Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry.     

酸水解法测定婴幼儿配方食品、乳和乳制品中二噁英类持久性有机污染物

建立适合婴幼儿配方食品、乳及乳制品中二噁英类持久性有机污染物(POPs)分析的前处理技术。方法 利用索氏抽提法和酸水解法分别提取婴幼儿配方食品、乳及乳制品中的脂肪,比较两种方法的差异。结果对于婴幼儿配方食品,两种萃取方法测得的结果差异不大;对于乳粉和液态鲜乳,索氏抽提法的结果与实际值偏差较大。结论 不同类型乳粉的组分(酪蛋白与乳清蛋白比例等)和加工工艺差异很大,酸水解法能有效地使乳及乳制品中的脂肪转变为游离态,提高了脂肪的提取率,为准确测定POPs奠定基础。     

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.     

Digestible reactive lysine in selected milk-based products

Reactive lysine contents, true ileal reactive lysine digestibility, and true ileal digestible reactive lysine contents were determined in a wide range of processed milk products. A previously validated assay based on determining reactive lysine in both food and ileal digesta, after reaction of these materials with O-methylisourea, was applied. Semisynthetic diets containing milk products as the sole sources of protein and including chromic oxide as an indigestible marker were fed to growing rats. Digesta from the terminal ileum were collected posteuthanasia and, with samples of the diets, analyzed for reactive lysine (homoarginine) contents. True reactive lysine digestibility was determined after correcting for endogenous lysine loss at the terminal ileum of rats fed an enzyme hydrolyzed casein-based diet, followed by ultrafiltration (5000 Da) of the digesta. Digestible total lysine (determined using conventional methods) was also determined. The true ileal reactive lysine digestibility was high (>91%) in all the milk products tested, but was highest in the UHT milk (100%) and lowest in the infant formulas (91 to 93%). Total lysine digestibility (conventional measurement) significantly underestimated reactive lysine digestibility for all the products tested. The mean underestimation ranged from 1.3 to 7.1% units. The mean digestible total lysine content was significantly different from the available lysine content for most of the products examined. In some cases this difference was small (<3%), but for a number of the products (evaporated milk, whole milk protein, lactose hydrolyzed milk powder, and a sports formula) the difference was greater (6.5 to 14%). This would suggest firstly that total lysine and total lysine digestibility determined using conventional methods were inaccurate when applied to some milk-based foods, and secondly that some of the milk products have undergone lysine modification. In general, milk proteins are a highly digestible source of amino acids and lysine.     

Validation of a PCR-RFLP based method for the identification of salmon species in food products

The use of a PCR-RFLP based method for the identification of salmon species in food products was investigated. The reliability and practicality of the method was tested by a collaborative study in which five European laboratories participated. Two unknown samples (a commercial product of known species composition and a mix of two salmon species) required identification by comparison with authentic reference species. From a total of 50?cases, 100% of authentic species were correctly assigned, with all unknown samples also correctly identified. A larger scale analysis of UK commercial products was also performed spanning the whole range of salmon products available. In almost all cases the salmon species declared was confirmed, although, a trout species was detected in one product declaring only the presence of salmon. The investigation confirms the reproducibility of the method in different laboratories, and its applicability for commercial product analysis.     

Electrophoretic techniques for species identification of fishery products

Summary At the present time species identification of fishery products is mainly performed by electrophoresis; in most cases isoelectric focusing (IEF) is given preference over other electrophoretic techniques. In this review the possibilities of application of IEF and other electrophoretic methods for analysis of raw, dried, salted, smoked, ripened, cooked or canned fish are discussed. It is shown that the protein patterns may be influenced by the type of muscle (light or dark), the freshness of fish or fillet, and by the conditions of frozen storage. Reference samples must often be used to obtain unequivocal results. A protein dry powder is introduced, which has been prepared from the sarcoplasmic fraction of many fish species yielding species-specific protein patterns. The powder is stable at room temperature and can be shipped without cooling.

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Elektrophoretische Methoden zur Bestimmung der Tierart in Fischereiprodukten

Zusammenfassung Zur Zeit erfolgt die Bestimmung der Tierart in Fischereiprodukten nahezu ausschließlich mit elektrophoretischen Methoden, vorzugsweise durch die isoelektrische Focussierung (IEF). In der vorliegenden Übersichtsarbeit werden die Anwendungsmöglichkeiten der IEF und anderer Elektrophoreseverfahren zur Analyse roher, getrockneter, gesalzener, geräucherter, gereifter, gegarter oder sterilisierter Fischereiprodukte diskutiert. Es wird aufgezeigt, in welchem Ausmaß die Proteinmuster durch die Art der Muskulatur (hell oder dunkel), den Frischegrad der Fische bzw. Filets und durch die Gefrierlagerbedingungen der Produkte beeinflußt werden. In vielen Fällen kann auf Referenzproben nicht verzichtet werden; es wird ein Proteinpräparat vorgestellt, das aus der sarkoplasmatischen Fraktion zahlreicher Fischarten isoliert wurde und Spezies-spezifische Proteinmuster lieferte. Das Präparat ist bei Raumtemperatur stabil und kann daher ohne Aufwand verschickt werden.

     

牛初乳中IgA的分离纯化及其产品免疫学检测方法的建立

用离子交换及分子筛的方法提纯牛ＩｇＡ，并制备抗血清。以此为基础，建立了测定牛初乳制品中ＩｇＡ含量的方法。该方法稍加改变后适用于各种产品中ＩｇＡ的免疫学测定及效用评价。     

New functional milk-based products in the Italian market

The aim of this study is to obtain an overview, from a chemical and a nutritional point of view, of functional products such as lactose-hydrolysed milk, milk with added probiotics, prebiotics and vitamins, probiotic fermented milk, and synbiotic fermented milk. For this purpose, six samples of lactose-hydrolysed UHT milk and two samples of milk with added functional ingredients, seven probiotic and four synbiotic fermented milk samples, have been studied. In lactose-hydrolysed milk samples, proximate composition, vitamins A, E and cholesterol contents have not shown significant differences, with the obvious exception of a skimmed milk sample containing small amounts of fat and related compounds. The other samples have shown a large compositional variability, and, in particular, cholesterol ranged from 3.6 to 12.7 mg/100 g, vitamin E ranged from 2.1 to 3300 μg/100 g and vitamin A ranged from 3.5 to 182.0 Retinol Equivalents (μg/100 g).     

A novel enzymatic method for the measurement of lactose in lactose-free products

The cover image is based on the Research Article A novel enzymatic method for the measurement of lactose in lactose-free products by David Mangan et al., DOI: 10.1002/jsfa.9317 . This cover was supported by Megazyme.

     

ELISA-test for cooked meat species identification on gelatine and gelatine products

The results of the ELISA-test with a commercial cooked meat species identification kit on gelatine and gelatine containing products can be influenced by different gelatine types and concentrations leading to false positive readings. The test cannot be applied especially to meat products containing relatively high amounts of soluble collagens because their extracts form solid gels in the test wells. In this case the different reagents added will penetrate the gels and react with each other. The kit may not speciate reliablely commercial gelatine in isolation or in products where gelatine is the sole animal protein source.     

Hormones in bovine milk and milk products: A survey

A survey of the published data on the occurrence of hormones in milk and milk products is presented. Bovine milk and colostrum contain a large number of hormones from either steroidic or peptidic origin. The main categories to which these molecules belong are gonadal (estrogens, progesterone, androgens), adrenal (glucocorticoids), pituitary (prolactin, growth hormone) and hypothalamic hormones (GRH, LH-RH, TRH). Other molecules, such as proteins related to the parathyroid hormone, insulin, somatostatin, calcitonin, bombesin, erythropoeitin and melatonin, are also of interest. The exact role of hormones in the development of the newborn is still not known, but it is believed that they may contribute to the growth of the newborn and to the development and maturation of its gastrointestinal and immune systems.     

发酵乳中双岐杆菌种类快速识别

建立发酵乳制品中双歧杆菌快速识别方法。采用酶解前处理法获取样品中菌体细胞,基于PCR-DGGE技术确定发酵乳中双歧杆菌种属。该方法能准确、快速鉴别双歧杆菌,检出限为105cfu/mL。该方法可用于发酵乳中双歧杆菌的准确识别。     

NOVATION系列产品-乳品稳定剂的新秀

一种新型的乳品稳定剂正在乳品行业悄然流行,这种稳定剂就是国民淀粉公司独创的产品-NOVATION系列。     

Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products

The aim of the present study was to evaluate an agar medium for the quantitative analysis of bifidobacteria in probiotic milk products on the market, and to investigate the identities of active strains. For this purpose, three sour milk products and a soft cheese brand were each analysed ten times with Wilkins-Chalgren agar (WCA) supplemented with mupirocin, and with MRS agar supplemented with dicloxacillin at two different concentrations. By statistical analysis of the bacterial counts obtained, WCA was shown to have the best detection rate. All the sour milk products examined conformed to Swiss food legislation and contained more than 10⁶ cfu of bifidobacteria per gram of living bifidobacteria. All the samples of soft cheese analysed revealed counts that were clearly (approximately 2 logs) below this minimum requirement. Isolated bifidobacteria strains were all identical with reference strains from producer companies shown by pulsed-field gel electrophoresis (PFGE) profiling. According to the labels, the three brands of sour milk examined contained different strains of bifidobacteria (Bifidobacterium lactis Bb 12, Bifidobacterium animalis DN 173010 and Bifidobacterium species 420). However, all the strains revealed repeatedly identical PFGE-patterns, thus showing a close relationship. These findings show that a better taxonomical definition of commercially used bifidobacteria strains is needed.     

Qualitative and quantitative identification of adulteration of milk powder using DNA extracted with a novel method

The extraction of high-quality DNA from processed dairy products is often the crucial step in an authentication process by PCR-based methods. In this study, we optimized a novel DNA extraction method for milk powder and used the extracted DNA for identification of milk powder based on PCR analysis. The DNA quality was assessed by amplifying target sequences from mitochondrial genes, as well as by monitoring the yield, purity, and integrity of the extracted DNA. In addition, a laboratory adulteration model of milk powder was detected by PCR-based methods (PCR and real-time PCR) using primers targeting the mitochondrial 12S rRNA gene. Results showed that a sufficient amount and quality of DNA could be isolated from milk powder with this method. Both PCR and real-time PCR detection of cow milk compositions in goat milk powder further confirmed the DNA extracted with this extraction method could be widely used in addressing milk powder adulterant by a PCR-based method.     

Development of a method for the identification of scombroid and common substitute species in seafood products by FINS

In the present work, a method for the authentication of scombroid products was developed, by means of FINS (Forensically Informative Nucleotide Sequencing) technique (Polymerase Chain Reaction (PCR) followed by phylogenetic analysis). The methodology developed allows the identification of most important scombroid species using the mitochondrial cytochrome b as molecular marker. Due to the different commercial value of the species belonging to this family, substitutions between species in seafood products can take place.     

Major advances in fresh milk and milk products: fluid milk products and frozen desserts

Major technological advances in the fluid milk processing industry in the last 25 yr include significant improvements in all the unit operations of separation, standardization, pasteurization, homogenization, and packaging. Many advancements have been directed toward production capacity, automation, and hygienic operation. Extended shelf-life milks are produced by high heat treatment, sometimes coupled with microfiltration or centrifugation. Other nonthermal methods have also been investigated. Flavored milk beverages have increased in popularity, as have milk beverages packaged in single-service, closeable plastic containers. Likewise, the frozen dairy processing industry has seen the development of large-capacity, automated processing equipment for a wide range of products designed to gain market share. Significant advancements in product quality have been made, many of these arising from improved knowledge of the functional properties of ingredients and their impact on structure and texture. Incidents of foodborne disease associated with dairy products continue to occur, necessitating even greater diligence in the control of pathogen transmission. Analytical techniques for the rapid detection of specific types of microorganisms have been developed and greatly improved during this time. Despite tremendous technological advancements for processors and a greater diversity of products for consumers, per capita consumption of fluid milk has declined and consumption of frozen dairy desserts has been steady during this 25-yr period.     

Recent aflatoxin survey data in milk and milk products: A review

Aflatoxin M₁ (AFM₁) occurrence in human and animal milk, infant formula, powdered milk, cheese and yoghurt represents a risk for health. The last four years (2010–2014) of data, as well as the most frequently and updated analytical methods applied for AFM₁ quantification, are evaluated. Aflatoxin B₁, considered the most potent toxic aflatoxin, is metabolised to form the monohydroxy derivative AFM₁. This metabolized, expressed in the milk, is relatively stable, and it is not eliminated by heat treatments or pasteurisation, and thus represents a serious health concern.