ISSN 2310-9599. Foods and Raw Materials, 2018, vol. 6, no. 1 TECHNOLOGICAL PROPERTIES OF MILK OF COWS WITH DIFFERENT GENOTYPES OF KAPPA-CASEIN AND BETA-LACTOGLOBULIN Sergey V. Tyulkina , Ramil’ R. Vafinb,* , Lenar R. Zagidullinc, Takhir M. Akhmetovc, Andrey N. Petrovd , and Friedhelm Diele a Kazan State Agricultural University, Ferma-2 Str., Kazan 420011, Russian Federation b Tatar Scientific Research Institute of Agriculture – Subdivision of FIC Kazan SC of Russian Academy of Sciences, Orenburgskiy trakt Str. 48, Kazan 420059, Russian Federation c Bauman Kazan State Academy of Veterinary Medicine, Sibirskiy trakt Str. 35, Kazan 420029, Russian Federation d All-Russian Research Institute of Canning Technology – Branch of V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Shkolnaya Str. 78, Vidnoe 142703, Russian Federation e Institute for Environment and Health, Petergasse 27, 36037 Fulda, Germany * e-mail: [email protected] Received March 20, 2018; Accepted in revised form April 24, 2018; Published June 20, 2018 Abstract: The presence of the desirable alleles and genotypes of casein and whey protein genes in the genome of cows affects the milk protein content, quality and technological properties of their milk. Two important properties of milk its producibility is judged on are cheeseability and heat resistance. The present studies aimed at estimating the technological properties of milk of black-motley × Holstein and Kholmogorskaya breeds cows of the Tatarstan type with different kappa-casein (CSN3) and beta-lactoglobulin (BLG) genotypes. The study was carried out using a sampling of the first-calf cows of 5 cattle-breeding farms of the Republic of Tatarstan. In animals, the CSN3 and BLG genotypes have been determined by a PCR-RFLP analysis. The cheeseability, heat resistance and thermostability of milk have been estimated using standard methods. The studies have established that the CSN3 and BLG genotypes of cows affected the condition of a casein clot and duration of milk clotting time. The best cheese-making properties of milk were inherent in the animals with the BB and AB genotypes of the CSN3 and BLG genes. They were superior to the coevals with the AA genotype in terms of the highest yield of the desired dense casein clot and the shortest duration of milk clotting time. The first-calf cows, which are the carriers of an A allele of the CSN3 gene, were superior to the animals with the BB genotype of the CSN3 gene on the thermostability of milk including that on the proportion of animals with this milk characteristic. The BLG genotype of the studied animals did not significantly affect the thermostability of milk. Moreover, the highest thermostability of milk was characteristic of black-motley × Holstein cows with the AA genotype. Keywords: Сow, milk, cheeseability, thermostability, allele, genotype, CSN3, BLG, PCR, RFLP DOI 10.21603/2308-4057-2018-1-154-162 Foods and Raw Materials, 2018, vol. 6, no. 1, pp. 154–162. INTRODUCTION dense casein clot, can be used when affected by a The manufacture of dairy products is impossible if rennet enzyme [2, 3]. dairy raw materials do not meet the requirements for The heat resistance of milk is the technological their development. In this context, attention should be property of milk to resist high temperatures without paid to two important properties of milk its protein coagulation [4]. This property of milk is an producibility, namely, the cheeseability and heat important condition for the development of sterilized resistance are judged on. products that are in high consumer demand due to their The cheeseability of milk is a set of indicators of long shelf life. To manufacture such products, milk is technological, physical and chemical and hygienic treated at high temperatures (110–160°C) [2, 3]. properties, as well as the chemical composition of Therefore, high requirements are imposed to milk milk [1]. To produce cheese and cottage cheese, only as the raw materials used for the manufacture of such milk, which can coagulate with the formation of a dairy products as cottage cheese, cheese, yogurt, Copyright © 2018, Tyulkin et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. This article is published with open access at http://jfrm.ru/. 154 ISSN 2310-9599. Foods and Raw Materials, 2018, vol. 6, no. 1 canned food, including gerodietic and functional The resulting homogenate is held in a thermostat at foods [5–11]. 95°C for 15 minutes with the lid of the tube open. The studies on the technological properties of milk In animals, the CSN3 and BLG genotypes have with the involvement of the modern molecular genetic been determined by a PCR-RFLP analysis. methods of diagnostics in cattle breeding are of The CSN3 gene was amplified using a Tertzik particular interest. A lot of countries currently use thermocycler (Russia) in volumes of reaction mixtures genetic markers that are related to the qualitative (20 μl) containing the appropriate buffer (60 mM Tris- features of dairy productivity [12]. HCl (pH 8.5), 1.5 mM MgCl2, 25 mM KCl, 10 mM The evidence has been presented that the presence of 2-Mercaptoethanol and 0.1 mM Triton X-100) 0.2 mM the "desirable" alleles and genotypes of casein (alpha- dNTPs, 1 U Taq DNA polymerase (SibEnzyme, casein [13], beta-casein [14] and kappa-casein [15–17]) Russia), 0.5 mkM of the oligonucleotide primers AB1 [18] and whey (beta-lactoglobulin [15, 19] and alpha- and AB2 [23] and 1 μl of a DNA sample as follows: lactalbumin [20]) milk proteins in the genome of cows have an effect on milk protein content, quality and × 1 : 94°С – 4 min; technological properties of their milk [3, 21, 22]. × 40 : 94°С – 10 sec, 63°С – 10 sec, 72°С – 10 sec; In this regard, the present studies aimed at × 1 : 72°С – 5 min; storage: 4°С [24]. estimating the technological properties of milk of cows of black-motley × Holstein and Kholmogorskaya The RFLP-identification of genotypes on the allelic breeds of the Tatarstan type with different kappa-casein variants A and B of the CSN3 gene was performed by (CSN3) and beta-lactoglobulin (BLG) genotypes. treating 20 μl of a PCR sample of 10 U of the In accordance with the aim of the study the restriction enzyme HinfI in the 1 × buffer "O" following tasks were being solved: (SibEnzyme, Russia) at 37°C overnight. – to genotype the studied sampling of first-calf cows in The BLG gene was amplified using a Tertzik several farms of the Republic of Tatarstan on the A and thermocycler (Russia) in volumes of reaction mixtures B alleles of the CSN3 and BLG genes by a PCR-RFLP (20 μl) containing the appropriate buffer (60 mM Tris- analysis; HCl (pH 8.5), 1.5 mM MgCl2, 25 mM KCl, 10 mM – to determine the cheeseability and thermostability of 2-Mercaptoethanol; 0.1 mM Triton X-100), 0.2 mM the milk of the studied sampling of first-calf cows dNTPs, 1 U of Taq DNA polymerase (SibEnzyme, depending on their genotype of the CSN3 and BLG Russia), 0.5 mkM of the oligonucleotide primers BLGP3 genes. and BLGP4 [25] and 1 μl of a DNA sample as follows: STUDY OBJECTS AND METHODS × 1 : 94°С – 4 min; The studies were carried out in Agricultural × 38 : 94°С – 10 sec, 60°С – 10 sec, 72°С – 10 sec; production cooperative named after Lenin and Dusym, × 1 : 72°С – 5 min; storage: 4°С [15]. LLC of Atninsky District, the LLC named after Tukay of Baltasinsky District, Biryulinskiy Stud Farm, OJSC The RFLP-identification of genotypes on the allelic and Hammer and sickle, LLC in Vysokogorsky District variants A and B of the BLG gene was performed by of the Republic of Tatarstan with 608 first-calf cows of treating 20 μl of a PCR sample of 5 U of the restriction the black-motley × Holstein breed and 265 first-calf enzyme HaeIII in the 1 × buffer "C" (SibEnzyme, cows of the Kholmogorskaya breed of the Tatarstan Russia) at 37°C overnight. type, respectively. Table 1 presents the spectrum of the genotype- To carry out molecular genetic studies in animal specific RFLP fragments generated during the reaction. were collected blood samples from the jugular vein. The cheeseability of milk was determined with the DNA was extracted from the samples of whole help of a rennet and rennet fermentation sample. preserved (10 mM of EDTA) blood using a combined Preparation of a rennet enzyme solution. 1 g of rennet alkaline method. DNA extraction procedure. 100 μl of powder with an activity of 100 thousand units is blood is mixed with 1 ml of dH2O and centrifuged at dissolved in a mixture of distilled water and glycerol of 10,000 rpm for 10 minutes. The resulting supernatant is an equal volume. After 24 hours, the solution is well discarded, and 50 μl of 0.2 M NaOH is added to the mixed, filtered through a paper filter, poured into dark precipitate and the mixture is thoroughly vortexed until dishes and stored in a fridge for no more than 5 days. the suspension is completely clarified. The resulting Immediately before use, the solution is diluted 25 times homogenate is thermostated at 60°C for 10 minutes. with distilled water. Then, 10 ml of the same sample of A proportional volume of 1 M Tris-HCl (pH 8.0) was the mixed milk is added into each of three tubes.