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ABSTRACT
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Lactic Acid Bacteria (LAB) commonly used as starter cultures in food technology are known to
manufacture antimicrobial products having great potential. The aim of this study is isolation of lactic
acid bacterial strain which has been potential for lactic acid production, to identify, characterize it and
to optimize culture conditions for the process. For this study, we have isolated different strains of
Lactic Acid Bacteria (LAB) from dairy sludge samples which were collected from 3 different dairy
plant of Gujarat, (India). Therefore, characterization of isolated strains through morphological,
physiological, biochemical and carbohydrate fermentation test were done. Lactic Acid Bacteria (LAB)
are a group of Gram-positive, non- spore forming, cocci or rod shaped, catalase-negative and fastidious
organisms, considered as Generally Recognized As Safe (GRAS) organism. For isolation, samples
were serially diluted and plated on MRS agar (DE MAN, ROGOSA and SHARPE agar. HI Media)
Plate. Well-isolated colonies with typical characteristics were picked from each plate and were further
sub cultured until pure isolates were obtained and transferred to MRS broth (HI Media) for further
experiments. Identification of lactic acid bacteria belongs to family Lactobacillaceae was done first
at genus level in the Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus.
Lactobacillus is rod shape and Streptococcus, Leuconostoc, Pediococcus are cocci shape. Most of
them are used in dairy starter culture, present in raw milk, milk-loving bacteria. Isolated colony was
identified by gram-staining, gram +ve and rod shape strain showed confirmation of lactobacillus .
L.bulgaricus and Streptococcus thermophilus were found as the most dominant species in common
sludge unit. While L.acidophillus was found as dominant species along with the L.bulgaricus and
Streptococcus thermophilus in the sludge of pro biotic acidophilus butter milk manufacturing unit
and L.casei, L.helveticus, L.brevis, L.lactis were present as the dominant species in the cheese
manufacturing unit along with the L.bulgaricus and Streptococcus thermophilus. Selected microbial
isolates obtained in this study will be used for production of lactic acid acts as monomer for the
synthesis of biodegradable polymer on my future study.
Key Words :LacticAcidBacteria(LAB),Isolation,Dairysludge,MRSagarplate,
MRS Broth, Bacterial strain
INTRODUCTION
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Spirillaceae
Pseudomonadaceae
Neisseriaceae
Enterobacteriaceae
Vibrionaceae
Rickettsiaceae
Microccaceae
Streptococcaceae
Bacillaceae
Lactobacillaceae
Propionibacteriaceae
Corynebacteriaceae
Actinomycetaceae
Mycobacteriaceae
Nocardiaceae
Genus
Campylobacter
Pseudomonas
Brucella
Acinetobacter
Moraxella-like organisms
Escherichia
Enterobacter
Salmonalla,Yersinia
Aeromonas
Chromobacterium
Flavobacterium
Vibrio
Coxiella
Micrococcus
Staphylococcus
Steptococcus
Leuconostoc
Bacillus
Clostridium
Lactobacillus
Listeria
Propionibacterium
Corynebacterium
Arthrobacter
Microbacterium
Actinomyces
Mycobacterium
Nocardia
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+ve
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ve
ve
Rods Lactoballius
ho mo fe r me nta tiv e
Coc c i
Coc c i
le uc o no sto r s
Tetrad
Growth @ 15C
+Ve
pediococcus
Roads
Lactobacillu Roads
he te r o te me nta tiv e
Ve
Ve
thermobacterium
+Ve
tr epto bacter ium
Gr owth@
45 C
Ve
+Ve
Growth@10C
+Ve
Lactococcus
+Ve
Enterococcus
Ve
Lactococcus
5
s
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Catalase test
A drop of 3% hydrogen peroxide was placed on
a clean microscopic slide. With a nicrome wire
loop pick up cells from the center of a well isolated
colony of the test culture and transfer them into
the drop of hydrogen peroxide. Both were mixed
and observed for gas bubble production. The
strains showing gram-positive and catalase
negative isolates were identified at species level.
Physiological and biochemical identification
Each isolate was activated in 5 ml MRS broth for
24 h at 30 C before use. Therefore, overnight
cultures were used during all the identification
procedures. Physiological and biochemical
identifications were performed according to the
methods and criteria of Sharpe and Fryer; Garvie,
Devriese et. al., Teuber.
For the identification of rod shaped isolates,
following tests were applied
1. Gas poduction from glucose
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Identification of Cocci
The characteristic used for the identification of
cocci shaped LAB in this study were presented.
Except for the arginine test, all the other tests
were the same as those for bacilli shaped LAB.
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Carbohydrates differentiation discs (from Himedia) was used for sugar fermentation test.
Andr ade peptone water, liquid media are
dispensed in 5 ml amount in test tube with
inverted Durhams tube for testing fermentation
Fig. 3 : Streptococcu
bacteria colony
thremophilus
Fig. 4 : Lactobacillus
bulgaricus
Table 3 : The percentage distribution of different genus of LAB in dairy sludge sample
and differential characteristics of lactic acid bacteria based on morphology
Lactic acid bacteria (%)
Sample
Dairy SludgeA1
40
15
35
30
35
10
25
Pediococcus
Lactobacillus
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45
50
50
10
55
45
10
55
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Morphology
Cocci
Cocci
Cocci
cocci in tetrads
Rods
10C
45C
6.5% NaCl
pH 4.4
pH 9.6
L, DL
D, L, DL
CO2fromglucose
Growth
Thermo bacterium
Strepto bacterium
Beta bacterium
Motility
Growth at 5c
Growth at 15c
Growth at 45c
Nitrate reduction
Homofermentation
Homofermentation
Heterofermentation
Fermentation
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Rods
Cocci
Cocci
Cocci
Lactobacillus spp.
Streptococcus spp.
Leuconostoc spp.
Pediococcus spp.
Rods
L. helviticus
Cocci
Rods
L. lactis
thermophillus
Rods
L. casei
Rods
Rods
L. acidophilus
L. brevis
Rods
Cell shape
L. bulgaricus
Species
G +ve
G +ve
G +ve
G +ve
G +ve
G +ve
G + ve
G+ ve
G +ve
G + ve
G + ve
reaction
stain
Gram
activity
Catalase
glucose
from
Acid
lactose
from
Acid
Cellular
Cocci
Single or in chain.
arrangement
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+
glucose
from
Co2
25-32C
15-35C
30-37C
30-50C
40-50C
30C
400-42C
40-43C
30C
35-38C
40C
temperature
Optimum
Table 4 : Morphological and simple physiological characterization of LAB isolated from the dairy sludge sample
L.lactis
L.helveticus
L.brevis
S. thermophilus
L.acidophilus
L.casei
L.bulgaricus
Lactic acid
S/N
10
Morpho 5C
logy
15C
Growth at
Mann
itol
Sorbi
tol
Treha
lose
Mal
tose
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Sugar fermentation
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+
Lact
ose
Gala
ctose
CONCLUSION
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RECOMMENDATION
REFERENCES
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