• Project presentation for B.Sc.

(H) Biotechnology
• 10 June 2020

Title: Studies on CM1 and GM1 probiotic bacteria of high altitudes of

Himachal Pradesh

Faculty of Applied Sciences and Biotechnology

Shoolini University, Solan (HP)
Supervisor- BY: PURVANSH BATSH
Prof. Anuradha Sourirajan (1711301002).
Dean Faculty of Applied Sciences and
Biotechnology,
VAISHALI
Shoolini University KANDARI(1711301004)
SHUBHAM
NARTA(1711301003)
 Introduction
•Probiotics are live microorganisms that provide certain health benefits when consumed.

• The term Probiotics is derived from a Greek word probios which means “for life”.

• Probiotics are often referred to as good or helpful bacteria because they help keep your gut healthy

by decreasing the number of bad bacteria in our gut.

•The currently available probiotic products include a wide array of bacterial and fungal species.

•The use of fermented milk and yogurt are a part of human history ,examples of probiotics.

•Consuming probiotics include improvement of intestinal health by the regulation of microbiota,

stimulation and development of the immune system, synthesizing the bioavailability of nutrients,

reducing the symptoms of lactose intolerance etc.

 History
In Japan, Minoru Shirota developed Yakult, a probiotic yogurt like product by
fermenting a mixture of skimmed milk with a special strain of Lactobacillus. casei
Shirota (Gilliland,1990).There are a large number of probiotics currently used and
available in dairy fermented foods, especially in yogurts. Current probiotic
research aims at the characterization of the normal, healthy gut microbiota in each
individual, assessing the species composition as well as the concentrations of
different bacteria in each part of the intestine, the target is to learn to understand
host–microbe interactions within the gut, microbe–microbe interactions within the
microbiota and the combined health effects of these interactions.

• The fermentation of dairy foods represents the techniques for food preservation
(Azizpour et al., 2009). In 1907, Metchnikoff suggested that it would be possible to modify
the gut flora and to replace harmful microbes with useful microbes called probiotics.
Described various organic and inorganic supplements that were believed to have the
ability to restore the health of patients. The survival of bacteria in the gastrointestinal
tract and the carrier food is necessary to have their beneficial effect on the host (Vilijoen
et al., 2001).
 Common Species and Strains
Common species of probiotics :
Lactobacillus (lactobacillus acidophilus and lactobacillus GG)- It is the one found in yogurt and
other fermented foods. It can help people suffering from diarrhea and people who cannot digest
lactose.[2] Hamasalim HJ. Synbiotic
Bifidobacterium (Bifidobacterium bifidus)- This can be found in some dairy products. It may
help in easing the symptoms of irritable bowel syndrome.
Common strains of probiotics :
B. Longum - Helps break down carbohydrates and also used as antioxidants.[3] Bongaerts et.al
L. Acidophilus- It is found in small intestine and the vagina. It helps in improving digestion and
fight off vaginal bacteria.
.Animalis - Helpful in fighting food-borne bacteria.
.Breve - Helps body absorb nutrients by fermenting sugar.
.Lactis -Derived from raw milk.
Reuteri - Found in intestine and mouth. It decreases the oral bacteria that causes toothdacay[4]
J .Nutr1995;125:1401e12
 Sources of Probiotics
1. Yoghurt
2. aged cheeses, such as cheddar, gouda, or mozzarella
3. kefir, a probiotic milk drink
4. traditional buttermilk (must not be cultured)
5. natto, a food made from fermented soybeans
6. fresh, sour dill pickles
7. kimchi
8. kombucha, a fermented tea
 Advantages of probiotics

• Probiotics plays an important role in development in

the immune system, digestion of food, production of
short chain fatty acids and essential vitamins.
Anti- carcinogenic effect of probiotics was studied in
both men and animals .Probiotics are beneficial on
host health, evaluating for their ability to survive and
grow. [1]

• [1]Bagchi T. Traditional food & modern lifestyle: impact of probiotics

• Cholesterol Reduction: Increasing blood cholesterol is a major risk factor for coronary heart diseases which
can be treated with the help of many cholesterol-lowering probiotics .[5]

• Anti-carcinogenic Properties: Probiotics like bifidobacteria or lactobacilli alone can alter colonic microbiota
and decrease the development of early preneoplastictumors and may lower the incidence of cancer.[6]

• Immunologic enhancement: Enhanced phagocytic activity of granulocyte, cytokine excretion in lymphocytes,

increased immunoglobulin secreting cells in blood are typical responses to probiotics, all of which are indicative
of changes in the immune system[7]

• Enhancement of short chain fatty acid production: Because the improved intestinal microbial communities
with probiotics primarily involve the stimulation of short chain fatty acid production is one of the essential
factors for the beneficial effects exerted by probiotics.

• Probiotics in diabetes and obesity: The gut flora enhances the body weight gain and
Increases the insulin resistance. Probiotics are novel gut flora modulators.[8]

[5],[6],[7] ,[ 8] Metchnikoff E, Mitchell PC, editors. Essaisoptimistes. London: Heinemann; 1907,

Sources of probiotics
 Mode of action
• Having in mind the use of micro-organisms of different origin with same useful
effects.

• Modes of action of probiotics will probably be based more than one principle ,the
alteration of the microbial population.

• The interaction between the probiotic strain and the intestinal microbiota may be
based on aggregation with pathogenic bacteria ,competative adhesion to
epithelial receptors, production of specific substances (organic acids,
bacteriocins , dipicolinic acid).

• As probably secondary effects, alteration of the structure and function of the

intestinal epithelium as well as of the immune response were described. (Simon
et al.,)

• The situation with regard to human nutrition was recently characterized in

nature(Abbott,2004)
Mechanisms of Action . (customprobiotics.com)
Review of literature
Sr. No Title Auther /Year/ Findings
Journal
1. •Total 11-15 strains were isolated
Screening of Probiotic Goat
from goat and cow milk.
Milk and Cow Milk Isolates
•Four goat milk isolate G4, G7,G8
for Acid Resistance,
Sridevi et al ., and G11, one cow milk isolate C12
Antagonistic Activity and
/2015/Int.J.Curr. exhibited acid resistance.
Tolerance to Antimicrobial
Microbiol.App.Sci •Screening on antimicrobial activity
Activity of Spices: Molecular
. all goat and cow milk isolates were
Identification of Potential
tolerate to chilly ginger, coriander
Probiotic Goat Milk Isolate,
and cinnamon
G8

2. Makete et al., 17 isolates were identified as

/2017/Probiotics lactobacillus. These isolates displased
Antimicrob phenotypic probiotic attributes
Isolation, Identification and Proteins including tolrence to acid and bile
Screening of Potential salts. These isolates have ability to
Probiotic Bacteria in Milk adhere to interstines and posseenssion
From South African Saanen of antagonistic activities against
Goats. Proteus vulgaris, Staphylococcus
aureus, Salmonella typhimurium,
Pseudomonas aeruginosa and
Escherichia coli
Methodology and
Results
 Methods
GM and CM was isolated By Pawan and Mousami (Bsc. Biotech student) in 2019
from goat and cow milk under the supervision of Prof. Anuradha Sourirajan

Revival of Bacterial strains from stock

We prepared MRS broth. ( De Man, Rogosa and Sharpe agar) media plates .
Then we took our strains from stoke plate.

Then we performed streaking of our strain in the plates and inoculate it at 30

degree C for 24hrs.

After that we performed some steps as to see the effect of temperature on

the strains .
 Methods
Effect of temperature
To determine the minimum and maximum temperature for the growth of bacterial isolate, GM and CM was
streaked on nutrient agar medium supplemented with 0.5% caffeine and incubated at different temperature
on 4 degree C, 15 degree C, 25 degree C, 30 degree C, 37 degree C, 45 degree C, 50 degree C
Steps
• Preparation of media- 2 plates of Agar. We took nutrient broth(N.B) 30ml for 10gm. For 1000ml it would
be 0.13gm.

Put 0.13 gm N.B into 10 ml distilled water.

Transfer it into three test tubes.

Do autoclave for 2 hours.

After autoclave put the samples into test tubes in laminar air flow.
 After autoclave put the samples into test tubes in
laminar air flow.

Inoculation should be done with the help of sterile loop.

Indicate the tube as GM and CM respectively.

Incubate it into shaker for 24 hours.

Growth can be seen after 24 hours due to turbidity in the test tube.
and Put those tubes into laminar. Pt the sample of GM and CM with
control.

Now prepare 75 ml of NB and put it into 15 tubes.

Take 2 pipette 1000ul and 100 ul.

Take 2 cubette and add 90 ml of GM and CM sample.

Add control to it hence dilution is done.

Now mark 5 as GM and 5 as CM and the rest 5 will be control.

Now take 100 ul pipette and add 100 ul of sample from the inoculated
tube to GM and CM respectively.

Put those samples at different temperature and note the readings after
24 hours
 RESULT: To determine the optimum
temperature for growth of CM1and GM1.

• We have observed the growth of strains (GM and CM) at different temperatures but 25
degree is the optimum growth temperature as shown in graph so from the experiment
that we have done we get to know that these strains grow best at 25 degree.
 Conclusion

• GM and CM was revived from glycerol stoke in MRS

media . Temperature optimization was done in liquid
medium and we observed that 25 ºC is the optimum
temperature.
 ACKNOWLEDGEMENT

Our parents and family

Vice-chancellor Prof. P.K. Khosla

Prof. Anuradha Sourirajan

Supervisor Dean Faculty of Applied Sciences
and Biotechnology

Prof. Kamal Dev and Er . Rajan Rolta

All Faculty members

Applied Sciences and Biotechnology

All Research Scholar of Yeast Biology Lab