Total antioxidant activity of bovine milk: Phenotypic variation and predictive ability of mid-infrared spectroscopy

The phenotypic variation of milk total antioxidant activity (TAA) and the ability of mid-infrared spectroscopy to predict this novel trait was investigated. Total antioxidant activity was measured through the reference spectrophotometric method on 1249 individual milk samples of Holstein Friesian cows. Sources of variation of milk TAA were investigated using a mixed model, which included the fixed effects of days in milk, parity and calving season, and the random effects of herd-test-date and error. Mid-infrared spectroscopy prediction models were developed using partial least squares regression approach. The average level of milk TAA was 6.93 mmoL L⁻¹ of Trolox equivalents; this exhibited a coefficient of variation of 15% and showed weak phenotypic correlations with milk quality traits. Values of TAA were lower in early lactation than in late lactation. Mid-infrared spectroscopy prediction models reached a coefficient of determination in external validation of 0.41, suggesting that they are not adequate for analytical purposes.     

Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese

The aim of this work was to develop an assay capable of detecting adulteration of soft goat, sheep and buffalo milk cheese with bovine milk from cheaper sources. A previously developed indirect competitive ELISA had a lower sensitivity when applied to cheese, compared with milk. A sandwich ELISA was developed utilising the same monoclonal antibody in combination with a polyclonal goat anti-bovine IgG antibody. Once optimised, the ELISA was found to be highly specific. Detection limits in milk were 0.001% cows’ milk adulteration of sheep or buffalo milk, and 0.01% cows’ milk adulteration of goat milk. Detection limits in soft cheese were 0.001% in goat cheese and 0.01% in sheep or buffalo cheese. The assay was highly reproducible with both intra- and inter-assay coefficient of variation <10%. The ELISA performance makes it suitable for development as a kit for use in routine surveillance of milk and soft cheese.     

Effect of antioxidant activity and functional properties of Chingshey purple sweet potato fermented milk by Lactobacillus acidophilus, L. delbrueckii subsp. lactis, and L. gasseri strains

In this study, individual selected lactic acid bacteria strains Lactobacillus acidophilus (LA), L. delbrueckii subsp. lactis (LDL), and L. gasseri (LGA) were grown in Chingshey purple sweet potato (CPSP) substrate/media. CPSP is rich in anthocyanin, which possesses antioxidant activity and in vitro cell assay. The antioxidant ability and functional properties of the fermented milk were examined. High-performance liquid chromatographic (HPLC) method was used to analyze the free amino acid, organic acids, and anthocyanin content. Total phenolic compounds, scavenging effects of 1,1-diphenyl-2-picyl-hydrazyl (DPPH) radicals, and scavenging effects of superoxide anion radicals were determined to evaluate the antioxidant ability of the samples. The cell proliferation of the fermented PSP milk was evaluated by 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl Tetrazolium Bromide (MTT) assay. The result indicated that the antioxidant ability of the fermented CPSP milk through LA, LDL, and LGA strains was significantly higher than CPSP. The main anthocyanins present in the samples are cyanidin and delphinidin. MTT assay has revealed that incubation with both PSP and fermented CPSP milk prevented the cell death of macrophage-like RAW264.7 cells. The potential health benefit of fermented PSP milk through LA, LDL, and LGA strains makes the further application of CPSP in health food highly worthwhile. PRACTICAL APPLICATION: (1) In our study, we have employed the γ-aminobutyric acid (GABA), organic acid contents, total phenol content, anthocyanins content, DPPH, oxygen radical absorbance capacity, superoxide dismutase activity assay, and cytotoxicity assay to assess the functional properties of fermented CPSP milk by different lactic acid bacteria. (2) Our results have revealed that the fermented CPSP milk samples possess high GABA concentrations, organic acid contents, anthocyanins contents, and antioxidant activity. This will provide potential opportunity to develop different functional food products from fermented CPSP milk. (3) The potential health benefit of fermented CPSP milk makes the further application of CPSP in health food highly worthwhile.     

黄山贡菊抗氧化活性成分提取工艺优化与HPLC-MS分析

探讨黄山贡菊的抗氧化活性化学成分。以FRAP及DPPH法分析抗氧化活性,采用响应面法优化黄山贡菊抗氧化活性成分的提取条件。在乙醇浓度60%,液料比35:1 (mL/g),超声时间25 min的条件下,黄山贡菊提取物的总抗氧化能力为(2.134±0.011) mmol/L(RSD=1.97%,n=3),与预测值(2.010 mmol/L)吻合很好。总黄酮当量为7.88 mg芦丁/L的黄山贡菊提取物的抗氧化能力相当于2.87 mmol/L亚铁离子的还原力;总黄酮当量为25.85 mg芦丁/L的提取物的DPPH自由基清除率为94.81%。采用HPLC-MS分析黄山贡菊提取物的抗氧化活性成分,共鉴定出了113个化合物,含黄酮类化合物72个,绿原酸及衍生物7个、香豆素类8个。这些化合物可能是黄山贡菊具备抗氧化活性的物质基础。     

Production of Caseinophosphopeptides from Na-Caseinates Prepared from the Milk of Several Species by a Proteinase of Lactobacillus helveticus PR4

The aim of this work was to produce caseinophosphopeptides (CPPs) from Na-caseinates prepared from the milk of six species (bovine, sheep, goat, pig, buffalo, human) hydrolyzed by a partially purified proteinase from Lactobacillus helveticus PR4 and to characterize the CPP preparations by RP-FPLC (reversed-phase fast-protein liquid chromatography) and for calcium solubilizing/binding activity. The yield of CPPs ranged from 0.60 to 1.86% of the original proteins. The calcium-solubilizing activity varied from 2.5 (human CPPs) to 11.4 (buffalo CPPs) mg Ca²⁺ mg^-1 CPP. Moreover, minor differences in calcium-binding ability between the CPP from different species were observed. This work showed that CPPs with different calcium-solubilizing activity can be produced from various Na-caseinates by a Lb. helveticus proteinase. Milks having casein-fractions with similar amino acid sequences (e.g., sheep and goat), showed CPPs with the same activity. The data generated in this study show the potential of different caseins to yield CPPs and may help in the selection of milk for the production of functional foods enriched in CPPs.     

甘薯茎叶中异槲皮苷及咖啡酰基奎宁酸类衍生物的抗氧化活性

通过测定还原能力、活性氧自由基的清除作用以及脂质过氧化抑制活性,对甘薯茎叶中含量较高的异槲皮苷(Q-Glu)和异绿原酸A,B,C(3,5-diCQA、3,4-diCQA、4,5-diCQA)4种多酚类化合物的抗氧化活性进行评估。结果显示：4种甘薯茎叶中的多酚类化合物均表现出较高的抗氧化能力,且抗氧化性与化合物浓度呈一定相关性：Q-Glu、3,5-diCQA、3,4-diCQA、4,5-diCQA对DPPH自由基的半清除浓度(IC50)分别为47.27、41.61、43.55、46.02μmol/L;对·OH的IC50值分别为190.99、151.20、151.24、217.31μmol/L;Q-Glu、3,5-diCQA、3,4-diCQA对O2－·的IC50值分别为0.81、0.46、0.71mmol/L;3,5-diCQA、3,4-diCQA、4,5-diCQA对脂质过氧化物的IC50值为3.13、2.53、4.44mmol/L,4,5-diCQA和Q-Glu在实验浓度范围内分别对O2－·清除率和对脂质过氧化物的抑制率低于50%。其中3,5-diCQA的还原能力和清除DPPH自由基、·OH、O2－·等活性氧和自由基的能力最强,3,4-diCQA对脂质过氧化反应的抑制作用最强。4种甘薯茎叶中的多酚类化合物均具有显著抗氧化能力。     

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.     

The effect of postharvest 1-MCP treatment and storage atmosphere on ‘Cripps Pink’ apple phenolics and antioxidant activity

This study investigated the effect of a postharvest 1-methylcyclopropene (1-MCP) treatment, controlled atmosphere and storage time on the total antioxidant activity (TAA) and phenolic compounds in the peel and flesh of ‘Cripps Pink’ apples (Malus domestica Borkh.). Preclimacteric apples were harvested and treated with 1-MCP then stored in normal atmosphere (NA) or controlled atmosphere (CA) at 0 °C for up to 160 days. In general, the level of phenolics decreased by 9% in the peel and significantly increased twofold in the flesh during cold storage, regardless of storage atmosphere or 1-MCP treatment. However, treatment with 1-MCP resulted in significantly lower concentrations of chlorogenic acid and procyanidin B2 in apple flesh, and catechin and epicatechin in the peel compared to the control fruits. There was no significant effect of CA on the phenolic compounds during long-term storage, except for quercetin 3-galactoside and quercetin 3-glucoside, which both significantly increased under CA storage. Total antioxidant activity (TAA) is an important nutritional attribute of apples in the human diet. The results showed that TAA in the peel tissue was about eight times higher than that of the flesh, with mean values of 4.75 g TE/kg FW and 0.56 TE/kg FW, respectively. The TAA in both the peel and flesh tissue increased significantly during storage by 40% and 70%, respectively. The storage atmosphere did not significantly affect TAA in either the peel or flesh, whilst the 1-MCP treatment significantly reduced the TAA in the peel tissue only. These results show the beneficial combined effects of pre-storage 1-MCP treatment and CA on ‘Cripps Pink’ apple phenolic composition and antioxidant capacity during long term storage.     

Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

Antioxidant and antiradical properties of cranberry juice and extracts

The antioxidant capacities of cranberry juice and three extracts isolated from frozen cranberries containing anthocyanins, water-soluble and apolar phenolic compounds, were evaluated at pH 2.5 and 7, respectively. The free radical-scavenging (FRS) and the lipid peroxidation inhibition (LPI) activities of each samples, and extracts, were studied using the N,N-diethyl-p-phenylenediamine (DPD) decoloration test and the thiobarbituric acid reactive substances (TBARS) assay, respectively. The cranberry phenols displayed good free radical-scavenging properties, but were less efficient at inhibiting the peroxidation of lipids. Of all the samples tested, the water-soluble phenolic compounds showed the greatest free radical-scavenging (68.2 mmol TE/mg phenol) and antioxidant (13.4 mmol TE phenol) activities. The polarity of the phenols, the pH of the medium and the juice process had a great influence on the antioxidant activities. The phenols isolated from cranberries with an aqueous solvent have greater antioxidant properties than those extracted with an organic solvent mixture. The antioxidant activity of the cranberry samples adjusted at pH 2.5 was greater than those adjusted at pH 7. Compared to the cranberry extracts, the juice exhibited a much lower antioxidant activity, especially when compared with the extract containing water-soluble compounds which the extraction conditions were similar to those used to obtain the juice.     

Antioxidant activity of commercial ready-to-drink orange juice and nectar

Total antioxidant activity (TAA), total phenolic compounds (TPC), and physicochemical characteristics of ready-to-drink orange juice and nectar from the most consumed brands available in Brazil were evaluated. TPC ranged from 18.7 to 54.2 mg of gallic acid/100 mL, and TAA varied from 57.88 to 349.32 μmol TEAC/100 mL ready-to-drink orange juice and nectar. The ascorbic acid content was the only physicochemical parameter that showed strong variation among packages and brands. Correlation of TPC with TAA showed that the higher the level of TPC the higher the TAA. Correlation of ascorbic acid content with TAA is higher for ready-to-drink orange juice than nectar. The same was found for the correlation of ascorbic acid content with TPC. The results confirm the contribution of the TPC to TAA.     

Molecular surveillance of Toxoplasma gondii in raw milk and Artisan cheese of sheep,goat, cow and water buffalo origin

This study is aimed at investigating the molecular prevalence of Toxoplasma gondii in raw milk and cheese of different animal species (sheep, goat, cow and water buffalo) in Kayseri Province, Türkiye, to provide a preliminary assessment for contamination risk. A total of 200 milk and cheese samples were analysed by real-time PCR. Toxoplasma gondii DNA was detected in two (8%) ewes and one (4%) goat raw milk sample, while none of the cheese samples were positive. These results indicated that the presence of T. gondii DNA in raw milk samples sold in Kayseri Province might be a risk factor for public health.     

Study on the Influence of Tea Extract on Probiotics in Skim Milk: From Probiotics Propagation to Metabolite

In this study, the influence of tea extract (TE) on the growth of probiotics in skim milk was examined. Lactobacillus plantarum ST‐III, Bifidobacterium bifidum Bb02, Lactobacillus acidophilus NCFM, and Lactobacillus rhamnosus GG were used in this study. The introduction of TE in milk significantly stimulated the propagation and acidification of L. rhamnosus GG and L. acidophilus NCFM. The antioxidant capacities and the total free amino acid contents of all fermented milk products were enhanced by the addition of TE; however, there were different antioxidant properties and free amino acid contents of fermented milk samples fermented by different bacteria. With a 9% (w/w) level, the fermentation with L. rhamnosus GG and L. acidophilus NCFM showed larger numbers of viable cells and faster acidifying rates, as well as excellent antioxidant capacity and abundant free amino acids.  The stimulative effects of TE on probiotics can be considered for industrial purposes and has practical implications for commercial applications.     

Concentrations of Volatile 4‐Alkyl‐Branched Fatty Acids in Sheep and Goat Milk and Dairy Products

Goat and sheep milk and dairy products thereof are characterized by a strong and unique flavor. In this context, the volatile minor fatty acid 4‐ethyloctanoic acid plays a prominent role along with 4‐methyloctanoic acid when both are present in free form. Using a novel GC/MS method in the selected ion‐monitoring mode, previously developed for sheep subcutaneous adipose tissue, we were able to analyze the total concentrations of these flavor‐relevant minor fatty acids as methyl esters in goat and sheep milk as well as in their products. Differences between the concentrations and ratios of 4‐methyloctanoic acid and 4‐ethyloctanoic acid in goat milk (n = 4), goat cheese (n = 4), sheep milk (n = 2), and sheep cheese (n = 4) were observed. Goat milk and cheese resulted in higher concentrations for both fatty acids (190 to 480 μg/g milk fat) and smaller 4‐Me‐8:0 to 4‐Et‐8:0 ratios (1.4 to 2.7) compared to sheep milk and cheese (78 to 220 μg/g milk fat; 4‐Me‐8:0 to 4‐Et‐8:0 ratio: 15 to 42). In all samples, the concentration of 4‐Me‐8:0 exceeded the one of 4‐Et‐8:0. However, due to its lower flavor threshold value the contribution of 4‐Et‐8:0 to the flavor was generally >76%. The calculated flavor values were >1400 for goat milk and cheeses and >200 for sheep milk and cheeses. In goat milk and its products, only a proportion of <0.1% 4‐alkyl‐branched fatty acids present in free form in the goat milk and <0.5% in the sheep samples would be sufficient to generate the characteristic goaty flavor. Parameters that promote or prevent the release of 4‐Me‐8:0, and especially 4‐Et‐8:0, will be decisive for the flavor in the resulting dairy product.     

不同浸渍时间嘉宝果浸泡酒的品质比较

以热带水果嘉宝果为原料制作嘉宝果浸泡酒,研究不同浸渍时间对嘉宝果浸泡酒的活性成分含量及抗氧化活性的影响,并对不同浸渍时间浸泡酒的挥发性成分进行分析,明确其特征风味物质。结果表明,浸泡酒中的酚类物质含量随浸渍时间延长而增加,浸泡3个月时嘉宝果浸泡酒的酚类物质含量已达到峰值,其总酚、总黄酮和原花青素含量分别为1581.11 mg GAE/L、352.20 mg CE/L和140.96 mg EPE/L,与浸泡6个月的含量无显著性差异。浸泡酒的抗氧化活性与酚类物质含量变化趋势一致,浸泡3个月的嘉宝果浸泡酒的ABTS、DPPH和FRAP抗氧化能力最强,含量分别为697.02 mmol TE/L,428.97μmol TE/L和19.97 mmol Fe²⁺/L。嘉宝果浸泡酒挥发性成分中主要鉴定出醇类、烃类、酯类和醛酮类等4类。苯乙醇、1-己醇、癸酸乙酯和辛酸乙酯等香气成分是嘉宝果浸泡酒的重要风味物质。综上所述,确定嘉宝果浸泡酒的最佳浸渍时间为3个月,此时浸泡酒的活性物质含量、抗氧化活性和风味物质种类均达到最好水平。     

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.     

Effect of olive leaves feeding on phenolic composition and lipolytic volatile profile in goat milk

The aim of this study was to evaluate phenolic composition, antioxidant potential, and lipolytic events in raw milk obtained from goat fed a dietary supplementation with olive leaves (OL), a by-product of the olive oil production chain. For this purpose, 30 Saanen goats were randomly allocated into 2 groups of 15 goats each: the control group received a standard diet that was prepared by taking into account the nutritional needs of lactating goats, whereas the experimental group (EG) was fed with an OL-supplemented diet (10% on a dry matter basis). At the end of the 30 d of the trial, the individual milk samples were collected and immediately analyzed for total phenolic content and antioxidant activity (AOA). Subsequently, the individual phenolic compounds have been identified and quantified through an ultra-high-performance liquid chromatography system and a characterization of free fatty acids released in milk has been performed. The results showed a positive effect of dietary OL supplementation in improving total phenolic content and AOA; furthermore, 19 phenolic compounds, including phenolic acids, flavonoids, simple phenols, and secoiridoids, have been identified in EG milk. In addition to this, a reduced accumulation of free fatty acids has been found in EG milk, and this finding leads us to hypothesize an inhibitory action of the identified phenolic compounds toward the enzymes responsible for lipolytic events. The use of the molecular docking approach verified the interactions, defining a fairly interesting framework for cinnamic acid, which should be able to noncovalently bind these enzymes, interfering with the recruitment of the substrate and therefore, slowing down their hydrolytic activity. In any case, this information will be subjected to in vitro evaluations for an accurate characterization of the biochemical mechanisms that can be established in milk naturally enriched with bioactive compounds.     

Short communication: A comparative analysis of recombinant chymosins

The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry.     

益生菌发酵乳模拟胃肠液环境下抗氧化活性

为间接评价益生菌发酵乳在人体内的抗氧化活性,分别采用嗜热链球菌（Streptococcus thermophilus）、德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）、动物双歧杆菌（Bifidobacterium）BB12及嗜酸乳杆菌（Lactobacillus acidophilus）制备SL、SL+BB12、SL+LA三种发酵乳,并对其冷藏过程中模拟胃肠液环境下对ABTS、DPPH自由基的清除能力进行了测定。结果表明,三种发酵乳随冷藏时间的延长其抗氧化活性都逐渐降低,添加菌种BB12和LA的发酵乳抗氧化活性下降的幅度小于SL发酵乳,在相同的冷藏时间下模拟胃肠液环境后抗氧化活性都较原发酵乳降低,但添加益生菌BB12和LA发酵乳在模拟胃肠液环境下抗氧化活性高于SL发酵乳,其中BB12高于LA。SL、SL+BB、SL+LA发酵乳冷藏7 d时对ABTS自由基最大清除率分别为46.21%、58.54%、51.99%,对DPPH自由基最大清除率分别为42.17%、53.34%、49.48%。     

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.