Two quantitative hexaplex real-time PCR systems for the detection and quantification of DNA from twelve allergens in food

According to the EU and Swiss legislation, allergens in food have to be labelled. Consumers, exhibiting allergic reaction, must be able to avoid such food and its products. In order to provide efficient and reliable methods, two novel hexaplex quantitative real-time polymerase chain reaction systems were developed and validated. The first system simultaneously determines DNA contents from cashew, peanut, hazelnut, celery, soy, mustard, whereas the second system determines DNA contents from milk (beef), egg (chicken), almonds, sesame, pistachio and walnut. The two tests exhibited a good specificity and a detection limit of at least 0.1?% for all analytes. This was additionally verified using samples from proficiency tests. Application on samples from the market revealed realistic and useful information about allergen contents. Quantitative results according to weight are not possible yet.     

Comparison between old and new methods for detection of allergenic substances (egg and milk)

The old ELISA method for detection of allergenic substances (egg and milk) in Kanagawa prefecture from 2003 to 2007, employed before improvement of the food allergen labeling system, yielded detection rates of 20% for egg and 30% for milk. In 2005, after improvement of the labeling system, the detection rate using the new ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol increased by about 10% for egg, but decreased by half for milk. There were 4 positive samples (over 10 μg/g) for both egg and milk proteins, on account of contamination by ingredients at the manufacturing line and the lack of proper food labeling. In 2009, the contamination levels of egg and milk proteins in labeled commercial foods were low. In a comparison between the new and old methods with the same samples, both the new ELISA and Western-blot analyses showed an increase in the detection rate of egg protein. In relation to milk protein, the detection rates were decreased with the new ELISA, although the ELISA detection rate and consistency rates with Western-blot analysis were increased. On the other hand, in the case of a protein content below 5 μg/g, it was impossible to determine ovomucoid and casein by Western-blot analysis.     

Analysis of erythromycin and oleandomycin residues in food by high-performance liquid chromatography with fluorometric detection.

A reliable and sensitive procedure is presented for the analysis of erythromycin (ERY) and oleandomycin (OLE) in food of animal origin, such as meat, liver, kidney, raw milk and egg. The method is based on a solid-phase extraction clean-up with a cation exchange cartridge, a 9-fluoromethylchloroformate (FMOC) precolumn derivatization and a separation by HPLC with fluorometric detection. The selectivity is satisfactory enough to control ERY and OLE residues as not many interfering peaks are observed for various food matrices. The macrolides recoveries of the total procedure were low, although >50%. However, addition of an internal standard (roxithromycin) corrected for recovery to give satisfactory quantitative results for repeatability, linearity, detection and quantification limits and mainly accuracy.     

Isolation and characterization by conventional methods and genetic transformation of Psychrobacter and Acinetobacter from fresh and spoiled meat, milk and cheese.

Of 126 samples of fresh and spoiled meat and dairy products, 40% were positive for the presence of Moraxella-like bacteria and 64% of Acinetobacter; 279 and 466 strains, respectively, were isolated and a part of these were tested by biochemical methods and DNA transformation assays. In some cases, the Moraxellaceae in the samples examined reached considerable quantitative levels, but their percentage in the microflora was generally low. Moraxella-like bacteria were predominant in fresh meat, Acinetobacter in spoiled meat and milk. Most acinetobacters belonged to biotype lwoffii (sensu lato) and all 90 strains tested were positive for DNA transformation with an auxotrophic Acinetobacter. Moraxella-like bacteria were identified as Psychrobacter immobilis in 96% of 103 transformation assays. Moraxellaceae show lipolytic activity but they are considered of low incidence in food spoilage. Only 3.7% of acinetobacters from dairy sources was able to produce ropy milk. Unlike strains from clinical isolates, psychrobacters and acinetobacters isolated from food often do not grow at 37 degrees C.     

Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR

The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used.     

Allergen analysis and authenticity control: Development of mass spectrometry-based methods for animal-derived food products

Food allergy and food fraud involving animal-derived products are two of the most significant issues in food markets. On one hand, immunoglobulin E (IgE)-mediated allergic reactions after ingestion of fish, crustaceans, eggs, or milk are among the most prevalent and can happen even after ingestion of trace amounts. On the other hand, new rules regarding product commercialization (e.g., novel food regulation) are more and more created while fraudulent species substitution in fishery products is very common. Sensitive and accurate analytical methods for allergen quantification and species identification in commercial food products are therefore urgently required whether to help food industries inform allergic consumers, to ensure the food compliance with new regulations or to combat food fraud. In the past few years, bottom-up proteomic techniques, which rely on the detection of peptide biomarkers resulting from a tryptic digest of food proteins using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been emerging in this field. The selection of reliable allergen-specific or species-specific peptide biomarkers is one of the most crucial steps when developing such methods whether for qualitative protein detection (i.e., screening analysis) or protein absolute quantification. The first part of this dissertation relates therefore to the selection of allergen peptide biomarkers for fish, invertebrates, eggs, and milk in an experimental way using a single chaotropic urea extraction buffer. The allergenic proteins responsible for those severe reactions are mainly parvalbumin, tropomyosin, ovalbumin, and caseins. The protein extraction was first assessed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the major allergens were well extracted. After that, data-dependent MS/MS spectra which were obtained using digest samples of authentic animals or pure allergenic ingredients were processed against a matching protein database, and identified peptides were filtered according to several criteria such as the sequence length, amino acid composition, specificity, reproducibility, and sensitivity. Myosin proteins were also a target of choice for fish detection due to their high prevalence and sequence homology even if they are non-allergenic. Contrary to fish and invertebrate databases which included entries for only one specific protein (i.e., parvalbumin, myosin, or tropomyosin), egg and milk databases contained all known allergenic proteins or even additional proteins for egg yolk. Two extra selection steps were achieved for egg and milk markers based on more stringent criteria regarding their sensitivity after targeting them in the corresponding allergenic ingredients. At this point, 17, 13, 10, 10 and 12 potential markers were respectively selected for fish, invertebrates, egg white, egg yolk and milk. An alignment algorithm was used for all those markers to get an idea about their biological specificity. The biological specificity was verified experimentally for fish and invertebrate potential markers by targeted analysis in digest samples of animal species that are relevant in the food industry (vertebrates and invertebrates). This verification was not done for egg and milk markers as it was not regarded as significant due to reported cross-reactivity among avian eggs and among mammalian milk. Two potential parvalbumin markers were detected in other nonfish vertebrates, while all potential tropomyosin markers except one were specific to at least an invertebrate class belonging to the same phylum. Parvalbumin and myosin markers as well as tropomyosin markers were exclusively found in vertebrates or invertebrates. Marker detectability was checked by analyzing processed fish products as well as cooked fish for potential parvalbumin and myosin markers, while commercial insect-based food products such as cereals bars or pasta were studied for potential tropomyosin markers. All expected fish and invertebrate markers were detectable in those complex food products. Detectability of egg and milk markers was assessed by analysis of bread, cookies, and chocolate samples contaminated at different stages of the sample preparation with trace amounts (100 pg/g) of eggs and milk (i.e, fortified, spiked, and incurred digest samples). Two egg white markers and seven milk markers were detected in all those samples. The most suitable markers in terms of sensitivity and specificity were finally chosen for each allergenic product. Thus, besides two myosin fish global markers, five parvalbumin markers were retained including at least one of the investigated fish species. In addition, five tropomyosin markers were chosen, their specificity allowing us to distinguish crustacean tropomyosin from that of insects/arachnids, or mollusks. At last, two ovalbumin markers and three casein markers were confirmed to be the most suitable allergen markers respectively for egg white and milk. All those retained markers could be compiled in a single multiplex method. The automation of the sample preparation could also be a promising improvement whether for qualitative or quantitative analysis.     

Iodine content in commonly consumed food in Hong Kong and its changes due to cooking

Levels of iodine of foods found in Hong Kong were analysed in 271 samples from 11 groups, including (i) cereals and grain products, (ii) legumes and vegetables, (iii) meat and poultry, (iv) egg and egg products, (v) milk and milk products, (vi) fish, (vii) crustaceans and mollusks, (viii) non-alcoholic beverages, (ix) condiments and sauces, (x) sashimi and (xi) seaweeds. All food samples were analysed individually as purchased. The iodine in all samples ranged from undetectable to 2.9?g?kg^?1. Seaweeds, iodised salt, seafood, milk and milk products as well as egg and egg products were rich sources of iodine. To estimate the influence of cooking on iodine levels in foods, a total of 15 individual samples were analysed as raw and respective cooked food. The influence of cooking on the iodine level was minimal, except for boiling, as iodine dissolved into the soup.     

食品中含硫氨基酸含量测定前处理条件的研究

为了建立食品中含硫氨基酸含量测定的前处理条件,该文选取了玉米粉、黄豆、鱼肉、婴幼儿配方乳粉、全蛋粉5种食品作为样品,对比了氧化剂不同配制比例、氧化时间和氧化温度对不同食品中含硫氨基酸测定结果的影响。结果发现,由于食品基质的不同,氧化剂配制比例、氧化时间和氧化温度对这5种食品中含硫氨基酸含量的测定结果产生了不同的影响。根据结果数据分析,在氧化剂配制比例为1∶9(体积比),氧化温度为0℃,氧化时间为16 h条件下,该文中提到5种食品都可以得到较好的实验结果。对于玉米粉、鱼肉和婴幼儿配方食品也可以选取氧化剂配制比例为1∶9(体积比),50℃下氧化5 min作为氧化条件。     

Sandwich Enzyme Immunoassay of Hen Egg Lysozyme in Foods

A sandwich enzyme immunoassay was developed to determine the concentration of hen egg lysozyme added as an antimicrobial agent to preserve food. The method enabled determination of egg lysozyme in beverages, milk and cheeses at a concentration as low as 1 ng/mL with an accuracy of ±12%. The recovery of the method was 85–105%. Human serum albumin (1%) was the most suitable protein for suppressing nonspecific binding.     

Study on detection of allergenic substances (egg and milk) in processed meat products and frozen foods

Allergenic substances (egg and milk) were measured in processed meat products and frozen foods of which the milk and egg ingredient ratios and manufacturing processes were clearly identified. An ovomucoid ELISA kit gave good results in the detection of egg ingredients. With an ovalbumin ELISA kit and egg ELISA kit, results for 6 of 16 foods were negative, but better results were obtained when a protein denaturant was added to the extraction buffer. Occurrence of contamination in the egg ingredient manufacturing line was confirmed in frozen foods. For milk ingredients, good results were obtained by ELISA using two kinds of kits (a casein ELISA kit and milk ELISA kit) described as being suitable for detection of allergenic substances. Allergenic substances were identified by Western blot analysis in all of the foods containing egg and milk ingredients.     

羊奶膻味脂肪酸代谢调控研究

山羊奶是一种营养完全的食品,含有能促进人类生长发育以及维持健康的必需营养成分,它所含各种营养成分的比例,大体适合人类的生理需要。发展山羊奶业不仅是对中国奶业数量上的补充,而且在品质上,与牛奶相比更具潜力。但是由于羊奶具有膻味,因此羊奶及其制品的市场占有率很低。研究证明羊奶中短、中链脂肪酸是造成膻味的主要原因。文章对近年来羊奶膻味与脂肪酸代谢及基因调控等的研究状况进行了综述。     

The nutritional value of dried skim milk in the diet of laying hens

An experiment is reported in which dried skim milk was used at levels of 0, 40, 80, 120 and 160 g kg^?1 of diet to replace mainly soyabean meal in the diets of two breeds of laying hens. AME and AME_n were significantly adversely affected by the two highest levels of added dried skim milk. Food conversion efficiency and bodyweight were unaffected by the added dried skim milk. Although overall food intake and egg number were not statistically significantly affected by dietary treatment there was a tendency towards a decline in both variables at the highest level of dried skim milk inclusion. The response of mean egg weight to increasing levels of dried skim milk was positively linear. Total egg weight was significantly affected by dietary treatment, the response being quadratic.     

Effect of food matrices on the biological activity of ricin

A cell-free translation assay was applied for the quick detection of ricin in food samples. Three economically important foods-ground beef, low-fat milk, and liquid chicken egg--were tested. The results indicated that ground beef had very little matrix effect on the assay, whereas low-fat milk and liquid chicken egg showed clear interference on the protein translation. A simple dilution in phosphate-buffered saline (PBS) effectively eliminated the translational inhibition from these foods. The concentrations inhibiting 50% of luciferase translation derived from the current study were 0.01 nM for the pure ricin A chain, 0.02 nM for pure ricin, and 0.087 nM for crude ricin in PBS. In most cases, the half inhibitory concentration values for ricin in food matrices were significantly lower than for those in PBS buffer, suggesting that some components in these food matrices might potentiate the activity of ricin. Thermal stability tests indicated that the ricin A chain was the least stable among the three forms of ricin in all matrices measured. The thermal stability of pure and crude ricins varied depending on the matrices. The specific activities of ricin in PBS buffer were confirmed by a neutralization test with ricin-specific and nonspecific antibodies. This study demonstrates that the cell-free translation assay is a rapid and sensitive method for detection of biologically active ricin toxin in ground beef, low-fat milk, and liquid chicken egg and that food matrices can greatly affect the thermal stability of ricin.     

鸡蛋蛋黄的功能及其制品

鸡蛋富含营养价值高的蛋白质，脂质，也是维生素和矿物质的良好来源，特别是其中的蛋白质人体吸收率达99．7％，且必需氨基酸十分丰富。鸡蛋不仅是个营养宝库，而且作为快餐食品，糕点和面包的食品原料，蛋白与蛋黄的热凝笥，蛋白的起泡性，蛋黄的乳化性等特性在食品工业上也得到普遍的应用。     

Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm.

Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine-stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm.     

Detection and quantitation of Escherichia coli O157 in raw milk by direct qPCR

An improved method was developed to detect and quantify Escherichia coli O157 in raw milk to support studies on the food safety implications of consumption of raw milk. Centrifugation coupled with ethylene diamine tetra acetic acid (EDTA), sodium dodecyl sulfate (SDS), DNase and trypsin treatments under optimized conditions was used to concentrate bacteria from 10 mL of raw milk and remove polymerase chain reaction (PCR) inhibitors. DNA from the entire sample pellet was efficiently extracted in a fast, single-tube step, and quantitated with a 5′ nuclease quantitative PCR assay. This approach represents a significant improvement over earlier methods, in ability to use large sample volumes, effectively remove inhibitors without use of toxic or flammable reagents, efficiently extract DNA and obtain rapid, quantitative results. E. coli O157:H7 inoculated into raw milk was detected at 1 cfu mL⁻¹ from a 10 mL sample in less than 3 h.     

Studies on organoleptic properties of food products from fresh egg and egg powder through principal component analysis

The population which is below the poverty line is devoid of nutritious diet. Egg and milk are categorized as complete foods. The defensive organizations are situated in such remote places where fresh food material is not available. Keeping in view these problems, the study of organoleptic variables, viz., color, appearance, aroma, texture and taste in the food products of cake, omelet doughnut, coconut macaroon and mayonnaise from fresh egg and egg powder, was conducted. Principal component analysis was carried out. Organoleptic properties of doughnut prepared from egg powder were superior compared to fresh egg which had better sensory traits for coconut macaroon. The sensory traits like taste, texture and aroma were the most influential traits studied to pronouncing as a panel decision. It is proposed that fresh egg and egg powder should be preferred in the process of preparation of coconut macaroon and doughnut, respectively.     

Determination of naturally occurring oestrogens and androgens in retail samples of milk and eggs.

The occurrence of the main steroid hormones (oestrone, 17alpha-oestradiol, 17beta-oestradiol, 17alpha-testosterone, 17beta-testosterone, dehydroepiandrosterone, 4-androstenedione), especially in milk and eggs, was investigated. An analytical method based on GC-MS/MS was developed for steroid measurement at an ultra-trace level in food products. The limits of detection for oestrogens were about 5 and 30 ng kg(-1) in milk and eggs, respectively. For androgens, the limits of detection were around 10 and 50 ng kg(-1) in milk and eggs, respectively. The method was applied to milk and egg samples collected in a French supermarket. In milk, oestrone was found at levels between 100 and 300 ng l(-1), while 17beta-oestradiol levels were estimated to be near 20 ng l(-1). 17alpha-testosterone was found to be from 50 ng l(-1) in skimmed milk to 85 ng l(-1) in whole milk. In egg samples, oestrone and 17beta-oestradiol were found at 1.5 and 0.9 microg kg(-1), respectively, while 17alpha-oestradiol was found to be in lower concentrations (i.e. around 0.55 microg kg(-1)). Regarding androgens, 17alpha- and 17beta-testosterone were estimated at 1.9 and 1.3 microg kg(-1), respectively. These results represent a first attempt to estimate the food exposure to steroid hormones. In the future, the collection of additional data should permit the comparison between this exogenous dietary intake and the daily endogenous production in pre-pubertal children as a basis of risk assessment regarding endocrine disruption linked to these molecules for this critical population.     

鸡蛋过敏原表位研究进展

鸡蛋过敏是一种常见的食物过敏反应,是人体对鸡蛋中蛋白质成分产生的一种变态反应。食物过敏反应的物质基础是抗原表位与抗体对位,解决食物过敏反应问题的关键一点即需要深入研究出过敏原的表位。本文介绍了鸡蛋中6种主要过敏原,简述了过敏原表位的定位方法,并详细综述了卵白蛋白、卵类粘蛋白、卵转铁蛋白和溶菌酶的过敏原表位研究进展。本文可为进一步鸡蛋过敏研究提供理论指导,也可为表位成分的定量检测以及无毒副作用鸡蛋过敏原疫苗的开发等方面提供一些参考。      

Comparison of extraction conditions for milk and hen's egg allergens

The evaluation of recovery rates by extracting milk powder and egg powder using eleven different extractants gave approximately similar results for both foods. Compared with the other extraction solutions investigated, ‘1% Tween 20® and 0.4% Triton X-100®’ and ‘4% SDS’ are the most suitable extractants to isolate proteins of hen's egg or milk. When comparing calculated protein recovery rates of egg and milk powder extracts, the results clearly indicated that the choice of a suitable extractant is of particular importance. Qualitative investigation of the extracts via LDS-PAGE followed by silver staining as well as immunoblotting confirmed the results of protein quantification. Hence, the immunoblots showed that the extraction agents had no negative influence on the antigenicity of the extracted allergenic proteins. In this study, variation of extraction temperature led neither to any benefit in extraction quality nor to degradation. Changing pH did not reveal any trends, but progressive protein hydrolysis under strong alkaline conditions. Evaluation of recovery rates as well as results of unspecific and specific staining of the extracts showed that an extraction time of 1?h is sufficient for an appropriate sample preparation. For investigations with and without food matrix different results were obtained. In summary, wheat starch did not influence the extraction quality within all examined materials and different extractants. In contrast, using fat powder and dry cake mix, respectively, led to different results in the extraction procedure. When fat powder and dry cake mix were used as food matrices, some protein recovery rates decreased and some increased depending on the allergen material. These results highlight the fact that the suitability of the extractant not only depends on the properties of the allergen but furthermore on the type of matrix containing the allergen.