novel synbiotics for urban lifestyle

“novel synbiotics for healthy urban lifestyle”

GUIDED BY-Dr. Pratima KhandelwalHead of Department Department of BiotechnologyNHCE, Bangalore-560103

Archita Biswas-1NH10BT007Ritasree Sengupta-1NH10BT039Shubhamita Saha-1NH10BT045Swayanka Biswas-1NH10BT049

Dept of Biotechnology, NHCEBangalore-560103

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CONTENTS1. Introduction2. Retrospection3. Lacunae4. Objectives5. Materials and Methodology6. Results and discussion7. Summary and conclusion8. Bibliography

VIII SEM PROJECT PRESENTATION, DEPT OF BIOTECHNOLOGY, NHCE, 2013-14

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INTRODUCTION What is healthy lifestyle???

The type of lifestyle that promotes human health and mental conditions

Factors effecting healthy lifestyle— Healthy food

Proper nutrition Personal hygiene

Environment Love and tenderness


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What is unhealthy lifestyle???The type of lifestyle that hampers the well-being of human

Conditions causing unhealthy life style--- Unhealthy food

Improper hygiene Abuse of substances like drugs, alcohol, tobacco

Excessive stress Unhealthy environment

Unhealthy habits


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Effects of unhealthy lifestyle—

Diabetics

Obesity

Cardiovascular diseases

Gastrointestinal diseases

Abnormal blood pressure


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PROBIOTICS…Mainly bacteria or yeastUsually present in the gut and intestinal

regionsHelps in digestion of the ingested foodEffects immune system



Pictures courtesy-Google images

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PREBIOTICS… Non-digestible food matters.

Helps in the growth of gut and intestinal

micro-organisms.

Doesn’t emphesize a specific bacterial group.

Increases the number and activity of the

probiotics mainly.


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Emergence of synbiotics……

probiotics prebiotics synbiotics


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SYNBIOTICS……

Contains nutritional supplements

Contains both probiotics and prebiotics

Usually remains in a synergism form


11FUNCTIONAL FOODS

NEW INGREDIE

NTS

FOOD

ADDING MORE

QUANTITY OF

EXISTING INGREDIE-

NTS


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STEVIA a sugar substitute.

taste has a slower onset and longer duration.

Some extracts have a bitter aftertaste at high

concentrations.

low carbohydrate.

has no effect on blood glucose.

glycoside extracts in Stevia have been recognized as

safe (GRAS) product.


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glycosides are non-fermentable.

Also pH and heat stable.

Approved by WHO and FDA.

Permitted daily intake of steviol glycoside is

up to 4mg per kg body weight.


Scientific classification

• Domain: Eukaryota

• Kingdom: Plantae

• (unranked):

Angiosperms

• (unranked): Eudicots

• (unranked): Asterids

• Order: Asterales

• Family: Asteraceae

• Tribe: Eupatorieae

• Genus: Stevia

STEVIA REBAUDIANA FLOWERSCourtesy- Google images


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BENEFITS OF STEVIA- TIMES OF INDIA

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NAME OF THE AUTHORS PAPER PUBLISHED

Hamilton et al.,2003 Some insights into the derivation and early uses of the word Probiotics.

Dilnawaz et al., 2011 Review on Probiotics

Schlundt et al., 2012. Health and nutritional properties of probiotics in food including powdered milk with live lactic acid bacteria.

Fuller et al., 1989 Probiotics in man and animals.

RETROSPECTION


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NAME OF THE AUTHOR PAPER PUBLISHED

Kumar et al.,2012 Traditional Indian fermented foods: a rich source of LAB

Maragkoudakis et al.,2005 Probiotic potential of Lactobacillus strains isolated from diary products.

Mc Caleb & Rob,1997 Controversial products in the natural food market.

Mishra et al.,2011 Anti-diabetic activity of medium-polar extract from the leaves of Stevia rebaudinia.


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Less existence of the indigenous brands to harness the powers of

probiotics in India.

No existence of non-dairy probiotic foods in Indian market till today.

Use of stevia in food products is still unpopular in major parts of the

world .

LACUNAE


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Food products having Probiotics and

Prebiotics are still yet to be found in the

Indian market.

Synbiotics together with natural sugar

replacers in food for dietetic purposes are

still unavailable in the market till today.


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OBJECTIVE

To develop acceptable, safe and stable confectionary and dairy synbiotic products for urban lifestyle.


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MATERIALS

•GENERA-Bifidobacterium, Lactobacillus

LAB[ LACTIC ACID

BACTERIA ]


STEVIA• Obtained from supplier• A sugar replacer• Used to make dietetic products

STEVIA SUPPLIER-AYUSHA laboratories Pvt Ltd.


Ayusha Herbs, Pvt. Ltd., Banshankhari 3rd stage, Bangalore

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SUNFIBER• SOURCE-TAIYO LUCID PVT LTD.• Prebiotic• Used to make dietetic and synbiotic

products


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DEVELOPED PRODUCTS

1. Synbiotic chocolates

2. Synbiotic kalakand and dietetic

kalakand

3. Synbiotic Biodips

4. Synbiotic Mayonnaise


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METHODOLOGYMORPHOLOGICAL TESTS•a. Gram staining

BIOCOMPATIBILITY TESTS•a. pH test•b. Bile test

CURDLING TESTS


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TESTS

•Products•0.85% saline•MRS Agar

Total Viable Count

•Yeast and mould count-PDA Agar

•E. Coli count-EMB AgarQuality tests for

Developed Products


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Biocompatibility TEST-Requirements

pH TEST

• 8M HCL• PBS Buffer• MRS Agar

BILE TEST

• 0.3% bile salt• MRS Agar


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pH TOLERANCE TEST

Pure culture of probiotics were grown overnight in 10ml of MRS broth

The bacteria was harvested by centrifugation at 10,000 rpm for 10 min

Pellet washed 3 times with PBS buffer


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The washed pellet was re-suspended in PBS buffer and was incubated at 37ºC for 2 hours

100 µl of the suspension was added to 10 ml of sterile PBS tubes(each adjusted to pH 2.0, 2.5, 3.0, 6.2) using 8M HCL

The tubes with the adjusted pH were further incubated for 20 min at 37ºC

The samples from each tubes were pour plated using MRS agar and they were incubated at 37ºC for 24 hrs


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BILE TEST1ml of overnight culture was inoculated in tubes containing 9ml of MRS

broth supplemented with 0.3% of Bile Salt.

Tubes were incubated at 37°C for 8hrs.

Cultures were monitored at every 45mins for growth by measuring absorbance of culture media at 630nm.

Bacterial growth was determined by means of measuring optical density at 620nm using spectrophotometer


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CURDLING TEST2 loops of bacterial colonies were inoculated into 10ml of MRS broth and

incubated overnight at 37°C.

Checked for turbidity, centrifuged at 10,000rpm for 10mins.

Pellet was inoculated into 10ml of sterilized milk and incubated for 6-8hrs at 37°C


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Curdled milk was centrifuged at 10,000rpm for 10mins.

Washed 4-5 times using Saline (0.85%).

Supernatant was discarded and pellet was used as a Probiotic Biomass.


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PROBIOTIC CHOCOLATES

INGREDIENTS 1. Cocoa powder 2. Milk powder 3. Cocoa butter 4. Stevia 5. Butter paper 6. Chocolate molds


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Indirect heating of cocoa butter was done till it melts

Mixing of cocoa powder in the melted butter

Then milk powder was added to the mixture


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The components were mixed thoroughly while heating indirectly

Stevia was added and mixed properly

small amount of melted butter was added to the chocolate molds

After which the probiotics were added to the chocolate, poured into the molds and refrigerated


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Indirect heating of

the mixture

Adding Stevia

Pouring into the molds


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SYNBIOTIC KALAKAND

INGREDIENTS1.Milk

2. Citric acid

3. Stevia

4. Sunfiber

5. Chocolate flavor


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PROCEDURE

250ml of the milk was boiled at the beginning

citric acid was added to the boiling milk

The milk got coagulated into the whey and the paneer


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After which the whey was drained out and only the paneer was taken

Again 250ml of milk was boiled and stirred till it was thickened

After which the drained paneer was added to the thickened milk

And stirred properly to eliminate all the clumps


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Stevia and cocoa powder was added in place of sugar

A butter paper was taken and kept on a tray

The hot kalakand was poured on the papered tray and cooled to 37ºc and probiotics was added to it


Making of the paneer


Separation of whey

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SYNBIOTIC BIODIPSIngredients

• Cocoa powder• Cocoa butter• Milk powder • Stevia• Sunfiber• Fruit pieces


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PROCEDURECocoa butter was indirectly heated and melted in a pan

Cocoa powder and milk solids were added and mixed properly

Stevia was added and properly mixed after which it was cooled and probiotics as well as prebiotics were added.


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Toothpicks were inserted in the fruit pieces

The fruit pieces were then dipped in the prepared chocolates

The dips then cooled and solidified by keeping in the refrigerator.


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SYNBIOTIC MAYONNAISEIngredients

• Milk-1 cup• Cornflour-4tbsp• Pepper powder-1/4tsp• Salt-1/4tsp• Olive oil-2tbsp• Mustard powder-1/4tsp• Sunfiber


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PROCEDUREMilk was taken and boiled till it gets slightly thick

Four spoons of cold milk was taken separately and cornflour was added to it and mixed properly

The mixture was then added to the boiling milk and stirred at low temperature


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Salt and pepper was added to enhance the taste

Mustard powder was added & the mixture was stirred in 1 spoon of vegetable oil

It was cooled to 37º C and probiotic was added to it


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RESULTS & DISCUSSIONGram’s staining test-presence of gram

positive bacteria


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OBTAINED SYNBIOTIC MAYONNAISE


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OBTAINED SYNBIOTIC BIODIPS


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OBTAINED SYNBIOTIC CHOCOLATES

SynbioticChocolates

ControlChocolates


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CONTROLPROBIOTIC

The developedKALAKAND


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Optimization results for Synbiotic Chocolates

The amount of stevia was optimized against the constant content of Cocoa Powder ,Milk Powder andTemperature (100ºC)


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Sl. No. Temperature (°C)

Amount of Cocoa powder in gm

Amount of Milk powder in gm

Amount of Stevia powder in gm

Remarks

1 100 4 2 0.0 Non grainy, bitter chocolate

2 100 4 2 0.1 Non grainy, bitter taste

3 100 4 2 0.2 Non grainy, bitter taste.

4 100 4 2 0.3 Non grainy sweet and bitter taste

5 100 4 2 0.35 Non grainy, sweet taste.

6 100 4 2 0.4 Grainy, bitter and sweet taste

7 100 4 2 0.5 Very grainy and bitter taste


Graph for Stevia optimization

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

2

4

6

8

10

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consumer acceptance

Consumer acceptance

Stevia content in grams

Consumer score

Optimized Ste-via content 0.35


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Bile acid tolerance test resultsTIME 0mins 45mins 90mins 135mins

Isolates from samples

O.D At 650 nm

Mango pickle 0.69 0.57 0.45 0.36

Sanna batter 0.45 0.56 0.75 0.58

Agathikeerai 0.21 0.30 0.45 0.61

Jowar koozh batter

0.22 0.42 0.62 0.72


Graph for bile tolerance test

0 20 40 60 80 100 120 140 1600

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

mango pickleSanna batteragathikeeraijowarkoozh

Time period (minutes)

OD at 630nm


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Curdling test results


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TVC Results


ColoniesObtained

60VIII SEM PROJECT PRESENTATION, DEPT OF BIOTECHNOLOGY, NHCE, 2013-14

Chocolates 8days 10^-13 Mayonnaise 8days 10^-13

Biodips 4days 10^-13 Kalakand 4days 10^-13

KALAKAND, Day 10

BIODIPS, Day 10

MAYONNAISE, Day 20

CHOCOLATE, Day 28

TVC, 10^-13

VIII SEM PROJECT PRESENTATION, DEPT OF BIOTECHNOLOGY, NHCE, 2013-14 61

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SAFETY AND EFFICACY TEST• EMB TEST- to check gram negative

lactose fermenting bacterial colonies (E.Coli)

• PDA TEST- to check the yeast and mould counts.


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MAYONNAISE, DAY 16

KALAKAND , DAY 8

EMB TEST


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BIODIPS, DAY 8

CHOCOLATE, DAY 28


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PDA Test


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SUMMARY & CONCLUSION

• Probiotic isolates were subjected to curdling tests, pH tests and bile acid tests in in-vitro conditions to check for their compatibility in intestinal microflora.

• Then these Probiotics were added to the products.• Addition of Stevia led to a certain color and texture

change, hence certain flavors were used to mask the color and taste of Stevia.

• There wasn’t any such change in taste of the products after adding probiotics.


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• Developed products carried highly desirable probiotic count (~ 10¹¹cfu per ml /g of the product).

• The probiotic isolates were processed to develop new dairy and non-dairy products with high acceptability scores.


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PATH FORWARD

• Molecular analysis - 16s RNA sequencing. • Marketing of developed probiotic foods.


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BIBLIOGRAPHY• Abdulla and jalaluddin, Anna olejnik and Anna Sip, 1998. Probiotics, prebiotics and

antioxidants as functional food. ActabiochimicalPolonica; V52(3). pp665-671.• Apostolou E, Pelto L, Kirjavainen PV.2001. Differences in the gut bacterial flora of

healthy and milk hypersensitive adults, as measured by fluorescence in-situ hybridization. FEMS Immunol Med Microbiol; V30.pp217-221.

• Chow and Wamer, Isenberg HD, Alperstein P, France K, Borenstein MT.1999. ingestion of yogurt containing Lactobacillus acidophilus as Prophylaxis for candidal vaginitis. Ann Intern Med. V116(5).pp 353-357.

• Corcoran,Stanton,C. Fitzgerald, F,C and Ross, R. P. 2005. Survival of Probiotic Lactobacilli in acidic environment is enhanced in the presence of metabolizable sugars environmental microbiology.V71.pp3060-3067.Ireland.

• Elina Tuomola, Ross Crittenden, Martin Playne, Erica Isolauri. February 2001. Quality asuurance criteria for Probiotic Bacteria. American Journal of Clinical Nutrition. V73.p393-398.

• Janofer U, Kavya G, Jnapti J, Dr Pratima Khandelwal. 2013. Probiotic Delivery through Foods for Healthy Lifestyle, eighth semester project report. Submitted to VTU.


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BIBLIOGRAPHY• Nehanarang.2011.An updated review on floating drug delivery system

(FDS).International Journal of Applied Pharmaceuticals.V3. Issue5.pp22-54.• Newmarker, Chris (18 December 2008). Federal regulators give OK for

Cargill's Truvia sweetener. Minneapolis / St. Paul Business Journal. Retrieved 18 December 2008.

• Pascal, Audet; Celine, Paguin; Christophe, Lacroix. 1991. Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in k-carrageenan of locust bean gum gel beads. Biotechnology techniques. Canada. V5, issue4. pp307-312.

• Pathan, Dilnawaz; Shakeel; Ziyaurrahman, A, P. 2011. Review on Probiotics. International research journal of pharmacy. India. V2. pp26-33.

• Pederson CS, Niketic G, Albury MN (1962). Fermentation of the Yugoslavian pickled cabbage. Applied microbiology 10 (1): 86–9.


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BIBLIOGRAPHY• Possemiers, S; Marzorati, M; Verstraete, W; Van, de, wiele. 2010. Bacteria and

chocolate. International journal of food microbiology. Belgium. V141. pp97-103. • Rosander A, Connolly E, Roos S (Oct 2008). Removal of antibiotic resistance gene-

carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938. Appl Environ Microbiol. 74 (19): 6032–40.

• Salminen,S,BouleyC,Boutron,R, 1998. Gastrointestinal physiology and function-targets for functional food development.Brazilian Journal Of Nutrition.V80.pp147-171.

• Satish, Kumar, R; Kanmani, P; Yuvaraj, N; Paari K, A; Pattukumar; Arul, V. 2012. Traditional Indian fermented foods: a rich source of lactic acid bacteria. International journal of food science and nutrients. India. V27. pp142-148.

• Shiby VK, Mishra HN (2013). Fermented milks and milk products as functional foods--a review. Critical reviews in food science and nutrition 53 (5): 482–96.

• Tanasupawat S, Thongsanit J, Okada S, Komagata K (2002). Lactic acid bacteria isolated from soy sauce mash in Thailand. Journal of General and Applied Microbiology (The Microbiology Research Foundation) 48 (4): 201–209.


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novel synbiotics for urban lifestyle

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