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HomePhotometry & ReflectometryDetermination of Sialic Acid in Milk

Determination of Sialic Acid in Milk as a Marker for Adulteration with Whey

Eddy Tan, Associate Senior Scientist
R&D APAC Lab (Singapore)

INTRODUCTION

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:

  1. Quantify the amount of sialic acid in the whey powder
  2. Quantify the amount of sialic acid in dairy milk by the standard addition method using the bovine whey powder from stage 1

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.

three dimensional chemical structure of sialic acid with solid wedge and dash bonds

Figure 1. Structure of a sialic acid (N-acetylneuraminic acid).

EXPERIMENTAL METHOD

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:

  • Brand 1 (from Denmark)
  • Brand 2 (from Singapore)
  • Brand 3 (from Thailand)

EXPERIMENTAL PROCEDURE

1. Standard Preparation for External Calibration Curve

  1. Sialic acid standard stock solution (~1000 mg/L)
  2. Weigh ~10 mg of the sialic acid analytical standard into a 10 mL volumetric flask
  3. Add ~6 mL of water into the flask and mix to dissolve completely
  4. Top up to mark and mix well before use. This is the 1000 mg/L sialic acid stock solution
  5. Prepare calibration standards in 15 mL centrifuge tubes from the stock solution of 1000 mg/L as in Table 1.
  6. See step 6 “Hydrolysis of GMP and derivatization of released sialic acid” for further steps

Standard

Volume of standard stock solution (µL)

Water

(µL)

Sialic acid (mg)

A

10

190

0.01

B

20

180

0.02

C

50

150

0.05

D

100

100

0.10

Table 1.Preparation of calibration standards

2. Reagent Preparation

  1. 0.1 M Sodium chloride pH 6.84: Weigh ~585 mg of sodium chloride into a 100 mL volumetric flask and dissolve it in 95 mL of water. Adjust pH to ~6.8 ±0.05 using 0.1 M hydrochloric acid or 0.1 M sodium hydroxide. Top up to mark with water.
  2.  240 g/L Trichloroacetic acid: Weigh ~7.2 g of trichloroacetic acid into a 100 mL glass bottle. Measure 30 mL of water using a measuring cylinder and add to the glass bottle. Mix well. 
  3. 200 g/L Phosphotungstic acid: Weigh ~2 g of phosphotungstic acid into a 25 mL glass bottle. Measure 10 mL of water using a measuring cylinder and add to the glass bottle. Mix well.
  4. Derivatization reagent: Weigh ~375 mg of ninhydrin into a 25 mL glass bottle and protect it from light with aluminum foil. Add 6 mL fuming 37% hydrochloric acid followed by 9 mL glacial acetic acid. Cap tightly and mix contents for 30 to 40 min on an orbital shaker.
  5. Ethanol:Water solution (95:5): With a 100 mL measuring cylinder, measure out 95 mL of ethanol. With a 10 mL measuring cylinder, measure out 5 mL of water. Pour both solvents into a 100 mL glass bottle and mix well.

**All reagents need to be freshly prepared on the day of use

3. Standard addition– Spiking of bovine whey powder in milk

  1. Weigh bovine whey powder into 50 mL centrifuge tubes according to Table 2. Record mass used.
  2. For the tube marked “Reagent blank”, add 8 mL of water to the centrifuge tube
  3. For the tube marked “Whey only”, pipette 8 mL of 0.1 M NaCl into the centrifuge tube
  4. For tubes marked Std Add 1 to Std Add 4, pipette 8 mL of milk into each centrifuge tube
  5. Mix well for all tubes and sonicate for 10 min

 

Samples for Standard Addition

Weight of bovine whey (mg)

Reagent blank

0

Whey only

50±2

Std Add 1

0

Std Add 2

50±2

Std Add 3

100±2

Std Add 4

150±2

Table 2.Weight of bovine whey powder

4. Precipitation and removal of milk solids

  1. Pipette 4 mL of 240 g/L trichloroacetic acid into each of the tubes from above and cap tightly
  2. Vortex or vigorously shake tubes for 10 seconds
  3. Allow the samples to stand at room temperature for 30 mins
  4. Vortex or vigorously shake tubes for 5 seconds
  5. Centrifuge sample tubes for 5 minutes at 7000 g
  6. Filter samples via 0.45 µm hydrophilic PTFE filter into a new 15 mL centrifuge tube

5. Precipitation and washing of Glycomacropeptide (GMP)

  1. Pipette 1.2 mL of 200 g/L phosphotungstic acid into each tube from 4.6.
  2. Vortex or vigorously shake the tube for 10 s
  3. Centrifuge sample tubes for 10 min at 7000 g
  4. Discard the supernatant and take care not to remove the sediment
  5. Pipette 5 mL of 95:5 ethanol:water solution into each tube
  6. Vortex or vigorously shake the tube for 10 seconds to redisperse precipitates (some precipitates will stick to the bottom tube wall)
  7. Ultrasonicate contents for 5 min to completely disperse precipitates (this step can be repeated to break down big particulates.)
  8. Centrifuge sample tubes for 10 min at 7000 g
  9. Carefully discard the supernatant and take care not to remove the sediment
  10. Repeat steps 4. to 8. for a second round of washing

6. Hydrolysis of GMP and derivatization of released sialic acid

  1. Pre-heat a water bath at 100 oC
  2. Wrap centrifugation tubes with aluminum foil to protect them from light
  3. Pipette 2 mL of glacial acetic acid into the 15 mL centrifuge tubes (external standards, reagent blank, whey only, and samples)
  4. Pipette 1 mL of acidic ninhydrin into the same tube and cap tightly
  5. Vortex or vigorously shake the tube for 10 s
  6. Ultrasonicate samples in a water bath for 5 min (this step can be repeated to break down big particulates)
  7. Remove the aluminum foil, vigorously shake tubes, and heat samples at 100 oC water bath for 10 min
  8. Remove tubes from the water bath (HOT!!), shake well, and cool down with room temperature water for 5 min

7. VIS Spectrophotometric measurement

  1. Set the spectrophotometer to the wavelength of 470 nm for measurement and zero with Zero Cells.
  2. Transfer the reagent blank into a 1 cm glass cuvette in the fume hood (fumes!)
  3. Tap the cuvette gently to remove air bubbles if any are present
  4. Wipe down cuvette sides with KimWipes and insert them into the sample chamber of the spectrophotometer
  5. Zero spectrophotometer with reagent blank
  6. Drain the reagent blank into a waste beaker
  7. Rinse the cuvette with a small volume of a sample before filling the cuvette
  8. Measurement of samples can be based according to increasing spiked whey concentration
  9. Record the sample absorbance measurements

Experimental Conditions

Method:

Spectrophotometric measurement in the visible range

Detection:

Vis @ 470 nm

Measuring range:

0.0 – 500 mg/L sialic acid in the 10-mm glass rectangular cell

Sample preparation:

See Sample Preparation Section (4.-6.)

Zero adjustment:

It is recommended to perform a zero adjustment for this method each new working day with water (e.g. from Milli-Q® System- Type I water)

Measurement procedure:

Select ad-hoc measurements and enter 470 nm as the wavelength of measurement

Perform the zero adjustment prior to the first measurement (procedure see "Zero adjustment").

In the fume hood, fill the pretreated sample into a 10-mm glass rectangular cell and place the cell into the cell compartment, the measurement is performed automatically.

The absorbance value appears in the display.

Note: The pretreated sample is to be filled in fume hood due to strong odor.

Table 3.Experimental conditions photometry overview

RESULTS AND DISCUSSION

Sialic Acid in Bovine Whey (External Calibration)

“Brand 1” Milk using the Spectroquant® Prove 600.

 

Amount Sailic Acid

(mg)

Averaged

Absorbance (n=3)

Reagent blank corrected Absorbance

Reagent blank

0

0.019

N.A.

Std 1

0.01035

0.096

0.077

Std 2

0.02070

0.175

0.156

Std 3

0.05175

0.395

0.376

Std 4

0.10350

0.736

0.717

Table 4.External Calibration Curve for (Sialic Acid)
A graphical plot between average absorbance and amount of sialic acid, measured in mg. This serves as an external calibration curve obtained for sialic acid in bovine whey, establishing the linear relationship in the plotted quantities. Furthermore, the linear equation is provided as y = 6.8441x – 0.0126, accompanied by an R² value of 0.9994.

Figure 2. External calibration curve for-Sialic acid.

Weight (mg)

Averaged Absorbance

Reagent blank corrected Absorbance

50.18

0.356

0.337

Table 5. Sialic acid content measured in bovine whey only sample against external calibration

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.

Sialic Acid in Milk (Standard Addition with Bovine Whey)

“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

Milk sample (Standard Addition)

Bovine Whey added (mg)

Sialic Acid in Bovine Whey added

(mg, calculated)

Std Add 1

0

0.0000

Std Add 2

50.21

0.0474

Std Add 3

101.94

0.0962

Std Add 4

151.05

0.1425

Table 6. Spike amount of bovine whey powder to milk and related sialic acid addition

Sample

Sialic acid Amount added (mg)

Averaged Absorbance (n=3)

Adjusted Absorbance

Std Add 1

0.0000

0.335

0.316

Std Add 2

0.0474

0.611

0.591

Std Add 3

0.0962

0.932

0.913

Std Add 4

0.1425

1.207

1.187

Table 7. Photometric results standard addition using bovine whey in milk samples
A graphical plot between average absorbance and amount of sialic acid, measured in mg. This serves as an standard addition curve obtained for sialic acid in bovine whey of brand 1, establishing the linear relationship in the plotted quantities. Furthermore, the linear equation is provided as y = 6.1653x – 0.3109, accompanied by an R² value of 0.9994.

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.

Determination of Sialic Acid in 3 Milk Brands (Standard Addition of Sialic acid using Bovine Whey)

The correlation of milk adulteration to its sialic acid content3 is classified in the following categories:*

Negative (Unadulterated):

≤ 5.14 mg/L sialic acid

Suspected: 

5.14 to 7.89 mg/L sialic acid

Highly probable:

> 7.89 mg/L sialic acid

Table 8 shows the results for for 3 milk samples prepared in duplicate and measured on Spectroquant® Prove 100 and 600 photometers using the same cuvette.

Milk Brand

Prove 100

Sialic Acid Content (mg/L)

Prove 600

Sialic Acid Content (mg/L)

Category

Experiment 1

Experiment 2

Experiment 1

Experiment 2

Brand “1”

6.30

6.42

6.30

6.37

Suspected

Brand “2”

7.07

6.82

7.06

6.91

Suspected

Brand “3”

7.22

7.00

7.16

7.42

Suspected

Table 8.Results from duplicate experiments measured on a Spectroquant® Prove 100 and 600

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.

CONCLUSION

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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References

1.
Neelima, Sharma R, Rajput YS, Mann B. 2013. Chemical and functional properties of glycomacropeptide (GMP) and its role in the detection of cheese whey adulteration in milk: a review. Dairy Sci. & Technol.. 93(1):21-43. https://doi.org/10.1007/s13594-012-0095-0
2.
Neelima, Rao PS, Sharma R, Rajput YS. 2012. Direct estimation of sialic acid in milk and milk products by fluorimetry and its application in detection of sweet whey adulteration in milk. Journal of Dairy Research. 79(4):495-501. https://doi.org/10.1017/s0022029912000441
3.
Fukuda SP, Roig SM, Prata LF. 2004. Correlation between acidic ninhydrin and HPLC methods to evaluate fraudulent addition of whey in milk. Lait. 84(5):501-512. https://doi.org/10.1051/lait:2004018
4.
Pelerine DH, Gomes MT. 2008. Whey proteins solubility curves at several temperature values. Ciência e Natura. 30(1):17–25. https://periodicos.ufsm.br/cienciaenatura/article/view/9740
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