Utilization of in vitro gastro intestinal system to check the - - PowerPoint PPT Presentation
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
Human an or animal l
gin Survive ive GI tract ct Benef eficial cial to the host Viable le during ng storage rage condition ition
Stress resistance stomach (pH 2.0 and digestive enzymes) small intestine (bile and pancreatic enzymes)
Regulate balance of gut microflora Reduce harmful bacteria Relieve diarrhea & constipatio n Strengthen immune system Increase availability
& nutrients Alleviate lactose intolerance
Lactobacillus species
Bifidobacterium species
Others
polymerization in alginate using calcium chloride
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
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.
Bacteria 0 hour 2 hour 4 hour OC1 310 X 105 773.5 x 103 530 x 103 OC2 667 X 105 140 x 104 82 x 104 OC3 28 x 107 244.2 x 101 223 x 101 OC4 383 X 106 434 x 101 97.7 x 101 OC5 100 x 107 695.5 x 103 535.5 x 103 OF3 165 X 107 445 HM2 2.4 x 108 80
2660 100 x 107 110 165
2056 44 x 107 197
2156 107 x 107
2083 122 x 107 1130
30 ml of starch (4%) in CalCl2 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
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 CaCl2 solution using metal spoon (sterilized by alcohol and UV). Capsules in CaCl2 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.
2660 386 x 106 242 x 101 239.5 x 101
2056 91.5 X 107 119.2 x 101 185.2 x 101
2156 243 X 107 932 145 x 101
2083 205 x 106