A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat,milk and cheese

Goat foodstuffs are considered as healthy foods with high nutritional value. This study demonstrated the development and validation of a triplex real-time PCR on the basis of species-specific and species-conservative TaqMan probes for the simultaneous identification of caprine and bovine DNA in meats, milk and cheeses with a prerequisite designed endogenous control. In this research, caprine and bovine meat, milk and cheese were specifically identified via developed primers and probes, and the limits of detection of this methodology were 0.005 and 0.01 ng DNA of milk and cheese from goat, and 0.01 and 0.05 ng DNA of milk and cheese from cow. Taken together, this approach was elaborated to address dairy adulteration issues to eliminate the fraud of economically motivated goat milk and cheese adulteration by adding cow milk.     

Nutritional and sensory characteristics of Minas fresh cheese made with goat milk,cow milk,or a mixture of both

This study aimed to assess and compare the nutritional, technological, and sensory characteristics of Minas fresh cheese made with goat milk, cow milk, or a mixture of the two stored in cold conditions for 21 d. The yield and centesimal composition of the cheeses were not affected by the type of milk used in their preparation. Reductions were observed in the moisture content, pH, proteolysis index, and instrumental hardness; moreover, increases were observed in the syneresis, acidity index, and depth of proteolysis index in all cheeses. The percentages of caprylic, capric, oleic, and linoleic fatty acids were higher in goat milk cheese and cheese made with a mixture of goat and cow milk compared with cow milk cheese, and a sensory evaluation revealed differences in color, flavor, and aroma between the cheeses. The preparation of Minas fresh cheese with a mixture of goat and cow milk can be a viable alternative for dairy products in the market that can be characterized as high-quality products that meet consumer demands.     

FATTY ACID COMPOSITION AND CONJUGATED LINOLEIC ACID CONTENT OF COW AND GOAT CHEESES FROM NORTHWEST ARGENTINA

ABSTRACT

In this study, we evaluated chemical characteristics, fatty acid composition and conjugated linoleic acid (CLA) content of cow and goat cheeses from Northwest Argentina. Similar chemical and fatty acid composition were determined in milk and cheese of both species. Palmitic, oleic and myristic acids were the most abundant fatty acids in dairy products. CLA level averaged 0.85 and 0.96 in milk and 0.76 and 1.04 g/100 g of fatty acids in cheese of cow and goat, respectively. Cis‐9,trans‐11 was the major isomer present in both species. Significant differences in CLA desaturase activity were observed, showing a value of 0.068 and 0.064 in milk, and 0.077 and 0.071 in cheese of cow and goats, respectively. Good nutritional properties were determined for cheeses of both species, which are fed on natural pasture during spring and summer seasons. Goat's cheese represents a higher source of CLA for human consumers than cow's cheese, offering from 156.6 to 222.6 mg/ 100 g of sample.

PRACTICAL APPLICATIONS

The present work shows the fatty acid composition and chemical characteristics of two fresh cheeses manufactured with cow and goat milk. Animals were fed on natural pasture during summer and spring seasons. It is known that pasture increases conjugated linoleic acid (CLA) concentration in milk fat, and the content in cheese is directly related to it. The CLA content of dairy products for the human consumers was analyzed, showing goat cheese with high polyunsaturated fatty acid content, including CLA. Cow and goat fresh cheese offer CLA as many ripening products of different countries, as cheddar or hard cheeses. Lipid composition of food is related to many illnesses, but some compounds are beneficial to human health. The main sources of CLA are milk and cheeses, and in Northwest of Argentina, no data are reported about it, where artisanal cheeses are consumed by the population. Therefore, the atherogenicity index was determined as well.     

Interlaboratory validation of two multiplex quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk proportions in cheese

Milk products like yogurt, flavoured milk-drinks, curd and cheese may be composed of milk different from cow, namely of ruminant species like sheep and goat. Such products experience an increasing demand in Europe and are recognised as healthy and naturally finished specialities. To verify declared milk compositions in these dairy products, two different quantitative multiplex PCR systems have been evaluated in a comparison test with eleven participating laboratories employing two unknown, traditionally manufactured cheeses with different degrees of ripening to determine milk fractions from cow, ewe and goat. Precision and accuracy was investigated by calibration to dilutions of DNA mixtures and to homologous matrix-adapted reference cheeses, respectively. As expected, independent of the particular method, best inter- and intra-laboratory accuracy has been achieved through the use of homologous reference cheese standards. Furthermore, it has been shown that cheese ripening and the concomitant DNA degradation exert an inverse effect on the method’s sensitivity and performance characteristics. Additionally, a broad market survey of different milk products demonstrated its applicability as an efficient analytical tool for food control laboratories to challenge the authenticity of milk and its products from small ruminants.     

Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism

A new polymerase chain reaction (PCR)-based method was developed to detect cows milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0.1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.     

Application of a polymerase chain reaction to detect adulteration of ovine cheeses with caprine milk

A polymerase chain reaction (PCR) procedure has been applied for the detection of caprine milk in ovine cheeses by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of a primer pair specific for goat in PCR analysis of cheese samples, enabled the amplification of a goat 122 bp fragment with a sensitivity threshold of approximately 1%, whereas no amplification signal was achieved with sheep's, cow's and water buffalo's milk DNA. This study demonstrates the usefulness of the proposed PCR assay for the qualitative detection of goats’ milk in ewes’ cheeses, and may therefore provide a simple and accurate approach applicable to the authentication of cheese or other dairy products in routine monitoring programs.     

蛋白质组学技术在不同物种乳掺假检测中的应用

乳品是人类重要的营养源，然而乳品掺假现象时常发生，近年来尤以向乳品中掺假动、植物蛋白，向特色畜乳中掺假牛乳等方式为主，这不仅损害了消费者的利益，甚至会危害消费者的健康。该研究总结了目前常见的掺假行为及相关检测方法，并介绍了蛋白质组学技术——一种通过确立特定生物标记物来区别不同物种乳的技术。作者查询了国内外近十年来牛乳和特色乳掺假方面的研究报道，关键词设置为“蛋白质组学”、“乳品”、“真实性”、“生物标记物”等，按照奶畜乳类别将所得文献进行分类。分别对奶牛乳、羊乳、驼乳、水牛乳、牦牛乳、驴乳的掺假物、潜在标记物和检出限等方面进行了总结和分析，以期为乳品真实性鉴定和保障乳品质量安全提供有效的工作思路。     

Robust PCR‐based method for quantification of bovine milk in cheeses made from caprine and ovine milk

To prevent fraud and enhance quality assurance, credible analysis of dairy products is crucial. A common problem is the addition of cheaper bovine milk to caprine and/or ovine dairy products and when not declared addition of bovine milk constitutes fraud. The aim was to develop a rapid, robust and sensitive method for the identification of adulteration of caprine and/or ovine cheeses with bovine milk. New quantitative real‐time polymerase (qPCR) assays were designed for the specific determination of bovine DNA (Cow1) and bovine, caprine and ovine DNA (BoCaOv). These were applied to 17 samples of caprine cheese and 24 of ovine cheese. Results showed that 17% (7/41) of these cheeses contained >5% bovine milk. As bovine milk was not declared as an ingredient in any of the samples, this represents adulteration. Other cheeses that contained detectable bovine milk at ≤5% (22%; 5/41) might pose a health risk to people allergic to bovine milk.     

Changes on expected taste perception of probiotic and conventional yogurts made from goat milk after rapidly repeated exposure

Goat milk yogurt is an excellent source of fatty acids, protein, and minerals; however, it is not well accepted by many consumers, due to its typical flavor derived from caprylic, capric, and caproic acids present in this milk and dairy products. Recently, the repeated-exposure test has been used to increase the consumption of particular foods. This methodology has been used to increase children’s willingness to eat food in some settings and has also been used to reduce sodium in soup. Based on these considerations, the aim of this study was to investigate whether repeated exposures may increase acceptance of both goat milk yogurt and probiotic goat milk yogurt. In a pre-exposure session, a total of 45 panelists (28 females and 17 males) from southeastern Brazil, who were not used to consuming dairy goat milk, evaluated the expected taste perception and the perceived liking after tasting 3 yogurt preparations. Then, consumers were randomly divided into 3 groups and participated in rapidly repeated exposure sessions performed within 6 d. Each panelist consumed only the yogurt that he or she would be exposed to. The day after the exposure sessions, all panelists returned to participate in the postexposure session and were asked to evaluate acceptance, familiarity, and the “goaty taste” characteristic of each yogurt. Regarding the expected liking before tasting, results showed higher expectations for cow milk yogurt compared with goat milk yogurt, which proved that consumers were not familiar with the goat milk yogurt. Likewise, only cow milk yogurt presented high acceptance and familiarity rates, confirming that these panelists were used to consuming cow milk products. With respect to the rapidly repeated exposure, 6 d were enough to significantly increase the consumers’ familiarity with goat milk yogurt and probiotic goat milk yogurt. However, this method was not suitable to significantly increase the acceptance of such products. Nonetheless, a correlation existed between the exposure sessions and the increase in acceptance of the exposure groups. Thus, hypothetically, the increasing of exposure sessions could be a strategy to increase goat milk product acceptance.     

Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay

The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.     

掺假羊乳及其制品中牛乳的检测技术研究进展

羊乳具有营养价值高、蛋白质组成更接近人乳、脂肪球直径小及致敏性低等优点, 更利于人体消化吸收, 受到消费者和乳品企业的青睐。近年来我国羊乳产业发展迅速且潜力巨大, 但由于受羊乳产量和养殖规模的限制, 羊乳价格昂贵, 市场中存在羊乳及其制品掺假牛乳的现象, 且掺假手段多样, 难以辨别。为了保证消费者的健康和权益, 保障羊乳市场良性发展, 羊乳及其制品的纯正性、真实性检测已经成为热点研究方向。本文通过分析基于乳中蛋白质、脂肪和核酸差异的羊乳中牛乳掺假检测技术的研究现状, 介绍和探讨了各检测技术的基本原理及其在应用中的优缺点, 同时展望羊乳掺假检测技术的发展方向, 旨在为牛羊乳混合掺假检测技术的进一步发展提供资料参考和思路。     

Proteolysis and Lipolysis of Goat Milk Cheese

Numerous varieties of goat milk cheeses are produced worldwide. Maturation or ripening of goat and other species milk cheeses is governed by interplay of many different factors. Proteolysis and lipolysis are two major biochemical processes in the multifaceted phenomenon of cheese aging, which involves a variety of chemical, physical, and microbiological changes under controlled environmental conditions. Proteolysis of cheeses in general is influenced by several factors including plasmin, chymosin, protease from starter and nonstarter bacteria, pH and moisture levels of the curds, storage temperature and time, salt content, salt-to-moisture ratio, and humidity. Primary factors affecting lipolysis in cheeses are fatty acid composition, lipolytic enzymes, lipolytic microorganisms, moisture, temperature, storage time, oxygen, and surface area, etc. Several analytical techniques have been used to measure proteolysis of goat and (or) cow milk cheeses during ripening, such as solubility of peptides and amino acids in various solvents or precipitants, liberation of reactive functional groups, various forms of chromatography, and different forms of electrophoresis. Lipolysis of goat milk cheeses has been estimated by acid degree value (ADV), acid value, and free fatty acid concentration, while lipid oxidation of dairy goat products can be determined by peroxide value, thiobarbuturic acid value (TBA). Recent reports have shown that goat cheeses had greater rates of protein degradation than cow counterparts, and that aging time and temperature synergistically elevated most of proteolytic and lipolytic indices in goat cheeses. This paper will further discuss proteolytic and lipolytic characteristics of goat milk cheeses.     

SDS-PAGE of Proteins in Goat Milk Cheeses Ripened under Different Conditions

Proteolytic characteristics of five varieties of commercial goat milk cheeses and a cow milk Cheddar aged under different conditions were evaluated by SDS-PAGE and an advanced densitometry system. All fresh goat cheeses had distinctively lower intensities of α_S1-casein (CN) bands than those of cow milk Cheddar, whereas intensities of β-CN were much greater in the goat cheeses. The PAGE patterns clearly displayed α_S2-CN in all goat cheese, but it was negligible in cow milk Cheddar. The greater protein degradation in hard goat cheeses than cow milk Cheddar at 4°C and 22°C strongly correlated with water-soluble nitrogen compound concentrations and densitometric values of corresponding cheeses.     

Characterization of major and trace minerals,fatty acid composition,and cholesterol content of Protected Designation of Origin cheeses

Cheese provides essential nutrients for human nutrition and health, such as minerals and fatty acids (FA). Its composition varies according to milk origin (e.g., species and breed), rearing conditions (e.g., feeding and management), and cheese-making technology (e.g., coagulation process, addition of salt, ripening period). In recent years, cheese production has increased worldwide. Italy is one of the main producers and exporters of cheese. This study aimed to describe mineral, FA, and cholesterol content of 133 samples from 18 commercial cheeses from 4 dairy species (buffalo, cow, goat, and sheep) and from 3 classes of moisture content (hard, <35% moisture; semi-hard, 35–45%; and soft, >45%). Mineral concentrations of cheese samples were determined by inductively coupled plasma optical emission spectrometry, and FA and cholesterol contents were determined by gas chromatography. Moisture and species had a significant effect on almost all traits: the highest levels of Na, Ca, and Fe were found in cheeses made from sheep milk; the greatest level of Cu was found in cow milk cheese, the lowest amount of K was found in buffalo milk cheese, and the lowest amount of Zn was found in goat cheeses. In all samples, Cr and Pb were not detected (below the level of detection). In general, total fat, protein, and minerals significantly increased when the moisture decreased. Buffalo and goat cheeses had the highest saturated FA content, and sheep cheeses showed the highest content of unsaturated and polyunsaturated FA, conjugated linoleic acid, and n-3 FA. Goat and sheep cheeses achieved higher proportions of minor FA than did cow and buffalo cheeses. Buffalo cheese exhibited the lowest cholesterol level. Our results confirm that cheese mineral content is mainly affected by the cheese-making process, whereas FA profile mainly reflects the FA composition of the source milk. This study allowed the characterization of mineral and FA composition and cholesterol content and revealed large variability among different commercial cheeses.     

Application of immunological methods for the detection of species adulteration in dairy products

A number of enzyme‐linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.     

High-Pressure Treatment and Freezing of Raw Goat Milk Curd for Cheese Manufacture: Effects on Cheese Characteristics

High-pressure treatment of raw goat milk curd was investigated as an alternative to thermal treatment of milk in cheese manufacture, and curd freezing as a procedure to surmount the seasonality of goat milk production. Experimental cheeses were made by mixing (70:30) fresh cow milk curd with frozen curd from pasteurized goat milk (PGC), frozen curd from raw goat milk (RGC), or frozen pressurized curd from raw goat milk (PRGC). Control cheese was made from a mixture (70:30) of pasteurized cow and goat milk. RGC cheese showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, whereas PRGC cheese had maximum aminopeptidase activity, esterase activity, and overall proteolysis. Control cheese exhibited the highest dry matter content and peptide levels, the lowest concentration of free amino acids, the highest concentration of volatile compounds such as free fatty acids, alcohols and esters, and the firmest texture. Differences in sensory characteristics between experimental and control cheeses were of minor importance. High-pressure treatment of curd allowed the production of cheese of bacteriological quality similar to that of control cheese made using pasteurized milk, while curd freezing did not alter the sensory characteristics of experimental cheeses with respect to control cheese.     

Fatty acid composition and CLA content in goat milk and cheese samples from Umbrian market

Goat dairy products are considered as a suitable alternative to cow milk products for some special categories of consumers (infants, old, and convalescent people). In the present research, a study on the lipid fraction of commercial goat milk and cheese samples purchased from Umbrian market was carried out. Particular attention has been paid to evaluating conjugated linoleic acid (CLA) content and determining some interesting nutritional indexes. The results of goat milk samples showed that saturated, monounsaturated and polyunsaturated fatty acid contents were on average 71.2, 23.4, and 3.8 %, respectively. CLA content (cis-9, trans-11 isomer) was on average 11.5 mg/100 g in milk. Considerable variability was observed for n6/n3 ratio that varied from 2.7 to 10.6 in the considered goat milk samples. With regard to goat cheese samples CLA concentration was on average 118.8 mg/100 g in fresh cheese and 217.7 mg/100 g in semi-hard cheese, and the difference was not significant. Interestingly, α-linolenic acid content was higher and n6/n3 ratio was lower in semi-hard with respect to fresh cheese samples (p < 0.05).     

Microbial dynamics during the ripening of a mixed cow and goat milk cheese manufactured using frozen goat milk curd

To overcome the seasonal shortage of goat milk in mixed milk cheese manufacture, pasteurized goat milk curd and high-pressure-treated raw goat milk curd manufactured in the spring were held at −24°C for 4 mo, thawed, and mixed with fresh cow milk curd for the manufacture of experimental cheeses. Control cheeses were made from a mixture of pasteurized cow and goat milk. The microbiota of experimental and control cheeses was studied using culture-dependent and culture-independent techniques. Bacterial enumeration by classical methods showed lactic acid bacteria to be the dominant population in both control and experimental cheeses. In total, 681 isolates were grouped by partial amplified rDNA restriction analysis (ARDRA) into 4 groups and identified by 16S rRNA gene sequencing as Lactococcus lactis ssp. lactis (563 isolates), Leuconostoc pseudomesenteroides (72 isolates), Lactobacillus spp. (34 isolates), and Lc. lactis ssp. cremoris (12 isolates). Temporal temperature gradient gel electrophoresis (TTGE) analysis of cheese showed (1) the predominance of Lc. lactis in all cheeses; (2) the presence of Leu. pseudomesenteroides population in all cheeses from d 15 onward; (3) the presence of a Lactobacillus plantarum population in control cheese until d 15 and in experimental cheeses throughout the ripening period. Due to the most diverse and complete set of peptidases present in the genus Lactobacillus, the prevalence of this population in experimental cheeses could give rise to differences in cheese flavor between experimental and control cheeses.     

Changes in texture,composition and sensory characteristics of Camembert cheese made from a mixture of goat milk and cow milk during ripening

The study was conducted to evaluate the nutritional, textural and sensory characteristics of Camembert cheese made from goat milk, cow milk and their blends. The type of milk used affected yield, physicochemical properties and fatty acid profile of the cheeses. The percentages of C6:0, C8:0 and C10:0 acids were higher in the cheeses containing ≥ 75% goat milk. The sensory evaluation results showed differences in texture, colour, aroma and flavour among the cheeses. Cheeses made with the mixture of cow and goat milk particularly 50% goat milk mixed with 50% cow milk (50G:50C) maintained particular positive nutritional characteristics especially with respect to the mineral contents, fatty acids and consumer acceptability.