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

• Dr. R. Jayabalan

Evaluation of viability of bacteria using in vitro gastro intestinal model and formulation of functional food with synbiotic microcapsules

Food Microbiology and Bioprocess Laboratory Department of Life Science National Institute of Technology Rourkela 769 008 Odisha, India jayabalanr@nitrkl.ac.in

Human an or animal l

• rigi

gin Survive ive GI tract Benef eficial cial to the host Viable le during ng storage rage condition ition

Expected characteristics and safety criteria of probiotics

Stress resistance  stomach (pH 2.0 and digestive enzymes)  small intestine (bile and pancreatic enzymes)

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

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 In vitro gastro intestinal model – one pot system – Poznan Univ. of Life Sciences

• 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. Importance of in vitro gastro intestinal model

Encapsulation

polymerization in alginate using calcium chloride

Cross linking –displacement of sodium ion by calcium ion to form calcium alginate

• 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

• 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

Microorganisms taken for study

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

• n 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

Free Bacteria (Code / Name) Log 10 number of cells 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.

Number of bacterial cells present before and after digestion

Encapsulation protocol

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

• 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 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.

Encapsulated Bacteria (Code / Name) Log 10 number of cells from microcapsules Log 10 number of cells released in to broth 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%)

Number of bacterial cells in microcapsules before and after digestion

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.

Image A : Microcapsules prepared by extrusion method, average size of beads ranged from 0.5mm to 1mm. Image B: Functional dry food product prepared by mixing dry food and synbiotic microcapsules.

Preparation of synbiotic microcapsules and its utilization for preparation of functional dry food product.

Prepare 2.0% Sodium alginate solution and add lyophilised probiotic bacteria to it and mix well in a magnetic stirrer, fill the mixture of sodium alginate and probiotic bacteria in a 1 ml capacity 31 gauge syringes. Drop wise add the mixture using the syringe into 0.075 mM cold CaCl2 solution with slow stirring. Minute beads will be formed instantly. Filter it and immerse them in cold 100mM CaCl2 solution for hardening. Lyophilize the microscopic beads and store at 4 C

Preparation of microcapsules by extrusion method

Post storage survivability study

Log10 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%)

Post storage survivability study of bacteria in microcapsules

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.

Log10 of number of cells (in synbiotic microcapsules incorporated dry food product) Bacteria Weak 1 Weak 2 Weak 3

• L. acidophilus NCIM

2660 2.52 + 0.2 2.21 + 0.2 (87.5%) 1.89 + 0.1 (72.4%)

• L. bulgaricus NCIM

2056 2.51 + 0.1 2.43 + 0.1 (97.0%) 2.18 + 0.1 (87.0%)

• L. fermentum NCIM

2156 2.56 + 0.3 2.13 + 0.1 (83.3%) 1.91 + 0.1 (74.5%)

• L. plantarum NCIM

2083 2.64 + 0.2 2.56 + 0.2 (97.1%) 2.23 + 0.2 (84.6%) 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.

Post storage survivability study of bacteria in synbiotic microcapsules

Conclusion

OC1 and OC2 – highly resistant OC5 – moderately resistant OC3, OC4, L. acidhophilus NCIM 2660 – slightly 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 Encapsulation protecting the bacteria against stress conditions Release of bacteria from capsules are observed Inclusion of mushroom extract in synbiotic microcapsules did not affect the survivability of bacteria OC1, OC2 and OC5 – should be studied for other probiotic characters Inclusion of mushroom extract did not affect the viability of bacteria in microcapsules

Acknowledgement

DST grant No. SERC/LS-156/2012 DBT grant No. 102/IFD/SAN/2770/2013-2014 TEQIP II Program, NIT Rourkela Poznan University of Life Sciences, Poland Centre for Nutraceuticals and Pharmaceuticals, Myongji University, Republic of Korea NIT Rourkela

Number of bacterial cells after digestion (CFU/mL)

Bacteria 0 hour 2 hour 4 hour OC1 3.10 x 107 ± 81.00 77.35 x 104 ± 95.18 53.0 x 104 ± 261.60 OC2 6.67 X 107 ± 140.00 140 x 104 ± 49.34 82.0 x 104 ± 26.20 OC3 28 x 107 ± 9.00 244.2 x 101 ± 37.40 225.5 x 101 ± 54.10 OC4 38.3 x 107 ± 139.50 384 x 101 ± 178.72 97.7 x 101 ± 18.70 OC5 100.25 x 107 ± 15.06 69.55 x 104 ± 48.23 53.55 x 104 ± 64.54 OF3 165 X 107 44.75 x 101 ± 3.70 HM2 24 x 107 ± 248.00 8 x 101 ± 1.63

• L. acidophilus NCIM

2660 100 x 107 ± 37.51 11 x 101 ± 2.4 16.8 x 101 ± 4.50

• L. bulgaricus NCIM

2056 44 x 107 ± 18.20 184.8 x 101 ± 9.20

• L. fermentum NCIM

2156 107 x 107 ± 23.30 1.5 x 101 ± 0.57

• L. plantarum NCIM

2083 122 x 107 ± 22.30 114.5 x 101 ± 4.50

Number of bacterial cells after digestion (CFU/mL)

Encapsulated Bacteria 0 hour 2 hour 4 hour OF3 37.0 x 107 73.50 x 102 31.00 x 102 HM2 78.2 x 107 19.50 x 102 14.50 x 102

• L. acidophilus NCIM

2660 38.6 x 107 24.20 x 102 23.95 x 102

• L. bulgaricus NCIM

2056 91.5 X 107 11.92 x 102 18.52 x 102

• L. fermentum NCIM

2156 243 X 107 9.32 x 102 14.50 x 102

• L. plantarum NCIM

2083 20.5 x 107

2:1 ratio of Pleurotus ostreatus extract- alginate mixture was used.

Table XX: Post storage survivability of bacteria in synbiotic microcapsules

Preparation of synbiotic microcapsules and post storage study

CFU/10 mg of synbiotic microcapsules Bacteria Week 1 Week 2 Week 3 Without extract With extract Without extract With extract Without extract With extract Lactobacillus acidophilus NCIM 2660

• 1. 22 x 102

5.22 3.37 x102 9.45 85 2.55 (30.3%) 1.63 x 102 3.33 (51.63%) 77 2.95 (36.8%) 68 5.97 (80%) Lactobacillus fermentum NCIM 2156 1.92 x 102 1.45 3.69 x 102 6.23 65 10.23 (61.4%) 1.38 x 102 12.45 (62.6%) 42 3.28 (78.1%) 82 4.49 (77.7%) Lactobacillus plantarum NCIM 2083 1.87 x 102 7.13 4.39 x 102 3.53 87 3.92 (53.4%) 3.68 x 102 29.42 (16.2%) 71 12.42 (62%) 1.72 x 102 9.98 (60.8%) Lactobacillus bulgaricus NCIM 2056 1.46 x 102 8.93 3.24 x 102 25.84 78 11.74 (46.5%) 2.73x 102 29.03 (15.7%) 62 15.83 (57.5%) 1.53 x 102 7.45 (52.8%)

Data represent mean ± standard deviation, n=3. Values in parenthesis represent percent reduction of bacterial cell numbers compared to week 1.

Viability count after three weeks storage (CFU/mg of synbiotic food)

Bacteria 1 WEEK 2 WEEK 3 WEEK

WE E WE E WE E

• L. acidophilus NCIM 2660
• 1. 22 x 102

5.22 3.37 x102 9.45 85 2.55 (30.3%) 1.63 x 102 3.33 (51.63%) 77 2.95 (36.8%) 68 5.97 (80%)

• L. bulgaricus NCIM 2056

1.46 x 102 8.93 3.24 x 102 25.84 78 11.74 (46.5%) 2.73x 102 29.03 (15.7%) 62 15.83 (57.5%) 1.53 x 102 7.45 (52.8%)

• L. fermentum NCIM 2156

1.92 x 102 1.45 3.69 x 102 6.23 65 10.23 (61.4%) 1.38 x 102 12.45 (62.6%) 42 3.28 (78.1%) 82 4.49 (77.7%)

• L. plantarum NCIM 2083

1.87 x 102 7.13 4.39 x 102 3.53 87 3.92 (53.4%) 3.68 x 102 29.42 (16.2%) 71 12.42 (62%) 1.72 x 102 9.98 (60.8%)

OC 4 (Isolated from Curd)

5.48 x 105 0.180 6.88 x 105 0.460 3.5 x 105 0.25 (39%) 5.63 x 105 0.251 (19%) 2.06 x 105 0.20 (62%) 3.9 x 105 0.450 (44%)

Data represent mean ± standard deviation, n=3. Values in parenthesis represent percent reduction of bacterial cell numbers compared to week 1

The microcapsules of bacteria with (E) and without mushroom extract (WE) was prepared and subjected to storage for 3 weeks, the encapsulated bacteria with extracts showed more viability.

Gastrointestinal models

• Solubility, release and availability for absorption of

pharmaceuticals within GI tract

• Cost-effective alternative for human and animal

trials, free from ethical constraints

• Testing toxic compounds, pathogens – without

harming animals

• Live micro-organisms which when administered in adequate amounts (≥ 106

CFU/ ml) confer health benefits on host (FAO of UN).

• Strain specific, population specific

Probiotics

Benefits

Regulate balance of gut microflora Reduce harmful bacteria Relieve diarrhea & constipation Strengthen immune system Increase availability

• f vitamins

& nutrients Alleviate lactose intolerance Strains Effective dose in CFU / day

Lactobacillus casei shirota 6.5 X 109

• L. rhamnosus

GG 109 X 1010

• L. plantarum

299 v 5 X 108

• L. acidophilus

NCFB 1748 3 X 1011

• L. reuteri

1 X 108-1011

• L. rhamnosus

DSM 6594 16 X 109

Probiotics - Emerge as Alternative Medicine

European Union, South Korea, China – banned antibiotics as growth promoters

Probiotic organisms currently used

Others

• Streptococcus thermophilus
• Lactococcus lactis ssp. Lactis
• Lactococcus lactis s ssp. Cremoris
• Enterococcus faecium
• Leuconostoc mesenteroides
• Propionibacterium freudenreichii
• Pediococcus acidilactici
• Saccharomyces boulardii

STRAINS

SOURCES

Lactobacillus acidophilus NCFM Danisco (Madison WI) Bifidobacterium infantis 35264 Procter & Gamble (Mason OH) Lactobacillus fermentum VR1003 (PCC) Probiomics, Eveleigh, Australia Lactobacillus rhamnosus R0011, L. acidophilus R0052 Institute Rosell (Montreal, Canada)

• L. casei Shirota, B. breve strain Yakult

Yakult (Tokyo, Japan)

• L. casei DN114001

Danone (Paris, France)

• L. reuteri RC-14
• Chr. Hansens (Milwaukee WI)
• L. rhamnosus GR1

Urex Biotech (London and Ontario)

• L. jhonsonii Lj-1

Nestle (Lausanne, Switzerland)

• L. plantarum

Probi AB (Lund, Sweden)

• L. salivarius UCC118

University College (Cork, Ireland)

• B. longum BB536

Morinaga Milk Industry Co., Ltd., (Zama City, Japan)

• B. Lactis HN019 (DR10)

Danisco (Madison WI)

• L. Paracasei F19

Medipharm (Des Moines, Iowa)