high product yield were selected for subsequent characterization. However, they could be
readily distinguished by microscopic examination. Identification of strains was further
investigated according to their morphological, cultural, physiological, and biochemical
characteristics by the procedures described in the Bergey’s Manual of Systematic
Bacteriology (Holt et al., 1994).
Table: 4.1 Percentage lactic acid yield and pH drop results of the 128 isolates
Isolate Percentage pH after Isolate Percentage pH after

Code yield of lactic six hours Code yield of lactic six hours
acid incubation acid incubation
production production
C1 31.45 5.65 C30 21.56 5.94
C2 27.66 5.74 C31 22.85 5.91
C3 25.31 5.84 C32 83.88 4.19
C4 22.96 6.21 C33 54.55 4.76
C5 33.12 5.55 C34 71.32 4.39
C6 15.56 6.21 C35 43.12 5.12
C7 22.13 5.97 G1 62.66 4.51
C8 44.21 5.09 G2 77.45 4.24
C9 39.32 5.27 G3 53.21 4.75
C10 77.88 4.24 G4 47.39 4.96
C11 47.96 4.97 G5 56.89 4.69
C12 55.17 4.78 G6 77.97 4.27
C13 22.17 5.94 G7 51.64 4.81
C14 40.44 5.21 G8 55.67 4.71
C15 71.64 4.44 G9 59.96 4.64
C16 77.85 4.29 G10 52.56 4.81
C17 19.23 6.08 G11 52.31 4.81
C18 81.88 4.21 G12 60.14 4.62
C19 21.23 5.98 G13 80.56 4.22
C20 29.36 5.64 G14 78.19 4.31
C21 79.11 4.25 G15 54.36 4.78
C22 80.23 4.21 G16 74.66 4.37
C23 46.57 4.97 G17 59.24 4.66
C24 69.77 4.33 G18 81.23 4.28
C25 48.14 4.82 G19 46.57 5.01
C26 22.75 5.92 G20 79.77 4.33
C27 66.53 4.51 G21 53.21 4.75
C28 56.11 4.71 G22 47.39 5.12
C29 23.27 5.94 B1 55.98 4.71
B2 51.23 4.82 CW7 12.66 6.52
B3 56.45 4.72 CW8 37.89 5.32
B4 61.23 4.63 CW9 31.67 5.62
B5 36.45 5.48 CW10 32.22 5.55
B6 59.24 4.68 CW11 27.51 5.74
B7 58.36 4.65 CW12 26.36 5.79
B8 57.29 4.68 CAM1 31.22 5.62
B9 59.27 4.6 CAM2 37.97 5.33
B10 58.36 4.68 CAM3 35.98 5.42
B11 54.22 4.77 CAM4 30.11 5.62
B12 68.21 4.49 CAM5 39.54 5.31
B13 61.23 4.59 CAM6 33.78 5.58
B14 56.22 4.69 CAM7 77.92 4.29
B15 45.69 5.06 GP1 21.61 5.97
B16 48.32 4.98 GP2 17.26 6.18
B17 49.02 4.92 GP3 21.39 6.02
B18 48.33 4.95 GP4 14.2 6.35
B19 55.89 4.77 GP5 19.58 6.08
B20 77.22 4.25 GP6 29.09 5.68
B21 58.69 4.67 S1 15.56 6.22
B22 43.22 5.09 S2 22.13 5.94
B23 62.59 4.58 S3 18.66 6.15
B24 57.97 4.71 S4 19.85 6.18
B25 51.64 4.82 S5 17.32 6.15
B26 59.24 4.62 S6 39.54 5.31
B27 58.3 4.68 S7 21.27 6.03
B28 48.22 4.92 S8 29.39 5.61
CW1 18.66 6.08 S9 22.51 5.92
CW2 19.87 6.02 S10 25.62 5.88
CW3 17.33 6.11 S11 12.67 6.48
CW4 18.65 6.09 S12 21.66 5.98
CW5 22.58 5.99 S13 17.25 6.16
CW6 25.69 5.85 S14 17.31 6.15
4.1.3 Morphological and Phenotypic characterization of Lactic acid bacteria
The single colonies of the sample isolates were grown on selective media.
Microscopic observation showed them to appear as short rods, long, thin and cocci shape
(Figure 4.1). Table 4.2 shows the morphological and physiological diversity of the 32
isolates strains giving details on cell morphology (rod, cocci, and ovoid shape) that were
subdivided into two groups: cocci (75%), and rods (25%) and on the basis of growth, and
gas production (homo- or hetero-fermentative) (Table 4.2). Isolates were further
subdivided on the basis of profuse growth and poor growth with 50 % (+++), 40 % (++),
and 10 % (+), respectively. Bacterial growth depends on culture medium with its ability
to produce a high concentration of biomass. The variation of different nutrients in the
medium are needed for lactic acid bacterial strains. The group of complex nutrients, i.e.,
skimmed milk, yeast extract, whey proteins, tryptone and peptone were used to satisfy the
complex demands of the bacteria. A complex medium, especially, M17 and MRS are
usually employed and supported the growth.
There were some strains showing good performance of homo-lactic acid

fermentative which were selected for further investigation as starter culture in the
subsequent fermentation processes.
Table: 4.2 Phenotypic characterization, colony observation and gas production
results of the selected 32 isolates.
STRAIN CELL TURBID GAS

COLOR FORM EVEVATION
CODE SHAPE GROWTH PRODUCTION
C10 Cocci White Circular Flat +++ -
C15 Cocci White Circular Raised ++ +
C16 Cocci White Circular Raised +++ -
C18 Rods White Punciform Raised +++ -
C24 Cocci Creamish Punciform Flat +++ +
C26 Cocci White Circular Flat +++ +
C27 Rods White Circular Raised ++ +
C32 Cocci Creamish Punciform Flat +++ -
G1 Cocci White Circular Flat ++ -
G2 Cocci White Circular Flat ++ -
G6 Cocci White Circular Raised +++ -
G8 Rods White Irregular Flat + +
G10 Cocci White Circular Raised ++ -
G13 Cocci White Circular Raised ++ +
G16 Cocci White Irregular Raised ++ -
G18 Cocci White Irregular Flat +++ -
G20 Cocci White Circular Flat ++ +
B4 Cocci White Circular Raised ++ +
B6 Cocci White Irregular Flat ++ -
B11 Rods Creamish Punciform Raised ++ +
B14 Rods Creamish Punciform Flat + +
B17 Cocci White Circular Flat ++ -
B20 Cocci White Circular Flat +++ -
B23 Rods White Irregular Raised ++ -
B27 Cocci White Circular Raised ++ -
CW11 Cocci White Circular Raised ++ -
S8 Cocci White Circular Raised ++ +
S15 Cocci White Circular Raised ++ -
CAM3 Rods Creamish Punciform Raised ++ +
CAM7 Rods Creamish Punciform Flat +++ -
Legend: profuse growth (+++), good growth (++), moderate to poor growth or a positive
reaction (+), no growth or no reaction (-).
After continuous sub-culturing of the selected strains, few strains showed poor cell
viability (<10% cell turbidity in broth) which were discarded. For further consideration,
isolates with gas production were discarded because they deemed to be hetero-
fermentative. Homo-fermentative strains with >40% viability on sub-culturing were
selected for further studies and designated according to their morphology. Isolates from
curd C10, C16, C21, C22 and C32 with morphology of cocci in chains (Streptococcus)
were re-designated as ST-100, ST-200, ST-300, ST-400 and ST-500 respectively while as
isolates from goat milk G6 and G18, from buffalo milk B20 were re-designated as ST-
600, ST-700 and ST-800 respectively. Isolates from curd C18 and from camel milk
CAM7 with rod shaped morphology were considered Lactobacillus and re-designated as
LB-100 and LB-200 respectively (Figure 4.2, 4.3).
4.1.3.1 Morphology
Table 4.3 showed general characteristics of the 10 isolated strains selected for
further studies. Most strains possessed cocci or short rods shape with their cell size
ranging from 0.2-1.0 μm. Morphological configuration showed white, creamy, and
circular colonies with entire margin. Surface was observed smooth with raised elevation.
All the isolates were subjected to Gram staining and they were examined under light
microscope. They stained blue- purple hence they all were Gram positive bacteria and
none of the isolate showed catalase activity. For physiological characterization most of
the strains grew well between 35-37°C except LB-100 growing well at 32° C only. ST-
500 showed a wide range of temperature compatibility from 32° C to 50° C. The ability to
grow at high temperature is a desirable trait as this could translate to increased rate of
growth and lactic acid production beneficial for fast curdling of milk and a high
fermentation temperature could reduce the chances of contamination by other
microorganisms (Mohd Adnan and Tan, 2007). Further, ST-400 and ST-500 were found
to tolerat to low pH range from 4.2-5.2. Growth at low pH for a starter is a bit dual
impact property as low pH may resist the growth of other pathogenic bacteria but can
also effect the shelf life and taste of the product due to continuous production of lactic
acid.
ST-500 was the only isolate found to tolerate 3% NaCl. Ability to grow at 3% and 5%
NaCl concentrations were tested for all isolates and only three of them were found to be
resistant to 3% NaCl concentration. Bacteria adapt to hyper-osmolarity by accumulation,
synthesis and transport of compatible solutes to restore turgor. It was well documented
that osmo-protectants (thermo-protection) could play additional positive role and
beneficial effects have been demonstrated on membrane integrity, protein folding and
stability, (Baliarda et al., 2003). Accumulation of osmo-protectants or compatible solutes
in the present studies had been considered to be the potential lactic acid bacteria.
Similarly, a higher tolerance to lactic acid was a desirable trait for an industrial strain of
LAB as it could produce more lactic acid in the fermentation broth without adversely
affecting itself.
The strain ST-500 grew at higher NaCl concentration compared with the other
isolates (Table 4.3). During fermentation process, lactic acid was produced by the cells
whereas alkali was pumped into the broth to prevent excessive reduction in pH. The free
acid would be converted to its salt lactate which would increase the osmotic pressure on
the cell. Therefore, a LAB strain with high osmo-tolerance would be desirable for
industrial application. These rapid screening processes resulted in domination of ST-500
in a wide range of tested conditions. Homo-fermentative lactic acid bacteria grew
substantially faster than other bacteria present in the same ecological niche. The higher
growth rate of lactic acid bacteria is a result of their simple primary metabolism, and their
ability in adaptation to rich environments
Table: 4.3 Complete morphological and phenotypical analysis of 10 selected strains
Code of Isolates
Characteristics ST-100 ST-200 ST-300 ST-400 ST-500
COLONY
MORPHOLOGY
Configuration Circular Circular Circular Circular Circular
Margin Entire Entire Entire Entire Entire
Elevation Raised Raised Raised Raised Raised
Surface Smooth Smooth Smooth Smooth Smooth
Pigment Cream Cream White White White
Opacity Opaque Opaque Opaque Opaque Opaque
CELL Cocci in Cocci in Cocci Cocci in Cocci in

MORPHOLOGY chains chains chains chains
Cell diameter Size 0.2- 0.4-0.6μm 0.6-0.8μm 0.4-0.6μm 0.4-
0.4μm 0.6μm
PHENOTYPIC
CHARACTERSTICS
Gram staining + + + + +
Spore Formation - - - - -
Motility - - - - -
Catalase Activity - - - - -
Fermentation Type Homo Homo Homo Homo Homo
Glucose Fermentation + + + + +
Nitrate Reduction - - - - -
Casein Hydrolysis + + + + +
Growth at 10o C - - - - -
Growth at 32o C - - - - +
Growth at 37°C + + + + +
Growth at 45o C + - - + +
Growth at 50 o C - - - - +
Growth with 1.5% Nacl + - + - +
Growth with 3% Nacl - - - - +
At pH 4.2 - - - + +
At pH 5.2 + + + + +
At pH 6.2 + + + + +
Code of Isolates
Characteristics ST-600 ST-700 ST-800 LB-100 LB-200
COLONY
MORPHOLOGY
Configuration Circular Circular Circular Circular Circular
Margin Entire Entire Entire Entire Entire
Elevation Raised Raised Raised Raised Raised
Surface Smooth Smooth Smooth Smooth Smooth
Pigment Cream Cream White White Cream
Opacity Opaque Opaque Opaque Opaque Opaque
CELL Cocci in Cocci in Cocci Rods Rods

MORPHOLOGY chains chains
Cell diameter Size 0.4- 0.5-0.6μm 0.4-0.5μm 0.6-0.9μm 0.5-
0.6μm 1μm
PHENOTYPIC
CHARACTERSTICS
Gram staining + + + + +
Spore Formation - - - - -
Motility - - - - -
Catalase Activity - - - - -
Fermentation Type Homo Homo Homo Homo Homo
Glucose Fermentation + + + + +
Nitrate Reduction - - - - -
Casein Hydrolysis + + + + -
Growth at 10o C - - - - -
Growth at 32o C - - - + +
Growth at 37°C + + + - +
Growth at 45o C + - - - -
Growth at 50 o C - - - - -
Growth with 1.5% Nacl + - + - -
Growth with 3% Nacl - - - + +
At pH 4.2 - - - + -
At pH 5.2 + + + + +
At pH 6.2 + + + + +
Figure 4.3: Microscopic views of ST-200 (A), ST-300(B), ST-400(C), ST-500(D),
LB-100(E) and LB-200(F)
4.2) BIOCHEMICAL CHARACTERIZATION OF ISOLATES
The type of carbohydrate and microorganism present in are the important criteria
in the resulting product. They contribute to the aroma, flavor development, and food
preservation (pH drop). The capabilities of LAB to grow at the expense of carbohydrates
and to produce fast acidification (lactic acid) are two criteria for the selection of suitable
candidates for starter strains. From a technological standpoint these criteria are important
for the inhibition of contaminating microflora, consistency of the final product, and
reduction in production time. Detailed physiological characterization of the sugar
fermentation pattern is an important requisite to classify new strains and select those
appropriate for maximal acidification or biomass production in a given carbon source.
Conventional techniques for studying sugar metabolism are usually very time-consuming,
needing long incubation periods and precise standard conditions. For identification of the
lactic acid bacteria, API 50 CH test kit (bioMerieuxΤΜ) was used. The API 50 CHL test
kit is a standard system associated with the study of the assimilation-fermentation of 49
different compounds (plus one control) as per manufacturer’s guideline. Lactic acid
bacteria could live in the absence as well as in the presence of the atmospheric oxygen
indicating that they are facultative anaerobes. All the isolates were tested against 42
sugars for their fermentation property (Table 4.4) and results obtained were used for
other purpose, for example, epidemiological grouping into types of the lactic acid
bacteria, taxonomical analysis of a group of lactic acid bacteria, and classification of an
unknown bacterial population into homogeneous groups. Among all the 42 sugars, almost
all the selected strains were observed positive fermentation against sucrose, lactose,
dextrose, maltose and fructose and negative against sorbitol, trehalose, raffinose, salicin,
glycogen, starch etc (Table 4.4)
Table 4.4 Fermentation results of selected isolates from different sugars
Code of Isolates
Sugars ST-100 ST-200 ST-300 ST-400 ST-500
D-arabinose - - - - -
L-arabinose - - - - -
D-xylose - - - - -
L-xylose - - - - -
D-galactose + + + + +
D-glucose + + + + +
D-fructose + + + + +
D-mannose - - - - -
D-mannitol + + + + +
D-sorbitol - - - - -
D-cellobiose + + + + +
D-sucrose + - - + +
D-trehalose - - - - -
D-melibiose + - + - -
D-lactose + + + + +
D-maltose - - - - -
D-raffinose - - - + +
D-melezitose + + + + +
D-turanose + + + + +
D-fucose - - - - -
D-arabitol - - - - -
L-arabitol - - - - -
Code of Isolates
Sugars ST-100 ST-200 ST-300 ST-400 ST-500
Dulcitol - - - - -
Erythritol - - - - -
Inositol + + - - -
Amygdalin - - - - -
Arbutin - - - - -
Glycerol - - - - -
Esculin + + + + +
Salicin - - - - -
Inulin + + + + +
Glycogen - - - - -
Xylitol + + + + +
Gentiobiose + - + - -
L-rhamnose - - - - -
N-acetylglucosamine + - + - -
Amidon (starch) - - - - -
Potassium gluconate - - - - -
potassium 2- - + - - -
ketogluconate
potassium 5- - - - - -
ketogluconate
Methyl-αD- - - - - -
Mannopyranoside
Glucopyranoside
Continued.
Sugars ST-600 ST-700 ST-800 LB-100 LB-200
D-arabinose - - - - -
L-arabinose - - - - -
D-xylose - - - - -
L-xylose - - - - -
D-galactose + + + + +
D-glucose + + + + +
D-fructose + + + + +
D-mannose - - - - -
D-mannitol + + + + +
D-sorbitol - - - - -
D-cellobiose + + + + +
D-sucrose + - - + +
D-trehalose - - - - -
D-melibiose + - + - -
D-lactose + + + + +
D-maltose - - - - -
D-raffinose - - - + +
D-melezitose + + + + +
D-turanose + + + + +
D-fucose - - - - -
D-arabitol - - - - -
L-arabitol - - - - -
` Continued
.
Code of Isolates
Sugars ST-600 ST-700 ST-800 LB-100 LB-200
Dulcitol - - - - -
Erythritol - - - - -
Inositol - - - + +
Amygdalin - - - - -
Arbutin - - - - -
Glycerol - - - - -
Esculin + + + + +
Salicin - - - - -
Inulin + + + + +
Glycogen - - - - -
Xylitol + + + + +
Gentiobiose + - + - -
L-rhamnose - - - - -
N-acetylglucosamine + - + - -
Amidon (starch) - - - - -
Potassium gluconate - - - - -
potassium 2- - + - - -
ketogluconate
potassium 5- - - - - -
ketogluconate
Mannopyranoside
Glucopyranoside
Legend: positive (+), negative (-).
Enzymatic activities of all the selected isolates were studied as there are few enzymes
from lactic acid bacteria having many dairy applications including lactase responsible for
the treatment of lactose intolerance. Lactase activity was observed in five out of ten
strains and all the lactase positive strains were streptococci. Phosphatase and glycosidase
activities were observed negative for all the strains (Table 4.5) as was also observed by
Tamang et al., 2007. They were all esculin hydrolase and arylamidase positive.
Table 4.5 Results of enzyme assays conducted on selected isolates
CODE OF ISOLATES
ST-
ENZYME ASSAYS ST-100 ST-200 ST-300 ST-400
500
PHOS (PHOSPHATASE) - - - - -
BGAL (BETA-
- - - - -
GALACTOSIDASE)
BMAN (BETA-
- - - - -
MANNOSIDASE)
LACTASE + + - - +
ProA (L-Proline
- - - - -
ARYLAMIDASE)
LeuA (Leucine
+ + - - +
ARYLAMIDASE)
TyrA (Tyrosine
+ - - - -
ARYLAMIDASE)
ESC (ESCULIN
+ + + + +
Hydrolase)
APPA (Ala-Phe-Pro-
+ + + + +
ARYLAMIDASE)
NAG (N-ACETYL-D-
- - - - -
GLUCOSAMINE)
BMAN (BETA-
- - - - -
MANNOSIDASE)
ProA (L-Proline
- - - - -
ARYLAMIDASE)
URE (UREASE) - - - - -
CODE OF ISOLATES
ENZYME ASSAYS ST-600 ST-700 ST-800 LB-100 LB-200

PHOS (PHOSPHATASE) - - - - -
BGAL (BETA-
- - - - -
GALACTOSIDASE)
BMAN (BETA-
- - - - -
MANNOSIDASE)
LACTASE + + - - -
ProA (L-Proline
- - - - -
ARYLAMIDASE)
LeuA (Leucine
+ + - - +
ARYLAMIDASE)
TyrA (Tyrosine
+ - - - -
ARYLAMIDASE)
ESC (ESCULIN
+ + + + +
Hydrolase)
APPA (Ala-Phe-Pro-
+ + + + +
ARYLAMIDASE)
NAG (N-ACETYL-D-
- - - - -
GLUCOSAMINE)
BMAN (BETA-
- - - - -
MANNOSIDASE)
ProA (L-Proline
- - - - -
ARYLAMIDASE)
URE (UREASE) - - - - -
ORIGINAL
COLOR OF POSITIVE NEGATIVE
S.N. TEST PRINCLIPLE
THE REACTION REACTION
MEDIUM
Detects acetonin Colourless/Light Pinkish Colourless/Slight
1 VP
production yellow red/Red copper
Esculin Detects acetonin
2 Cream Black Cream
Hydrolysis production
Detects PYR enzyme
3 PYR Cream Cherry Red Cream
activity
Detects β -galactosidase
4 ONPG Colourless Yellow Colourless
activity
No change in
Arginine Detects arginine Olive green to
5 dark Purple colour or yellow
utilization decarboxylation light purple
colour
Carbohydrate
6 Glucose Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
7 Lactose Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
8 Arabinose Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
9 Sucrose Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
10 Sorbitol Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
11 Mannitol Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Carbohydrate
12 Raffinose Pinkish red/Red Yellow Red/Pinkish red
Ulilization
Figure: 4.4 Analysis of biochemical results of all selected 10 strains by Kit

menthod
MR-VP test differentiates bacteria upon the fermentative end product they
produce. Some bacterial strains produce large amount of acids and some produce a
neutral acetoin as the end product. MR-VP tests were conducted for all isolates and it was
observed that all the isolates were MR -positive and VP-negative (Table 4.6) (Figure 4.5,
4.6).
Table: 4.6 Biochemical characterization results on the basis of different biochemical

test analysis
CODE OF ISOLATES
BIOCHEMICAL TEST ST-100 ST-200 ST-300 ST-400 ST-500

Methyl Red Test + + + + +
Voges Proskauer Test - - - - -
Starch Hydrolysis - - - - -
Nitrate Reduction Test - - - - -
Urea Test - - - - -
Catalase Test - - - - -
CODE OF ISOLATES
BIOCHEMICAL TEST ST-600 ST-700 ST-800 LB-100 LB-200

Methyl Red Test + + + + +
Voges Proskauer Test - - - - -
Starch Hydrolysis - - - - -
Nitrate Reduction Test - - - - -
Urea Test - - - - -
Catalase Test - - - - -
Casein hydrolysis was also conducted in which skim milk powder was used as
casein source; all the strains produced a clear zone around the area of bacterial contact
indicating the presence of caseinase enzyme. Starch hydrolyzing test was also conducted
in which maize and potato starch were used along with nitrogen and carbon sources to
check the hydrolysis. All the selected isolates were negative against starch hydrolysis.
The strains were urease and catalase negative indicating that the strains belong to LAB
origin.
Chapter 5
Molecular Characterization
5.1 MOLECULAR CHARACTERIZATION
Despite the wide application of lactic acid bacteria, they are currently
differentiated by the determination of DNA-DNA similarity, their G+C content and, by
physiological characterization (Tanasupawat et al., 1993). Biochemical identification is
not accurate for determining the genotypic differences of microorganisms. The molecular
technique (genotypic characterization) has been applied to characterize the LAB bacteria.
Molecular technique is fast, practical and accurate. The use of 16S rRNA gene sequences
to study bacterial phylogeny and taxonomy has been by far the most common
housekeeping genetic marker used for a number of reasons. These reasons include (i) its
presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the
function of the 16S rRNA gene over time has not changed, suggesting that random
sequence changes are a more accurate measure of time (evolution); and (iii) the 16S
rRNA gene (1,500 bp) is large enough for informatics purposes. A preliminary estimation
of taxonomic distribution was used as criteria in the selection for further investigations.
Identification by using selective PCR amplification and subsequent sequencing of the
16S rDNA genes directly from the isolated LAB was carried out. Ten selected strains
were identified on the basis of their 16S rDNA homologies with entries in the GenBank-
EMBL databases used as an additional taxonomic criterion for distinguishing these
bacteria from other members of the family (genotypic characterization). DNA were
extracted, and used as template for PCR amplification (Figure 5.1). PCR products were
detected for the partial nucleotide sequences. 16S rDNA gene sequences have been
performed to infer organismal phylogeny reflecting the organismal evolutionary history.
DNA quantity and purity of the sample isolates was estimated using nanodrop
spectrophotometer (Table 5.1).
Figure: 5.1 Digest results of the 16s rDNA genes of representative isolates along
with the reference. Sample IDs of the different lanes are mentioned on
the top of the lane along with the last lane of Ladder (L) Hind III
digested lambda ladder.
Table: 5.1 Quality Control reports of all of sample isolates. DNA Concentration and
purity of samples estimated using nanodrop spectrophotometer.
ABSORBA
ABSORBANC DNA TOTAL QC
S. SAMPLE NCE QC QC
E VALUE CONCENTRA YIELD in INTEGRIT
No NAME VALUE PURITY YIELD
260/230 TION ng/µl ng Y
. 260/280
1 ST-100 2.13 2.21 3677.29 91932.2 Optimal Optimal Optimal
9 LB-100 1.02 1.15 2217.23 88763.9 Optimal Optimal Optimal
10 LB-200 1.07 1.18 4048.37 103582.1 Optimal Optimal Optimal
All the samples were found suitable for Sanger sequencing
5.1 16s rDNA SEQUENCING

Molecular characterization of all the ten isolates was evaluated on the basis of 16s
rDNA sequencing results. Seven strains with ST designations were identified as
Streptococcus thermophilis with percentage similarity from 96.66% to 100% in
GenBank-EMBL databases. Two strains with LB designations were L. acidophilus and L.
indicus with 99 and 96% similarity in databeses respectively.
5.1.1 16s rDNA sequencing results for ST-100
SANGER TRACE FOR ST-100 PRIMER P1 (AGAGTTTGATCCTGGCTCAG)
Best BLAST Hit Results Streptococcus thermophilus ATCC19258
Score Expect Identities Gaps Strand
2571 bits(1374) 0.0 1391/1398(99%) 5/1398(0%) Plus/Plus
Query 27 ATA-ATGC-AGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG 84
||| |||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17864 ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG
17923
Query 85 GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA 144

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17924 GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA
17983
Query 145 ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC 204

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17984 ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC
18043
Query 205 AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA 264

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 18044 AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA
18103
FASTA SEQUENCE FOR ST-100
GGGAAGGGTCAGTACGCTCTGCTGTTATAATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCG
AACGGGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAAT
GGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGA
GGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAG
AAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGGTAGC
TTACCAGAAAGGGACGGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTG
GGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGG
SANGER TRACE FOR ST-200 PRIMER P1(AGAGTTTGATCCTGGCTCAG)
Best BLAST Hit Results Streptococcus thermophilus CNRZ1066
2601 bits(1388) 0.0 1423/1437(99%) 13/1437(0%) Plus/Plus
Query 30 ATA-ATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG 88
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17864 ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG
17923

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17924 GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA
17983

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 17984 ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC
18043

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 18044 AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA
18103
TGGTGGCGTTGTTTCTATCATGCGGTGCTATAATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT
TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCAT
AACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGT
AGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC
GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGA
GTGAAGAAGGTTTTCGGATCGTAAAGCTCTTGTTTGTAAGTCAAGGAACGGGGTGGTGAGAGGTGGAAAGTTCACACT
GTGGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAAAGGCTGTGGCTCAACCATAGTT
CGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATAT
GGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGG
ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGG
Best BLAST Hit Results Streptococcus thermophilus MN-ZLW-002
2564 bits(717) 0.0 739/748(99%) 7/748(0%) Plus/Plus

Query 1 AACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCCAAGAACC 60
|||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| |
Sbjct 18215 AACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTC-AAGAA-C
18272
Query 61 GGGGTGTGAGGAGGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAAGGGACGGCT 120

|||||||| || |||||||||||||||||||||||||||||||||| ||||||||||||
Sbjct 18273 -GGGTGTGA-GA-GTGGAAAGTTCACACTGTGACGGTAGCTTACCAG-AAAGGGACGGCT
18328
Query 121 AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 18329 AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGG
18388
Query 181 CGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 18389 CGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAG
18448
GGAGGGGGGGCTTTGTTGTCTACTGCACTCTGCACTTTACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGA
ACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAA
TGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTG
AGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCC
AGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAA
GAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCCAAGAACCGGGGTGTGAGGAGGTGGAAAGTTCACACTGTGACG
GTAGCTTACCAGAAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATT
TATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAA
ACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACAC
CGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGC
Figure: 5.2 Results of phylogenetic analysis of ST-100, ST-200 and ST-300 in the
form of Phylogenetic tree
1646 bits(891) 0.0 989/1029(96%) 36/1029(0%) Plus/Plus
Query 8 TGTAAGTC-AG-ACCGGTGTGAGAGTGGAA-GTTCAC-TTG-GACGGTAGC-TA-CAG-A 59
|||||||| || || ||||||||||||||| |||||| || ||||||||| || ||| |
Sbjct 372 TGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAA 431
Query 60 A-GGAC-GCT-ACTACGTG-CAGCAG-CGC-GT-ATACGTA-GT-CCGAGCG-TGT-C-G 107

| |||| ||| |||||||| |||||| ||| || ||||||| || ||||||| ||| | |
Sbjct 432 AGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGG 491
Query 108 A-TTA-T-GGCGT-AAGCGAGCGCA-GCCG-TTGAT-AGTCTG-AG-TAAA-GCTGT-GC 156

| ||| | ||||| ||||||||||| || | ||||| |||||| || |||| ||||| ||
Sbjct 492 ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGC 551
Query 157 TC-ACCATAG-TCGCTTT-GAAACTGTCAAAC-TGAGTGCAGAAGGGGAGAGTGGAATTC 212

|| ||||||| ||||||| ||||||||||||| |||||||||||||||||||||||||||
Sbjct 552 TCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTC 611

ACGCTCGTGTAAGTCAGACCGGTGTGAGAGTGGAAGTTCACTTGGACGGTAGCTACAGAAGGACGCTACTACGTGCAG
CAGCGCGTATACGTAGTCCGAGCGTGTCGATTATGGCGTAAGCGAGCGCAGCCGTTGATAGTCTGAGTAAAGCTGTGCT
CACCATAGTCGCTTTGAAACTGTCAAACTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTA
GATATATGGAGGAACACCCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGC
GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCC
GAAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGC
ACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTA
GAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA
AGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGG
EZTAXON RESULTS
Pairwise Diff/Total megaBLAST BLASTN
Rank Name/Title Authors Strain Accession
Similarity nt score score
Streptococcus (Orla-Jensen
ATCC
1 salivarius subsp. 1919) Farrow AY188354 96.660 34/1018 1679 1592
19258(T)
thermophilus and Collins 1984
Streptococcus Whiley and ATCC
2 GL831116 96.464 36/1018 1671 1584
vestibularis Hardie 1988 49124(T)
Streptococcus
Andrewes and ATCC
3 salivarius subsp. AY188352 96.464 36/1018 1671 1584
Horder 1906 7073(T)
salivarius
ATCC
Streptococcus Kawamura et al.
4 700780( GL732464 94.401 57/1018 1514 1461
peroris 1998
T)
Streptococcus 682-
5 Vela et al. 2011 FN643224 94.401 57/1018 1493 1429
porcorum 03(T)
Best BLAST Hit Results Streptococcus thermophilus MN-ZLW-002
2564 bits(1421) 0.0 1424/1425(99%) 3/1425(0%) Plus/Plus

Query 1 ATACATGC-AGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG 59
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 44 ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG 103

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 104 GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA 163

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 164 ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC 223

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 224 AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA 283

ATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAA
CCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTG
AAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGAC
GATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG
TAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCT
CTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACT
ACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTT
TGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAG
AGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGG
Best BLAST Hit Results Streptococcus infantarius
2564 bits(1421) 0.0 1424/1425(99%) 3/1425(0%) Plus/Plus
Query 1 ATACATGCAAGTAGAACGCTGAAGACTTTTCGCTTGGCTAAAGTTGGAAGAAGTTGCGAA 60
|||||||||||||||||||||||||||||| |||| |||||||||||||| |||||||||
Sbjct 24 ATACATGCAAGTAGAACGCTGAAGACTTTTAGCTT-GCTAAAGTTGGAAG-AGTTGCGAA 81
Query 61 CGGGTGAGTAACGCGTAGGTAACCTAGCCTACTAGCGGGGGATAACTATTGGAAACGATA 120

||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||
Sbjct 82 CGGGTGAGTAACGCGTAGGTAACCT-GCCTACTAGCGGGGGATAACTATTGGAAACGATA 140
Query 121 GCTAATACCGCATAACAGCATTTAACCCATGTTAGATGCTTGAAAGGAGCAATTGCTTCA 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 141 GCTAATACCGCATAACAGCATTTAACCCATGTTAGATGCTTGAAAGGAGCAATTGCTTCA 200
Query 181 CTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 201 CTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC 260

ATACATGCAAGTAGAACGCTGAAGACTTTTCGCTTGGCTAAAGTTGGAAGAAGTTGCGAACGGGTGAGTAACGCGTAG
GTAACCTAGCCTACTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAGCATTTAACCCATGTTAGAT
GCTTGAAAGGAGCAATTGCTTCACTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
CGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA
GCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAA
AGCTCTGTTGTAAGAGAAGAACGTGTGTGAGAGTGGAAAGTTCACACAGTGACGGTAACTTACCAGAAAGGGACGGCT
AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGC
GGTTTAATAAGTCTGAAGTTAAAGGCAGTGGCTTAACCATTGTTCGCTTTGGAAACTGTTAGACTTGAGTGCAGAAGGG
AGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAAC
mega
Pairwise BLASTN
Rank Name/Title Authors Strain Accession Diff/Total nt BLAST
Similarity score
score
UNCULTURE Lin A et al.,

1 BD10692 JQ187473 99.99 1/1417 1679 1214
D BD10692 2013
Streptococcus
Schlegel et HDP9024
2 infantarius AF429763 99.929 2/1417 2737 1276
al. 2003 6(T)
subsp. coli
Streptococcus Poyart et al. CIP
3 DQ232532 99.859 2/1418 2739 1264
lutetiensis 2002 106849(T)
Streptococcus
ATCC
infantarius Schlegel et
4 BAA- ABJK02000017 99.718 4/1418 2724 1258
subsp. al. 2003
102(T)
infantarius
Andrewes
Streptococcus ATCC
5 and Horder GL698435 99.647 5/1417 2706 1244
equinus 9812(T)
1906
2643 bits(1421) 0.0 1424/1425(99%) 3/1425(0%) Plus/Plus
Query 5 GGGCGTGCCTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT 64
||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 7 GGGCGTG-CTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT 65
Query 65 TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAA 124

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 66 TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAA 125
Query 125 CGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTG 184

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 126 CGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTG 185
Query 185 CTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAG 244

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 186 CTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAG 245

TTGCGGGCGTGCCTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAG
TAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACA
CATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGG
CTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTA
CGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTC
GGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAA
GGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCG
AGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGT
GCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGG
CTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGG
Best BLAST Hit Results Streptococcus thermophilus CGLBL208
1454 bits(1421) 0.0 1424/1425(99%) 3/1425(0%) Plus/Plus
Query 38 ACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGG 97
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 30 ACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGG 89
Query 98 TAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATG 157

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 90 TAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATG 149
Query 158 GATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTT 217

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 150 GATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTT 209
Query 218 GTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGA 277

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 210 GTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGA 269
Query 278 GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG 337

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 270 GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG 329
Query 338 GGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTT 397

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 330 GGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTT 389

TGGGGAGTTGCTTACATCTTATTATTATTCTCTATTCACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACG
GGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGG
ATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGG
TAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGA
CTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAA
GGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTAC
CAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGT
AAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAA
CTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG
Pairwise Diff/Total mega BLAST BLAST N

Rank Name/Title Authors Strain Accession
Similarity nt score score
Streptococcus Cruciata
1 thermophilus et al., CGLBL208 KF286609.1 100 0/1454 1454 1214
CGLBL208 2014
Streptococcus
Settani et
2 thermophilus FMA196 HQ721271.1 100 0/1454 2737 1276
al., 2011
FMA196
5.1.9 16s rDNA sequencing results for LB-100
SANGER TRACE FOR LB-100 PRIMER P3 (TACGGTTACCTTGTTACGACTT)
Best BLAST Hit Results Lactobacillus acidophilus
1408 bits(762) 0.0 761/762(99%) 1/762(0%) Plus/Plus
Query 1 CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1439 CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA 1380
Query 61 TTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1379 TTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGC 1320
Query 121 AGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTCGCTTGCCTTCGCAGGCTTGCTC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1319 AGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTCGCTTGCCTTCGCAGGCTTGCTC 1260
Query 181 CTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTT 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1259 CTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTT 1200
FASTA SEQUENCE FOR LB-100

CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGAT
CCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTC
GCTTGCCTTCGCAGGCTTGCTCCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGA
CTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATGA
CAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAG
TGTCCCCGAAGGGAACTCCGTATCTCTACGGATTGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAA
TTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCG
GAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACT
ACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCA
GTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTC
5.1.10 16s rDNA sequencing results for LB-200
SANGER TRACE FOR LB-200 PRIMER P3 (TACGGTTACCTTGTTACGACTT)
Best BLAST Hit Results Lactobacillus indicus
1583 bits(857) 0.0 953/995(96%) 23/995(2%) Plus/Minus
Query 1 TATCCCACCGACTTTGGGCATTGCAAACTTCCATGGGGGGACGGGCGGGGTGTACAAGGC 60
||||||||||||||||||||||||| |||||||||| | ||||||||| |||||||||||
Sbjct 1466 TATCCCACCGACTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGC 1407
Query 61 CCGGGAACGTATTCACCGCGGCGGGCTGATCCGCGATTACTAGCGATTCCAGCTTCGGGC 120

||||||||||||||||||||||| ||||||||||||||||||||||||||||||||| ||
Sbjct 1406 CCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGC 1347
Query 121 AGGCGAGTTGCAGCCTGCAGTCCGAACTGAAAACAGCTTTAAGAGATCCGCTTACCCTCG 180

|||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||
Sbjct 1346 AGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACAGCTTTAAGAGATCCGCTTACCCTCG 1287
Query 181 GGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGG 240

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1286 CGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGG 1227
FASTA SEQUENCE FOR LB-200

TATCCCACCGACTTTGGGCATTGCAAACTTCCATGGGGGGACGGGCGGGGTGTACAAGGCCCGGGAACGTATTCACCG
CGGCGGGCTGATCCGCGATTACTAGCGATTCCAGCTTCGGGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAAAACAG
CTTTAAGAGATCCGCTTACCCTCGGGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAA
GGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCTCTAGAGGGCCCAACTTAATGA
TGGCAACTAAAGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACAACAGCCATGC
ACCACCTGTCTCTGCGTCCCCGAAGGGAACCACCTATCTCTAGGTGTAGCGCAGGATGTCAAGACCTGGTAAGGTTCTT
CGCGTTGCTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTC
GTACTCCCCAGGCGGAGCGCTTAATGCGTTTGCTGCGGCACTGAGGACCGGAAAGTCCCCAACACCTAGCGCTCATCGT
TTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCAAGCCTCAGCGTCAGTTGCAAACCAAAG
AGCCGCCTTCGCCACTGGGGTTCTTCCATATATCTACGCAT
Table: 5.2 16s rDNA sequencing results of selected 10 isolates with best hit
similarities at data in NCBI
PERCENTAGE QUERY
STRAIN STRAIN ACCESSION
S. No.
DESIGNATION HOMOLOGY SIMILARITY COVER
Streptococcus
1 ST-100 thermophilus strain 99% 96% NR 042778.1
ATCC 19258
Streptococcus
2 ST-200 thermophilus strain MN- 99% 96% NR 074827.1
ZLW-002
Streptococcus
ZLW-002
Streptococcus
salivarius subsp
4 ST-400
thermophilus strain
96.66% 95% AY188354
ATCC 19258(T)
Streptococcus
ZLW-002
Streptococcus
6 ST-600
infantarius subsp. coli
99.92% 97% AF429763
Streptococcus
7 ST-700 thermophilus strain 99% 98% NR 042778.1
ATCC 19258
Streptococcus
8 ST-800 thermophilus 100% 97% KF286609.1
CGLBL208
Lactobacillus
9 LB-100 acidophilus NCFM 99% 99% NR 075045.1
strain
Lactobacillus
10 LB-200 delbrueckii subsp. 96% 100% NR 029106.1
indicus NCC725
Figure 5.3 Results of phylogenetic analysis of ST-400, ST-500 and ST-700 in the
form of Phylogenetic tree
Figure 5.4 Results of phylogenetic analysis of LB-100 and LB-200 in the form of
Phylogenetic tree
ST-200, ST-300 and ST-500 were found similar to Streptococcus thermophilus
strain MN-ZLW-002 (accession NR 074827.1) with percentage similarity of 99% and
query cover of 97% (Table 5.2). The phylogenetic trees for all the isolates were prepared
(Figure 5.2, 5.3 and 5.4). ST-100 (96%), ST-200 (96%) and ST-700 (97%) were found
similar Streptococcus thermophilus strain ATCC 19258 (accession NR 042778.1) with
maximum query cover of 98%. ST-600 was identified as Streptococcus infantarius subsp.
coli (99.92% similarity) and was excluded form further studies. Isolate LB-100 was
identified as Lactobacillus acidophilus and observed 99% similar with Lactobacillus
acidophilus NCFM strain (accession NR 075045.1). LB-200 was identified as L. indicus
with similarty of 96% with Lactobacillus delbrueckii subsp. indicus NCC725.
ST-500 was selected for whole genome sequencing on the basis of its dairy
applications and efficient performance as starter for curd and yogurt. The strain was
submitted to NCIM (National Collection of Industrial Microorganisms) facility, Pune,
under safe deposit agreement with accession number NCIM 5539.
5.2 WHOLE GENOME SEQUENCING OF ST-500 (STREPTOCOCCUS

THERMOPHILUS NCIM 5539)
Total genomic DNA of the selected strain was prepared essentially by using
Cardinal et al., 1994 procedure. DNA (214 ng/µl) was subjected to PCR utilizing primer
1 (AGAGTTTGATCCTGGCTCAG) and primer 2 BOX A1R (TACGGTTACCTTGTTACGACTT) as per
procedure described by Versalovic et al.1994.
Each 27 µl PCR reaction contained 5 µl 5× Gitschier buffer (1 M (NH4)2SO4, 1 M

Tris-HCl (pH 8.8), 1 M MgCl2, 0.5 M EDTA (pH 8.8) and 14.4 M β -mercapto-ethanol
and volume is maintained to 200 mL with double distilled), 0.6 mg/ mL BSA (Sigma, A-
7906), 100% DMSO (Sigma, D-8418), 0.2 mM dNTP (Sigma, D7295), 0.5 µM
oligonucleotide primer, 1 unit of Taq DNA polymerase (Sigma, D1806) and distilled
water. PCR amplifications were performed in a DNA thermal cycler with an initial
denaturation step (95°C, 7 min), followed by 30 cycles of denaturation (94°C, 1 min),
annealing (53°C, 1 min) and extension (65°C, 8 min), and a single final extension step
(65°C, 16 min). The amplified fragments were fractionated on a 1.5% w/v agarose gel
during 200 min at a constant voltage of 40 V in 0.5×TAE (Tris-Acetat EDTA) at 4°C. A
10-kb reference marker (Sigma, D7058) was used to allow standardization, followed by
staining with ethidium bromide and visualization. The 16s rDNA study of the strain
suggests its similarity of 99% with MN-ZLW-002 Streptococcus salivarius subsp.
thermophilus and was described with the help of circular genomic map (Figure 5.6).
The De-novo sequencing and assembly of novel strain of ST-500 (Streptococcus

thermophilus) on Ion torrent platform. Following are the whole genome sequencing
details:
1.6 ug of Qubit quantified DNA was sonicated for 300sec on Covaris. 950 ng of DNA
was taken for library preparation. End-repair and adapter ligation was done according to
the protocol. The sample was bar coded at this step. The sample was cleaned using
Ampure XP beads. Sample was size selected using 2% Low melting agarose gel. The gel
purified sample was amplified as per the protocol. The amplified product was cleaned up
using Ampure XP beads. The sample was run on Bioanalyzer HS chip to check the
distribution and was quantified using Qubit Fluorometer. A final yield of 1567.2 pg was
obtained (Table 5.3). WGS ePCR results showed the highest peak range of 10380-12152
bp (Figure 5.5).
Table: 5.3 Qubit values of prepared library
Sample Qubit reading (pg/ul) Yield (pg) Ion Xpress

Barcode
ST-500 78.36 1567.2 8
Figure 5.5: Figure presents the library preparation report with peak indicating
the ST-500 library optimal of sequencing
Figure 5.6: Circular genomic map of ST-500 (Streptococcus thermophilus) in
comparison with Streptococcus thermophilus MN-ZLW-002
Table: 5.4 Summary of the platforms and alignments of WGS of ST-500
Sample Sequence Data Information:

Mean Read length ~231 bp
Type of sequencing: Single/Paired end sequencing Single end
Sequenced using <Illumina GAII Analyzer/454 GS FLX/5500 Ion Torrent

SOLiD System>
Reference Sequence Data Information:
Organism Name Streptococcus

Type of Sequence: Genomic/Transcriptomic Genomic
No. of Chromosomes/Transcripts NA
Analysis Steps:
QC and Raw data processing
QC Program used SeqQC-V2.0
High Quality Reads filtering: Yes/No NO
Vector (Adapter/Primer) contaminated reads filtering: Yes/No NO
B block trimming NO
Alignment Information
Alignment Tool used tmap- v3
Assembly tool used Mira_3.9.15

and CAP3
Table: 5.5 Details of alignments and Raw data sequences obtained from WGS
Raw Data SeqQC

IonXpress_001_R_2013_07_10_13_14_04_user_JAG-
Fastq file name
77_Auto_user_JAG-77_82.fastq
Fastq file size 2.49 GB
Time taken for Analysis 1.55 Minutes
Maximum Read Length 462
Minimum Read Length 8
Mean Read Length 231
Total Number of Reads 5505428 (5.51 millions)

Total Number of HQ
5487966 (5.49 millions)
Reads 1*
Percentage of HQ Reads 99.683%
Total Number of Bases 1273865252 bases

Total Number of Bases
1273.86525 Mb
in Mb
Total Number of HQ
1268504974 bases
Bases 2*
Total Number of HQ
1268.50497 Mb
Bases in Mb
Percentage of HQ Bases 99.579%

Table: 5.6 Alignment statics of ST-500 in comparison with S. thermophilus MN-
ZLW-002 and S. thermophilus MTCC_5461
Alignment Statistics : Sample ST500
Sample: ST500 S. thermophilus MN-ZLW- S.

002 thermophilus
MTCC_5461
Total number of reads 837186 836570
Total number of reads aligned 779518 724777
%Reads aligned to reference 93.11 86.64
Reference sequence length (bp) 1848520 1619896
Reference sequence covered (bp) 1848467 1598418
Percentage 1X coverage 100.00 98.67
Average Read Depth 66.26 67.52
GOAL:-
To identify lactic acid genes (at protein level) in the sequenced sample ST500
METHODS:-
1 Predicted proteins of Sample ST500 were subjected to homology search
(BLAST) against Uniprot bacterial proteins (reviewed) involved in lactic acid
metabolism.
2 BLAST output were filtered to pass 30% Identity and 30% Query*/Subject**
coverage
*Query: Predicted proteins
**Subject: Bacterial Lactic Acid proteins (Uniprot reviewed)
Results:
Total Predicted Protein 2912
Total Predicted proteins passing the filters 907
Chapter 6
Media Standardization
6.1 NUTRITIONAL REQUIREMENTS OF BACTERIA
The ultimate expression of microbial organization is the self replication. The

chemicals from the environment that are utilized for bacterial growth are called nutrients.
During anabolism, nutrients are taken up and are changed into cell constituents in an
energy depending process. Different nutrient groups exist and are broadly classified into
those providing energy such as carbohydrates, and those used as components in cellular
structures such as peptides. Most of the microorganisms require an organic compound as
their carbon source including carbohydrates, peptides or amino acids, fatty acids, organic
acids, nitrogen bases and aromatic compounds (Ganzle et al., 2007; Brant et al., 2006 ).
LAB has numerous nutritional requirements for growth, especially nitrogen sources. The
general assumption is that biomass synthesis in lactic acid bacteria is predominantly from
building blocks present in the culture medium. The most important application of LAB is
their use as starter strains in the manufacture of various fermented products. LAB are
fastidious microorganisms that require an exogenous source of amino acids or peptides,
which are provided by the proteolysis of the proteins present in raw material. Nitrogen is
the second most abundant element in the cell after carbon. A typical bacterial cell is about
12% nitrogen (by dry weight) and it is a main constituent of protein, nucleic acids, and
several other constituents in the cell.
Medium optimization generally involves determining the appropriate nutrients

and establishing the concentrations of these nutrients that result in maximum levels of
biomass or targeted microbial products. Due to a lack of various biosynthetic pathways,
lactic acid bacteria generally require media rich in nutrients. A rich nutrient environment
can be provided by complex media comprised mostly of complex components (such as
yeast extract, peptone, or tryptone), by semi-defined media formulated mostly with
defined chemicals (except for one or two complex nutrients), and by CDMs (synthetic
media) containing no complex components (Zhang and Greasham, 1999). Although in
most cases complex and semi-defined media provide greater biomass yields than CDMs,
using these types of media in physiological studies focusing on metabolism and
regulation makes data more difficult to interpret (Cocaign et al., 1995). For this reason, a
CDM that supports reasonable cell growth can be very useful in studies of gene
regulation, protein expression, and metabolic fluxes.
By systematically adding or removing components from a CDM formulation, the specific
nutritional and regulatory requirements for growth and targeted metabolic pathways can
be determined. The cheaper sources of media are more useful for commercial production
or mass production of bacteria. Among the ten isolated strains, ST-500 was observed
with potential for commercialization on the basis of its performance in dairy applications
and was selected for experiments on media standardization. Lactic acid production have
been studied under the control of various operating factors and media components with
the objective to achieve high product concentration and low fermentation cost
6.2 EFFECT OF DIFFERENT SOURCES OF NUTRITION ON ST-500

(STREPTOCOCCUS THERMOPHILUS NCIM 5539)
6.2.1 Carbon Sources

The lactic acid bacteria for industrial process have to metabolize the
carbohydrates into optically pure lactic acid through homo-fermentative pathway and
required a simple medium composition. Moreover, S. thermophilus could also produce
lactic acid with a high cell mass yield if the appropriate carbohydrates are used as carbon
sources. The homo-fermentative characteristics of carbohydrate metabolism of S.
thermophilus were identified. Lactic acid fermentation by using various carbohydrates as
the main carbon source were investigated in modified MRS broth containing 10, 20, 30,
40 g/L of the different carbon sources: lactose, sucrose, maltose, xylose, dextrose and
fructose, respectively (Yun et al.,2003). Several carbon sources were tested in order to
get the suitable carbon source for cell mass production. First set of experiment was
started with 10 g/L of dextrose, sucrose, maltose, xylose, lactose and fructose
individually with modified MRS broth contents (Figure 6.1A).
Figure 6.1: (A) Influence of carbon sources on lactic acid fermentation for mass
cell production supplied with modified MRS broth and 1% of the
selected carbon sources. All the carbon sources were tested indiviually
with their 10g/L content in media.
Figure 6.1: (B) Influence of carbon sources on lactic acid fermentation for mass
with their 20g/L content in media.
Figure 6.1: (C) Influence of carbon sources on lactic acid fermentation for mass
with their 30g/L content in media
Figure 6.1: (D) Influence of carbon sources on lactic acid fermentation for mass
with their 40g/L content in media
Prepared media was inoculated and incubated for 24 hours at 42 °C. The
experiment was conducted in duplicates and media samples were tested after 24 hours,
fresh cell mass calculation, optical density and percentage yield of lactic acid was
recorded. The maximum cell growth and the highest lactic acid concentration were
obtained after 24 h of the fermentation time. These experiments were operated with non
controlled pH: therefore; pH of fermentation broth rapidly decreased due to accumulation
of lactic acid. Interestingly, sucrose showed better results than lactose and other carbon
sources with more than 2% fresh cell mass and 34% lactic acid yield (Figure 6.1C).
Sucrose was followed by lactose with 1.6% cell mass yield and 26% lactic acid yield.
Other carbon sources including dextrose, xylose, maltose and fructose showed 1.4%,
0.8%, 1.1% and 1.2% cell mass yield, respectively.
All the carbon sources were increased in quantity with modified MRS broth. The
experiments were conducted in duplicates and observed that sucrose showed much better
result compared to other carbon sources. Both fresh cell mass yield and lactic acid yield
was increased (Figure 6.1D). There was a slight fall in growth in trials when there was
more than 4% sucrose was added in media. A fresh cell mass yield of 3.4% was achieved
with 2% sucrose content which was increased to 4.5% with 3% sucrose and 5.2% with
4% sucrose content in media. Percentage concentration of lactic acid was also recorded
and a symmetrical increase was observed with the increase in cell mass viz. 44%, 55%
and 81% lactic acid with 2, 3 and 4% sucrose content, respectively. Lactose also played a
crucial role in maintaining viable cell mass with 2.3%, 3.1%, and 3.8% fresh cell mass
yield with 2, 3 and 4% lactose in media. Other carbon sources were found to be
unsuitable for fast multiplication and fermentation of S. thermophilus. The percentage
cell mass yield for xylose, dextrose, fructose and maltose was 1.8%, 2.9%, 2.8% and
2.7%, respectively. As a result, the suitable carbon source for (ST-500) S. thermophilus
was in the order of sucrose > lactose > dextrose > fructose > maltose > xylose
respectively (Figure 6.1D).
6.2.2 Effect of Nitrogen Sources
Inexpensive and effective nitrogen source is also necessary to reduce the

fermentation cost. Supplementation of nitrogen source into the fermentation medium is
important in order to maintain quality high yield. Peptone was used as the sole nitrogen
source to improve the nutritional quality of the fermentation medium. Peptone contains
growth promoting compounds in addition to organic nitrogenand along with other carbon
compounds.
Effect of various nitrogen sources supplemented in modified MRS broth

containing 10, 20, 30, 40 g/L of tryptone, proteose peptone, beef extract, meat extract,
soya peptone and casein peptone were used and the result obtained are presented in
Figure 6.2 A. Higher supplementation of peptide nitrogen greatly influenced the cell
multiplication and fermentation. Considering this fact, special attention was provided to
the maximum and minimum limits of the sources to make an efficient media for optimal
growth. First set of experiment was started with 10 g/L of tryptone, proteose peptone,
beef extract, meat extract, soya peptone and casein peptone individually with modified
MRS broth contents. Prepared media was inoculated and incubated for 24 hours at 42 °C.
The experiment was conducted in duplicates and media samples were observed after 24
hours by their optical density, fresh cell mass calculation and percentage yield of lactic
acid.
The LAB are able to respond to changes in nitrogen availability by regulating the
activity of the proteolytic system to ensure proper nitrogen balance in the cell. It was
found that the synthesis of many exoproteins influenced, in part at least, by the level of
individual nutrients in the extracellular environment (Rollan et al., 1998). Cell
multiplication rate and fresh weight was observed higher in tryptone (casein enzyme
hydrolysate) and casein peptone compared to other nitrogen sources. A cell mass yield of
4.1% with 1% tryptone was observed which was increased to 5.1% with 2%, 6% with 3%
and 7.6% with 4% tryptone in media, (Figure 6.2 D) while casein peptone produced
3.4%, 4.4%, 5.3% and 6.6% with 1, 2, 3 and 4% concentration, respectively.
Figure 6.2: (A) Effect of nitrogen sources on lactic acid fermentation for mass cell
production supplied with modified MRS broth and 1% of the selected
nitrogen sources. All the nitrogen sources were tested indiviually with
their 10g/L content in media
Figure 6.2: (B) Effect of nitrogen sources on lactic acid fermentation for mass cell
production of supplied with modified MRS broth and 2% of the
selected nitrogen sources. All the nitrogen sources were tested
indiviually with their 20g/L content in media
Figure 6.2: (C) Effect of nitrogen sources on lactic acid fermentation for mass cell
their 30g/L content in media
Figure 6.2: (D) Effect of nitrogen sources on lactic acid fermentation for mass cell
their 40 g/L content in media
This reflected the complex nutritional demand of this fastidious lactic acid
bacterium due to its limited biosynthesis capacity. The percentage cell mass yield
observed for proteose peptone, meat extract, beef extract and soya peptone was 4%,
4.1%, 4.6% and 4.3%, respectively. The suitable carbon source for (ST-500) S.
thermophilus was in the order of tryptone > casein peptone > beef extract > meat extract
> soya peptone > proteose peptone respectively (Figure 62 D).
6.2.3 Effect of Vegetable protein and Whey protein sources
Whey, a biological by-product of the cheese industry is usually disposed as waste.

It is a source of functional valuable proteins. Addition of 1 or 2% of a whey protein
concentrate (WPC) to a whey-based medium used for fermentation with Lactobacillus
delbrueckii subsp. bulgaricus 11842 or Streptococcus thermophilus ST 20 has produced
significantly higher bacterial count and much faster acidity development than the control
whey or whey UF permeate media. Industrial whey fractions of • -lactalbumin or β -
lactoglobulin used as nutrient supplements in the same concentrations as the WPC
resulted in smaller but still noticeable effects (Bury et al., 1998). Vegetable protein
sources are also useful in the medium to provide the content of amino acids required by
bacterial cells for multiplication. Common vegetable protein sources used in bacterial
mass production medium are soy protein and wheat peptones. Milk protein sources
include a number of proteins like whey proteins concentrate, sweet whey powder, skim
milk powder, casein proteins etc.
The protein concentrations were autoclaved separately and mixed at the time of
inoculation with the other ingredients of modified MRS. Selected protein sources were:
whey proteins concentrate, sweet whey powder, soy protein, wheat peptones, casein
proteins and vegetable casein protein. 2% and 4% quantity was selected for all the protein
sources and results of the study revealed that whey protein concentrate is a much better
option to carry forward followed by casein protein showing good results with 2% content
in media (Figure 6.3A). Whey protein concentrate showed 4.5% cell mass yield and 30%
lactic acid yield with 2% concentration and 5.7% cell mass yield and 40% lactic acid
yield with 4% concentration in medium. Casein protein showed 4.8% cell mass yield and
34% lactic acid yield with 2% concentration and 5.6% cell mass yield and 39% lactic
acid yield with 4% concentration in medium.
Figure 6.3: (A) Effect of vegetable protein and milk protein sources on lactic acid
fermentation for mass cell production supplied with modified MRS
broth and 2% of the selected sources. All the sources were tested
Figure 6.3: (B) Effect of vegetable protein and milk protein sources on lactic acid
broth and 4% of the selected sources. All the sources were tested
The percentage cell mass yield observed for sweet whey powder, soy protein,
wheat peptone, veg casein peptone was 4.8%, 4%, 2.2% and 3.7% respectively. As a
result, the suitable source for (ST-500) S. thermophilus was in the order of whey protein
concentrate > casein protein > sweet whey powder > soy protein > veg casein peptone >
wheat peptone respectively (Figure 6.3 B).
6.2.4 Effect of Yeast Extract, Buffers and other salts used
Normally, commercial yeast extract possesses 18 essential elements such as Al,

Ba, Cd, Co, Cr, Cu, Fe, Ga, Mg, Mn, Mo, Ni, Pb, Sn, Sr, Ti, V, and Zn, respectively and
is found to be essential for growth.
Figure 6.4: Influence of sodium, potassium, ammonium and magnesium salts on

lactic acid fermentation for mass cell production supplied with
modified MRS broth and were tested indiviually with their mentioned
content in media.
Moreover, it also contains nutritious substances such as amino acids, peptides,

vitamins, and several organic acids including pyruvic and glyceric acid (Jiang et al.,
2010). Initially, the influence of yeast extract concentrations on lactic acid fermentation
and cell mass production from modified MRS medium were investigated by
supplementing with the concentration between 1-6 g/L, lactic acid fermentations were
performed using 250 mL shake flasks at 30 °C with pH 6.0. At 1 g/L of yeast extract,
lactic acid fermentation was not completed, and the cell growth was almost stopped after
12 h because the nutrient was depleted. On the other hand, the maximum lactic acid
productivity and cell mass was obtained at 5 g/L whereas fresh cell mass weight (28 g/L)
was obtained at 6 g/L of yeast extract showing a decrease in cell mass production
compared to 5g/L yeast extract which was 33g/L (Figure 6.5).
Figure 6.5: Influence of yeast extract (amino acid source) on lactic acid
broth and were tested indiviually with 1g/L to 5g/L content in media.
The increase of yeast extract concentration between 4 and 6 g/L had very little
effect on increasing the lactic acid production. Bacterial Cells were also provided with
sodium, potassium, ammonium and magnesium for their survival. Di potassium hydrogen
sulphate, hydrogen potassium suplhate, sodium acetate, tri-ammonium citrate,
magnesium sulphate, manganous sulphate were added to modified MRS broth in different
concentrations. Cell mass of 4.6% along with lactic acid percentage of 60% was obtained
from 5 g/L sodium acetate followed by 2g/L K2HPO4 with 4.5% fresh cell mass yield
along with 55% of lactic acid yield (Figure 6.4). It was observed that all the buffers had
its own role in media with different conditions provided to the cells and media. Based on
the experimental research conducted on different ingredients in different concentrations,
media formulations were designed and tested for combined action of ingredients with
standardized concentrations.
Table: 6.1 Results of different media compositions concluded after individual

studies of media ingredients for mass cell production of Streptococcus thermophilus
NCIM 5539
Lactic acid
Media Fresh Cell
Nutrient Supplementation (g/L) concentration
Designation Mass yield g/L
g/L
Yeast Extract 5g/L, Tryptone 20g/L,
1 61.22 ± 0.15 78.62 ± 0.16
Sucrose 30g/L, K2HPO4 2g/L
2 Sucrose 30g/L, Lactose 10g/L, 63.67 ± 0.22 79.99 ± 0.19
K2HPO4 2g/L
3 Sucrose 30g/L, Lactose 10g/L, 64.29 ± 0.13 81.22 ± 0.23
Sodium acetate 5g/L, K2HPO4 2g/L
Sucrose 30g/L, Lactose 10g/L, WPC
4 53.78 ± 0.11 67.47 ± 0.15
10g/L, Sodium acetate 5g/L,
K2HPO4 2g/L
Sucrose 40g/L, Lactose 15g/L,
5 73.37 ± 0.26 88.34 ± 0.07
Sodium acetate 5g/L, K2HPO4 2g/L,
MgSO4 0.1g/L.
Sucrose 40g/L, Lactose 20g/L,
6 68.26 ± 0.11 84.32 ± 0.17
Sodium acetate 5g/L, K2HPO4 2g/L,
MnSO4 0.05g/L
Media 5 was selected for further studies as the output observed from this media
was far better compared to other minimal media and readymade media available in
market. Media 5 was also observed better compared to MRS and M17 broth media for the
identified isolated strain.
6.3 INFLUENCE OF FACTORS ON LACTIC ACID BACTERIA

FERMENTATION
6.3.1 Effect of various temperatures on cell mass production
Temperature adaptation of bacterial growth under different temperature regimes

was studied. Understanding of the optimal temperature required on lactic acid
fermentation will facilitate improvement of the production process. The temperature were
monitored and controlled during the fermentation process at 30, 37, 42, 45 and 50°C with
fermentation time 30 h as shown in Figure 6.6. In industrial fermentation processes, the
operating temperature of the fermenter is often raised to optimum level to increase
microbial activity depending on the characteristics of the microorganism used along with
the other environmental conditions. The cell count showed a similar trend. The results
indicated that temperature had a pronounced influence on the growth of these
microorganisms during lactic acid fermentation. Moreover, the temperature affects the
rate of biochemical reactions, the activity of extracellular enzymes, the generation time,
and the activity of the microorganisms involved.
.
Figure 6.6: The influence of temperature on cell mass yield on self designed
Media 5 by Streptococcus thermophilus NCIM 5539
The optimum temperature and time for high cell mass yield was 42°C for 24
hours on the basis of cell mass obtained by centrifugation and absorbance of the samples.
Temperature is the most important factor on nutrient utilization and cell viability. Higher
temperature stimulated the rapid growth of lactic acid bacteria resulting in a rapid decline
in pH, and consequently suppressed the growth of S. thermophilus. Although, S.
thermophilus could grow at 50 °C, the optimum temperatures for its growth were
observed between 40-45 °C. Some literatures reported that the rate of reaction for
microorganism increases with increasing temperature until a limiting maximum
temperature is reached (55°C), after which the growth rate decreases very rapidly (Peleg,
1996).
However, when the temperature of the medium is above or below that required for
optimum growth, the microbial activity is substantially reduced and the organisms may
eventually die. The pH value of the fermentation broth decreased from 6.0 to values less
than 4.5 as fermentation proceeded reaching conditions unfavorable for cell growth. Cell
population was a measure of the increase in biomass over time and it was determined
from the exponential phase. Growth rate was one important way of expressing the
relative ecological success of a species or strain in adapting to its natural environment.
The duration of exponential phase in cultures depended upon the size of the inoculums,
the growth rate and the capacity of the medium and culturing conditions to support cell
growth. At 37 °C and 42 °C, the yield and productivity of lactic acid were also similar.
The production yield and volumetric productivity were calculated with the time at the
exponential phase of the cell growth. It was, therefore, important that the fermentation
temperature must be maintained as stable as possible since bacteria grow optimally
within a narrow temperature range. In general, subsequent experiments were conducted at
42 °C in order to reduce the cost of electricity of the production process. For this reason,
the control of temperatures were necessary during lactic acid fermentation for lactic acid
production.
6.3.2 Effect of pH on cell mass production
The effect of different pH on cell growth, and substrate metabolism were

performed in batch fermentation. The initial pH of the medium was maintained at 4.0,
5.0, 6.0, 7.0, or 8.0, respectively, with 5M NH4OH and 1N Hydrochloric acid after the pH
dropped to these points due to the production of lactic acid. Fermentation was completed
in 18 h with complete utilization of glucose. In addition, different fermentation
parameters viz. pH in relation to lactic acid production were studied (Figure 6.7)
Figure 6.7: The influence of initial set point of pH on cell mass yield on self
designed Media 5 by Streptococcus thermophilus NCIM 5539
Figure 6.8: Reducing sugar concentrations (Sucrose) during batch fermentation

of Streptococcus thermophilus NCIM 5539
This was in accordance since the μmax obtained early in the fermentation where
the lactic acid concentration was very low. There are some reportes where low level of
biomass and lactic acid concentration were synthesized at pH values lower to 5.5 (Bai et
al., 2004) which could be due to partial loss of the enzymatic activity involved in the
biosynthesis of biomass and lactic acid. Our results showed the high level of biomass and
lactic acid when pH was maintained in the pH range 5.0-7.0 with the optimum at pH 6.0.
Further, at each pH value, the reciprocal of the productivity of lactic acid was linearly
correlated to the specific growth rate. Maximum fresh cell mass was obtained from initial
pH range of 6-7 with a maximum cell yield of 70-75 g/L. The cell mass obtained for the
different pH values did not present significant variations (Goncalves et al., 1997). The
optimum pH for cell growth of S. thermophilus appeared to be 6.0; lactic acid
fermentation at pH 6.0 was completed faster than other pH. The microorganism,
especially, lactic acid bacteria, did not alter its metabolic pathway in the pH range of 5.0-
7.0. Increase in initial pH beyond 6.5 did not improve the lactic acid production. The
higher initial pH brings excessive stress on the microorganism metabolic abilities
(Vijayakumar et al., 2008). The lactic acid productivity has been considered as one of
the critical factors for economical lactic acid fermentation (pH 6.0). S. thermophilus
appears to be suitable LAB for further study on lactic acid fermentation at pH 6.0 and
temperature 42 °C.
6.3.3 Effect of various agitation speeds on cell mass production
Agitation is important for adequate mixing, mass transfer and heat transfer,
respectively. It assists not only the mass transfer between the different phases presented
in the culture, but also maintains homogeneous chemical and physical conditions in the
culture by means of mixing, eliminating gradients of concentration. However, at high
agitation speed the biomass concentration may decrease because of cell damage, cell loss
by the impeller and shear force (Senthuran et al., 1999), while at low agitation speed, the
liquid or fermentation broth could not homogeneous mix well and the substrate could not
be utilized completely.
The effect of agitation speed on cell mass production were studied at three
different agitation speeds (100, 200, 300 rpm). The influence of cultivation factors on the
lactic acid fermentations was observed for cell growth, substrate and product
concentrations, respectively. 100 rpm agitation speed significantly affected the
fermentation performance.
(A)
(B)
(C)
Figure 6.9: Effect of agitation speed on the cell growth, reducing sugar
concentration and mass production by Streptococcus thermophilus
NCIM 5539: 100 rpm (A), 200 rpm (B), and 300 rpm (C).
6.4 MEDIA COSTING
Interest in the physiology of lactic acid bacteria has been stimulated by the
industrial importance of these bacteria and the potential use of genetic engineering in
strain optimization. For metabolic investigations, a defined growth medium for these
bacteria is desirable which (i) supports growth at a reasonably high rate and (ii) allows
for exponential growth over a wide range of cell concentrations. Fermentation medium
can represent almost 30% of the cost for a microbial fermentation (Hofvendahl and
Hahn-Hagerdal, 2000). Complex media commonly employed for growth of lactic acid
bacteria are not economically attractive due to their high amount of expensive nutrients
such as yeast extract, peptone and salts (Jensen and Hammer, 1993).
Cost of media ingredients is one of the major hurdles which are continuously
creating a barrier for dairy industries to launch their starter cultures in market. In current
study, it was observed that the isolated strains were not performing well on economical
ingredients of media. The cost of ingredients based on the composition of MRS or M17
was also too high. Himedia, India was selected for price comparison of media
ingredients because of its consistent product quality and easy availability. Few other
media ingredient vendors were also tested for their availability and price like
• Qualigens Fisher Scientific, India

• Rankem Synergy Scientific, India
• Sigma-Aldrich, United States
• Central Drug House (CDH) Chemicals, India
• Merck, Germany
There is a minor difference in the prices of media ingredients locally available in

India, but chemicals from Sigma-Aldrich and Merck were observed to be on higher side
for cost with low ratio of availability in stock. MRS and M17 are well known medium for
cultivation and fermentation of lactic acid bacteria (Khedid et al., 2009). The costing of
the media was compared between MRS and M17.
Table: 6.2 Media per litre cost comparison between MRS and M17 on individual
ingredient basis from Himedia, India catalogue.
MEDIA MRS M-17 Catalogue Price Price/ Kg Price/ Kg

COMPONENTS for Himedia for MRS for M17
2013-14
Yeast Extract 5g/L 2.5g/L 3000/ Kg 15/- 7.5/-
Protease Peptone 10g/L Nil 3580/ Kg 35.8/- Nil
Dextrose 20g/L Nil 450/ Kg 9/- Nil
Lactose Nil Nil 650/ Kg Nil Nil
Sucrose Nil Nil 250/ Kg Nil Nil
Beef Extract 10g/L 5g/L 3300/ Kg 33/- 16.5/-
Sodium Acetate 5g/L Nil 418/ Kg 2.09/- Nil
Ammonium 2g/L Nil 1892/ Kg 3.78/- Nil
Citrate
Di Potassium 2g/L Nil 830/ Kg 1.66/- Nil
Phosphate
Magnesium 0.1g/L 0.25g/L 360/ Kg 0.036/- 0.054/-
Sulphate
Manganese 0.05g/L Nil 780/ Kg 0.39/- Nil
Sulphate
Ascorbic Acid Nil 0.5g/L 5700/ Kg Nil 2.85/-
Agar 12g/L 15g/L 4676/ Kg 55.75/- 69.68/-
Disodium Nil 19g/L 13130/ Kg Nil 249.47/-
Glycero
Phosphate
Casein Peptone Nil 2.5g/L 3870/ Kg Nil 9.67/-
Soya Peptone Nil 5g/L 4064/ Kg Nil 20.32/-
Meat Peptone Nil 5g/L 3550/ Kg Nil 17.75
TOTAL PER LITRE COST OF MRS & M17 AGAR 156.50/- 393.79
TOTAL PER LITRE COST OF MRS & M17 BROTH 100.75/- 324.11/-
Both the media are not economically viable for commercial production of starter
culture. In order to reduce the production cost, the media was modified to increase cell
mass. Several experiments were conducted for optimization as per variation in methods
described in Chapter 5. We developed a medium, self optimized media MEDIA 5 (M5),
and the cost was calculated on the basis of prices/Kg of different ingradients from
Himedia (Table 6.3). The cost per ingredient provided in Himedia catalogue was for
laboratory scale experimental use only.
Table: 6.3 Costing of the designed media on the basis of ingredient prices available
with Himedia
S. No. Media Ingredient Amount Catalogue Price from Price / L

g/L Himedia 2013-14
1 Yeast Extract 5g/L 3000/ Kg 15/-
2 Sucrose 40g/L 250/ Kg 10/-
3 Lactose 15g/L 650/ Kg 9.75/-
4 Sodium Acetate 5g/L 418/ Kg 2.09/-
5 Tryptone 20g/L 4728/Kg 94.56/-
6 K2HPO4 2g/L 830/ Kg 1.66/-
7 MgSO4 0.1g/L 360/ Kg 0.036/-
TOTAL PER LITRE COST OF MEDIA 5 133.09/-
Himedia was later contacted for their commercial prices. Surprisingly, the total cost per
litre for media 5 was reduced to almost 50% by new prices of ingredients from Himedia
(Table 6.4). Still other Indian suppliers of media ingredients were contacted for their
samples in order to reduce the cost of the media. Suppliers contacted were:
• Chaitanya Chemicals, Maharashtra, India
• Titan Biotech, Delhi, India
• Warkem Biotech, Mumbai, India
Samples from these companies were tested on the designed parameters for their
efficiency to produce cell mass along with the price evaluation and found that Warkem
Biotech, Mumbai, India has low commercial price with better quality of raw material. Per
Litre media cost from Warkem was around INR 55±2 with 60g/L fresh mass yield.
Table: 6.4 Costing of the designed media on the basis of commercial prices provided
by Himedia
Amount Commercial Price from

S. No. Media Ingredient Price / L
g/L Himedia 2013-14
1 Yeast Extract 5g/L 1550/ Kg 7.75/-
2 Sucrose 40g/L 125/ Kg 5/-
3 Lactose 15g/L 450/ Kg 6.75/-
4 Sodium Acetate 5g/L 336/ Kg 1.68/-
5 Tryptone 20g/L 2410/Kg 48.2/-
6 K2HPO4 2g/L 680/ Kg 1.36/-
7 MgSO4 0.1g/L 274/ Kg 0.027/-
TOTAL PER LITRE COST OF MEDIA 5 70.76/-
The prices of media ingredients from Warkem Biotech, India were also evaluated
and implemented on the compositions of MRS and M17 media. Some difference was
observed between the cost/L of MRS and cost/L of media 5 (Figure 6.10) but huge
difference in overall fresh weight was observed in both media. The morphology and
characterization of ST-500 was also critically studied to notice the impact of new
ingredients and performance of the same strain in its dairy applications.
Figure 6.10: Price & Fresh cell mass weight g/L comparison of media from
different vendors.
Figure 6.11: Price & Fresh cell mass weight g/L comparison of media M17, MRS
and Media 5 from same vendor.
The overall performance of the strain ST-500 was studied with new nutritional
ingredients and it was observed that highest cell mass of 65g/L was obtained from
ingredients of pilot scale (Laboratory grade) due to their content purity. Price variation
will depend on the size and quality of packaging. Himedia, India are very economical
(Figure 6.11) as compared to lab grade ingredients. But the issue with price was still
there.
The commercial prices of Chaitanya Chemicals, Maharashtra, India, Titan Biotech,
Delhi, India and Warkem Biotech, Mumbai, India were tested. The individual ingredient
price issue was not mentioned due to some confidential issues with the dealer. But the
final price was calculated to be INR 55 and the yield of fresh cell mass obtained from the
same was 58 g/L.
6.5 BATCH FERMENTATION OF ST-500 AND PROCESS
STANDARDIZATION
In a bioprocess development, two main streams involved are upstream and

downstream processes. The work focused on multiplication and mass production of cell
mass from fermentation broth. ST-500 (Streptococcus thermophilus) was successfully
isolated and characterized in the laboratory. The time course of pH, cell concentration,
reducing sugar concentration, and lactic acid concentration during fermentation were
studied. Batch fermentations was first performed in 2 and 5 L fermenters (Biostat A plus,
Sartorius stedim) with working volume 1.5 and 4 L, respectively. Up-scaling was
standardized in 1.5 L and 4 L media in lab scale. M5 was prepared by mixing all the
ingredients uniformly and autoclaved within the fermenter vessel (Figure 6.12, 6.13, 6.14
and 6.15). 5N sodium hydroxide and 1N Hydrochloric acid was prepared for maintaining
the pH balance during fermentation. 10% (v/v) inoculum grown in M5 broth for 24 h
was aseptically inoculated into the fermenter. Turbidity in batch was observed after 8
hours (Figure 6.16) and regularly monitored up to 24 hours followed by batch closing and
cell mass collection in sterile flasks (Figure 6.17, 6.18 and 6.19).
The resulted standardized conditions were provided during the batch. Standard conditions
provided were:
Set temperature : 42º C

Set RPM : 200
Set pH : 6.5
Inocolum : 10%
Base for pH : 5N Naoh
Acid for pH : 1N Hcl
Antifoam : Not Required
Sterility : Fermenter was autoclaved at 15 psi
Sucrose concentration rapidly decreased during the first 16 h followed by a

much slower decreasing rate. Utilization of sucrose nearly ceased after 24 h of operation
with the remaining concentration of less than 1 g/L. It was observed that S. thermophilus
possessed a relatively short lag phase followed by exponential phase. The growth entered
its stationary phase at 22 h where cell concentration reached plateau of approximately 64
g/L until the end of the fermentation process (Figure 6.22). Amount of acid-base
consumption during the batch run was also recorded by Batch Master Software (Sartorius
stedim) and a history plot of the batch was created for evaluation (Figure 6.20).
Cell multiplication rate of ST-500 in 5 L fermenter was observed higher

compared to 2 L fermenter, by analyzing OD at 660 nm after every four hour interval
(Figure 6.21). Controlled fermentation operation offer special advantages over
uncontrolled fermentations in shaker incubators where the pH drops continuously
inhibiting the cell growth after 12-14 hours of incubation, where as in fermenter the pH
was regularly maintained by continuous feed of buffers.
Figure 6.20: History plot of the 5 L batch of Streptococcus thermophilus showing
the change in pH and consumption of acid and base for maintaining
the set pH balance.
Figure 6.21: Fermentation profile of ST-500 recorded on the basis of OD at 660nm
Figure 6.22: Batch fermentation profile of ST-500 in 5L fermenter showing cell

mass concentration, lactic acid percentage and reducing sugar
concentration
On the basis of results from 2L and 5L vessel size fermentation processes, the up-scaling
was done on 50L and 300L fermenters. 240 L media was prepared in sterile buckets
followed by steam sterilization of vessel and media at 121°C (Figure 6.24). The plant
steam pressure was maintained at 3.5 kg/ cm2. 50L fermenter was utilized as seed
fermenter to prepare inocolum which was directly transferred to 300L fermenter. 1
micron steam filters were used to maintain aseptic conditions during sample collection
between the run. The major issue is to maintain sterility of the batch as a minor chance of
contamination can lead to batch failure. The optical density was recoreded to a maximum
of 1.324 (Figure 6.25) which was comparatively much higher than 0.934 recorded in 5 L
batch at Lab scale.
Figure 6.25: Fermentation profile of ST-500 recorded on 300L fermenter on the

basis of OD at 660nm
Upstreaming of ST-500 on different vessel sizes (2L, 5L, 50L and 300L) of
fermenter was evaluated on the basis of final cell mass (g/L), OD, lactic acid content and
cell viability. Maximum cell mass and maximum OD was obtained from 300L vessel but
maximum cell viability was observed in 5L vessel size of fermenter (Table 6.5). Cell
viability and OD from 5L vessel was further explored on the basis of incubation hours
(Table 6.6).
Table: 6.5 Comparison of Batch results with complete up-stream analysis of ST-500
500 mL 2L 5L 50L 300L

Particulates
Flask Fermenter Fermenter Fermenter Fermenter
Media Media 5 Media 5 Media 5 Media 5 Media 5
Final pH on Batch
4.7 6.54 6.58 6.47 6.42
closing
End Sugar Content 0.82% 0.37% 0.28% 0.22% 0.15%
Lactic acid
23% 52% 55% 64% 71%
Percentage
Fresh Cell Mass
12g 44g 47g 67g 72 g
g/L
Optical Density 0.317 0.911 0.946 1.236 1.267
Cell Viability
776×105 790×105 825×105 812×105 817×105
(CFU/mL)
Base consumption N/A 60 mL 136 mL 1032 mL 8026 L
Acid Consumption N/A 4 mL 9 mL 120 mL 1200 mL
Table: 6.6 Cell growth rate with respect to incubation hours
Incubation hours Maximum OD at Colony forming

660nm units per mL sample
8 0.634 339×105
12 0.694 371×105
16 0.755 489×105
20 0.841 592×105
24 0.946 825×105
CFU of the inocolum tube recorded by 285×105, 0.2% EDTA was blank for OD
6.6 DOWN STREAM PROCESSING OF ST-500
Cells were sedimented by centrifugation and high speed of centrifugation cause

some cells to loose viability. It was, therefore, thought appropriate to standardize
centrifugation to minimize the cell loss. It was observed that 5000 rpm is appropriate
with CFU of 7.56×107 and viability loss of 2×106. The batch was centrifuged at 5000 rpm
for 10 minutes followed by washing of the pellet with normal saline at 5000 rpm for 5
minutes. Washing also cause viability loss of 1.1×106. A total loss of 3.1×106 cell
viability during centrifugation was obsereved.
6.6.1 Lyophilization
All the compounds used as lyoprotectants in this study were found to be effective
in most of the cases, providing protection to various lactic acid bacteria (Carvalho et al.
2004). Eighteen lyoprotectants were used separately as medium to provide protection
from high vacuum and freeze shock during lyophilization along with a blank sample.
Skim milk powder was used in sample preparation before freezing because of its property
of preventing cellular injury by stabilizing the cell membrane constituents and creating a
porous structure in the freeze dried product (Castro et al., 1996; Salmer-Olsen et al.,
1999). Skim Milk also contains proteins that provide a coating to the bacterial cells
(Abadias et al., 2001). On testing the viability of S. thermophilus with skim milk (10%
w/w) shows highest cell viability of 74% when observed in dry form. On the other hand
sugar and sugar derivatives were also used for their protective effect during lyophilization
and also during after lyophilization storage (Carvalho et al., 2002, 2003c). Sugar alcohol
like sorbitol has found to be one of the strong protective agent during lyophilization and
storage of L. bulgaricus, L. plantarum, L. rhamnosus, E. faecalis and E. durans
(Carvalho et al., 2003c), but in current study, its performance as protective agent for
lyophilization of S. thermophilus was dull and discouraging. Infect cheaper sugar sources
like sucrose (72%), glucose (51%), lactose (66%) and maltodextrine (67%) showed better
results compared to high cost sugars like mannitol (44%), fructose (45%), maltose (32%),
ribose (39%) and arabinose (28%) as high cost compounds would likely to restrict their
large scale industrial use. The ability of mono sodium glutamate (MSG) to protect
microbial cells during lyophilization and cryopreservation is also described by several
workers (Font de Valdez et al., 1983; Martos et al., 1999). Use of 1% (w/w) MSG for S.
thermophilus also showed protective effect with 57% viability, while as more than 1%
quantity showed viability loss. Lyophilization of ST-500 with all the seven lyophilization
media was conducted at 0.004 mbar vacuum pressure individually in plates and vials
(Figure 6.27).
Table: 6.7 Combinations of protectants along with the ratio of addition
Lyophilization Constituents in percentage Proportion of addition

Medium (mL of cell inocolum :
protective medium)
1 Skim Milk 10%, Sucrose 5%, Sodium
Caseinate 5% 1:5
2 Skim Milk 10%, Lactose 5%, Whey

Protein Concentrate 5% 1:7
3 Skim Milk 10%, Maltodextrine 5%,

Sweet whey Powder 5% 1:5
4 Sodium Caseinate 10%, Skim Milk 5%,

Maltodextrine 5%, Mono sodium 1:8
glutamate 1%
5 Sodium Caseinate 5%, Whey Protein
concentrate 5% 1:8
Maltodextrine 10%, Meso-inositol 0.5%,
Mono sodium glutamate 1%
6 Sodium Caseinate 5%, Lactose 5%,
Mono sodium glutamate 1%, Skim Milk 1:5
5%
Sodium Caseinate 10%, Skim Milk 5%,
7 Sucrose 5% 1:5
Mono sodium glutamate 1%
The effect of K2HPO4 and KH2PO4 on cell viability of B. bifidum has been
studied by Qin et al. (2013) showing cell survival viability up to 77.80% with KH2PO4
and 79.82% with K2HPO4. But studying the effect of these compounds on S.
thermophilus reveals opposite results with K2HPO4 (1% w/w) 21% and KH2PO4 (1%
w/w) 17%. Milk proteins present in skim milk leads to stabilization of protein structures
via reactions between the amino group of the microbial cell proteins and with protectant.
Other mixtures with casein protein and whey proteins were also tested revealing
interesting results by providing high cell viability to S. thermophilus. Sodium caseinate
resulted in highest viability of 81% while as whey protein concentrate (WPC) with 65%
and sweet whey powder (SWP) 58% (Figure 6.26).
Figure 6.26: Effect of different lyoprotectants on the viability after lyophilization.

If not other indicated 10% (w/w) solution were used. The error bars
show the standard deviation
A total of 40 combinations were prepared based on the results obtained on their role as
individual protectant in providing the viability to S. thermophilus on lyophilization.
After initial studies combinations producing more than 50% viability were considered for
further studies in which only 7 combinations were observed significant with high
viability (Table 6.7). The ratio of addition was also studied and implemented on the basis
of thickness and viscosity of the prepared lyophilization medium. Lyophilization medium
(LM) 2, 4, 6 and 7 were found efficient in maintaining the cell viability both during
freezing and drying state of lyophilization. LM 7 was observed as unique viability
escalator providing better results compared to other lyophilization media (Figure 6.29).
The reason behind the success of LM 7 is the role of protective milk proteins which are in
abundance in this particular formulation playing a role in stabilizing the protein structures
of this microbe and sugar source playing a crucial part in maintaining the physical state
of the membrane lipids and enzyme level. All the protective compounds in lyophilization
medium 7 have their unique role in maintaining the cell viability, also the medium is cost
efficient and can easily be implemented on large scale industrial production of S.
thermophilus for its role in starter culture. After final lab scale evaluation, lyophilization
media 7 was used for lyophilization of 300 L batch at TBI (Technology Based Incubator),
Delhi University with output of 82% viability (Figure 6.28).
Figure 6.29: Effect of different Lyophilization media on the viability after

lyophilization. The error bars show the standard deviation
Studies on lyophilization of lactic acid bacteria suggest that the stability of

lyophilized cells decreases during storage at -8° C. Up to 5% viability loss was observed
on re-examining the stored vials of S. thermophilus after a period of 180 days. On the
other hand lyophilized vials of the same microbe stored at -60° C showed higher survival
rate with less than 1% viability loss. An organism which survives the various steps of
freezing, drying and storage may, nevertheless, lose its viability during rehydration.
Therefore, rehydration is a critical step in the recovery of freeze-dried microorganisms,
because cells that were subjected to sub lethal injury may not be able to repair said
damage if they are rehydrated under inappropriate conditions.
Table: 6.8 Viability loss during up-downstream bio-processing
Process Measured with Colony forming Maximum OD at Viability Loss

parameters units per mL 660nm
sample
Complete fermentation at 7.76×107 0.924 Nil
optimized conditions
Centrifugation at 5000 7.56×107 0.915 2×106
(rpm) for 10 minutes
Centrifugal washing of 7.45×107 0.904 1.1×106
pellet with saline water at
5000 (rpm) for 5 minutes
Pre-Freezing for 7.39×107 0.899 6×105
Lyophilization at -60°C
Freeze drying at 0.04 7.15×107 0.884 2.4×106
mbar vacuum pressure
Total Viability Loss 0.040 6.1×106
Process viability loss during the up-stream and down-stream processing was also
recorded by taking samples after regular intervals from fermentation to freeze drying. In-
process, samples were diluted four times to equalize the proportion of fresh liquid culture
and pellet. Optical density was recorded at 660 nm and CFU (colony forming units) were
calculated by serially diluting the samples followed by pour-plate method and colony
counting. An average viability loss of 1.5×106±2 was observed after each standard step
process towards down-streaming with a total viability loss of 6.1×106 (Table 6.8). Cell
multiplication ratio in the final product was also recorded. It was observed that
lyophilized culture with viability of CFU size of 5×109 was sufficient enough to work as
an inocolum in 1L milk for curd preparation. On inoculation of 4 hours at 42°C the initial
CFU of 5×109 was increased to a prebiotic level of 2×1014 with per curd CFU content in
curd of 2×1011.
Chapter 7
Dairy Applications
7.1 DAIRY APPLICATIONS
The present study relates to the dairy applications of bacterium Streptococcus

thermophilus. S. thermophilus is essentially a lactic acid bacterium widely used as a
starter culture for the production of dairy products viz. production of hard cheese of Swiss
type, soft cheese, and yogurt (Falentin et al., 2010; Hao et al., 2011). S. thermophilus is
classified as a nonpathogenic, single streptococcus species to possess a generally
recognized as safe status (Burton et al., 2006). It is also considered as “the second most
important industrial dairy starter after Lactococcus lactis” (Mayo et al., 2008). Along
with the use in manufacturing fermented food, streptococcus is reported to posses
probiotic properties in adequate amount conferring a health benefit to the host (Labeer et
al., 2008; Khalil 2009).
As noted above, a healthy population of beneficial, mutualistic, and commensal

microorganisms in the digestive tract plays a substantial role in maintaining the health
and welfare of the host organism. Such microorganisms create benefits to their hosts in
many ways through competition with pathogenic microorganisms, aiding in the digestion
and absorption of food, helping with vitamin synthesis, and regulating immune responses.
Therefore, healthy individuals often display a robust collection of beneficial
microorganisms in their digestive systems, which aid them in maintaining a disease free
state, and further contribute to the overall well being of the individual.
Bacterial isolate ST-500 was tested for various dairy applications like curd,
fermented milk, cheese, mishthi dahi and frozen fruit dessert. During milk fermentation,
organic acids, mainly lactic acid, are produced by the microorganisms lowering the pH
value. This acid production can be used to characterize milk fermentation and is therefore
an important method to test the activity of starter cultures. Curd is an important part of
Indian diet. In most Indian homes curd is prepared almost every day. Homemade curd is
not only very simple to prepare but is also delicious.
The isolate was tested for following applications:
• Curd
• Yogurt
• Flavored yogurt
• Mishti Dahi (Sweet Curd)
• Butter Milk
• Fruit dessert
• Yogurt Softy
• Cheese
7.1.1 Application testing for curd, yogurt, butter milk, mishti dahi
Pasteurized milk was allowed to cool up to 42°C followed by stirring the milk
slowly to distribute the culture organism (ST-500) uniformly throughout the milk and the
inoculated milk was poured into 100 mL capacity sterile plastic cups, which were then
incubated at 42°C for about 6 hours for curd. (a) Incubation period of 12 hours for butter
milk, (b) 7 hours for misthti dahi (10% sugar), (c) Incubation period of 6 hours for
yogurt, (d) Incubation period of 7 hours for flavored yogurt. For preparation of yogurt
LB-200 and ST-500 were used in combination with ratio of 1:1 mixed culture. Flavored
yogurt (set) and flavored yogurt (stirred) were also prepared in 100 mL volume cups with
same combination of St-500 and LB-200. Mango, orange, strawberry, pineapple,
blueberry and lichi flavors were prepared using both fruit pulp and natural flavors
available in market. After fermentation, the cups were shifted to refrigerator for
overnight. All the fermented products were then analyzed for sensory quality, rheological
attributes and physico-chemical attributes (Table 7.1).
7.1.2 Selected Parameters for application testing

7.1.2.1 Texture of Curd
The texture of curd depends mainly upon the heat treatment given to milk. Cow
milk (3.5% fat and 8.5% SNF) was subjected to two separate treatments: (1) heating at
63oC for 30 min and (2) boiling treatment without holding period. The texture of the
prepared curd was smooth with a thick gel (Figure 7.1), while as texture of yogurt was
little loose and flow able. A rough and grainy texture was observed in mishti dahi (Table
7.1).
7.1.2.2 Sensory Evaluation
The chilled curd, yogurt, butter milk and mishti dahi was served to a panel of
seven judges and its colour and appearance, flavor, body and texture and overall
acceptance were evaluated on 9- point Hedonic scale (Amerine et al., 1965). The scores
awarded by the judges were better compared to standard curd available in market. The
apperence of yogurt, flavored yogurt (Figure 7.2) and mishti dahi were very much liked
and appreciated by tasting panel.
7.1.2.3 Syneresis
The set curd at 5°C was slowly transferred to 15 mL capacity centrifuge tubes
causing minimum disturbance to the coagulum. The centrifuge tubes were balanced by
adjusting their weights and centrifuged at 2000 rpm in a Remi centrifuge for 5 min. The
quantity of whey separated at the top of the coagulum inside centrifuge tubes was
recorded as milliliters. The higher the volume of whey separated, the higher was the
syneresis and vice versa. Syneresis in case of curd and mishti dahi prepared from ST-500
was observed up to mark. Syneresis was not checked for buttermilk as the product is a
noncoagulum type fermented drink.
7.1.2.4 Acidity
The acidity of the curd samples was analyzed by BIS method and expressed as
per cent lactic acid (Bureau of Indian Standards, 1981).
7.1.2.5 pH
pH was determined by potentiometric method i.e. by potential difference between
the sample and electrolyte solution present inside the electrode of pH meter, using digital
pH meter (Systronic Co., Bangalore). The electrode of the pH meter was directly dipped
in the set curd and the pH was recorded (5°C). pH was recorded to check the lactic acid
secreting capability of the culture in specific duration with favorable temperature. Curd
was prepared in cups with 100 mL volume and was incubated. pH was recorded after 3,
3.5, 4, 4.5, 5, 5.5, 6 hours on incubation from different cups of same experiment, so that
the coagulation will not get disturbed (Figure 7.3). Curd prepared with different fat
concentrations was also checked for coagulation and pH drop during incubation (Figure
7.5)
Figure 7.3 Graphical presentation of pH drop rate after inoculation of ST-500 with
different fruit and sugar percentages on incubation at 42°C
Figure 7.5: Graphical presentation of pH drop rate after inoculation of ST-500
with different percentage of milk fat on incubation at 42°
7.2) APPLICATION TESTING FOR CHEESE PRODUCTION FROM ST-500
Firstly S. thermophilus used for the manufacture of fermented milks such as

yoghurt, it is now increasingly used in cheese production, for example, in production of
cheeses that was formerly made with Lactococci bacteria, such as Lactococcus
lactis or Lactococcus cremoris. This bacterium converts lactose in milk into lactic acid by
acidifing the milk. In the case of cheeses, this acidification not only encourages the action
of the coagulant and the synaeresis of the curds, but also inhibits the growth of many
undesirable bacteria, certain of which are pathogenic bacteria, and allows their
elimination at a greater or lesser speed. On an industrial scale, the hydrolysis of urea
by Streptococcus thermophilus poses a number of problems. This is because, in cheese
manufacturing for example, the technological operations (cutting of the curds, stirring,
etc.) must take place at given values of pH, but in practice these operations are generally
carried out at predetermined times. Therefore, the variations in acidifying activity due to
urea hydrolysis may lead to defects and variability in the texture, moisture level, and
ripening properties of the resulting cheeses. Moreover, because ammonia is basic, the
production of ammonia increases the time necessary to reach a given pH. This results in
the cheese-making equipment being tied up for longer and in an increase of the risk of
contamination by undesirable micro-organisms. It is desirable that the cheese-making
whey does not contain an excessive amount of ammonia, because this whey is often used
as an ingredient in human food and animal feed. The production of ammonia from urea is
difficult to control, in part because the urea content of milk is variable (for example, from
2 to 8 mM) and depends in part on the diet of the livestock that produce the milk. In
certain aspects, methods of producing reduced open-texture cheese comprising: a)
contacting milk with: i) urease positive Streptococcus thermophilus bacteria and a urease
inhibitor, and/or ii) urease negative Streptococcus thermophilus bacteria, which are not
able to release ammonia from urea at same level as wild-type bacteria; and b) fermenting
the milk under conditions such that initial cheese is produced; and c) aging the initial
cheese for a period of time such that reduced-texture cheese is produced which has a
reduced amount of open-texture compared to control cheese, wherein the control cheese
is produced in the same manner as the reduced open-texture cheese but employs the
urease positive Streptococcus thermophilus bacteria without the urease inhibitor are
provided.
Samples of fresh 1% fat milk were treated with various combinations of ST-500
(S. thermophilus NCIM 5539) bacteria. In each testing, an acidification curve was
determined by measuring the pH of the milk from the time of addition until 250 minutes
after addition. Milk from one source was used as the starting material for each
experiment. The temperature of the milk was held at 35° C. for the duration of each
experiment. The activity of the S. thermophilus bacteria was correlated to the amount of
time that it takes for the pH of the milk to reach a particular level. S. thermophilus, is
anaerobic and has little or no activity in the presence of oxygen. When it is active,
pyruvate formate lyase is believed to produce formate. When oxygen is present, S.
thermophilus activity is believed to decrease because the amount of formate produced by
pyruvate formate lyase is reduced. When an external formate source, such as sodium
formate, is added, the activity of S. thermophilus is increased. Formate sources other than
sodium formate, such as formic acid, potassium formate, magnesium formate, calcium
formate, or any other acceptable formate, may also be used for this purpose.
In current study, ST-500 took a much longer time to coagulate which was
different according to fat concentrations of the milk. The acidity on coagulation was on
much higher level and observed between 4-5 final pH. The texture, taste and pH of the
cheese were observed on aging period of 10, 20 and 30 days. The taste of the cheese was
found increasingly better with aging. The final product obtained after 30 days was with a
little loose texture as compared to other lactic cheese available in the market.
Table: 7.1 Results of application testing done on the basis of certain parameters
Parameters tested
S.N Applicatio
. n Tested Post Gel Taste Water/Whe
Fina Textur
Acidificatio Firmnes Flavor/Arom y
l pH e
n s a Separation
Quality
Creamy
Testing of Smoot
1 4.45 4.45 ± 0.1 High Mild Curd Nil
ST-500 for h
flavor
Curd
Quality
Cream
Testing of
2 4.92 4.92 ± 0.1 Loose Low yogurt flavor Low
ST-500 for
and aroma
yogurt
Quality
Testing of Sweet
Smoot
3 ST-500 for 4.53 4.53 ± 0.2 High creamy Dahi Low
h
Mishti aroma
Dahi
Quality
Sour and
Testing of
4 4.87 4.87 ± 0.4 Grainy N/A fatty Cheese Nil
ST-500 for
aroma
Cheese
Quality
Testing of Creamy
5 ST-500 for 5.02 5.02 ± 0.2 Loose Low Fruit flavor Low
Fruit/Flavo and aroma
r Yogurt
Quality
Testing of
Very Very Salty butter
6 ST-500 for 4.44 4.44 ± 0.1 N/A
Loose Low Curd flavor
Butter
Milk
Quality
Rich
Testing of Smoot
Creamy
7 ST-500 for 4.88 4.88 h and N/A N/A
yogurt flavor
yogurt Soft
and aroma
softy
7.3.1 Chemical & Physiological Parameters for Fermented Milk (set curd)
 Acidity: Acid production by Starter Culture is assessed by pH measurement of

inoculated milk after incubation. Acidity in terms of pH measurement of
fermented milk should be between 4.5 to 5.
 Post Acidification: Post acidification should be very low.
 Flavor: Like Indian Traditional Curd (Mild acidic).
 Rheology: Firmness, Texture and viscosity are important rheological or textural
parameters that govern the quality of Fermented milk (Set Curd).
 Gel Firmness: Firmness is the quality of steadiness or immovability (fixed).
 Texture: Texture is the appearance, or consistency of surface of a Substance.
Fermented milk (Set Curd) should be Smooth & Soft in appearance & have the
proper cutting ability.
 Taste: Sour (Mild Acidic) & slightly buttery in taste (Creamy).
 Water/Whey Separation: Another Parameter which governs the quality of set
curd is the separation of water/whey from set curd and it should be very Low after
curdling (4 hour).
 Heat treatment: Milk is heated before the culture inoculation. If pasteurized milk
is used, it is heated up to 42-43°c.
7.4.1 TESTING OF ST-500 (STREPTOCOCCUS THERMOPHILUS NCIM 5539)

STARTER CULTURE AGAINST AVAILABLE CULTURES IN THE
MARKET FOR CURD PREPARATION
Market samples of starter culture were brought to laboratory for comparative

testing against ST-500. All the samples were maintained at -20° C storage conditions
before use. 100 mg starter culture sample of JAMA (CHR. Hansen, Denmark),
FlavoGard (Danisco Dupont, Copenhagen) and ST-500 was collected separately and
added in 10 mL of milk. All the three 10 mL milk stock samples were mixed uniformly.
A 0.3 mL sample was dispensed from all the three stocks and inoculated in three beakers
respectively containing 200 mL milk at 40° C with standard parameters (Table 7.2).
Table: 7.2 Selected Parameters and standards for comparative analysis
S.N. PARAMETER STANDARDS USED
1 Percentage of Fat in Milk 4%
2 Percentage of SNF* in Milk 9%
3 Inoculation temperature 40
4 Incubation temperature 42
5 Incubation Period 4-5 hours
Additional Fortification of
6 None
milk
7 Heat Treatment 70 for 10 minutes
8 Starter culture used JAMA, ST-500, Flavogard
9 Amount of Culture used Equal
Table: 7.3 Results of comparative testing done on selected parameters
Parameters tested
Starter Water/
S.N.
Testing Final Post Gel Taste Whey
Texture
pH Acidification Firmness Flavor/Aroma Separat
ion
Creamy Mild
1 ST-500 4.45 4.45 ± 0.1 Smooth High Nil
Curd flavor
Creamy Mild
2 JAMA 4.48 4.48 ± 0.1 Smooth High Nil
Curd flavor
Creamy
3 Flavogard 4.63 4.63 ± 0.1 Smooth High Nil
yogurt flavor
Final product ST-500 starter culture was observed effective against all the major
applications of dairy industries. Most importantly, the application of its use as starter
culture for Indian traditional curd is of great commercial and industrial value. As
described in Table 7.3 the curd prepared from ST-500 was observed almost equivalent to
the curds prepared by the available starter cultures in the market. The other dairy
applications were also observed significant in terms of its commercial use.
The culture showed tremendous potential in yogurt production also when analyzed
with LB-200. Both the bacteria worked in synchronization to produce both flavored and
plain yogurt with rich and creamy taste and texture. However, this synchronization of ST-
500 and LB-100 is a matter of more experimental study which can be the future prospect.
Chapter 8
Summary & Conclusion
The requirement for making a good meal is to have ingredients of a good quality
available, and to have sufficient variety to generate interesting taste and flavor. This is,
however, not as simple as it sounds, as a large number of our basic food items are
perishable, and quality is also not always easy to recognize. In the industrialized world,
the fraction of our food prepared outside the private kitchen is exceeding 50% and this
fraction is still rising. With the knowledge about the genetics and the physiology of lactic
acid bacteria we now possess, it is possible to engineer starters for a variety of purposes
where a suitable starter culture cannot easily be found in nature.
Lactic acid bacteria obtained by biotechnological process is preferred for

industrial applications, especially, dairy industry. Lactic acid bacteria are good organisms
for lactic acid fermentation. Streptococcus thermophilus (NCIM 5539) is a homo-
fermentative LAB isolated from curd is extensively explored in this study. It exhibited
more than 84 % lactic acid production yield along with cell mass yield of more than 6.5%
was achieved which is desirable for industrial application. The intention of the present
investigation was to isolate, characterise and study various parameters of different
environmental conditions to optimize the production of starter culture of Streptococcus
thermophilus (NCIM 5539). The isolated strain was further identified from MTCC
(Microbial Type Culture Collection) CSIR Labs, Chandigarh, India for genuine
confirmation of our identification results. The strain was later deposited to NCIM
(National Collection of Industrial Microorganisms), Pune, India, under safe deposit with
accession number NCIM 5539. This deposition was followed by Whole Genome
Sequencing of the strain conducted by Genotypic, Bangalore, India.
In order to characerise the isolated strain of Streptococcus thermophilus (NCIM

5539), phenotypic (morphology, physiological and biochemical tests) and genotypic
methods (16s rDNA sequencing identification and WGS) were performed. One hundred
and twenty eight isolates were identified as homo-fermentative and all were studied for
their fermentation, multiplication and lactic acid production capabilities. On the basis of
result obtained, 32 were selected for complete phenotypic, morphological and
fermentation analysis. Ten isolates with very strong potential as starter culture and other
dairy industrial uses were further selected for complete analysis and characterization
along with dairy applications of their final product. This selection was made on the basis
of biochemical analysis including sugar fermentation, casein hydrolysis, catalase test,
starch hydrolysis, urease activity, MRVP test etc.
Yeast extract and peptone were used in the fermentative media as nitrogen
source for production of lactic acid bacteria though expensive for commercial production.
The efficiency of this process could be improved within limits by varying culture
conditions and medium composition. Commercial grade sucrose as carbon source and
tryptone as nitrogen source were found to reduce the nutrients cost by 60%. This study
suggests that sucrose, tryptone and yeast extract could be used as relatively cheaper
nutritional source in the fermentation medium for production of biomass of S.
thermophilus.
Ten selected strains were, further, identified at molecular level (16s rDNA
sequencing) and found that seven strains are of cocci type (chain) in morphology
belonging to Streptococcus thermophilus , one strain belong to Streptococcus infantarius
subsp. coli and two strains with short and long rod morphology belong to Lactobacillus
acidophilus and Lactobacillus delbrueckii subsp indicus, respectively. Phylogenetic tree
were prepared for all 10 strains with the help of BLAST at NCBI for best hit results.
Whole genome sequencing of one selected strain of S. thermophilus was studied to
observe the overall genetic expression of the strain. The selected strain is the new
discovery in the world as the ninth strain of S. thermophilus for which the complete
genomic profile has been investigated (data will be submitted to NCBI after patenting).
Batch fermentation was conducted with maximum cell mass achieved (7%).
More than 100 lab scale fermenter batches (2L & 5L) and 25 pilot scale batches (50L to
300L) were prepared and performed for detailed analysis of cell multiplication,
fermentation, freezing and lyophilization. The final starter culture produced after
complete up and down stream processing was tested for its efficiency in dairy industrial
application of fermented food products. Curd prepared from S. thermophilus starter was
observed completely fine on parameter testing. Other fermented foods like buttermilk,
flavored/ plain yogurt, mishti dahi, frozen dessert, yogurt softy and cheese were prepared
with fine taste, texture, creaminess, flavor, aroma and low post acidification. Finally, the
culture market can also be increased by expanding the application of cultures and by
increasing the value of the products. In both cases, the cultures developed will contribute
a larger part of the value of the final product compared to the current applications.
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 Publications
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Rohit Sharma, Bhagwan S Sanodiya, Gulab S Thakur,

Pallavi Jaiswal, Sangeeta Pal, Anjana Sharma and
British
Prakash S Bisen (2013) Characterization of lactic acid
Microbiology
1 bacteria from raw milk samples of cow, goat, sheep, 1
Research Journal
camel and buffalo with special elucidation to lactic acid
Science Domain
production. British Microbiology Research Journal 3(4):
743-752.
Rohit Sharma, Bhagwan S Sanodiya, Deepika Bagrodia,
Mukeshwar Pandey, Anjana Sharma and Prakash S Bisen International
2 (2012) Efficacy and Potential of Lactic acid bacteria Journal of Pharma 3
modulating human health. International Journal of and Bio Sciences
Pharma and Bio Sciences. 3(4): (B) 935-948.
Rohit Sharma, Bhuvan Bhaskar, Bhagwan S Sanodiya,
Gulab S Thakur, Pallavi Jaiswal, Nitin Yadav, Anjana IOSR Journal of
Sharma, Prakash S Bisen (2014) Probiotic Efficacy and Pharmacy and
3 0
Potential of Streptococcus thermophilus modulating Biological
human health: A synoptic review. IOSR Journal of Sciences.
Pharmacy and Biological Sciences. 9(3): 52-58
Rohit Sharma, Bhuvan Bhaskar, Bhagwan S Sanodiya,
Gulab S Thakur, Pallavi Jaiswal, Anjana Sharma, Prakash Microbiology
4 S Bisen. Standardization of Lyophilization medium for Research 0
Streptococcus thermophilus subjected to viability Page Press
escalation on freeze drying. (accepted)
Mousumi Debnath, Mukeshwar Pandey, Rohit Sharma,
Journal of
Gulab S Thakur and Pushpa Lal (2010). Biotechnological
Medicinal Plants
5 intervention of Agave sisalana: A unique fiber yielding 15
and Research
plant with medicinal property. Journal of Medicinal
Plants and Research. 4(3): 177-187.
Rohit Sharma, Gulab S Thakur, Bhagwan S Sanodiya,
IOSR Journal of
Ashish Savita, Anjana Sharma, Prakash S Bisen (2012)
Pharmacy and
6 Therapeutic potential of Calotropis procera: A giant 4
Biological
milkweed. IOSR Journal of Pharmacy and Biological
Sciences.
Sciences. 4(2): 45-57.
Rohit Sharma, Gulab S Thakur, Bhagwan S Sanodiya, Global Journal of
Mukeshwar Pandey and Prakash S Bisen (2012). Saponin: Research on
7 A wonder drug from Chlorophytum species. Global Medicinal Plants 3
Journal of Research on Medicinal Plants and Indigenous and Indigenous
Medicine. 1(10): 503-515 Medicine
Rohit Sharma, Saxena Nidhi, Thakur Gulab S, Sanodiya Global Journal of
8 1
Bhagwan, Jaiswal Pallavi, Conventional method for Research on
saponin extraction from Chlorophytum borivilianum Medicinal Plants
(2014) Global Journal of research on medicinal plant and and Indigenous
indigenous medicine, 3(2): 33-39. Medicine
Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya et

al. (2013) In vitro induction of tuber formation for the
synthesis of secondary metabolites in C.borivilianum sant African journal of
9 0
ed. Fernand. African journal of Biotechnology 12(20)- Biotechnology
2900-2907
Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya,

Mukeshwar Pandey, GBKS Prasad and Prakash S Bisen Journal of Applied
(2012) Gymnema sylvestre: An alternative therapeutic and
10 2
agent for management of diabetes. Journal of Applied and Pharmaceutical
Pharmaceutical Science. 2(12): 001-006. Science.
Mukeshwar Pandey, Surendra K Chikara, Manoj K Vyas,

Rohit Sharma, Gulab S Thakur, Prakash S Bisen (2012) International
11 Tinospora cardifolia: A climbing shrub in Health care Journal of Pharma 2
management. International Journal of Pharma and Bio and Bio Sciences
Sciences. 3(4): (P) 612-628.
Mukeshwar Pandey, Rakesh Baghel, Astha Gupta, GBKS
Prasad and Prakash S Bisen (2011). High Frequency in- African Journal of
12 1
vitro shoots regeneration of Momordica balsamina, an Biotechnology.
important medicinal and nutritional plant. African Journal
of Biotechnology. 10(70): 15808-15812.
Global Journal of
Rohit Sharma (2014) Micropropagation: An essential tool Research on
13 to flourish endangered medicinal plants. Global J Res. Medicinal Plants 0
Med. Plants & Indigen. Med. 3 (6), 252-262 and Indigenous
Medicine
Mukeshwar Pandey, GBKS Prasad and Prakash S Bisen
(2011). Factors effecting in-vitro propagation of
14 Springer 1
Momordica balsamina:a medicinal and nutritional
climber. Physiology and molecular biology of plants.
17(2): 193-197
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“Selective Enumeration, Isolation and Identiﬁcation of Streptococcus

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Chapter 1 Introduction Page

Chapter 2 Literature Review

Chapter 5 Molecular Characterization

Chapter 6 Media Standardization

Chapter 7 Dairy Applications

Chapter 8 Summary & Conclusion 205

2 Figure 2.2 Characterization of Lactic acid bacteria

Hetero-fermentative pathway showing the production of

Phylogenetic tree and important commercial importance

6 Figure 2.6 Silent features of lactic acid bacteria

Various applications of Lactic acid bacteria modulating

Electron microscopic image of S. thermophilus (Source:

Phylogenetic tree of Lactobacillales based on

Flow diagram of the main steps involved in the

11 Figure: 2.11 Flow sheet for the preparation of acidophilus milk

12 Figure: 2.12 Flow sheet for the preparation of Curd

13 Figure: 3.1 Step elaboration of Inocolum preparation from 1st. 2nd

Circular genomic map of ST-500 (Streptococcus

Influence of carbon sources on lactic acid fermentation

Influence of carbon sources on lactic acid fermentation

Influence of carbon sources on lactic acid fermentation

Effect of nitrogen sources on lactic acid fermentation for

Effect of nitrogen sources on lactic acid fermentation for

Effect of nitrogen sources on lactic acid fermentation for

Effect of vegetable protein and milk protein sources on

Effect of vegetable protein and milk protein sources on

Influence of sodium, potassium, ammonium and

Influence of yeast extract (amino acid source) on lactic

47 Figure: 6.14 Assembling of fermenter for inoculation

48 Figure: 6.15 Completely assembled fermenter ready for inoculation

49 Figure: 6.16 Turbidity observed after 16 hours of incubation

51 Figure: 6.18 Collection of turbid broth in sterile flasks for further

Front view of BioSata A plus lyophilizer, (B, C, D)

(A) presents the commercial model lyophilizer for large

Effect of different Lyophilization media on the viability

A, B, C presenting the texture, cut ability, thickness,

A and C presenting the texture, cut ability, thickness,

Graphical presentation of pH drop rate after inoculation

Application testing results of ST-500 (Streptococcus

Graphical presentation of pH drop rate after inoculation

Selected Parameters and standards for comparative

Results of comparative testing done on selected

AAD : Antibiotic associated diarrhea

1.1 SIGNIFICANCE OF THE STUDY

1.2 RESEARCH OBJECTIVES

This research work focuses on isolation of Streptococcus thermophilus and

• To isolate and identify potential Streptococcus thermophilus strain capable of fast

1.3 RESEARCH HYPOTHESIS

1.4 SCOPE AND LIMITATION OF THE STUDY

Present work involves investigation about production of starter culture by

1.5 EXPECTED RESULTS

2.1 MICROBES AND FERMENTATION

2.1.1 Industrial microbiology

2.1.1.1 Industrial Microbes

Products Bacteria Yeast and Filamentous

Camembert-type cheeses Agaricus bisporus,

Sufu (soya bean curd) Acetobacter species Mucor sp.

Nucleotides Bacillus subtilis,