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Utilization of in vitro gastro intestinal system to check the survivability of free and encapsulated bacteria under gastro intestinal stress conditions Dr. R. Jayabalan Research Training: Dr. Kamila Goderska Department of Fermentation and Biosynthesis Faculty of Food Science and Nutrition Poznan University of Life Sciences Poznan, Poland

Objective  To check the survivability of free and encapsulated bacteria under gastro intestinal stress conditions by utilizing in vitro gastro intestinal model. Expected characteristics and safety criteria of probiotics Human an or Viable le during ng Survive ive GI Benef eficial cial animal l storage rage tract ct to the host origi gin condition ition Stress resistance  stomach (pH 2.0 and digestive enzymes)  small intestine (bile and pancreatic enzymes)

Probiotics • Live micro-organisms which when administered in adequate amounts (≥ 10 6 CFU/ ml) confer health benefits on host (FAO of UN). • Strain specific, population specific Regulate balance of gut microflora Alleviate Reduce lactose harmful intolerance bacteria Benefits Increase Relieve availability diarrhea & of vitamins constipatio & nutrients n Strengthen immune system

Probiotic organisms currently used Lactobacillus species Others • • L. acidophilus Streptococcus thermophilus • L. plantarum • • Lactococcus lactis ssp. Lactis L. casei ssp. rhamnosus • L. fermentum • Lactococcus lactis s ssp. Cremoris • L. delbreuckii ssp. bulgaricus • L. helveticus • Enterococcus faecium • Leuconostoc mesenteroides Bifidobacterium species • Propionibacterium freudenreichii • B. adolescentis • • B. bifidum Pediococcus acidilactici • B. longum • • Saccharomyces boulardii B. infantis • B. breve

In vitro gastro intestinal model  The assembled in vitro gastro intestinal model consists of 3 compartments (i) Automatic pH controller with acid and alkali dispenser (ii) Digestion vessel and (iii) Water bath with magnetic stirrer

Importance of in vitro gastro intestinal model  To study the behavior of microorganisms under the stress conditions of stomach and small intestine  The test organism in a relevant media undergo simulated digestion by: – pepsin under acidic conditions (stomach) – pancreatin & bile under neutral pH (small intestine) – Pancreatin – mixture of amylase, trypsin, lipase, ribonuclease and protease.

Encapsulation Cross linking – displacement of sodium ion by calcium ion to form calcium alginate polymerization in alginate using calcium chloride

Importance of Encapsulation  High resistance to stress in GIT  High fermentation rate  High substrate utilization  Less inhibition by product  Longer survivability in storage  Maintain high cell density per volume  Reduces the possibility of contamination  Cost effective

Microorganisms taken for study • Isolated organisms (from Omfed curd, Rourkela and home made curds collected from Rourkela)  OC1, OC2, OC3, OC4, OC5, OF3, HM2 • Purchased from NCIM Pune:  Lactobacillus acidophilus NCIM 2660  L. bulgaricus NCIM 2056  L. fermentum NCIM 2156  L. plantarum NCIM 2083

Methodology to study the free cells ability to tolerate stress conditions 10 ml saline added to cell pellet and mixed thoroughly. 1 ml of this solution serially diluted in saline up to 10 -8 & 1 ml of dilution plated on MRS agar by pour plate method. 9 ml of cell pellet solution added to 200 ml MRS broth in digestion vessel & mixed thoroughly. Magnetic bead added. The cap with sampling tube & thermometer assembled. The digestion unit kept in water bath. Sterile pH probe inserted in the cap of digestion vessel. Acid & alkali tubes filled with respective solutions using syringe and attached to the vessel using needles. The automatic pH controller switched on & adjusted to pH 2.0. Pepsin solution (2 ml) added to MRS broth at pH 2.0 via the sampling tube. 2 ml of sample taken after 2 hours in sterilized tube & pH adjusted to 6.0. 1 ml of sample plated on MRS agar plate & remaining 1 ml diluted up to 10 -8 using saline solution & 1 ml of the dilutions plated on MRS agar by pour plate method. The plates incubated at 37°C for 48 hours & colonies counted. Bile solution (10 ml) added to MRS broth via sampling tube at pH 6.0 & pH was adjusted to 7.4. 2 ml of sample taken after 2 hours in sterilized tube & the digester was switched off. 1 ml of sample plated on MRS agar plate & remaining 1 ml diluted up to 10 -8 using saline solution & 1 ml of the dilutions plated on MRS agar by pour plate method. The plates incubated at 37°C for 48 hours & colonies counted.

Number of bacterial cells after digestion (CFU/mL) Bacteria 0 hour 2 hour 4 hour 773.5 x 10 3 310 X 10 5 530 x 10 3 OC1 667 X 10 5 140 x 10 4 82 x 10 4 OC2 28 x 10 7 244.2 x 10 1 223 x 10 1 OC3 383 X 10 6 434 x 10 1 97.7 x 10 1 OC4 100 x 10 7 695.5 x 10 3 535.5 x 10 3 OC5 165 X 10 7 OF3 0 445 2.4 x 10 8 HM2 0 80 100 x 10 7 L. acidophilus NCIM 110 165 2660 44 x 10 7 L. bulgaricus NCIM 0 197 2056 L. fermentum NCIM 107 x 10 7 0 0 2156 122 x 10 7 L. plantarum NCIM 0 1130 2083

Encapsulation protocol 30 ml of starch (4%) in CalCl 2 solution (0.61%) added to cell pellet & mixed thoroughly. 1 ml solution taken for serial dilution (in saline 0.85% ) up to 10 -8 . 1 ml of dilution plated on MRS agar by pour plate method. Cells counted after incubation at 37°C for 48 hours. Cell pellet solution taken in syringe with needle & added drop wise to sodium alginate solution (0.6%) while mixing in magnetic stirrer. Formed capsules filtered using stainless steel sieve (sterilized by UV in laminar chamber) & then transferred to CaCl 2 solution using metal spoon (sterilized by alcohol and UV). Capsules in CaCl 2 solution (1.22%) mixed using magnetic stirrer for 15 minutes & then filtered using stainless steel sieve. Capsules were transferred to falcon tubes using metal spoon and stored in refrigerator.

Number of bacterial cells after digestion (CFU/mL) Encapsulated 0 hour 2 hour 4 hour Bacteria 37 x 10 7 73.5 x 10 2 31 x 10 2 OF3 78.2 x 10 7 19.5 x 10 2 14.5 x 10 2 HM2 386 x 10 6 242 x 10 1 239.5 x 10 1 L. acidophilus NCIM 2660 91.5 X 10 7 119.2 x 10 1 185.2 x 10 1 L. bulgaricus NCIM 2056 L. fermentum NCIM 243 X 10 7 932 145 x 10 1 2156 L. plantarum NCIM 205 x 10 6 0 0 2083

Conclusion OC2 – highly resistant OC1 and OC5 – moderately resistant OC3, OC4, L. acidhophilus NCIM 2660 – lower resistant OF3, HM2, L. bulgaricus NCIM 2056, L. fermentum NCIM 2156, and L. plantarum NCIM 2083 – Not tolerating the stress conditions Encapsulated OF3, HM2, L. bulgaricus NCIM 2056 and L. fermentum NCIM 2156 – moderately resistant Encapsulated L. plantarum NCIM 2083 – not tolerant to stress conditions – should be repeated OC2, OC1 and OC5 – should be studied for other probiotic characters