Testing procedures and stability of cultures. B. coagulans 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. B. coagulans 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 Bacilllus coagulans spore count, estimation of the lactic acid producing capacity, determining loss on drying and ensuring the absence of contaminants. 1. Identification tests: a) Description: White to off white powder with characteristic odour. b) Microscopic examination: Suspend a small quantity of Bacillus coagulans 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 B. coagulans 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 B. coagulans powder sample into a presterilized volumetric flask containing 100ml of physiological saline, mix thoroughly by keeping volumetric flask in sonicator for 10min. Make up the volume to 250ml with sterilized physiological saline. Transfer l ml of this suspension into a 9.0 ml sterile physiological saline in an autoclaved Test 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 75° 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 37°C for 48 hours. (3) Counting The plates showing 30 - 300 colonies are ideal for counting. Select and count up to five 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 = 250x106, The viable spore count is 30x250x106 = 6000 million viable spores per gram. Preparation of diluent: Physiological saline is prepared by dissolving 9 gm sodium chloride in 1000 ml of distilled water. The solution is sterilized with steam at 1.2 Kg/cm2 pressure at 121°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 6.0 - 6.2 using 1N HCL / 1N NaOH solution using pH meter and sterilize the medium with steam at 1.2 Kg/cm2 pressure at 121° C for 15 minutes. 3. Lactic acid producing capacity To an accurately weighed 1.0 gm of B. coagulans 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 B. coagulans 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 Test for E.coli: Enrichment: Primary test : (Acid and gas test) Secondary test: (Indole test) If a red colour is noticed in the reagent layer, indole is present. The presence of acid, gas and indole in the secondary test (Indole test) confirms the presence of E.coli. 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 LactoSpore®. a) Sample used Bacillus coagulans powder (6000 million viable spores per gram) Method: Accelerated stability studies of B. coagulans 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). B. coagulans 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 LactoSpore® 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 Bacillus coagulans (by viable plate counts) in LactoSpore® powder at 45°C + 1°C.
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| * The taxonomical classification was revised in 1939 in the seventh edition of the Bergey’s Manual of Determinative Bacteriology to B. coagulans, although some researchers continued to use the original name. | |||||||||||||||||||||||||||
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