QUANTIFICATION OF L(+) LACTIC ACID AND D(-) LACTIC ACID
To quantify the amount of L(+) and D(-) Lactic acid produced by LactoSpore®.
The production of L (+) and D (-) Lactic acid produced by LactoSpore® in the broth is quantified using Megazyme assay procedures K-DLATE 10/04.
Megazyme assay kit (K-DLATE 10/04), Micropipettes, Conical flasks, Pipettes, Test tubes, UV spectrophotometer (UV-1601PC), pH meters, 2M Sodium hydroxide, Sterile GYEL - Glucose Yeast Extract Acetate Broth (HiMedia-M964), What man No. 41 filter paper.
1. 1 gm of 15 Billion Units/g of LactoSpore® is added to 100 ml of sterile GYEL broth (HiMedia-M964). This is incubated at 37° C in a shaker at 150 rpm for 48 hrs.
2. A sterile GYEL broth without LactoSpore® is kept as a blank and incubated under the same conditions, simultaneously.
3. After incubation period, 1.0 ml of the broth is diluted to 100 ml using D.M water. This sample solution is used for the determination of L(+) Lactic acid. This dilution is based to obtain a lactic acid concentration between 0.5 to 30 microgram/ml).
4. The Megazyme assay protocol is then followed further. In the protocol, as mentioned in the point#3, (General consideration), the sample pH is adjusted to a value of 10.0 using 2M NaOH solution. Similarly the diluted blank solution pH is also adjusted to 10.0.
5. For D (-) Lactic acid determination, the sample is diluted 10 folds (i.e. 10 ml of the sample is diluted to a total volume of 100 ml using D.M. Water). The procedure followed above for L(+) lactic acid is then followed for D(-) lactic acid estimation.
6. The content of specific isomer is then calculated based on the Calculation given in the assay protocol.
7. The value obtained is in terms of g/Lit. This is then converted to total lactic acid content & the (%) of the individual isomers are derived accordingly.
LactoSpore® produces predominantly 99.82% of L (+) Lactic acid; 0.18% of D (-) Lactic acid in the broth.
D-Lactic acid (D-Lactate) and L-Lactic acid (L-Lactate) Assay procedures,