L Lactic acid

Question 1

Describe lactic acid production in cheese making

Answer 1

During cheese making, lactose present in milk is converted into lactic acid by fermentation of lactic acid bacteria.
Lactic acid content range from 0.2% (camembert) to 1.3% (cheddar).
Most of the lactic acid is present in L(+) isomer while as much as 50% may be present in the D(-) optimal isomer.

Question 2

What did we do in this practical?

Answer 2

We used different types of cheese and add 80ml of water to the cheese and heat at 60 degree Celsius for 15 minutes .
We cooled the solution down and fill up to 100ml.
To obtain fat separation, keep the flask in refrigerator for 15 minutes and filter into 50ml tube. Filtrate is kept.
Solution 1,2 and suspension solution is added to the filtrate.
Absorbance is read at 340nm of blank and sample after 3 minutes.
Solution 4 is added after 3 minutes and 2nd absorbance is read after 10 minutes.

Question 3

Explain the principle of the assay

Answer 3

There is 2 main enzymes reactions in the assay.
The 1st reaction is catalyzed by L-lactate dehydrogenase . L lactate + NAD+ -> Pyruvate + NADH + H+ via L-LDH..
The reaction is reversible and the equilibrium favors the L=lactic acid and NAD+.
The 2nd reaction is to trap the pyruvate product. This is achieved by D-GPT D-glutamate-pyruvate transaminase in the presence of excess D-glutamate.
Pyruvate + D-glutamate -> D-alanine + 2-oxoglutarate via D-GPT.
The amount of NADH formed is stoichiometric with the amount of L-lactic acid. NADH is measured by the increase in absorbance at 340nm.

Question 4

What are the solution in the assay?

Answer 4

Solution 1 is glycylglycine buffer, D-glutamate and Sodium azide as preservative
Solution 2 is NAD+
Solution 3 is GPT or D-Glutamate-pyruvate transaminase suspension
Solution 4 is L-Lactate dehydrogenase suspension
Solution 5 is L-Lactic acid standard solution with sodium azide as preservative.

Question 5

Determine the absorbance difference for both the blank and the sample

Answer 5

∆A = (A2 - A1)sample - (A2 - A1)blank

Question 6

Calculate the mass of lactic acid in 1g of sample of cheese

Answer 6

(Cuvette volume x Molecular weight of lactic acid (90.1))/ (extinction coefficient of NADH x light path(1cm) x Sample volume x 1000)
Then multiply by absorbance difference
Final volume refers to cuvette volume
Sample volume refers to sample volume added to cuvette