Comparative Analysis of Simplex and Duplex PCR for Detection of Adulteration of Goat Milk and Its Heat-Treated Products

Introduction: Ensuring the safety and authenticity of milk and technological products of its processing is the primary task of the dairy sector of the industry. Modern molecular genetic technologies make it possible to ensure effective detection of adulterated dairy products, namely to assess the presence of substitution of one type of milk for another. However, there is a significant lack of studies focused on the molecular identification of dairy products that have undergone various thermal processing regimes. In this regard, the influence of milk heating processes on the degradation of nucleic acids and their subsequent analysis using PCR technologies to determine the species composition in the food industry is becoming an actual direction.

Purpose: To conduct a comparative analysis of the effectiveness of simplex and duplex polymerase chain reaction (PCR) methods for determining the origin of milk and milk products subjected to different thermal treatments.

Materials and Methods: The work was carried out in the laboratory of applied microbiology and genomics of microorganisms of the All-Russian Research Institute of Dairy Industry. The objects of the study were raw, pasteurized, sterilized milk, fermented milk products on yogurt starter and binary milk mixtures of cattle and small ruminants obtained on their basis. This study aims to apply PCR technologies to solve the problem of determining the species composition of milk obtained from cow (Bos taurus) and goat (Capra hircus) and products based on them. Total DNA was extracted from food samples for subsequent analysis by simplex and duplex PCR using a set of species-specific oligonucleotide primers.

Results: The sensitivity of simplex and duplex PCR assays for milk-based products was compared and it was found that the relative detection limit for bovine DNA using duplex PCR was lower than simplex PCR and was 50 % for raw milk, 10 % for pasteurized milk and yoghurt starter sour milk. The sensitivity of detection of goat DNA by duplex and simplex PCR was at the level of 1 % except for sterilized milk mixtures: when duplex PCR was used, the detection limit for goat DNA was lower and amounted to 5 %.

Conclusion: Molecular genetic methods using mitochondrial targets make it possible to determine the origin of milk in dairy products. The possibilities of PCR application in the analysis of heat-treated dairy products are limited by the size of the amplicons obtained. PCR-based test systems provide a wide range of opportunities for composition and adulteration detection in the dairy industry.

1. Agarkov K. V., Pryanichnikova N. S. (2023). Relevance of the development of new types of milk-based dry mixtures // Food innovations and biotechnologies: Collection of abstracts of the XI All-Russian (national) scientific conference of students, graduate students and young scientists / under the general editorship of A.Yu. Prosekova. Kemerovo: Kemerovo State University, 94-96.

2. Assembly, G. (2017). Resolution adopted by the General Assembly on 6 July 2017. In Technical Report A/RES/71/313.

3. Caldwell, J.M., Pérez-Díaz, I.M., Sandeep, K., Simunovic, J., Harris, K., Osborne, J.A. and Hassan, H.M. (2015), Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials. Journal of Food Science, 80: M1804-M1814. https://doi.org/10.1111/1750-3841.12937

4. Charykov, V. I., & Zlydnev, A. N. (2017). Analysis of electrophysical methods of milk pasteurisation. In Priority directions of energy development in agro-industrial complex (pp. 34-38).

5. De, S., Brahma, B., Polley, S., Mukherjee, A., Banerjee, D., Gohaina, M., ... & Goswami, S. L. (2011). Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese. Food Control, 22(5), 690-696.

6. Deng, L., Li, A., Gao, Y. et al. Detection of the Bovine Milk Adulterated in Camel, Horse, and Goat Milk Using Duplex PCR. Food Anal. Methods 13, 560–567 (2020). https://doi.org/10.1007/s12161-019-01678-2

7. FAO. (2023). Contribution of Terrestrial Animal Source Food to Healthy Diets for Improved Nutrition and Health Outcomes–an Evidence and Policy Overview on the State of Knowledge and Gaps.

8. Galal‐Khallaf, A., Hussein, D., & El‐Sayed Hassab El‐Nabi, S. (2021). Single nucleotide polymorphism‐based methodology for authentication of bovine, caprine, ovine, camel, and donkey meat cuts. Journal of Food Science, 86(10), 4444-4456.

9. Gilmanov, Kh. Kh., Vafin, R. R., Bliadze, V. G., & Mikhailova, I. Yu. (2020). The problem of falsification of milk species. Current issues in the dairy industry, cross-industry technologies and quality management systems, 1(1), 125-129. https://doi.org/10.37442/978-5-6043854-1-8-2020-1-125-129

10. Golinelli, L. P., Carvalho, A. C., Casaes, R. S., Lopes, C. S. C., Deliza, R., Paschoalin, V. M. F., & Silva, J. T. (2014). Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese. Journal of Dairy Science, 97(11), 6693-6699.

11. Guo, L., Qian, J. P., Guo, Y. S., Hai, X., Liu, G. Q., Luo, J. X., & Ya, M. (2018). Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique. Journal of dairy science, 101(8), 6776-6786.

12. Hazra, T., Sharma, V., Sharma, R., & Arora, S. (2016). Simplex PCR assay for detection of cow milk presence in goat milk. Indian Journal of Dairy Science, 69(5), 621-625.

13. Hird, H., Chisholm, J., Sánchez, A., Hernandez, M., Goodier, R., Schneede, K., ... & Popping, B. (2006). Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction. Food additives and contaminants, 23(7), 645-650.

14. Kourkouli, A., Thomaidis, N., Dasenaki, M., & Markou, A. (2024). Novel and sensitive touchdown polymerase chain reaction assays for the detection of goat and sheep milk adulteration with cow milk. Molecules, 29(8), 1820.

15. López-Calleja, I., González, I., Fajardo, V., Rodríguez, M. A., Hernández, P. E., García, T., & Martín, R. (2004). Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique. Journal of Dairy Science, 87(9), 2839-2845. https://doi.org/10.3168/jds.S0022-0302(04)73412-8.

16. Meldenberg, D. N., Polyakova, O. S., Semyonova, E. S., & Yurova, E. A. (2020). Development of a comprehensive assessment of the protein composition of raw milk from various farm animals for the development of functional products. Storage and processing of agricultural raw materials, (3), 118-133. https://doi.org/10.36107/spfp.2020.352

17. Merkusheva, I. N., Petrichenko, S. P., & Kozhukhova, M. A. (2005). Nutritional and biological value of goat milk. News from universities. Food technology, 2-3.

18. Milknews (2023). What's happening in the cow's milk market. Retrieved from https://milknews.ru/longridy/Chto-proishodit-na-rynke-kozego-moloka.html

19. Pokorska, J., Kułaj, D., Dusza, M., Żychlińska-Buczek, J., Makulska, J. New Rapid Method of DNA Isolation from Milk Somatic Cells // Anim Biotechnol. - 2016. - Vol. 27, No. 2. - P. 113-117. - DOI: 10.1080/10495398.2015.1116446

20. Putri, A. E., Farajallah, A., & Perwitasari, D. (2019). The origin of pesisir cattle based on D-loop mitochondrial DNA. Biodiversitas Journal of Biological Diversity, 20(9).

21. Rodrigues, N. P. A., Givisiez, P. E. N., Queiroga, R. C. R. E., Azevedo, P. S., Gebreyes, W. A., & Oliveira, C. J. B. (2012). Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay. Journal of dairy science, 95(5), 2749-2752.

22. Shegidevich, E. D. (2021). Changes in protein composition of dairy raw materials during mechanical and thermal processing. In Youth in science-2021 (pp. 131-133).

23. Shuvarikov, A. S., Kanina, K. A., Robkova, T. O., & Yurova, E. A. (2018). On the issue of assessing the composition of sheep, goat and cow milk. Sheep, goats, wool, 1(1), 20-22.

24. Shuvarikov, A. S., Yurova, E. A., & Pastukh, O. N. (2017). Qualitative indicators of cow, goat and camel milk, taking into account allergenicity. News of the Timiryazev Agricultural Academy, (5), 115-123. https://doi.org/10.26897/0021-342X-2017-5-115-123

25. Stackebrandt, E. (2009). Phylogeny based on 16S rRNA/DNA. Encyclopedia of Life Sciences (ELS). John Wiley & Sons.

26. Statista. (2024). Dairy products & eggs worldwide: Statista's forecast. Получено из https://www.statista.com/outlook/cmo/food/dairy-products-eggs/worldwide

27. Tuncay, R. M., & Sancak, Y. C. (2022). Comparison of PCR Methods for Determination of Different Types of Milk Added to Goat Milk. Balıkesir Sağlık Bilimleri Dergisi, 11(3), 509-514.

28. Wang, Z., Li, T., Yu, W., Qiao, L., Liu, R., Li, S., Zhao, Y., Yang, S., & Chen, A. (2020). Determination of content of camel milk in adulterated milk samples by normalized real-time polymerase chain reaction system based on single-copy nuclear genes. Journal of the Science of Food and Agriculture, 100(8), 3465-3470. https://doi.org/10.1002/jsfa.10382.

29. Yurova, E. A., Filchakova, S. A., & Zhizhin, N. A. (2020). Application of the PCR analysis method to determine the species composition of dairy raw materials. News of TSKhA, 6(6). https://doi.org/10.26897/0021-342X-2020-6-16-25

30. Zakharova, I.N., & Sugyan, N.G. (2021). The use of goat's milk in the nutrition of young children (clinical examples). Medical Council, (17), 175-181. https://doi.org/10.21518/2079-701X-2021-17-175-181

31. Zimnyakov, V. M., Ilyina, G. V., Ilyin, D. Yu., & Zimnyakov, A. M. (2023). State, problems and prospects of milk production in Russia. Machinery and technologies in livestock, (1 (49)), 4-10. https://doi.org/10.22314/27132064-2023-1-4

32. Zobkova, Z. S., Fursova, T. P., & Zenina, D. V. (2018). Selection of protein ingredients that enrich and modify the structure of fermented milk drinks. Beverage Industry Current Issues, (2), 64-69. https://doi.org/10.21323/978-5-6041190-3-7-2018-2-64-69.