Characterization of the genetic polymorphism linked to the ß-casein A1/A2 alleles using different molecular and biochemical methods.

The 2 major subvariants of ß-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of ß-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.

Effects of preservative, storage time, and temperature of analysis on detailed milk protein composition determined by reversed-phase high-performance liquid chromatography.

Milk preservative and freezing are used as strategies to prevent microbial growth and milk degradation, especially when immediate analytical processing is not feasible. The effects of the addition of preservative and freezing procedures have been investigated mainly in relation to milk gross chemical composition predicted through mid-infrared spectroscopy. This study aimed to determine whether different preservatives (i.e., no preservative, hydrogen peroxide, Bronopol, and Azidiol), freezing times (i.e., 0, 7, and 30 d), and temperatures of analysis (i.e., 5 and 21°C) influence the composition of milk protein fractions determined through reversed-phase HPLC. Bulk milk samples for the analysis of protein profile were collected from 5 commercial dairy farms. Data were analyzed with a linear mixed model, which included type of preservative, time of storage, temperature of analysis, and the interaction between type of preservative and time of storage as fixed effects, with the farm and the residual as random effects. Samples with no preservative had the greatest amount of all protein fractions, whereas Bronopol-preserved milk had the lowest amount. Increasing storage time under freezing conditions had a nonlinear detrimental effect on milk protein fractions. The temperature of analysis significantly contributed to the variation of κ-casein, ß-casein, αS1-casein, ß-lactoglobulin, and α-lactalbumin fractions. The z-scores were calculated to evaluate the similarity between detailed protein profile of fresh milk without preservative analyzed at 5°C and detailed protein profile of milk treated according to the tested conditions. Overall results suggested a good agreement between different analytical conditions. Still, short storage time under freezing conditions is recommended to avoid degradation of milk protein fractions and consequent analytical underestimation.

Detailed comparison between organic and conventional milk from Holstein-Friesian dairy herds in Italy.

Several studies have reported gross composition differences between organic and conventional milk; however, most studies have not considered other factors such as breed and diet ingredients, which are known to influence milk composition. Thus, this study aimed to provide a detailed characterization of Holstein-Friesian cow milk from organic (ORG) and conventional (CONV) herds with similar diet ingredients and in the same geographic area. Bulk milk samples (n = 225) of 12 ORG and 12 CONV farms were collected from September 2019 to August 2020. Farms were located in Northern Italy, included corn (meal, silage, or both) in the lactating diets, and had similar management conditions, but ORG herds spent a period on pasture. Factors affecting milk composition were tested using a linear mixed model, which included calendar month, farming system (ORG and CONV), and their interactions as fixed effects, and farm nested within farming system as random effect. Results showed that total fat, lactose, vitamin E, and AA did not significantly differ between farming systems. Total protein and casein contents were significantly lower in ORG than CONV herds, and somatic cell score (SCS) was greater in ORG than CONV. Among minerals, differences were observed for Fe, K, Mg, and S in some months, being lower in ORG than CONV for K, Mg, and S and greater or lower for Fe depending on the month. Among fatty acid (FA) groups, index, and ratios, only polyunsaturated FA and n-3 FA tended to be greater in ORG than CONV, and cis-FA were greater in ORG than CONV during October. Among the most abundant individual FA, only C16:1n-9 differed, being lower in ORG than CONV. The calendar month (and hence seasonal feed ration) was significant for milk gross composition, SCS, vitamin E, mineral profile (except for Mo, Sr, and Zn), AA profile, FA groups (except for medium-chain FA), FA index and ratios, and individual FA (except C16:0). We conclude that the overall milk composition was quite similar between the 2 farming systems. This could be related to the similarity of the selected farms, the Holstein-Friesian breed, and generally high level of intensity in both farming systems.

Technical note: Repeatability and reproducibility of curd yield and composition in a miniaturized coagulation model.

Miniaturized coagulation (MC) models have been proposed for the evaluation of curd yield (CY) in individual milk samples of different dairy species and breeds, and for the analysis of cheese microstructure and texture. It is still unclear if MC using less than 50 mL of milk is suitable to evaluate CY and chemical composition, and if preservative added to raw milk may interfere with MC process. Therefore, this study aimed at evaluating repeatability and reproducibility of CY, curd moisture, and fat and protein content on curd dry matter (DM) from MC trials using 40 g of milk. Miniaturized coagulations were performed by 3 different operators on 3 consecutive days, using raw milk (RM) and raw milk added with preservative (RMP). Repeatability of CY, calculated as relative standard deviation on 6 miniaturized curds obtained within a day by the same operator, was below 5% for MC carried out with both RM and RMP. The Horwitz ratio, which is the ratio between measured and expected reproducibility, highlighted good reproducibility for CY from RM and fair reproducibility for CY from RMP. The same ratio highlighted lower accuracies for curd moisture and fat and protein content on curd DM, especially for MC trials carried out with RMP. The z-test was performed to evaluate the similarity between curds manufactured with RM and RMP in terms of average yield and chemical composition; z-scores did not highlight significant differences between values obtained from MC carried out with RM and RMP. It can be concluded that preservative had negligible effects on MC, giving the opportunity to extend milk physical and chemical stability, to schedule laboratory trials on longer time span, and to broaden the sample size within a batch of analyses.

Effects of animal versus vegetal rennet on milk coagulation traits in Mediterranean buffalo bulk milk.

Given consumer interest in Mozzarella di latte di Bufala and other cheeses, and the growing interest of the cheese industry in offering products adequate for lactovegetarian consumers, this study aimed to compare clotting capacity of vegetal and animal rennet in buffalo milk. Milk coagulation properties of 1,261 buffalo bulk milk samples collected during milk quality testing were assessed by lactodynamography using commercial animal (75% chymosin and 25% bovine pepsin) and vegetal (Cynara cardunculus) rennets. Chemical composition of milk samples was predicted by MilkoScan (Foss Analytics, Hillerød, Denmark) calibrated with specific buffalo standards. Rennet effect (animal versus vegetal) was statistically analyzed with a paired t-test. Fat, protein, and lactose contents of milk samples were 7.94%, 4.52%, and 4.80%, respectively. A similar variability of milk coagulation properties was observed with both rennets, with the exception of greater variability of curd firmness at 30 min after the addition of vegetal rennet compared with animal rennet (73 and 26%, respectively). On average, when using plant rennet, milk started to coagulate and reached the 20-mm coagulum 12 ± 0.22 min and 1.9 ± 0.20 min, respectively, later than with animal rennet. Thirty minutes after rennet addition, curds were almost twice as firm in animal as in vegetal rennet (difference of 23.92 ± 0.66 mm). However, curd firmness at 60 min was only 1.21 ± 0.39 mm thicker with vegetal than with animal rennet. Moreover, when using animal rennet, 99.52% of samples started coagulating within the first 30 min of analysis, whereas only 70.42% did so when using vegetal rennet. We conclude that vegetal rennet has the capacity to coagulate buffalo milk, achieving a similar curd firmness to that of animal rennet at 60 min. Further studies are needed to evaluate the sensory characteristics and consumer acceptability of Mozzarella di latte di Bufala processed with vegetal rennet.

Validation of a gold standard method for iodine quantification in raw and processed milk, and its variation in different dairy species.

Adequate milk consumption significantly contributes to meeting the human iodine recommended daily intake, which ranges from 70 µg/d for infants to 200 µg/d for lactating women. The fulfilment of iodine recommended daily intake is fundamental to prevent serious clinical diseases such as cretinism in infants and goiter in adults. In the present study iodine content was measured in raw and processed commercial cow milk, as well as in raw buffalo, goat, sheep, and donkey milk. Iodine extraction was based on 0.6% (vol/vol) ammonia, whereas iodine detection and quantification were carried out through an inductively coupled plasma mass spectrometer analyzer. Among processed commercial cow milk, partially skimmed pasteurized milk had the greatest iodine content (359.42 µg/kg) and raw milk the lowest (166.92 µg/kg). With regard to the other dairy species, the greatest iodine content was found in raw goat milk (575.42 µg/kg), followed by raw buffalo (229.82 µg/kg), sheep (192.64 µg/kg), and donkey milk (7.06 µg/kg). Repeatability of milk iodine content, calculated as relative standard deviation of 5 measurements within a day or operator, ranged from 0.96 to 1.84% and 0.72 to 1.16%, respectively. The overall reproducibility of milk iodine content, calculated as relative standard deviation of 45 measurements across 3 d of analyses and 3 operators, was 4.01%. These results underline the precision of the proposed analytical method for the determination of iodine content in milk.