Eddy Tan, Associate Senior Scientist
R&D APAC Lab (Singapore)
The food industry worldwide is required to demonstrate food authenticity and the quality control of milk is one example. Unethical producers can add nitrogen-rich compounds to correct the milk protein content. Adulteration of dairy milk with cheese whey can reduce its production cost significantly (4 to 5 times). Cheese whey does not change the sensory perception nor adversely affects health1. Another common milk adulterant is sweet whey2.
Glycomacropeptide (GMP) is a casein peptide found in dairy products like cheese whey. GMP is released in whey by rennet during cheese making. GMP is rich in sialic acid and this compound can therefore be used as an indicator to determine the adulteration of milk by whey.
The detection of adulterated milk is based on the detection of sialic acid bound in whey. This determination was carried out in 2 stages:
GMP is first precipitated with trichloroacetic acid and phosphotungstic acid. Sialic acid is released from GMP and derivatized with the acidic ninhydrin method3 with the spectrophotometric measurement at 470 nm.
Figure 1. Structure of a sialic acid (N-acetylneuraminic acid).
The standard addition method was chosen as milk can be a highly variable matrix – dependent on the source origin and processing employed. In addition, milk can be classified by different amounts of fat (full fat, skim milk, half and half), type of heat treatment (pasteurization, extended shelf life treatment, ESL, ultra-high temperature, UHT), and also the additives used (fortified with vitamins, etc.).
Cheese whey was not available locally so bovine whey was used instead.
Both the Spectroquant® Prove 100 and Prove 600 spectrophotometers were used for measurements.
Milk Samples
Three brands of dairy milk that are locally popular were selected:
**All reagents need to be freshly prepared on the day of use
Figure 2. External calibration curve for-Sialic acid.
From the plot, the sialic acid content in 50.18 mg bovine whey is calculated using the equation:
y = 6.8441x + 0.0126
x = (0.337-0.0126)/6.8441 = 0.0473 mg sialic acid
This represents a sialic acid concentration in bovine whey of 0.00094 mg/mg.
“Brand 1” Milk by Spectroquant® Prove 600
The amount of sialic acid in bovine whey as determined earlier is 0.00094 mg per mg bovine whey
Figure 3.Standard calibration curve- Bovine whey in Brand 1 milk
From the standard addition plot, we have the equation
y = 6.1653x + 0.3109
We set y = 0 and solve for x.
x= |-0.3109/6.1653|
x = 0.05043 mg of sialic acid and this is from 8 mL of milk.
This represents a sialic acid concentration in the milk of 6.30 mg/L.
The correlation of milk adulteration to its sialic acid content3 is classified in the following categories:*
From our results, all three milk brands are in the “Suspected” category.
*It is however important to note that the sialic acid content is affected by many parameters – one being the breed of dairy cows.2 Therefore, the defined categories above should be taken as guidance only.
The procedure described here is a modified version of the method published by Fukuda SP et.al. 20043 and allows the determination of sialic acid in bovine milk. It offers a practical alternative to screen for Glycomacropeptide (GMP) content.
See more applications for photometry and reflectrometry at SigmaAldrich.com/wfa-applications
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