Evaluation of viability of bacteria using in vitro gastro intestinal - PowerPoint PPT Presentation

Evaluation of viability of bacteria using in vitro gastro intestinal model and formulation of functional food with synbiotic microcapsules Dr. R. Jayabalan Food Microbiology and Bioprocess Laboratory Department of Life Science National Institute of Technology Rourkela 769 008 Odisha, India jayabalanr@nitrkl.ac.in

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

In vitro gastro intestinal model – one pot system – Poznan Univ. of Life Sciences The assembled in vitro gastro intestinal model - 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, pH 2.0) – pancreatin & bile under neutral pH (small intestine) – Pancreatin – mixture of amylase, trypsin, lipase, and ribonuclease.

Encapsulation Cross linking – displacement of sodium ion by calcium ion to form calcium alginate polymerization in alginate using calcium chloride  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 before and after digestion Number of bacterial cells present before and after digestion Log 10 number of cells Free Bacteria (Code / Name) 0 hour 2 hours 4 hours OC1 7.5 0.1 5.9 0.1 (78.7%) 5.7 0.2 (76.0%) OC2 7.8 0.1 6.1 0.2 (78.2%) 5.9 0.2 (75.6%) OC3 8.4 0.1 3.4 0.1 (40.4%) 3.3 0.1 (39.2%) OC4 8.6 0.2 3.5 0.2 (40.7%) 3.0 0.1 (34.8%) OC5 9.0 0.1 5.8 0.1 (64.4%) 5.7 0.1 (63.3%) OF3 9.2 0.1 0.0 (0%) 2.6 0.1 (28.2%) HM2 8.0 0.7 0.0 (0%) 1.9 0.1 (23.7%) L. acidophilus NCIM 2660 9.0 0.2 2.0 0.1 (22.2%) 2.2 0.1 (24.4%) L. bulgaricus NCIM 2056 8.6 0.2 0.0 (0%) 3.3 0.1 (38.3%) L. fermentum NCIM 2156 9.0 0.1 0.0 (0%) 1.2 0.2 (13.3%) L. plantarum NCIM 2083 9.1 0.1 0.0 (0%) 3.1 0.1 (34.0%) Values represent mean standard deviation; n = 4 (duplicates of two dilutions) Cells are counted from 1 mL of broth taken at 2 and 4 hours interval. Values in parenthesis represent the percentage of original cells remaining.

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 in microcapsules before and after digestion Log 10 number of cells from Log 10 number of cells released in to Encapsulated microcapsules broth Bacteria (Code / Name) 0 hour 4 hours 2 hours 4 hours OF3 8.5 0.2 1.9 0.2 (22.4%) 3.8 0.1 (44.7%) 3.5 0.1 (41.1%) HM2 8.9 0.1 2.9 0.1 (32.6%) 3.3 0.2 (37.0%) 3.1 0.1 (34.8%) L. acidophilus NCIM 2660 8.6 0.1 2.6 0.5 (30.2%) 3.4 0.1 (39.5%) 3.4 0.1 (40.0%) L. bulgaricus NCIM 2056 8.9 0.1 3.3 0.3 (37.0%) 3.2 0.1 (36.0%) 3.3 0.1 (37.0%) L. fermentum NCIM 2156 9.4 0.1 3.1 0.1 (33.0%) 3.0 0.1 (32.0) 2.3 0.1 (24.4%) L. plantarum NCIM 2083 8.0 0.1 0.0 (0%) 0.0 (0%) 0.0 (0%) Values represent mean standard deviation; n = 4 (duplicates of two dilutions) Cells are counted from 1 mL of broth / digested microcapsules. Values in parenthesis represent the percentage of original cells remaining.

Preparation of synbiotic microcapsules and its utilization for preparation of functional dry food product. Preparation of microcapsules by extrusion method Prepare 2.0% Sodium alginate solution and add Drop wise add the mixture using the syringe into lyophilised probiotic bacteria to it and mix well 0.075 mM cold CaCl2 solution with slow stirring. in a magnetic stirrer, fill the mixture of sodium Minute beads will be formed instantly. Filter it and alginate and probiotic bacteria in a 1 ml immerse them in cold 100mM CaCl2 solution for capacity 31 gauge syringes. hardening. Lyophilize the microscopic beads and store at 4 C Image A : Microcapsules prepared by Image B: Functional dry food product extrusion method, average size of prepared by mixing dry food and beads ranged from 0.5mm to 1mm. synbiotic microcapsules.

Post storage survivability study Post storage survivability study of bacteria in microcapsules Log 10 of number of cells (in microcapsules) Bacteria Weak 1 Weak 2 Weak 3 L. acidophilus NCIM 2660 2.08 + 0.2 1.92 + 0.1 (92.4%) 1.88 + 0.2 (90.4%) L. bulgaricus NCIM 2056 2.16 + 0.1 1.89 + 0.1 (87.4%) 1.79 + 0.3 (82.8%) L. fermentum NCIM 2156 2.28 + 0.3 1.81 + 0.2 (79.3%) 1.62 + 0.4 (71.0%) L. plantarum NCIM 2083 2.27 + 0.2 1.93 + 0.3 (85.3%) 1.81 + 0.2 (81.4%) OC4 5.73 + 0.2 5.54 + 0.1 (96.6%) 5.31 + 0.1 (92.5%) Cells are counted from 10 mg of microcapsules. Values represent mean standard deviation; n = 4 (duplicates of two dilutions) Values in parenthesis represent the percentage of original cells remaining.

Post storage survivability study of bacteria in synbiotic microcapsules Log 10 of number of cells (in synbiotic microcapsules incorporated dry food product) Bacteria Weak 1 Weak 2 Weak 3 L. acidophilus NCIM 2.52 + 0.2 2.21 + 0.2 (87.5%) 1.89 + 0.1 (72.4%) 2660 L. bulgaricus NCIM 2.51 + 0.1 2.43 + 0.1 (97.0%) 2.18 + 0.1 (87.0%) 2056 L. fermentum NCIM 2.56 + 0.3 2.13 + 0.1 (83.3%) 1.91 + 0.1 (74.5%) 2156 L. plantarum NCIM 2.64 + 0.2 2.56 + 0.2 (97.1%) 2.23 + 0.2 (84.6%) 2083 OC4 5.83 + 0.1 5.75 + 0.3 (98.5%) 5.59 + 0.1 (95.7%) 2:1 ratio of Pleurotus ostreatus extract-alginate mixture was used. Cells are counted from 10 mg of microcapsules. Values represent mean standard deviation; n = 4 (duplicates of two dilutions) Values in parenthesis represent the percentage of original cells remaining.