Milk is one of the products that can be adulterated in many ways affecting the quality of this and its derivatives. Glucomacropeptide (GMP) is a protein that is found only in the whey from the production of fresh cheese, enzymatically obtained from the coagulation of casein and which is commonly used to adulterate fresh or powdered milk. The aim of this study was to determine the adulteration of milk with cheese whey thought a molecular approach, where the glucomacropeptide was collected by sequential precipitation with trichloroacetic acid (ATC) and detected by polyacrylamidododecylsulfate gel electrophoresis (PAGE-SDS), using samples of fresh milk, intentionally adulterated with serum in the proportion of 0%, 1%, 5%, 10% and 15%. The results obtained showed that the detection of glucomacropeptide by electrophoresis was positive in all samples of adulterated milk, evidencing a band of 20.9 kDa in the reading, corresponding to the molecular weight of the GMP, showing that the technique used determines the adulteration in the milk, in a specific and sensitive way, also shows that in the evaluation of physical-chemical and microbiological parameters of milk, there are no significant differences between treatments, except for the pH that tends to decrease as the percentage of serum in the milk increases.
For Codex Stan 206 [
One of the main components of milk is protein, which together with enzymes are composed of amino acids [
The enzymatic pathway of coagulation is given by renin or chymosin (produced in the abomasum of lactating ruminants) or other coagulant or proteolytic enzymes (pepsin, bacterial and plant enzymes) [
GMP is therefore a 64-amino acid peptide, present exclusively in whey cheese, released after the specific cleavage of κ-casein by chymosin; consists of two non-glycosylated isoforms (aGMP A and aGMP B) and their different glycosylated forms (gGMP A and gGMP B), the most predominant carbohydrate of gGMP being N-acetylneuraminic acid (sialic acid) [
Studies have shown that GMP helps the production of cytokines, regulating immune system interactions [
Therefore, the adulteration of milk with whey from cheeses is not a public health problem; however it is considered a fraud [
There are several methods to determine adulteration of milk with cheese whey, among which we can mention indirect methods that are based on the determination of the proportion of whey casein present in milk, which is done through: quantification of no casein nitrogen analysis, quantification of sulfhydryl groups per gram of protein, determination of ammonium levels in milk using a potentiometer, and determination of the cysteine-casein complex, using a polarographic method [
The objective of this investigation was to detect GMP in milk adulterated with whey through sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE) and to determine whether there is or not alteration of the physico- chemical and bacteriological properties of adulterated milk.
The importance of the study is to verify and thus to provide the dairy industry with a reliable method for determining the adulteration of raw milk with cheese whey (the practice of which is very common), since this constitutes cheating and fraud for the buyer, which directly affects the production of dairy products, due to the reduction of natural components of milk, which are necessary to improve performance in the dairy industry, avoiding economic losses to it.
This research was carried out at the Polytechnic School of Chimborazo, located in the Riobamba city (to 190 km from Quito), province of Chimborazo-Ecuador.
In the present study, milk from a single commercial house and dairy cheese whey was used, based on the NTE INEN 4 [
・ T0: Fresh milk without adulteration;
・ T1: Milk adulterated with 1% whey;
・ T2: Milk adulterated with 5% whey;
・ T3: Milk adulterated with 10% whey;
・ T4: Milk adulterated with 15% whey.
The variables that were evaluated were: milk adulteration determination by GMP detection, physical chemical characteristics (protein, fat, pH and density) and microbiological characteristics (reductase).
The detection of GMP by electrophoresis was performed as described by Galindo,
Wheylevels | Code | Repets | SEU* | SEU/Treatment |
---|---|---|---|---|
Control | T0 | 4 | 1 | 4 |
1% | T1 | 4 | 1 | 4 |
5% | T2 | 4 | 1 | 4 |
10% | T3 | 4 | 1 | 4 |
15% | T4 | 4 | 1 | 4 |
Total | 20 |
*Size of the Experimental Unit.
et al. [
Milk protein: crude protein (CP) titration by the Kjeldahl method, based on the NTE INEN 16:84 [
Milk fat: the evaluation was made based on the Gerber Method, proposed in the NTE INEN 12:73 [
Density of the milk: it was made according to the established in the NTE 11:84 [
pH of the milk: the determination was made by direct reading by introducing the electrode of a pH meter, previously adjusted with buffers of known pH 4.00 and 7.00, in the milk previously heated and homogenized at 40˚C (to disperse the fat) and then cooled to 20˚C, based on the NTE 1500 [
Reductase test: according to NTE INEN 18:73 [
The physicochemical and microbiological characteristics were evaluated by ana- lysis of variance and Duncan at 5%.
The presence of Glucomacropeptide in the milk of the control treatment (T0 [negative control]) was negative, whereas in the milks in which the different whey levels were applied (1%, 5%, 10% and 15%) was positive in all cases, evidencing a band of 20.9 kDa in the electrophoresis.
The average protein of the milk obtained in the present investigation was 3.24%, with a coefficient of variation of 2.36%, when performing the analysis of variance it was possible to determine there was no statistical difference between the treatments.
Milk including milk whey on average recorded 3.86% of fat and a coefficient of variation of 1.38%, when performing analysis of variance did not determine significant differences between different levels of whey in adulterated milk. When analyzing
The milk pH included cheese whey on average was 6.67, with a coefficient of variation of 0.58%; by subjecting the experimental results to the analysis of variance, it was possible to determine that there are significant differences between the different levels of whey. The use of 1% and 5% of whey allowed to register 6.73 and 6.70 of pH, which differ significantly from the rest of whey milk levels,
mainly of 15% whey with which 6.60 of pH was reached. In
On average, the milk density included cheese whey registered 1033 g/ml with a coefficient of variation of 0.04%; when the experimental results were submitted to the analysis of variance, it was possible to determine that no significant differences between the treatments were observed. When analyzing the experimental results, according to
According to the reductase analysis, the average milk recovered normal color at 3.94 hours, with a coefficient of variation of 1.12%; when submitting the experimental results it was possible to notice that there are significant differences between the different treatments. When analyzing the milk without the application of whey, it takes 4.07 hours to recover its normal color, a value that differs significantly from other treatments, mainly 15% whey, which reached 3.86 hours in taking the natural color. According to
Milk is one of the most complete foods that exist in nature and has been consumed by man for many years. Since the milk industrialization and its derivatives began, the adulteration of this food was increased in order to obtain greater revenue. One of the most commonly used forms of milk adulteration is the addition of water and/or whey from the manufacture in fresh cheese rich in GMP.
This study shows that there was no presence of GMP in unadulterated raw milk belong to control group (To) due to the absence of this adulteration indicator that only allows identify the presence of serum in milk, as indicated by Alcázar, C., et al. [
Other studies also determine the presence of GMP in milk, but by reverse phase high-performance liquid chromatography with mass spectrometry detection [
On the other hand, other studies indicate the presence of bands of 20.1 and 14 kDa corresponding to samples of pure GMP, the original immunogen, in different states of aggregation [
The average milk protein obtained in this research and shown in
Variables | Wheylevels | CV% | Average | Sign | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Control | 1% | 5% | 10% | 15% | |||||||||
GMP | N | b | P | a | P | a | P | a | P | A | * | ||
Proteins (%) | 3.28 | a | 3.20 | a | 3.20 | a | 3.30 | a | 3.20 | A | 2.36 | 3.24 | Ns |
Fat (%) | 3.90 | a | 3.88 | a | 3.88 | a | 3.80 | a | 3.83 | A | 1.38 | 3.86 | Ns |
Ph | 6.68 | ab | 6.73 | a | 6.70 | a | 6.63 | bc | 6.60 | C | 0.58 | 6.67 | ** |
Density (g/ml) | 1.033 | a | 1.033 | a | 1.033 | a | 1.032 | a | 1.032 | A | 0.04 | 1.033 | Ns |
Reductase test (h) | 4.07 | a | 3.99 | b | 3.91 | c | 3.88 | C | 3.86 | C | 1.12 | 3.94 | ** |
Color (points) | 5.00 | a | 4.91 | a | 4.84 | a | 4.50 | B | 4.13 | C | 2.03 | 4.68 | ** |
Taste (points) | 4.99 | a | 4.90 | a | 4.83 | a | 4.49 | B | 4.12 | C | 2.04 | 4.67 | ** |
Smell (puntos) | 4.97 | a | 4.88 | a | 4.81 | a | 4.47 | B | 4.10 | C | 2.04 | 4.65 | ** |
Total | 14.96 | a | 14.69 | ab | 14.49 | ab | 13.46 | B | 12.34 | B | 2.04 | 13.99 | ** |
Equal letters do not differ significantly according to Duncan at 5%. CV%: Coefficient of variation. N: Negative. P: Positive, Sign: Significance is defined as the ratio of the standard deviation to the mean. Ns: There are no significant differences (P > 0.05). *: Significant differences (P > 0.05). **: Highly significant differences (P > 0.05).
Other studies also show that milk adulterated with whey has average values of total protein (3.25% - 3.75%), total solids (11.9% - 13%) and other physicochemical parameters that are within normal limits established by Ecuadorian legislation [
In relation to the average fat of the milk obtained in this study (
The results obtained in this study coincide with the results published by Araújo [
As for the average of the results in relation to pH (
In the study by Araújo [
The average milk density obtained in this research and shown in
Our results coincide with those obtained in a study of presence of GMP in milk by liquid chromatography [
All microorganisms have a reducing power because they can eliminate oxygen, being the main ones Lactococcus, coliforms and Bacillus [
The results obtained in our research coincide with those recorded by Araújo [
The results obtained showed that the detection of glucomacropeptide by electrophoresis was positive in all samples of adulterated milk, evidencing a band of 20.9 kDa in the reading, corresponding to the molecular weight of the GMP. The present study determined that the detection of GMP by electrophoresis is a sensitive and specific method able to detect GMP in adulterations with concentrations of up to 1% of whey, showing to be a robust method that could be used in the routine of the dairy industry, as well as evidenced that the milk adulterated with whey maintains constant its microbiological, physical and chemical characteristics without denoting the possible presence of adulterations; there are no significant differences between treatments, except for the pH that tends to decrease as the percentage of serum in the milk increases.
To the Polytechnic School of Chimborazo, mainly to the teachers of the College of Animal Sciences. To the College of Veterinary Medicine of the Central University of Ecuador, to their teachers and authorities.
Puga-Torres, B.H., Morales-Arciniega, S.M., Naranjo, L.F.N., De la Torre-Duque, D.I., Campos-Vallejo, R.M., Santander-Parra, S.H. and Vayas-Ma- chado, E.C. (2017) Detection of Glucomacropeptide in Raw Milk Adulterated with Cheese Whey in Ecuador. Food and Nutrition Sciences, 8, 579-590. https://doi.org/10.4236/fns.2017.86040