TECHNOLOGICAL PROPERTIES OF MILK OF COWS WITH DIFFERENT GENOTYPES OF KAPPA-CASEIN AND BETA-LACTOGLOBULIN

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.


INTRODUCTION
The manufacture of dairy products is impossible if dairy raw materials do not meet the requirements for their development. In this context, attention should be paid to two important properties of milk its producibility, namely, the cheeseability and heat resistance are judged on.
The cheeseability of milk is a set of indicators of technological, physical and chemical and hygienic properties, as well as the chemical composition of milk [1].
To produce cheese and cottage cheese, only milk, which can coagulate with the formation of a dense casein clot, can be used when affected by a rennet enzyme [2,3].
The heat resistance of milk is the technological property of milk to resist high temperatures without protein coagulation [4]. This property of milk is an important condition for the development of sterilized products that are in high consumer demand due to their long shelf life. To manufacture such products, milk is treated at high temperatures (110-160°C) [2,3].
The studies on the technological properties of milk with the involvement of the modern molecular genetic methods of diagnostics in cattle breeding are of particular interest. A lot of countries currently use genetic markers that are related to the qualitative features of dairy productivity [12].
In this regard, the present studies aimed at estimating the technological properties of milk of cows of black-motley × Holstein and Kholmogorskaya breeds of the Tatarstan type with different kappa-casein (CSN3) and beta-lactoglobulin (BLG) genotypes.
In accordance with the aim of the study the following tasks were being solved: -to genotype the studied sampling of first-calf cows in several farms of the Republic of Tatarstan on the A and B alleles of the CSN3 and BLG genes by a PCR-RFLP analysis; -to determine the cheeseability and thermostability of the milk of the studied sampling of first-calf cows depending on their genotype of the CSN3 and BLG genes.

STUDY OBJECTS AND METHODS
The studies were carried out in Agricultural production cooperative named after Lenin and Dusym, LLC of Atninsky District, the LLC named after Tukay of Baltasinsky District, Biryulinskiy Stud Farm, OJSC and Hammer and sickle, LLC in Vysokogorsky District of the Republic of Tatarstan with 608 first-calf cows of the black-motley × Holstein breed and 265 first-calf cows of the Kholmogorskaya breed of the Tatarstan type, respectively.
To carry out molecular genetic studies in animal were collected blood samples from the jugular vein. DNA was extracted from the samples of whole preserved (10 mM of EDTA) blood using a combined alkaline method. DNA extraction procedure. 100 μl of blood is mixed with 1 ml of dH 2 O and centrifuged at 10,000 rpm for 10 minutes. The resulting supernatant is discarded, and 50 μl of 0.2 M NaOH is added to the precipitate and the mixture is thoroughly vortexed until the suspension is completely clarified. The resulting homogenate is thermostated at 60°C for 10 minutes. A proportional volume of 1 M Tris-HCl (pH 8.0) was added to the lysate followed by the careful vortexing of the mixture. 500 μl of 96% ethanol are added to the resulting homogenate followed by holding the mixture in a freezer (-20°C) for 30 minutes. The nucleoprotein complex is precipitated by centrifugation at 12,000 rpm for 10 minutes. The supernatant is discarded, and the residue is dried at 60°C for 12 minutes by opening the lid of the tube. 100 μl of 10% ammonia are added to the dried precipitate, the mixture is vortexed carefully and thermostated at 60°C for 10 minutes, then vortexed again and held in a thermostat at 60°C for 10 minutes.
The resulting homogenate is held in a thermostat at 95°C for 15 minutes with the lid of the tube open.
In animals, the CSN3 and BLG genotypes have been determined by a PCR-RFLP analysis.
The RFLP-identification of genotypes on the allelic variants A and B of the CSN3 gene was performed by treating 20 μl of a PCR sample of 10 U of the restriction enzyme HinfI in the 1 × buffer "O" (SibEnzyme, Russia) at 37°C overnight.
The RFLP-identification of genotypes on the allelic variants A and B of the BLG gene was performed by treating 20 μl of a PCR sample of 5 U of the restriction enzyme HaeIII in the 1 × buffer "C" (SibEnzyme, Russia) at 37°C overnight. Table 1 presents the spectrum of the genotypespecific RFLP fragments generated during the reaction.
The cheeseability of milk was determined with the help of a rennet and rennet fermentation sample. Preparation of a rennet enzyme solution. 1 g of rennet powder with an activity of 100 thousand units is dissolved in a mixture of distilled water and glycerol of an equal volume. After 24 hours, the solution is well mixed, filtered through a paper filter, poured into dark dishes and stored in a fridge for no more than 5 days. Immediately before use, the solution is diluted 25 times with distilled water. Then, 10 ml of the same sample of the mixed milk is added into each of three tubes. The tubes with milk are put in a water bath at 35°C, a thermometer is placed in one tube to monitor the water temperature. The milk temperature is brought to 35°C, then 1 ml of the diluted rennet enzyme solution of the same temperature is added into two tubes. The content of the two tubes is quickly mixed and placed in the water bath fixing the time. The temperature is maintained at 35°C. The duration of milk clotting time is determined in minutes, taking into account the time interval from the addition of the rennet to the formation of a dense clot. The heat resistance of milk was determined with the help of a thermal (crucible) sample. Setting a crucible sample. 2 ml of milk is added into each of molybdenum glass tubes. The tubes with milk are put in an ultrathermostat and heated to a temperature of 135°C fixing the time. If the consistency of milk does not change within 5 minutes, then it is considered heatresistant.
The thermostability of milk was also determined taking into account the time interval from the moment the tubes were placed in the ultrathermostat until the first signs of protein coagulation.
The variational statistical analysis of the results of the studies was carried out using the biometric method [26]. The reliability of the obtained results of the studies was confirmed by the tabular data of Student's criterion.

RESULTS AND DISCUSSION
The results of cattle genotyping on the A and B alleles of the CSN3 and BLG genes with the used sets of primers and restriction endonucleases for a PCR-RFLP analysis are satisfactory in terms of the reproducibility and identification of genotypes.
The primers BLGP3 and BLGP4 initiate the amplification of the BLG gene locus of cattle with a length of 262 bp, and the HaeIII-RFLP analysis of the generated genotype-specific fragments (AA = 153/109 bp, BB = 109/79/74 bp and AB = 153/109/79/74 bp) provides a correct genotyping procedure (Fig. 2).
The rationality of the use of whole milk for manufacturing protein-milk products, including cheese, is affected by its technological properties, such as coagulability under the influence of a rennet enzyme, the density of the formed casein clot and duration of milk clotting time.  The study has determined that the kappa-casein (CSN3) genotype of cows is associated both with the condition of a casein clot and with duration of milk clotting time. In all three samples, the milk from the cows of the Kholmogorskaya breed of the Tatarstan type with the AA genotype of the kappa-casein gene had the worst cheeseability properties. Both friable and flabby casein clots (Tables 2, 3, 4) were obtained from the milk of the cows (46.8-48.6%) of this genotype.
The presence of the allele B of the kappa-casein gene in the animal genome significantly affected the improvement of the condition of a casein clot. The proportion of milk with the condition of a casein clot characterized as dense in the cows of the homozygous genotype BB was 100%, and in the cows with the heterozygous genotype AB -81.8-84. 1%.
The most desirable in cheese-making is milk the clotting time of which when treated with a rennet enzyme is within the range of 15-40 minutes. If the milk clotting time is more than 40 minutes, there is a large loss of raw materials with a low yield of cheese due to a disruption in the manufacturing process. The best indicators on duration of milk clotting time have been noted in the first-calf cows with the genotype BB of the kappa-casein gene. The milk of these animals coagulated in the period with the lowest time interval -16.9-18.2 min. The milk clotting time in the animals with the AA genotype turned out to be longer and was 30.4-31.3 minutes (P < 0.001).
The similar studies carried out using a single sampling of black-motley × Holstein cows with different genotypes of the kappa-casein gene also showed that there are intergroup differences in the cheese-making properties of milk. The groups of the cows carrying the allele A of the kappa-casein gene in their genotype had a higher proportion of animals with the worst condition of a casein clot. Both friable and flabby casein clots were obtained from the milk of 50.0% of the cows with the AA genotype ( Table 5).
The presence of the allele B of the kappa-casein gene in the animal genome had a significant effect on the condition of a casein clot. The proportion of milk with the condition of a casein clot characterized as dense in the cows with the heterozygous genotype AB was 80.6%, and in the cows with the homozygous genotype BB was equal to 100.0% (Table 5).      The best indicators on duration of milk clotting time were characteristic of the first-calf cows with the genotype BB of the CSN3 gene. The clotting time of their milk was the shortest -18.9 min. The longest clotting time was noted for the milk of the cows with the AA genotype and was equal to 31.7 minutes. In this case, the milk from the animals with the heterozygous genotype AB was at the intermediate level of the analyzed indicator -25.4 min. The first-calf cows carrying the allele B of the CSN3 gene in their genome were favorably inferior to their coevals with the AA genotype by 6.3-12.8 min (Table 5).
Similar results were obtained when carrying out a rennet test of the milk of the cows with different CSN3 genotypes in the studies of animals of the Yaroslavl breed [27], of the holsteinized Kholmogorskaya breed of the "Tsentralny" type [28], the Samara type of blackmotley cattle [29], of the Ural black-motley breed [17], the red-motley breed of the created Volga type [30], the Volga type of the red-motley breed [31], the Simmental and red-motley breeds [16], the Italian Holstein breed [32], the Danish Jersey and Holstein breeds [33], the dairy breeds of different ecological zones of the Siberia, Sakha (Yakutia) and Macedonia, namely black-motley, Holstein, red steppe and Simmental [34], the Sicilian Cinisara breed [35], Estonian Holstein, red-motley Holstein, Estonian red, the Estonian native breed [36] and the Macedonian Holstein breed [37]. In their studies, the milk from the cows with the AB and BB genotypes of the CSN3 gene compared to the milk from the animals with the AA genotype when affected by the enzyme had shorter coagulation periods. However, the studies of Norwegian red cattle have provided some other results. Thus, the duration of milk clotting time when affected by a rennet enzyme from the animals with different genotypes of the kappa-casein gene was in the following order: АВ<АА<ВE<ВВ [38].
It is believed that the whey protein betalactoglobulin, like the other protein fractions of whey, does not lend itself to rennet coagulation, and therefore they are absent in cheese mass. Nevertheless, the genetic types of this protein can affect the process of isolating whey from a casein clot and thereby improve the quality of cheese mass [3].
The study revealed that of 2 sampling of blackmotley × Holstein first-calf cows with different betalactoglobulin (BLG) genotypes, the milk of the firstcalf cows with the BB genotype had the best cheesemaking properties. When affected by a rennet enzyme, a dense casein clot was obtained from the milk of 78.8%-80.0% of cows, and a flabby clot -from only 6.1%-6.7%, respectively (Tables 6 and 7).
The ability of milk to coagulate proved to be worse in the animals with the genotypes AB and AA of the beta-lactoglobulin gene. Thus, the yield of a dense and flabby clot was 56.6%-57.5% and 6.7%-8.2% (the genotype AB), as well as 42.8%-52.0% and 14.4%-16.0% (the genotype AA), respectively.
Most of the processing lines for cheese production are designed for the duration of the process of milk clotting to 40 minutes. The increase in milk clotting time leads to an increase in the losses of raw materials and, respectively, to a low cheese yield. The best indicators on clotting time were characteristic of the cows with the genotypes AB and BB of the beta-lactoglobulin gene. In these groups of animals, the milk clotting occurred for 27.3-29.1 min. This indicator in the cows with the AA genotype was the worst and was 29.8-33.0 minutes, which is higher than that in the animals carrying the allele B of the BLG gene by 0.7-4.8 min.
Similar results were obtained when carrying out a rennet test of the milk of the cows with different BLG genotypes in the studies of the domestic Kholmogorskaya breed [3], the Ukrainian black-motley breed [39], the Danish Jersey and Holstein breeds [33], Norwegian red cattle [38], the Russian black-motley and Bestuzhev breeds [22]. In their studies, the milk from the cows with the genotypes AB and BB of the BLG gene, in comparison with the milk from the animals with the genotype AA, had shorter coagulation periods when effected by an enzyme. However, different results were obtained in the studies of the Swedish red and Holstein breeds [40], the Estonian Holstein, red-motley Holstein, Estonian red and Estonian local breeds [36] and the French Holstein breed [41]. Thus, duration of milk clotting time when affected by a rennet enzyme from the animals with different BLG genotypes was in the following order: АА<АВ<ВВ. The studies of individuals of the Sicilian Cinisara breed with different genotypes of the beta-lactoglobulin gene also showed that the coagulation properties when affected by a rennet enzyme corresponded to the following sequence BB<AA<AB in terms of duration [35].
The long-term storage of milk and dairy products is impossible without high-temperature treatment (63-150°C) which is used for pasteurization, sterilization, thickening and drying. When treated by high temperatures, the product often undergoes irreversible protein coagulation and rapid milk coagulation. Therefore, the solution to the problem related to an increase in the heat resistance of milk is of high practical importance. In this regard, we have studied the heat resistance of milk of the cows with different genotypes of the kappa-casein gene.
The study has determined that the milk of three sampling of first-calf cows of the Tatarstan type with the BB genotype of the CSN3 gene had a lower thermostability (33.1-35.2 min), with the AA genotype -a high thermostability (60.1-65.8 min), and that with the genotype AB showed an intermediate value (57.5-62.9 min) ( Table 8).
It has been determined in the studies of the thermostability of milk of a single sampling of blackmotley × Holstein first-calf cows with different CSN3 genotypes that the milk of cows with the BB genotype had a lower thermostability (39.3 min), with the AA genotype -increased thermostability (57.2 min), and with the genotype AB -an intermediate value (56.5 min). The first-calf cows with the genotype AA of the CSN3 gene were superior to the coevals with the genotypes BB and AB by 17.9 min and 0.7 min, respectively (Table 9).
Similar results on the heat resistance of milk from the cows with different CSN3 genotypes were obtained in the studies of Holstein, Ayrshire, Kholmogorskaya and Kholmogor × Holstein cross-breeds of domestic lineage [3], the red-motley breed and the created Volga type of Russian breeding [30]. In their studies, the milk from the cows with the genotypes AA and AB of the CSN3 gene had a higher thermostability compared with the milk from the animals with the BB genotype. However, the studies of the Bestuzhev breed and domestic Bestuzhev × Ayrshire cross-breeds [3] gave some other results. Thus, the milk from the cows with the genotypes AB and BB of the CSN3 gene was more heat resistance compared to the milk from the animals with the AA genotype.   We also carried out a study of the thermostability of milk of two sampling of black-motley × Holstein cows with different BLG genotypes. The study showed that the thermostability of milk of first-calf cows with different BLG genotypes was within the ranges of 52.7-56.0 min (the genotype BB) and 58.5-58.9 min (the genotype AA). The animals carrying the B allele of the BLG gene were inferior in this indicator to the coevals with the genotype AA by 1.5-6.2 min (Table 10).
Similar results on the thermostability of milk of cows with different BLG genotypes were obtained in the studies of animals of the Russian black-motley breed [19], the Ukrainian black-motley breed [39] and the domestic black-motley and Bestuzhev breeds [22]. In their studies, the milk from the cows with the genotypes AA and AB of the BLG gene had a higher heat resistance compared to the milk from the animals with the genotype BB. However, the studies of the Holstein, Ayrshire and Kholmogorskaya breeds and Bestuzhev × Ayrshire cross-breeds of domestic selection [3] gave some other results. Thus, the thermostability of milk with different BLG genotypes was expressed in the following order: AA<AB<BB, while the order for the Bestuzhev breed and domestic Kholmogor × Holstein cross-breeds was AB<AA<BB, respectively.

CONCLUSIONS
The selected systems of cattle genotyping on the A and B alleles of the CSN3 and BLG genes by a PCR-RFLP analysis allowed us to genotype correctly the sampling of first-calf cows in several cattlebreeding farms in the Republic of Tatarstan.
The study of first-calf cows of the Kholmogorskaya breed of the Tatarstan type and black-motley × Holstein cows has shown that the best cheese-making properties of milk are inherent in the animals with the genotype BB and AB of the CSN3 gene. Their milk had the highest yield of the desired dense casein clot, as well as the shortest duration of milk clotting time, and they were significantly superior to their analogs with the AA genotype on these indicators. As for the BLG gene, the first-calf cows with the genotype BB and AB had the best cheese-making properties of milk. These animals were superior to their 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 of the Kholmogorskaya breed of the Tatarstan type and black-motley × Holstein 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 the black-motley × Holstein cows with the AA genotype.
It is advisable to use the milk from the cows with the genotypes BB and AB of the CSN3 and BLG genes that has the best cheese-making properties for manufacturing cheeses and products of lactic acid fermentation. When processing the milk from the cows with the AA and AB genotypes of the CSN3 gene as the most heat-resistant, it is advisable to use it to produce drinking pasteurized and sterilized milk with a long shelf life and canned milk. It is ineffective to differentiate cow milk on heat resistance with considering of the BLG genotype.