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Testing procedures and stability of cultures. L. sporogenes cells are protected from destruction by environmental factors by the naturally-present microencapsulation system, the spore coat. In probiotic therapy, for successful implantation in the gastrointestinal tract, a very large number of viable lactobacilli is essential. In the case of non-sporulated species, attempts are made to retain the viability of the vegetative cells during prolonged storage by freeze-drying. These lyophilized cells have to be stored under adequate refrigeration and are susceptible to environmental damage. L. sporogenes spores can be stored at room temperature without loss of viability. The methodology employed for testing the viability of these spores and conformance to specifications is outlined in the following pages: The tests involved include: Identification tests, viable lactobacillus spore count, estimation of the lactic acid producing capacity, determining loss on drying and ensuring the absence of contaminants. 1. Identification tests: a) Description: A white to grayish powder with characteristic odor and slightly sweet taste. b) Microscopic examination: Suspend a small quantity of L. sporogenes powder in about 5 ml sterile normal saline solution in a test tube and mix well. Prepare a wet mount on a glass slide by using one loopful of suspension as prepared above; cover with a coverslip. Examine the wet mount using a phase contrast microscope. The spores are seen as small, terminal , oval shaped refractile bodies within the dark vegetative cells. c) Qualitative test for lactic acid production: As described under "viable spore count" determination (see below), after incubation at 37°C for 48 hours, select the colonies with a platinum wire loop and transfer aseptically to a tube containing 20 ml of previously sterilized and cooled glucose yeast extract liquid medium. Incubate the tube at 37°C for 48 hours and then centrifuge at 2500-3000 rpm for 10 minutes. Transfer the clear supernatant liquid to a separatory funnel and extract by adding 5 ml. of dilute sulfuric acid (10%) and 50 ml of ether. Collect the ether layer, evaporate in a water bath carefully to dryness and dissolve the residue in 5 ml of water. Add the solution drop-wise to Uffelman’s reagent, prepared by adding two drops of 1N ferric chloride to 10 ml of 1% phenol solution. The color of the solution turns from bluish violet to yellow, indicating the presence of lactic acid. 2. Viable lactobacillus spore count: The number of viable spores in a sample is determined by the following procedure: (1) Dilution and heat-treatment Weigh out 1 gm L. sporogenes powder sample into a surface-sterilized homogenizing container (jar), add 200 ml of sterile physiological saline and homogenize at about 12000 - 15000 rpm for 5 minutes. Transfer l ml of the homogenized suspension into a 9.0 ml sterile physiological saline in a screw capped tube (25mm by 150mm size) and mix thoroughly. Repeat this serial dilution until a dilution of 10-6 dilutions are obtained. This is called the dilution factor. Allow the final tube to stand in a water bath at 70° C for 30 minutes and then cool immediately to about 45° C. (2) Plating Liquify GYE (Glucose Yeast Extract) agar medium and cool to 45°C in a water bath. Set out sterile petri dishes, five per sample to be tested. Add l ml from the heat treated final dilution tube into each petri dish and then pour 15 ml of the molten medium into each of the petri dishes and mix thoroughly. When solidified, incubate the plates in an inverted position at 40°C for 48 hours. (3) Counting The plates showing 30 - 300 colonies are ideal for counting. Select and count up to six plates and average the count per plate. Calculate the number of viable cells per gram of sample by multiplying the average number of colonies counted per plate by the reciprocal of the dilution factor. For example, Average number of colonies per plate =30 Final dilution factor = 200x10, The viable spore count is 30x200x10 = 6000 million viable spores per gram. Preparation of diluent: Physiological saline is prepared by dissolving 8.5 gm sodium chloride and 15 mg sodium lauryl sulfate in 1000 ml of distilled water. The solution is sterilized with steam at 1.2 kg/cm2 pressure at 120°C for 15 minutes and then cooled. Preparation of GYE agar medium: Glucose Yeast Extract agar medium has the following composition:
Adjust pH of the medium to 6.3 using 1N HCl / 1N NaOH solution using pH meter and sterilize medium with steam at 1.2 kg/cm2 pressure at 120° C for 15 minutes. Preparation of trace mineral solution:
Weigh accurately the required quantity of salts and add a small quantity of distilled water and dissolve well. Raise the volume to 50ml in a volumetric flask. The solution will attain a pink color. This solution can be kept in a refrigerator for 2 months. 3. Lactic acid producing capacity To an accurately weighed 1.0 gm of L.sporogenes powder, add exactly 100 ml of sterile normal saline solution and homogenize at about 1200-1500 rpm. for 7-10 minutes. This makes a test solution of 1 : 100 dilution. Transfer a small quantity of the test solution (about 10 ml) into a sterile test tube (25 mm x 150 mm) and allow to stand in a water bath at 75°C for 30 minutes. Cool immediately to 45-50°C. Pipette exactly 1.0 ml of the solution in 10 ml of GYE liquid medium previously sterilized and cooled to room temperature. Incubate the tubes at 37° C for 48 hours. Duplicate tubes are used for this purpose. After incubation, titrate the lactic acid produced with 0.05N sodium hydroxide using bromothymol blue neutral red indicator. To meet specifications, not less than 10 ml of 0.05 N sodium hydroxide must be consumed in the titration. 4. Loss on drying: A one gram sample of L. sporogenes powder, when dried at 100° for 3 hours should lose not more than 8 % of its weight. 5. Detection of E .coli and other coliform bacteria in LactosporeÒ powder Procedure Weigh l.0 gm dry L.sporogenes powder and mix with 10 ml of sterile water in a 20 ml tube (25mm x 150 mm size), using a blender. Add l ml of this suspension into a petri dish and add about 10 ml of PC agar medium, mix well and allow to set. Then add 10 ml of PC agar medium to cover the earlier layer, spread evenly and allow to set and then incubate at 35°-37° C for 24 hours. Run the assay in triplicate. Results If there is E.coli contamination or if there are other coliform bacteria, they can be detected due to change in color of the medium. Proteus and bacteria in intestinal flora produce pink colonies. E.coli produces dark red colonies. Preparation of P.C. medium: Prepare P.C. medium of following composition:
Add peptone, NaCI, lactose, ferric ammonium citrate, KH2PO4 and neutral red to about 800 ml of water and dissolve them well. Separately dissolve Na-desoxycholate in an adequate amount of water and dissolve well. Combine both the solutions and raise the volume up to 1000 ml. Adjust the pH to 7.3± 0.l and add agar. Dissolve by heating with constant stirring. Then pour 20ml of the medium into each test tube and sterilize at 100°C for 30 minutes, cool and store away from light (to avoid photo - reaction). 6. Accelerated stability studies at elevated temperature To assess the viability of spores over prolonged storage periods, accelerated stability tests have been performed on samples of LactosporeÒ. a) Sample used Lactobacillus sporogenes (6000 million viable spores per gram) Method: Accelerated stability studies of L. sporogenes powder were carried out by incubating the powder in a brown bottle, sealed with a polyethylene plug cap, at 45°C + 1°C for 90 days (considered to be equivalent to two years storage at room temperature). L. sporogenes powder was used for this purpose (25 grams). The powder contained 7260 million viable spores per gram (claimed as not less than 6000 million viable spores per gram). Glucose yeast extract agar medium was used, according to the method specified for L. sporogenes count by M/s Metchnikoff Biosystems Pvt. Ltd., Medchal, A.P., India. The details are described above. The results obtained with a sample of LactosporeÒ are indicated in Figure 6.1. It is evident that the spores remain viable and conform to specifications even after a storage period equivalent to two years at room temperature. Viability of Lactobacillus sporogenes (by viable plate counts) in LACTOSPOREÒ powder at 45°C + 1°C.
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