Detection and Validation of A2 Milk Suitable for Consumers Having Milk Intolerance by ELISA Method

Year 2023, Volume: 9 Issue: 4, 881 - 892, 22.12.2023

Mediha Esra Yayla

https://doi.org/10.28979/jarnas.1254155

Abstract

Casein proteins, which make up 80% of the total proteins in cow's milk, consist of mainly A1 and A2 genetic types which differ by a mutation that causes conversion from proline to histidine. Histidine-containing A1 protein undergoes proteolytic degradation in the gastrointestinal system, while this is not observed during the digestion of A2 protein. A1 milk consumption causes bloating, gas, discomfort and symptoms confused with lactose intolerance. Studies showed that A1 milk consumption may cause diabetes, coronary heart disease, arteriosclerosis, sudden infant death, and is associated with autism and schizophrenia. With an increasing trend in the world, A2A2 milk (milk without A1 protein) production is becoming widespread with consumer preferences; and, A2 milk takes its place on the market shelves. With the onset of this trend, the need for a new analysis on food safety became evident. It will be required by food control laboratories to test the absence of A1 protein in milk to be labeled as A2 milk. In this study, the quantitative analysis and validation of β-casein A1 and A2 proteins in cow's milk by EnzymeLinked ImmunoSorbent Analysis (ELISA) method was investigated. The methods have detection limits of 1.8 and 0.8 ppm, and quantitation limits of 17 and 2.4 ppm for A1 and A2, respectively.

Keywords

β-casein A2 protein, cow milk, ELISA, food safety, method validation

Thanks

The work on this study was carried out in TUBITAK Marmara Research Center, Food Institute, Food Molecular Biology Laboratory. The Laboratory staff Mr Serkan Savsar and Mr Fuat Arslan is thanked for their support and technical assistance. The author would also like to express her gratitude to Uluova Dairy Company, which provided the milk samples used in this study.

References

• Anna, M., Salvatore, S., Omar, B. and Eugenio, M. (2016). Detecting-casein variation in bovine milk. Molecules, 21(141):1-7.
• Asledottir, T., Le, T., Petrat-Melin, B., Devold, T., Larsen, L. and Vegarud, G. (2017). Identification of bioactive peptides and quantification of β-casomorphin-7 from bovine β-casein A1, A2 and I after ex vivo gastrointestinal digestion. International Dairy Journal, 71:98-106.
• Biswas, S., & Saha, M.K. 2015. Uncertainty of Measurement for ELISA in a Serological Testing Laboratory. Immunochemistry & Immunopathology: Open Access, 1(2), 1-5. DOI: http://dx.doi.org/10.4172/icoa. 1000109 Blues, J., Bayliss, D., Buckley, M. (2004). The calibration and use of piston pipettes. National Physical Laboratory Teddington, Middlesex, United Kingdom.
• Bonfatti, V., Grigoletto, L., Cecchinato, A., Gallo, L. and Carnier, P. (2008). Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants. Journal of Chromatography A, 1195 (1):101-106.
• Boro, P., Naha, B., Saikia, D. and Prakash, C. (2016). A1 and A2 Milk & Its Impact On Human Health. International Journal of Science and Nutrition, 7(1):01-05.
• Boutrou, R., Gaudichon, C., Dupont, D., Jardin, J., Airinei, G., Marsset-Baglieri, A., Benamouzig, R., Tomé, D. and Leonil, J. (2013). Sequential release of milk protein–derived bioactive peptides in the jejunum in healthy humans. American Journal of Clinical Nutrition, 97:1314–23.
• Brooke-Taylor, S., Dwyer, K., Woodford, K. and Kost, N. (2017). Systematic Review of the Gastrointestinal Effects of A1 Compared with A2 β-Casein. Advances in Nutrition, 8(5):739-748.
• De Noni, I. (2008). Release of beta-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine beta-casein variants and milk-based infant formulas. Food Chemistry, 110 (4):897-903.
• De Noni, R., FitzGerald, H., Korhonen, Y., Le Roux, C., Livesey, I., Thorsdottir, D. and Tomé, R. (2009). Scientific report of EFSA prepared by a DATEX working group on the potential health impact of β-casomorphins and related peptides. EFSA Scientific Reports, 231:1–107.
• Duarte-Vázquez, M., García-Ugalde, C., Álvarez, B., Villegas, L., García-Almendárez, B., Rosado, J. and Regalado, C. (2018). Use of urea-polyacrylamide electrophoresis for discrimination of A1 and A2 beta casein variants in raw cow’s milk. Journal of Food Science and Technology, 55(5):1942-1947.
• Ellison, S.L.R., Williams, A. (2007). EURACHEM/CITAC Guide: Use of uncertainty information in compliance assessment. 1st edn. Geneva, Switzerland. EURACHEM/CITAC. (2001). EURACHEM/CITAC Guide: Quantifying Uncertainty in Analytical Measurement. 3rd edn. Teddington, England.
• FDA ORA. (2020). Methods, Method Verification and Validation. ORA Laboratory Manual Volume II. Retrieved from https://www.fda.gov/media/73920/download.
• GeneTel Laboratories LLC (n.d.). A1 ELISA Kit Protocol. Http://www.Genetel-Lab.com. Retrieved March 15, 2021, from http://www.genetel-lab.com/images/A1%20ELISA%20Kit%20Simple %20Test%20 SOP.pdf
• GeneTel Laboratories LLC (n.d.). A2 ELISA Kit Protocol. Http://www.Genetel-Lab.com. Retrieved March 15, 2021, from http://www.genetel-lab.com/images/A2%20ELISA%20Kit%20Simple %20Test%20 SOP.pdf
• Givens, I., Aikman, P., Gibson, T. and Brown, R. (2013). Proportions of A1, A2, B and C β-casein protein variants in retail milk in the UK. Food Chemistry, 139(1):549-552.
• Hang, T. (2018). Vinamilk tiên phong với sữa A2. Retrieved from http://vietnamnet.vn/vn/kinh-doanh/vinamilk-tien-phong-voi-sua-a2-458398.html
• Hawkes C. (2007). Regulating food marketing to young people worldwide: trends and policy drivers. American Journal of Public Health, 97(11):1962–1973.
• ISO. (1995). Evaluation of measurement data — Guide to the expression of uncertainty in measurement. International Organization for Standardization (ISO), Geneva, Switzerland.
• ISO/CASCO, (2018). General requirements for the competence of testing and calibration laboratories (ISO/IEC 17025:2017). Retrieved from https://www.iso.org/standard/66912.html.
• Jain, V. and Gupta K. (2005). Food and Nutritional Analysis: Overview. Encyclopedia of Analytical Science (Second Edition), 202-211.
• Kamiński, S., Cieslińska, A. and Kostyra, E. (2007). Polymorphism of bovine beta-casein and its potential effect on human health. Journal of Applied Genetics, 48:189–98.
• Kudlejova, L. and Risticevic, S. (2012). 9 - Application of Solid-Phase Microextraction in Food and Fragrance Analysis. Handbook of Solid Phase Microextraction, 291-334.
• National Institute of Standards and Technology, (1995). Nomenclature in evaluation of analytical methods, including detection and quantification capabilities (IUPAC Recommendations 1995), Pure and Applied Chemistry, 67(10), 1699-1723.
• Nestle M. (2002). Food Politics. Berkeley and Los Angeles: University of California Press. Pal, S., Woodford, K., Kukuljan, S. and Ho, S. (2015). Milk intolerance, beta-casein and lactose. Nutrients , 7:7285–97.