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.     

A competitive enzyme-linked immunosorbent assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine beta-casein.

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine beta-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine beta-casein. The bovine caseins in milk or cheese samples compete with the bovine beta-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine beta-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.     

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.     

Feta cheese texture: the effect of caprine and ovine milk concentration

Feta cheese was produced commercially with different caprine to ovine milk ratios. Milk fat concentrations, moisture and salt contents were similar for all the batches. However, the hardness and adhesive characteristics of the cheeses differed in relation to the milk ratio. The hardness of the cheese appeared to be correlated to increased goat milk content. Cryo-scanning electron microscopy (cryo-SEM) of the cheese samples showed that feta cheese with a higher proportion of caprine milk had a more compact and less porous appearance than feta produced from purely ovine milk. This difference in cheese structure helps to explain the difference in hardness between the samples.     

Isolation and characterisation of caseinmacropeptide from bovine,ovine, and caprine cheese whey

Based on the thermostability of caseinmacropeptide (CMP) and on the differences in molecular weight of its polymeric and monomeric forms, we have developed a method of isolating CMP from whey protein concentrate (WPC) and from liquid sweet cheese whey, particularly suited to large-scale industrial production. This procedure includes acidification and heating and ultrafiltration of cheese whey to give a CMP powder with a protein content from 75 to 79%. CMP obtained from WPC and from pure bovine, ovine, and caprine cheese whey were characterized. The CMP recovery was close to 71-76% and the purity determined by RP-HPLC ranged from 75 to 90%.     

Oxidative stability and antioxidant activity of bovine,caprine, ovine and asinine milk

This study compares the oxidative stability of fat isolated from bovine, caprine, ovine and asinine milk as well as the antioxidant activity of casein and whey from these milks. Fat from bovine and asinine milk showed the highest oxidative stability. The antioxidant activity of casein and whey was examined before and after an in vitro digestion process. Whey from asinine milk showed the highest antioxidant activity. The antioxidant activity of whey and casein increased after simulated intestinal digestion. In addition, the results of this study showed that asinine whey exhibited radical scavenging activity comparable with the strong synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).     

荧光定量PCR法计数农家干酪中动物双歧杆菌乳酸亚种

分别利用荧光定量PCR及平板菌落计数法计数农家干酪中双歧杆菌的菌体数,通过对结果的比较分析,建立一种适用于快速、敏感、特异的检测干酪中双歧杆菌活菌数的方法。利用传统工艺制备双歧杆菌农家干酪,在其贮存期间分别采用荧光定量PCR及平板菌落计数法计数干酪中双歧杆菌的数量。其中,在利用荧光定量PCR法检测时,对影响PCR定量准确的因素进行系统研究,包括设计双歧杆菌引物,并对引物特异性进行评价、考察,从干酪基质中提取DNA的数量和质量,建立标准曲线。引物特异性验证结果表明,引物专一性强。采用试剂盒法从干酪样品中提取DNA的纯度较好,OD_(260)/OD_(280)均在1.75~1.82之间。除贮存第1天外,荧光定量PCR法计数结果比平板计数法高0.39%~2.25%,未见显著差异。荧光定量PCR具有灵敏、特异、简便和快速的特点,可用于干酪中双歧杆菌的定量检测。     

Comparison of the characteristics of halloumi cheese made from ovine milk, caprine milk or mixtures of these milks

The aim of this work was to study the rheological, physicochemical and organoleptic characteristics of four types of halloumi cheese made from pure ovine milk, pure caprine milk or mixtures containing 15 or 30% caprine milk. All the milks were standardized for casein to fat ratio. Pure ovine milk gave the highest yield of halloumi cheese and caprine the lowest. No significant differences in acidity, pH, moisture, total protein and ash contents of the four types of cheese were found. In contrast, significant differences in soluble proteins, non-protein nitrogen, acid degree value and calcium contents were noted. Rheological examinations showed the hardness and the force at the point of fracture of caprine halloumi cheese to be lower than those of the other types of cheese. The same cheese presented the highest compression at the point of fracture and cohesiveness, while there were no differences in gumminess or chewiness. Members of the assessment panel showed a preference for halloumi cheese made from 30% caprine milk and 70% ovine milk.     

Qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milks using native-PAGE

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milk using whole milk samples as well as their whey protein fraction. Quantification was based on measuring band intensity of bovine β-lactoglobulins in all milk mixtures and bovine α-lactalbumin in caprine/bovine milk blends. Linear relationships were established between the band intensity of bovine β-lactoglobulins and α-lactalbumin vs. volume percentage of added bovine milk in all milk analysed, with the correlation coefficient from 0.9950 to 0.9998. These correlations enabling the quantification of bovine milk percentage within the wide range from 3% or 5% to 90% in caprine/bovine and ovine/bovine milk blends, respectively. The differences between the actual percentages of bovine milk present in the adulterated milk samples and those calculated using the regression lines were less than or equal to 5% for all samples. This method offers a rapid determination combined with unequivocal identification of the bovine whey proteins in almost every caprine/bovine or ovine/bovine milk mixtures.     

A study of the growth of Lactobacillus acidophilus in bovine, ovine and caprine milk

The fermentation of reconstituted bovine, ovine and caprine milks with Lactobacillus acidophilus was investigated. Better growth of Lb. acidophilus was observed in ovine milk, but higher acidities developed in caprine milk. The high acidity produced after 12 h of fermentation (pH 3.9) created a hostile environment for the survival of the microorganism in caprine milk.     

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography–mass spectrometry (HPLC–MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

HPLC and GLC analysis of the triglyceride composition of bovine, ovine and caprine milk fat

A total of 116 molecular species of triglycerides were identified in milk fat using a combination of HPLC and GLC. Triglyceride composition was predicted from the random composition, which was calculated on the basis of the mole fractions of the main fatty acids making up the total triglyceride fraction. The qualitative composition of the milk fat was similar in cows', ewes' and goats' milk. In all three milks the partition number of the main triglycerides was 46, but the proportions of the triglycerides with partition numbers of 34, 38, 42, and 48 exhibited substantial differences among the milks of the three species.     

Comparison of Coxiella burnetii shedding in milk of dairy bovine, caprine, and ovine herds

The shedding of Coxiella burnetii in bovine, caprine, and ovine milk was measured using PCR, in 3 herds for each species, the bulk tank milk samples of which were positive at the time of their selection. Milk samples of 95 cows, 120 goats, and 90 ewes were sampled over 16 wk, as was the bulk tank milk. The shedding of C. burnetii in vaginal mucus and feces was checked at the beginning of the experiment and 2 mo later. The clinical signs in the selected herds as well as the duration and the shedding routes differed among the 3 species. The cows were asymptomatic and shed C. burnetii almost exclusively in milk. In one of the caprine herds, abortions due to C. burnetii were reported. The goats excreted the bacteria mainly in milk. In contrast, the ewes, which came from flocks with abortions due to Q fever (C. burnetii infection), shed the bacteria mostly in feces and in vaginal mucus. This could explain why human outbreaks of Q fever are more often related to ovine flocks than to bovine herds. These excretions did not seem more frequent when the samples were taken close to parturition. The samples were taken from 0 to 421 d after parturition in bovine herds and from 5 to 119 d and 11 to 238 d after parturition in the caprine and ovine herds, respectively. The shedding in milk was sometimes intermittent, and several animals shed the bacteria but were negative by ELISA: 80% of the ewes were seronegative, underscoring the lack of sensitivity of the ELISA tests available for veterinary diagnosis. The detection of antibodies in milk seems more sensitive than it is in serum.     

Influence of ovine milk in mixture with bovine milk on the quality of reduced fat Muenster-type cheese

Reduced fat Muenster-type cheeses were manufactured from a mixture of bovine skim milk and ovine whole milk and from bovine milk only (control). Cheeses were evaluated at 15, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids. alpha(s1)-Casein degradation was similar for both cheeses during the aging period, but beta-casein degradation proceeded at a faster rate in the control cheese. The total amounts of free fatty acids remained constant throughout the ripening time; however, the cheeses produced with bovine/ovine milk yielded a significantly larger amount of caprylic (C8:0) and capric (C10:0) acids compared with the bovine milk cheeses. Lactobacilli increased during the aging period, while the populations of lactic acid bacteria, yeast and molds, and lipolytic organisms did not increase. Both cheeses had comparable cheese flavor intensity, but the bovine/ovine milk cheese had a greater occurrence of off flavors. The bovine/ovine milk cheeses were firmer than the bovine cheeses throughout the aging period.     

Changes during ripening in chemical composition,proteolysis, volatile composition and texture in Kashar cheese made using raw bovine,ovine or caprine milk

Kashar cheeses were manufactured from pure ovine (OV), bovine (BV) and caprine (CP) milk, and the chemical composition, cheese yield, proteolysis, hardness, meltability and volatile composition were studied during 90 days. Gross chemical composition, cheese yield and level of proteolysis were higher in OV cheeses than those of BV or CP cheeses. Glu, Val, Leu, Phe and Lys were the most abundant free amino acids (FAA) in the samples, and the concentrations of individual FAA were at the highest levels in OV cheeses with following BV and CP cheeses. Urea‐PAGE patterns and RP‐HPLC peptide profiles of the BV cheeses were completely different from the small ruminants’ milk cheeses (OV or CP). Higher and lower hardness and meltability values were observed in CP cheeses, respectively. OV cheeses resulted in higher levels of the major volatile compounds. In conclusion, the Kashar cheese made using OV milk can be recommended due to high meltability, proteolysis and volatiles.     

Transfer of oxytetracycline from ovine spiked milk to whey and cheese

Cheese-making trials with ovine milk spiked with oxytetracycline (OTC) at maximum residue limit (MRL) level were performed to study the OTC distribution between whey and cheese, and assess its effect in cheese-making process and cheese composition. An in-house validated method, based on liquid chromatography-tandem mass spectrometry, was adopted to detect OTC in milk, whey and cheeses. The MRL OTC spike induced a delay in the pH lowering causing a higher moisture content in 1-day OTC cheese compared with the control cheese. No effects of OTC were observed on the mass balance of the process, on the recovery of fat and protein and, in general, on physico-chemical parameters and gross composition of obtained cheeses. OTC was mainly recovered in the 1-day cheeses (61 ± 5%). Despite a 17 ± 8% OTC reduction observed during cheese ripening, probably due to partial degradation, remaining residues were not negligible and could contribute to reach the acceptable daily intake.     

Development of two screening duplex PCR assays for genetically modified organism quantification using multiplex real-time PCR master mixes

Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development, allowing for the detection of a large number of GMOs. To improve GMO detection efficiency, using specific multiplex master mixes, we developed two real-time PCR screening duplex PCR assays for the detection of P35S/Tnos and Pnos/T35S sequences. By combining these tests, we were able to reduce the time and cost of analysis. For the Pnos/T35S duplex, good sensitivity was obtained using one of the mixes compared to the others. Both duplexes had 100% specificity when tested on DNA from GM maize, rapeseed and soybean. When the duplexes were tested on DNA containing various amounts of GM maize and soybean, the corresponding targets were detected. The detection limit of our methods was found to be between 2 and 8 haploid genome copies for both P35S/Tnos and Pnos/T35S tests. In summary, with high efficiency and good linearity, the proposed two screening duplexes allow for more efficient GMO detection.     

The quality of milk in relation to cheese manufacture*

The gross composition and bacteriological quality of milk used in cheese manufacture can have a significant influence on both the yield and the quality of the cheese produced. Within the past JO years there have been changes in terms of milk production and utilization in the dairy industry in the United Kingdom which could influence both the composition of milk and its bacteriological quality. Methods to eliminate any undesirable effects of changes in milk quality are available. The advantages of standardizing the casein to fat ratio in milk and extending the storage life of milk by thermization and deep cooling are discussed in relation to cheese manufacture.