**5. GMP as an indicator of milk adulteration with cheese whey**

One of the problems presented by the dairy industry is the adulteration of milk with whey cheese, which is very cheap and not detected by sensorial tests. Cheese whey does not cause harm to health, however, it affects milk-derived products manufacturers financially and can affect the consumers nutritionally, so the addition of cheese whey is considered a fraud. Due to GMP present in cheese whey, the detection of this peptide may indicate the addition of cheese whey to milk. Some of the methods that detect GMP as an indicator of the presence of cheese whey are described below.

### **5.1 Electrophoretic methods**

 *Sodium dodecyl sulfate−polyacrylamide gel* (*SDS-PAGE*): This method was standardized to analyze pasteurized milk and milk powder. Samples has to be previously treated with 24% trichloroacetic acid (TCA) to eliminate interfering k-casein and later with 50% TCA to precipitate GMP, which is resuspended in Tris-buffer 0.05 M HCl, 1 mM EDTA, pH 7.2 [82]. Analyzing under these conditions cheese whey and milk added with cheese whey, a protein fraction of 20.8 kDa is identified corresponding to GMP that allowed to detect adulteration with whey up to 1%. This protein fraction is not detected in samples of acid whey or in raw milk. This technique had been previously used to analyze milk drinks that were distributed in schools as part of a program of the Brazilian government [83]. However, the method showed sensitivity to detect 5% of added whey, probably because they did not treat the sample with TCA. In later works, it was able to detect 40 and 20 μg of GMP in samples analyzed by electrophoresis in SDS-PAGE and in cellulose acetate strips, respectively, due to the use of thiobarbituric acid and malachite green dye reactions instead of Coomassie blue as developing agents [84].

*Capillary electrophoresis* (*CE*): A variant of electrophoresis is CE, a technique that has the advantage of allowing a rapid detection of GMP. This method has been used to identify GMP as an indicator of the presence of cheese whey in buttermilk powder and skim milk. An advantage of this method is that it is usually reproducible, repeatable and sensitive; however, the interpretation of the results is difficult [85].

#### **5.2 Chromatographic method**

 High performance liquid chromatography (HPLC) has been widely used to identify GMP as indicative of milk adulteration with cheese whey. In order to carry out the analysis, it is necessary to pre-treat the samples with TCA to precipitate proteins that can interfere (k-casein) and to concentrate GMP [86]. Similarly, a rapid and sensitive HPLC method on a gel permeation column was developed to detect GMP to follow the hydrolysis of k-casein by chymosin in milk [87]. The only pretreatment given to samples was addition of TCA (final concentration 8%) to precipitate the interfering caseins and whey proteins. This method was widely used by several researchers to analyze different samples, such as skimmed milk powder [88]. Cation-exchange chromatography has also been used to detect GMP, previously removing caseins from whey samples by precipitation with HCl at pH 4.6, neutralizing with TCA at 2–8% and analyzing supernatants [89]. On the other hand, a Reversed-Phase HPLC (RP-HPLC) method was developed and validated to separate and quantify GMP and was demonstrated to be precise, sensitive, and reliable [90].

*Glycomacropeptide: Biological Activities and Uses DOI: http://dx.doi.org/10.5772/intechopen.82144* 

The determinations were performed in the linear range of 15–200 μg/mL and the detection limit was 2 μg/mL. The method was applied to the analysis of rennet and acid whey, whey protein concentrates produced by the dairy industry, and also for the detection of rennet whey in powdered milks.

 The European Commission uses two methods to detect the presence of cheese whey in milk: a gel permeation chromatography and subsequently a RP-HPLC as a confirmatory test [91]. However, it has been shown that the sensitivity of this method is affected by the presence of acidified rennet whey, which makes it difficult to detect the addition of whey [92]. Besides, the HPLC methodology used to analyze compounds like GMP in dairy products usually includes extractions with solvents, sample's preparation require a lot of time and reactives, the equipment is very sophisticated and demands trained personal.

## **5.3 Spectroscopy methods**

Spectroscopy has also been used to detect GMP. The medium infrared spectroscopy (MIR) was used to analyze milk powder in order to detect GMP as adulteration parameter. Although this method is fast, it is not widely used because derived spectra are not very easy to interpret, in addition to its high cost [93]. On the other hand, by liquid chromatography/electrospray coupled to mass spectrometry, milk products were analyzed and it was able to quantify GMP from concentration of 10 pmol, although the method was not used to detect milk adulteration [94].

### **5.4 Immunochemical methods**

Immunoassays are analytical methods of great application in the food area, and have the advantages that they are quick, sensitive, and that the sample to be analyzed requires little or no treatment. Several immunochemical methods have been developed in order to identify and quantify GMP in milk. Firstly, it is necessary to produce antibodies against GMP and later, these antibodies can be used for the development of the different immunochemical methods that detect it. Some of these assays are described below:

*Enzyme-linked immunosorbent assay* (*ELISA*): It is an immunoassay widely used to analyze foods. It has the advantage that is simple, sensitive, and fast, in addition to being inexpensive. Two main ELISA assays has been developed to detect GMP in milk samples. An inhibition ELISA method was performed to detect bovine rennet whey solids in skim milk powders that presented a detection limit of 0.1% (w/w) and used enzyme-labeled monoclonal antibodies against bovine k-casein [95]. On the other hand, Chávez and co-workers [96] developed a sandwich ELISA using polyclonal antibodies against GMP, that showed a limit of detection of 0.047% (w/w).

*Western blot assay*: As ELISA, this technique is an immunoassay designed to detect proteins in complex samples and also has great specificity. Using the same polyclonal antibodies against GMP previously mentioned, Chávez and co-workers developed a western blot system to detect GMP [97]. When analyzing cheese whey, this antibody recognized three protein fractions of 20.1, 14, and 45 kDa. The detection limit of the test was 0.5% (v/v) to liquid cheese whey and 0.001% (w/w) to whey powder.

 *Immunochromatographic lateral-flow assay*: The development of immunochromatographic systems for quality control is a relatively new field of research and has been applied to milk. There are commercial immunochromatographic sticks which contain monoclonal antibodies specific to GMP labeled with colloidal particles that present a limit detection of 4% (v/v) of milk whey. Using these immunosticks, it has been possible to identify GMP in different samples of commercial milks [98].

Besides, it has been developed an immunochromatographic lateral-flow test that used two specific anti-bovine κ-casein monoclonal antibodies, with a detection limit of 15 ng/ml of GMP and 1% (v/v) of cheese whey [99].

 In summary, different techniques and methods have been developed and used to detect GMP as an index of adulteration of milk with cheese whey. Some of them can also be used to quantify GMP in food products. The aim of this area of research is to achieve one that bring together being cheap, fast, easy to develop, and to interpret the results, with high sensitivity and a limited sample processing. These characteristics will allow people to use them at the time and place of milk reception.
