# A Review of Milk Frauds and Adulterations from a Technological Perspective
## 3 Results and Discussion
### Methods for Detecting Adulterants in Milk
#### 3.2.3. Instrumental Methods (Quantitative)
PCR is a detection technique that can be used to identify adulterants in milk, including milk from various sources, in both qualitative and quantitative ways. The several PCR versions, such as multiplex PCR, real-time PCR, restriction fragment length polymorphism (RFLP), etc., are used to identify microbiological and exogenous milk from diverse sources in raw and processed milk. Due to a few limitations, PCR is not currently used as a standard procedure for detecting adulterants in milk. The potential use of PCR as a detection tool is restricted by its incapacity to detect adulterants in milk without DNA and its inhibition by high concentrations of compounds like fat and protein [3; 11; 12]. According to EU legislation, isoelectric focusing of \(\gamma\)-cazeins after plasmolysis should be used as a reference method to ensure that the products obtained are exactly sheep's milk, goat's milk, or buffalo milk, or a mixture of sheep's, goat's, and buffalo milk. The method may be used to detect either raw or heat-treated and caseinized cow's milk in fresh or ripened sheep's, goat's, and buffalo's milk cheeses or mixtures thereof [3]. Since the late 1980s, ELISA has been utilized for species identification in milk and dairy products. Recently, a commercially available kit has been developed that is based on a competitive ELISA using a mouse monoclonal antibody raised against bovine \(\kappa\)-casein and allows screening of both raw milk and heat-treated cow and buffalo milk in milk and cheese from other species and sources. The enzyme-linked immunosorbent assay, or ELISA, is a widely used laboratory procedure. The possibility of using ELISA to identify adulteration using relatively thermostable proteins s caseins has been raised. In a study by [PERSON] et al., a quick ELISA technique for the identification of cow milk in yak milk that has been tampered with was created utilizing a high-affinity anti-bovine-casein monoclonal antibody. As a result of the test, the minimal detection limit was 1% (\(\
u/\
u\)) yak milk containing cow milk, and the linear range for adulteration detection was 1 to 80%, which is a wide enough range for regular adulteration testing. This test was not influenced by a series of treatments applied to the milk, such as acidification, thermal treatment, or the addition of rennet [3; 11; 12; 42; 84]. In addition, biosensors may give quick, quantitative, and direct determinations and are thus frequently used in the field of food testing. In their study, [PERSON] et al. developed a label-free optical immunosensor that utilized Mach-Zehnder interferometry to identify milk adulteration, employing a competitive immunoassay to quantify bovine k-casein in sheep and goat milk. With a total testing period of 12 min, a limit of detection of 0.04 g/mL concerning bovine k-casein was achieved, which corresponds to roughly 0.06% cow milk in undiluted ewe/goat milk. Several factors were tuned in order to produce this immunoassay, including the exact amount of immobilized bovine k-casein, the amount of anti-k-casein antibodies, the prior incubation period, and the effect of the matrix. As a result of the outstanding analytical efficiency of the proposed assay as well as the portability of the sensor system, this device is appropriate for on-site detection of milk adulteration [85]. In another study, [PERSON] et al. evaluated the performance of the quartz crystal microbalance (QCM) covered with polystyrene to detect the adulteration of milk of different species with cow's milk. The technique was created based on the difference in protein profilesbetween cow's milk and goat's milk. The recently introduced QCM immunosensor to recognize cow milk presence has a limit of detection of 1 ppm. These detection limits were satisfactory for employing the biosensor for the detection of adulteration of goat's milk with cow's milk [86].