Thermostable muscle antigens suitable for use in enzyme immunoassays for identification of meat from various species

A method for the extraction of thermostable muscle antigens for use in enzyme immunoassays is described. The method yields antigens devoid of contaminating proteins which reduce the adsorption of the antigens on to the plate. The effect of such proteins is stimulated by the addition of gelatin. Gelatin (5 mg ml^?1) results in 100% inhibition of the antigen adsorption on to the plate.     

Studies on characterisation of thermostable antigens of adrenals and muscle tissues of meat animals

The characteristics of heat-stable, ethanol-precipitable antigens (BE) of adrenals and muscle tissues of cattle, buffalo, sheep, goat and pig were studied. The antigens were subjected to protein analysis. The distribution patterns of the antigens employing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as well as polyacrylarnide gel isoelectric focusing revealed that the heat-stable antigens of a dreds contained only one component corresponding to troponin T, whereas muscle BE showed associated low molecular weight components along with troponin T as major component. Negative results of thymol-H₂SO₄ staining of SDS-PAGE gels indicated that the thermostable antigens did not belong to the mucoprotein class.     

Identification of meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

Survey of authenticity of meat species in food products subjected to different technological processes,by means of PCR-RFLP analysis

Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFLP) was considered for exploring the incidence of incorrect labelling in food products containing one or more meat species. Universal primers CYT b1/CYT b2, which amplify a variable region of the mitochondrial cytochrome b of vertebrates, and endonucleases PalI, MboI, HinfI and AluI were used for this purpose. Fifty food products, nine of them raw or cured and the other 41 subjected to a variety of technological processes such as pre-cooking and freezing, cooking and smoking, dehydration or sterilisation, were investigated. Twenty of the 50 products declared mixtures of meat species on their labels. Fifteen (30%) of the 50 food samples investigated displayed an incorrect qualitative labelling. While this affected only one (11.1%) of the nine raw/cured products, 14 (34.2%) of the 41 products subjected to some type of heat-processing were not correctly labelled. The undeclared presence of turkey was the most frequent concern, since it was detected in seven food products. The complete absence of a declared species of high commercial value—such as beef or roe-deer—was observed in another four cases. The PCR-RFLP method used here proved to be a rapid and easy-to-perform two-step analytical approach to achieve qualitative meat species identification in raw and cooked food products containing one or more different species.     

A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences

Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

高效液相色谱法同时测定熟肉制品中日落黄、 胭脂红和诱惑红

目的 建立对熟肉制品中日落黄、胭脂红和诱惑红三种合成色素同时测定的方法。方法 样品采用无水乙醇-氨水-水(7: 2: 1)提取, 加正己烷去除脂肪, 氮吹后定容, 用高效液相色谱法测定。经Symmetry Shield RP18柱分离, 甲醇-0.02 mol/L乙酸铵溶液梯度洗脱, 检测波长为508 nm。结果 本方法的相对标准偏差(n=6)在0.92%～3.2%之间, 平均回收率在86.4%～99.1%之间, 检出限在0.005～0.008 mg/kg之间。结论 本方法灵敏度高, 分离效果好, 结果稳定可靠, 适用于熟肉制品中日落黄、胭脂红和诱惑红的同时测定。     

北京市S区居民熟肉制品中亚硝酸盐膳食暴露风险评估

目的 了解北京市S区居民通过熟肉制品暴露亚硝酸盐的潜在健康风险及影响因素。方法 采用分光光度法对S区2013—2019年市售535份熟肉制品中的亚硝酸盐含量进行监测,结合S区不同年龄组居民肉制品的个体消费量数据,并与联合国粮农组织/世界卫生组织的食品添加剂联合专家委员会（FAO/WHO JECFA）规定的亚硝酸盐每日允许摄入量（ADI）0.07 mg/kg·BW进行比较,评价其膳食暴露健康风险。结果 北京市S区市售熟肉制品中亚硝酸盐的检出率为94.02%,总体超标率为9.16%。2岁以上人群通过熟肉制品的亚硝酸盐每日暴露量范围为0.000 0~0.390 0 mg/kg·BW,平均暴露量为0.018 8 mg/kg·BW,其中有2.5%人群的暴露量超过了ADI。除50~64岁组和≥80岁组外,其他年龄组人群熟肉制品来源亚硝酸盐日暴露量均超过了ADI。2~5岁组和6~12岁组熟肉制品来源亚硝酸盐日暴露量在P₉₅百分位上超过了ADI,处在不可接受水平;13~17岁组熟肉制品来源亚硝酸盐日暴露量在P₉₅百分位上等于ADI。结论 S区市售熟肉制品中亚硝酸盐残留有导致部分人群存在慢性毒害的风险,应加强监管和抽检力度,向公众普及亚硝酸盐相关知识,最大限度地减少外源性亚硝酸盐的添加和内源性亚硝酸盐的产生。     

Duplex PCR approach for the detection and quantification of donkey,horse and mule in raw and heat‐processed meat products

Donkey‐related products have been paid more attention for their high nutritional value to human beings. Due to donkey resource scarcity, coupled with gradually increasing market demand, adulterated donkey meat products with other low‐cost animal meat, especially with the similar species horse and mule, are often found in market. Therefore, detection of species fraud in donkey meat products is important for consumer protection and food industries. In this study, a simple and highly specific duplex PCR method, based on the simultaneous amplification of fragments of the mitochondrial ATP synthase subunit 8/6 and ND2, was developed and optimised for the identification of horse, donkey and mule species in raw and heat‐processed meat products. To the best of our knowledge, it was the first time for this strategy applied to these three genetically related animal meat products differentiation to date. The duplex PCR generated a 153‐bp and 83‐bp amplification products for horse and donkey, respectively. While for mule, both of the two length amplification products are appeared on the agarose gel. Target meat species could be detected at a level of 1%, and the results indicated that the duplex PCR assay could be used in the authentification of donkey‐related products with high specificity, cost‐effectiveness and simplicity.     

Species identification by oligonucleotide hybridisation: the influence of processing of meat products

We have evaluated the influence of meat processing on the results obtained with a species identification test by DNA oligonucleotide hybridisation. Freezing and thawing of meat did not cause a substantial reduction in the hybridisation signal. Heating of meat at 100°C or 120°C, however, led to signal reduction caused by DNA degradation, but identification was still possible. The signal is highly influenced by the kind of tissues processed. Four extensively processed products with 50 g kg⁻¹ species admixtures were tested. Admixtures could be detected in three products but no hybridisation signal was observed with corned beef. We conclude that the quantification of admixtures by hybridisation is not better than with most alternative methods of species identification, as the strength of the signal depends on factors such as tissue origin and sample processing. © 1999 Society of Chemical Industry     

Identification of meat species by PCR-RFLP of the mitochondrial COI gene

Meat authenticity verification is pertinent for economical, religious or public health concerns. The present study investigates the use of PCR-RFLP of a part of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene for identification of species origin of raw meat samples of cow, chicken, turkey, sheep, pig, buffalo, camel and donkey. PCR yielded a 710-bp fragment in all species. The amplicons were digested with seven restriction endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I and Xba I) that were selected based on the preliminary in silico analysis. Different levels of polymorphism were detected among samples. The level of COI variation revealed using only Hpa II was sufficient to generate easily analyzable species-specific restriction profiles that could distinguish unambiguously all targeted species. Compared to previously published reports for the determination of meat origin at the molecular level, the approach developed here is much cheaper and faster for routine identification of meats in food control laboratories.     

高效液相色谱法测定熟肉制品中苯甲酸、山梨酸、胭脂红和诱惑红含量

目的建立高效液相色谱法测定熟肉制品中食品防腐剂(苯甲酸和山梨酸)和色素(胭脂红和诱惑红)的分析方法。方法样品经组织捣碎、超声提取、过滤、定容,利用高效液相色谱法检测,甲醇水混合溶液作为流动相,二极管阵列检测器(photo-diode array,PDA)检测,苯甲酸和山梨酸的检测波长为230nm,胭脂红和诱惑红的检测波长为508nm。结果苯甲酸和山梨酸在0.05~0.30mg/mL,胭脂红和诱惑红在2.5~12.5μg/mL范围内,均具有良好的线性关系良好,相关系数r均0.9999。苯甲酸和山梨酸的最低检出限(limit of detection,LOD)范围为1.2~1.8 mg/kg,最低定量限(limit of quantification,LOQ)范围为3.6~5.4mg/kg;胭脂红和诱惑红的LOD范围为0.05~0.1mg/kg,LOQ范围为0.15~0.3 mg/kg。平均加标回收率为94.8%~96.9%,相对标准偏差(relative standard deviation,RSD)为2.4%~4.1%。100份熟肉制品样品中,5份有山梨酸检出,检出率为5.0%,检测结果范围为0.030~0.65g/kg;1份有胭脂红检出,检出率为1.0%,检测结果为0.0023g/kg;3份有诱惑红检出,检出率为3.0%,检测结果范围为0.0036~0.0083g/kg;苯甲酸在所有熟肉制品样品中均未检出。结论该方法灵敏度高,回收率高,检测限低,符合分析要求,适合检测熟肉制品中的苯甲酸、山梨酸、胭脂红和诱惑红。     

Modeling and predicting spoilage of cooked, cured meat products by multivariate analysis

A cooked, cured meat product is a perishable product spoiled mainly by lactic acid bacteria (LAB). LAB cause discoloration, slime formation, off-odors and off-flavors as the result of their metabolic activity producing various products. These microbial products in conjunction with the microbial population could be used to assess the degree of spoilage of this type of product. The spoilage evaluation was achieved by following a multivariate approach. Cluster analysis, principal component analysis and partial least square regression were employed to associate spoilage with microbiological and physicochemical parameters. The developed model was capable of giving accurate predictions of spoilage describing the spoilage associations. The study might contribute to the improvement of quality assurance systems of meat enterprises.     

Lead and cadmium in meat and meat products consumed by the population in Tenerife Island, Spain

The aim of this study was to determine the levels of lead and cadmium in chicken, pork, beef, lamb and turkey samples (both meat and meat products), collected in the island of Tenerife (Spain). Lead and cadmium were measured by graphite furnace atomic absorption spectrometry (GFAAS). Mean concentrations of lead and cadmium were 6.94 and 1.68 µg kg^-1 in chicken meat, 5.00 and 5.49 µg kg^-1 in pork meat, 1.91 and 1.90 µg kg^-1 in beef meat and 1.35 and 1.22 µg kg^-1 in lamb meat samples, respectively. Lead was below the detection limit in turkey samples and mean cadmium concentration was 5.49 µg kg^-1. Mean concentrations of lead and cadmium in chicken meat product samples were 3.16 and 4.15 µg kg^-1, 4.89 and 6.50 µg kg^-1 in pork meat product, 6.72 and 4.76 µg kg^-1 in beef meat product and 9.12 and 5.98 µg kg^-1 in turkey meat product samples, respectively. The percentage contribution of the two considered metals to provisional tolerable weekly intake (PTWI) was calculated for meat and meat products. Statistically significant differences were found for lead content in meats between the chicken and pork groups and the turkey and beef groups, whereas for cadmium concentrations in meats, significant differences were observed between the turkey and chicken, beef and lamb groups. In meat products, no clear differences were observed for lead and cadmium between the various groups.     

预包装熟肉制品生产过程微生物污染状况监测及分子溯源分析

目的 了解河南省预包装熟肉制品生产加工过程中微生物污染状况,分析污染高风险环节及关键控制点,降低食源性疾病的发生概率。方法 样品的采集及检测参照《国家食品污染和有害因素风险监测工作手册》进行,阳性菌株的血清学分型及分子分型分析参照《国家食源性疾病监测工作手册》进行。结果 共监测样品168份,其中环境样品104份(生区4份,熟区100份),检出单核细胞增生李斯特菌8株;熟肉相关样品60份,检出单核细胞增生李斯特菌24株,沙门菌2株。2株沙门菌血清型均为肠炎沙门菌,经XbaⅠ酶切与脉冲场凝胶电泳后获得2种带型,相似度为92.9%。32株单核细胞增生李斯特菌分属6个血清型(1/2a、1/2b、1/2c、4ab、3a和4b),1/2a为优势血清型;经AscⅠ酶切与脉冲场凝胶电泳后获得17种带型,每种带型包括1～10株菌,相似度为55.8%～100.0%。监测生产用水4份,菌落总数和大肠菌群均＜1 CFU/mL,均符合国家饮用水标准要求。结论 生区环境与食品及食品与食品之间存在交叉污染现象,产品经蒸煮环节后微生物污染可得到有效控制,建议进一步严格产品分区管理,加强地面和操作人员的消毒处理,规定合理的生产加工及保存期限。     

多维灰聚类法与定性分析在熟肉制品安全风险 评估中的应用

目的 对2015年某省内生产销售的熟肉制品进行检验,并对检验结果进行安全风险评估。方法 对熟肉制品生产过程中容易出现的风险点进行定性分析, 从而确定熟肉制品的风险因素, 利用多维灰色聚类法对影响食品安全的主要风险因素进行定量评估, 探讨影响熟肉制品有害因素的主次关系。结果 评估结果显示抽检中不合格样品大多属于低风险类, 可以得出熟肉制品的食品安全状况属于安全可控状态。结论 该法明确了食品安全风险的等级, 为食品安全控制做出前瞻性、有效性的技术支撑, 对今后风险评估工作的开展具有切实可行的实践意义。     

Identification of cattle, llama and horse meat by near infrared reflectance or transflectance spectroscopy

Visible and near infrared reflectance spectroscopy (VIS-NIRS) was used to discriminate meat and meat juices from three livestock species. In a first trial, samples of Longissimus lumborum muscle, corresponding to beef (31) llamas (21) and horses (27), were homogenised and their spectra collected in reflectance (NIRSystems 6500 scanning monochromator, in the range of 400-2500 nm). In the second trial, samples of meat juice (same muscle) from the same species (20 beef, 19 llama and 19 horse) were scanned in folded transmission (transflectance). Discriminating models (PLS regression) were developed against “dummy” variables, testing different mathematical treatments of the spectra. Best models indentified the species of almost all samples by their meat (reflectance) or meat juice (transflectance) spectra. A few (three of beef and one of llama, for meat samples; one of beef and one of horse, for juice samples) were classified as uncertain. It is concluded that NIRS is an effective tool to recognise meat and meat juice from beef, llama and horses.     

2015~2017 年烟台市某肉制品生产企业的生产 加工环节致病菌检测及分布特征分析

目的 了解2015~2017 年烟台市熟肉制品各生产环节致病菌污染状况及病原分布特征。方法 监测项目为菌落总数、大肠菌群、金黄色葡萄球菌、李斯特菌属、沙门氏菌, 检测方法依据《食品安全国家标准 食品微生物学检验》各项标准进行, 空气参照GB/T 16294-2010《医药工业洁净室沉降菌的测试方法》。结果 427份样品中, 检出致病菌75 株, 致病菌总体污染率为17.56%。单核细胞增生李斯特氏菌35 株, 其中25 株来源于环境样品, 占总检出率的71.42%; 金黄色葡萄球菌26 株, 16 株来源于原辅料, 占总检出率为23.19%; 沙门 菌19 株, 全部来源于原辅料。结论 金黄色葡萄球菌、沙门氏菌的主要检出来源于原辅料样品,单核细胞增生李斯特氏菌主要检出来源于环境样品,因此提高原辅料采购管理要求,并加强原辅料质量管理,同时加强对生产环境消毒力度。     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

环介导等温扩增与qPCR用于检测肉制品中狗源性成分的比较

目的 建立基于实时荧光PCR和环介导等温扩增检测肉制品中狗源性成分的检测方法并比较2种方法的检测效能.方法 针对狗线粒体CYTB基因保守序列,采用Primer Explorer Version 4软件设计环介导等温扩增引物,Primer Express 3.0.1软件设计实时荧光PCR引物及探针.提取狗肉基因组DNA作...