Detection of adulteration of milk in special milk based on quantitative real-time PCR

Abstract

Abstract: With the increasing demand for special milk, low value milk is often mixed into special dairy products, and the authenticity of dairy products attracts more and more attention. In this study, the adulterated milk components in special milk and its heat-treated products were detected based on SYBR Green quantitative real-time PCR and TaqMan quantitative real-time PCR, and the influence of different heat treatment methods on adulteration detection is discussed to meet the detection requirements of different commercial special dairy products. The results show that the designed bovine specific primers can amplify DNA in cow milk and have high specificity without cross-reactivity with non-target animals. The minimum detection limits of the two quantitative real-time PCR methods for milk DNA were 1 pg (SYBR Green) and 10 pg (TaqMan), respectively, and both of them could detect at least 0.1% (w/w) of milk adulteration in the special milk mixture. In order to evaluate the reproducibility of the adulterated simulation, a total of 9 repeated tests were carried out in 3 days to verify the high reproducibility of the method. In addition, the established quantitative real-time PCR method was used to analyze the authenticity of 20 commercial processed special dairy products. There are 7 products were detected to contain milk components, indicating deliberate adulteration. The experimental method is feasible for the authenticity detection of dairy products.

Key words: Special milk, Quantitative real-time PCR, Food adulteration, Authenticity analysis

CLC Number:

TS252.7

Wei LI Guo-Dong SUN Jie yang. Detection of adulteration of milk in special milk based on quantitative real-time PCR[J]. FOOD SCIENCE.

References

[1]Visciano Pierina,Schirone Maria. Food frauds: Global incidents and misleading situations[J]. Trends in Food Science & Technology,2021,114.
[2]BROOKS C, PARR L, SMITH J M, et al. A review of food fraud and food authenticity across the food supply chain, with an examination of the impact of the COVID-19 pandemic and Brexit on food industry [J]. Food Control, 2021, 130: 108171-108185.
[3]Alok Kumar Yadav, Rakesh Kumar, Lakshmi Priyadarshini, et al. Composition and medicinal properties of camel milk: A Review[J]. Asian Journal of Dairy and Food Research,2015,34(2).
[4]Martini M , Altomonte I , D Tricò, et al. Current Knowledge on Functionality and Potential Therapeutic Uses of Donkey Milk[J]. Animals, 2021, 11(5):1382.
[5]Seyiti S , Kelimu A . Donkey Industry in China: Current Aspects, Suggestions and Future Challenges[J]. Journal of Equine Veterinary Science, 2021, 102:103642.
[6]Vincenzetti S , Santini G , Polzonetti V , et al. Vitamins in Human and Donkey Milk: Functional and Nutritional Role[J]. Nutrients, 2021, 13(5):1509.
[7]Gracy K V V. Medeiros, Rita C R E . Queiroga, Whyara K A Costa, et al. Proteomic of goat milk whey and its bacteriostatic and antitumour potential[J]. International Journal of Biological Macromolecules,2018,113.
[8]Shariatikia Malihe, Behbahani Mandana, Mohabatkar Hassan. Anticancer activity of cow, sheep, goat, mare, donkey and camel milks and their caseins and whey proteins and in silico comparison of the caseins.[J]. Molecular biology research communications,2017,6(2).
[9]Angela Di Pinto, Valentina Terio, Patrizia Marchetti, et al. DNA-based approach for species identification of goat-milk products[J]. Food Chemistry,2017,229.
[10]WARREN C M, AGRAWAL A, GANDHI D, et al. The US population-level burden of cow's milk allergy [J]. World Allergy Organization Journal, 2022, 15(4): 100644-100660.
[11]YOSHINO S, HIRAGUCHI Y, TATEKAWA M, et al. Vaccine-induced anaphylaxis in a child with cow's milk allergy: Lactose hydrate was the culprit [J]. Journal of Allergy and Clinical Immunology: Global, 2022: 1-3.
[12]付尚辰, 李玲, 郑卫民, 等. 掺假羊乳及其制品中牛乳的检测技术研究进展 [J]. 食品安全质量检测学报, 2021, 12(8): 3000-3007.
[13]ABBAS O, ZADRAVEC M, BAETEN V, et al. Analytical methods used for the authentication of food of animal origin [J]. Food Chemistry, 2018, 246: 6-17.
[14]Jeffrey T. LeJeune, P?ivi J. Rajala-Schultz. Unpasteurized Milk a Continued Public Health Threat[J]. Clinical Infectious Diseases,2009,48(1).
[15]AGREGAN R, ECHEGARAY N, LOPEZ-PEDROUSO M, et al. Proteomic Advances in Milk and Dairy Products [J]. Molecules, 2021, 26(13): 3832-3852.
[16]Hicham Benabdelkamel, Afshan Masood, Ibrahim O Alanazi, et al. Proteomic Profiling Comparing the Effects of Different Heat Treatments on Camel (Camelus dromedarius) Milk Whey Proteins[J]. International Journal of Molecular Sciences,2017,18(4).
[17]J Liao, L. Yang, A M Sheppard, Y F Liu. Comparison of DNA quality in raw and reconstituted milk during sterilization[J]. Journal of Dairy Science,2018,101(1).
[18]Marek ?nirc, Tomá? Fekete, ?ubomír Belej, et al. Detection of ovine milk adulteration using taqman real-time pcr assay[J]. Potravinarstvo,2017,11(1).
[19]BAPTISTA M, CUNHA J T, DOMINGUES L. DNA-based approaches for dairy products authentication: A review and perspectives [J]. Trends in Food Science & Technology, 2021, 109: 386-397.
[20]DENG L, LI A L, GAO Y, et al. Detection of the Bovine Milk Adulterated in Camel, Horse, and Goat Milk Using Duplex PCR [J]. Food Analytical Methods, 2019, 13(2): 560-567.
[21]Abdel-Rahman S M, Elmaghraby A M, Haggag A S. (2015) Fast and sensitive determination of camel’s and goat’s meat and milk using species-specific genetic markers. Biotechnol Anim HusbandryBiotehnol Stoc 31:415–423.
[22]DI PINTO A, TERIO V, MARCHETTI P, et al. DNA-based approach for species identification of goat-milk products [J]. Food Chemistry, 2017, 229: 93-97.
[23]Haddad M, Abu-Romman S . PCR-based identification of bovine milk used in goat and sheep local dairy products marketed in Jordan[J]. Eurasian Journal of Biosciences, 2020, 14:5267-5272.
[24]Randiwela R , Mangalika U , Adikari A , et al. PCR based assay for the detection of cow milk adulteration in buffalo milk[J]. Indian Journal of Animal Research, 2018, 52(3) : 383-387.
[25]Guo L , Qian J P , Guo Y S , et al. Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique.[J]. Journal of Dairy Science, 2018, 101(8):6776-6786.
[26]Guo L , Ya M , Hai X , et al. A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat, milk and cheese[J]. International Dairy Journal, 2019,95 : 58-64.
[27]Brenn C , Schrder U , Hanel R , et al. A multiplex real-time PCR screening assay for routine species identification of four commercially relevant crustaceans[J]. Food Control, 2021, 125(3):107986.
[28]Khatun M A , Hossain A , Hossain M S , et al. Detection of species adulteration in meat products and Mozzarella-type cheeses using duplex PCR of mitochondrial cytb gene: A food safety concern in Bangladesh[J]. Food Chemistry: Molecular Sciences, 2021.
[29]Tsakali E , Agkastra C , Koliaki C , et al. Milk Adulteration: Detection of Bovine Milk in Caprine Dairy Products by Real Time PCR[J]. Journal of Food Research, 2019, 8(4):52.
[30]Tsakali E , Agkastra C , Koliaki C , et al. Milk Adulteration: Detection of Bovine Milk in Caprine Dairy Products by Real Time PCR[J]. Journal of Food Research, 2019, 8(4):52.
[31]王一村, 李娜, 翟中华, 等. 数字 PCR 技术的发展及其在食品源性成分鉴定中的应用 [J]. 山东农业科学, 2018, 50(4): 160-165.
[32]金鹭, 陈传君, 林华, 等. 基于实时荧光 PCR 对肉制品中羊肉的精确定量 [J]. 食品与发酵工业, 2020, 46(8): 246-253.
[33]陈传君, 金鹭, 林华, 等. 食品中羊肉源性成分微滴数字 PCR 定量方法的建立 [J]. 食品与发酵工业, 2020, 46(6): 229-237.
[34]Cunha J T , Ribeiro T I B , Rocha J B , et al. RAPD and SCAR markers as potential tools for detection of milk origin in dairy products: Adulterant sheep breeds in Serra da Estrela cheese production[J]. Food Chemistry, 2016, 211(nov.15):631-636.
[35]LIAO J, LIU Y F, KU T, et al. Qualitative and quantitative identification of adulteration of milk powder using DNA extracted with a novel method [J]. Journal of Dairy Science, 2017, 100: 1657- 1663.
[36]GUO L, QIAN J P, GUO Y S, et al. Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique [J]. Journal of Dairy Science, 2018, 101: 6776-6786.
[37]HAI X, LIU G Q, LUO J X, et al. Triplex real-time PCR assay for the authentication of camel derived dairy and meat products [J]. Journal of Dairy Science, 2020, 103(11): 9841-9850.
[38]WANG Z Y, LI T T, YU W J, et al. Determination of content of camel milk in adulterated milk samples by normalized real-time polymerase chain reaction system based on single-copy nuclear genes [J]. journal of the science of food and agriculture, 2020, 100(8): 3465-3470.
[39]Maria Feligini et al. Detection of Adulteration in Italian Mozzarella Cheese Using Mitochondrial DNA Templates as Biomarkers[J]. Food Technology and Biotechnology, 2005, 43(1).
[40]]I López-Calleja, Alonso I G , Fajardo V , et al. PCR detection of cows' milk in water buffalo milk and mozzarella cheese[J]. International Dairy Journal, 2005, 15(11):1122-1129.
[41][41] Golinelli L P , Carvalho A C , Casaes R S , et al. Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese[J]. Journal of Dairy ence, 2014, 97(11):1-7.
[42]LI T T, WANG J S, WANG Z Y, et al. Quantitative determination of mutton adulteration with single-copy nuclear genes by real-time PCR [J]. Food Chemistry, 2021, 344: 128622-128630.
[43]WANG W J, LIU J J, ZHANG Q D, et al. Multiplex PCR assay for identification and quantification of bovine and equine in minced meats using novel specific nuclear DNA sequences [J]. Food Control, 2019, 105: 29-37.
[44]WANG W, FU M, ZHANG Q, et al. A novel quantitative real-time PCR method for the detection of mammalian and poultry species based on a shared single-copy nuclear DNA sequence [J]. Food Chemistry, 2021, 341(Pt 2): 128170-128179.