Lactic acid Producing Capacity

STANDARD OPERATING PROCEDURE

Requirements

1. Laboratory weighing balance-having good precision.
2. Homogenizer with jar or sonicator
3. Shaker
4. Incubator capable of being maintained at 37°C
5. pH meter
6. Autoclave

Diluent

Sterile saline solution 0.9% (9 g Sodium Chloride dissolved in 1000 mL of distilled water).

Reagents

Sodium hydroxide 0.05N NaOH
Bromo Thymol Blue neutral red indicator

Medium

Glucose Yeast Extract Acetate Broth.

Procedure

1. Weigh accurately 1g of Bacillus coagulans powder and transfer it into a sterile 250 mL volumetric flask containing about 100 mL of sterile saline solution, cover the volumetric flask with a stopper and sonicate for 10 to 15 minutes. Aseptically make up the volume to the mark with sterile saline solution. This makes TEST SOLUTION of 1:250 dilution.

STANDARD OPERATING PROCEDURE

• Transfer a small quantity (about 10 mL) of TEST SOLUTION (1:250 dilution) into a sterile dilution tube and allow standing the tube in a water bath at 70o to 75o C for 30 minutes as heat-shock.
• After heat-shock, cool the test solution immediately to 45°C and pipette out exactly 2.5 mL of the test solution in a sterile solution tube containing 10 mL of Glucose Yeast Extract Acetate Broth, previously sterilized and cooled to room temperature. Use duplicate tubes for this purpose.
• Incubate the tubes at 37°C for 48 hours and then titrate the Lactic acid so produced with 0.05N NaOH adding 2 drops of Bromo Thymol Blue Neutral Red indicator.
• Perform blank in the same way omitting sample addition.

Limits:   Not less than 10 mL of 0.05N NaOH is consumed.

Calculation:

Volume of NaOH consumed (Titration volume – Blank) x Exact Normality of NaOH    =      mL 0.05