Multiplex PCR method for the simultaneous detection of histamine-, tyramine-, and putrescine-producing lactic acid bacteria in foods

In a screening of primers, we have selected three pairs of primers for a multiplex PCR assay for the simultaneous detection of lactic acid bacteria (LAB) strains, which potentially produce histamine, tyramine, and putrescine on fermented foods. These primers were based on sequences from histidine, tyrosine, and ornithine decarboxylases from LAB. Under the optimized conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases, a 924-bp fragment from tyrosine decarboxylases, and a 1,446-bp fragment from ornithine decarboxylases. When the DNAs of several target organisms were included in the same reaction, two or three corresponding amplicons of different sizes were observed. This assay was useful for the detection of amine-producing bacteria in control collection strains and in a LAB collection. No amplification was observed with DNA from nonproducing LAB strains. This article is the first describing a multiplex PCR approach for the simultaneous detection of potentially amine-producing LAB in foods. It can be easily incorporated into the routine screening for the accurate selection of starter LAB and in food control laboratories.     

食品中产组胺微生物及其控制的研究进展

组胺是食品中游离组氨酸经组氨酸脱羧酶分解产生的一种胺类物质,也是一些出口水产食品中一项重要的质量检测指标。分析比较了国内外几类易产组胺食品中的产组胺微生物分布情况,并综述了组胺形成和控制方法的研究进展,由此可为食品中组胺的预防及控制提供基础,以确保食品的安全性。     

Histamine fish poisoning revisited

Histamine (or scombroid) fish poisoning (HFP) is reviewed in a risk-assessment framework in an attempt to arrive at an informed characterisation of risk. Histamine is the main toxin involved in HFP, but the disease is not uncomplicated histamine poisoning. Although it is generally associated with high levels of histamine (> or =50 mg/100 g) in bacterially contaminated fish of particular species, the pathogenesis of HFP has not been clearly elucidated. Various hypotheses have been put forward to explain why histamine consumed in spoiled fish is more toxic than pure histamine taken orally, but none has proved totally satisfactory. Urocanic acid, like histamine, an imidazole compound derived from histidine in spoiling fish, may be the "missing factor" in HFP. cis-Urocanic acid has recently been recognised as a mast cell degranulator, and endogenous histamine from mast cell degranulation may augment the exogenous histamine consumed in spoiled fish. HFP is a mild disease, but is important in relation to food safety and international trade. Consumers are becoming more demanding, and litigation following food poisoning incidents is becoming more common. Producers, distributors and restaurants are increasingly held liable for the quality of the products they handle and sell. Many countries have set guidelines for maximum permitted levels of histamine in fish. However, histamine concentrations within a spoiled fish are extremely variable, as is the threshold toxic dose. Until the identity, levels and potency of possible potentiators and/or mast-cell-degranulating factors are elucidated, it is difficult to establish regulatory limits for histamine in foods on the basis of potential health hazard. Histidine decarboxylating bacteria produce histamine from free histidine in spoiling fish. Although some are present in the normal microbial flora of live fish, most seem to be derived from post-catching contamination on board fishing vessels, at the processing plant or in the distribution system, or in restaurants or homes. The key to keeping bacterial numbers and histamine levels low is the rapid cooling of fish after catching and the maintenance of adequate refrigeration during handling and storage. Despite the huge expansion in trade in recent years, great progress has been made in ensuring the quality and safety of fish products. This is largely the result of the introduction of international standards of food hygiene and the application of risk analysis and hazard analysis and critical control point (HACCP) principles.     

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.     

Activity of two histidine decarboxylases from Photobacterium phosphoreum at different temperatures, pHs, and NaCl concentrations

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible enzymes. This article reports the effect of various growth and reaction conditions, such as temperature, pH, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P. phosphoreum. The histidine decarboxylase activity of the cell-free extracts was highest in 7 degrees C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at pH 6.0. Moreover, percent activity of the constitutive and inducible enzymes was highest for the inducible enzyme in cultures grown at 7 degrees C and pH 7.5 and in 5% NaCl. The temperature and pH dependences of histidine decarboxylase differed between the constitutive and inducible enzymes; that is, the activity of histidine decarboxylases was optimum at 30 degrees C and pH 6.5 for the inducible enzyme and 40 degrees C and pH 6.0 for the constitutive enzyme. The differences in the temperature and pH dependences between the two enzymes extended the activity range of histidine decarboxylase under reaction conditions. On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two enzymes. Additionally, the effects of reaction temperature, pH, and NaCl concentration on the constitutive enzyme activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive enzyme activity reflected the whole histidine decarboxylase activity.     

Improved enzymatic method for the rapid determination of histamine in wine.

Fermented foods are frequently contaminated by histamine generated by microorganisms possessing histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in man. Thus, it becomes important to set a reliable method for rapid histamine quantification in foods. The detection of bacteria exhibiting histidine decarboxylase activity is also important to estimate the risk of contamination of food. Previous enzymatic methods used to quantify histamine in fish gave erroneously high values due to interference when applied to wine. A new enzymatic method is described that allows the direct determination of histamine concentrations in this type of sample. It can be used for the detection of histamine in synthetic media, grape must or wine (white, rose, red) without polyphenols or sugar interferences. This new enzymatic method shows a good correlation (R2 = 0.996, p < 0.001) between the histamine concentrations and absorbances in the interval 0.4-160 mg l(-1). Comparison between this enzymatic method and a high-performance liquid chromatography method showed a high correlation (R2 =0.9987, p<0.001). A miniaturized enzymatic method is also proposed, which is particularly useful when high numbers of samples must be analysed.     

Control of biogenic amines in food--existing and emerging approaches

Biogenic amines have been reported in a variety of foods, such as fish, meat, cheese, vegetables, and wines. They are described as low molecular weight organic bases with aliphatic, aromatic, and heterocyclic structures. The most common biogenic amines found in foods are histamine, tyramine, cadaverine, 2-phenylethylamine, spermine, spermidine, putrescine, tryptamine, and agmatine. In addition octopamine and dopamine have been found in meat and meat products and fish. The formation of biogenic amines in food by the microbial decarboxylation of amino acids can result in consumers suffering allergic reactions, characterized by difficulty in breathing, itching, rash, vomiting, fever, and hypertension. Traditionally, biogenic amine formation in food has been prevented, primarily by limiting microbial growth through chilling and freezing. However, for many fishing based subsistence populations, such measures are not practical. Therefore, secondary control measures to prevent biogenic amine formation in foods or to reduce their levels once formed need to be considered as alternatives. Such approaches to limit microbial growth may include hydrostatic pressures, irradiation, controlled atmosphere packaging, or the use of food additives. Histamine may potentially be degraded by the use of bacterial amine oxidase or amine-negative bacteria. Only some will be cost-effective and practical for use in subsistence populations.     

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time <2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.     

Tyramine and phenylethylamine biosynthesis by food bacteria

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.     

Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the C_t values and histamine concentrations determined by HPLC.     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

食品中的生物胺及其检测方法

生物胺普遍存在于生物体中,有重要的生理功能.许多天然食品中都含有生物胺.摄入大量生物胺会引起身体不适,严重的还可能危及生命.引起食物中毒的生物胺主要包括组胺和酪胺.此外,存在大量微生物的食品中,生物胺的含量较高,同类食品中生物胺的种类和含量又有很大的变化,这一现象受许多因素影响,如微生物的种类和数量、温度、pH值等.生物胺含量还与食品的腐败变质有关,是食品品质的指示器,因此对于生物胺的检测受到重视.目前检测食品中生物胺含量的方法主要有薄层色谱、气相色谱、毛细管电泳和高效液相色谱.     

Histamine stability in Rihaakuru at -80, 4 and 30°C

Rihaakuru is a shelf stable fish paste product formed from a fish soup prepared from tuna. Histamine contamination is a food safety issue with this product that is manufactured from tuna fish that has been temperature abused. Histamine concentrations decreased between 31% and 73% in Rihaakuru stored for 10months at either -80, 4 or 30°C. This appears to be a property of the product as histamine solutions are reported to be stable, at least under frozen storage. The risk of histamine food poisoning due to Rihaakuru may reduce during the storage of the product.     

Molecular methods for the detection of biogenic amine-producing bacteria on foods

Biogenic amines are low molecular weight organic bases that can be detected in raw and processed foods. Several toxicological problems resulting from the ingestion of food containing biogenic amines have been described. Biogenic amines are mainly produced by the decarboxylation of certain amino acids by microbial action. Since the ability of microorganisms to decarboxylate amino acid is highly variable, being in most cases strain-specific, the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the risk of biogenic amine food content and to prevent biogenic amine accumulation in food products. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. PCR methods offer the advantages of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes. Moreover, these molecular methods detect potential biogenic amine risk formation in food before the amine is produced. The aim of the present review is to give a complete overview of the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria. These genetic procedures allow the introduction of early control measures to avoid the development of these bacteria.     

Cloning and sequencing of the histidine decarboxylase gene from Photobacterium phosphoreum and its functional expression in Escherichia coli

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5'-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.     

A review: Current analytical methods for the determination of biogenic amines in foods

Analysis of biogenic amines (BA) in foods was reviewed. Biogenic amines are natural antinutrition factors and are important from a hygienic point of view as they have been implicated as the causative agents in a number of food poisoning episodes, and they are able to initiate various pharmacological reactions. Histamine, putrescine, cadaverine, tyramine, tryptamine, β-phenylethylamine, spermine, and spermidine are considered to be the most important biogenic amines occurring in foods. Analysis of BA is important because of their toxicity and their usage as indicators of the degree of freshness or spoilage of food. Several methods have been developed for determination of biogenic amines in food. The analytical methods used for quantification of BA are mainly based on chromatographic methods: thin layer chromatography (TLC), gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). HPLC is most often used for the analysis method of BAs. Due to low volatility and lack of chromophores of most BA, UV-spectrometric detectors cannot be used. The large majority of assays employs fluorimetric detection with precolumn or postcolumn derivatization techniques. This review shows that these methods allow quantitative determination of biogenic amines, individually or simultaneously in foods.     

组胺分子印迹聚合物中模板分子制备比例的紫外光谱学研究

组胺广泛存在于各类食物当中,在利用组胺分子印迹聚合物传感器对组胺进行快速检测的过程中,模板分子与功能单体的配比对于最终合成的印迹聚合物的特异吸附能力影响显著。组胺和2-（三氟甲基）丙烯酸分别被选作模板分子和功能单体。为探究二者的配比,利用紫外光谱扫描的方法分别检测了组胺与2-（三氟甲基）丙烯酸的最大吸收峰,并通过差式紫外扫描以及对功能单体不同加入比例的紫外扫描,发现组胺与2-（三氟甲基）丙烯酸的比例至少为1∶4时才能形成稳定的预组装复合体系,而比例为1∶6时,最大吸收峰显著下降且最大吸收峰波长红移,整个体系达到最稳定的状态。所得的结果对于制备组胺分子印迹聚合物传感器对组胺进行快速检测有着较好的指导性意义。     

2,5-diketopiperazines in food and beverages: Taste and bioactivity

2,5-Diketopiperazines (2,5-DKPs) have been found to occur in a wide range of food and beverages, and display an array of chemesthetic effects (bitter, astringent, metallic, and umami) that can contribute to the taste of a variety of foods. These smallest cyclic peptides also occur as natural products and have been found to display a variety of bioactivities from antibacterial, antifungal, to anthroprotective effects and have the potential to be used in the development of new functional foods. An overview of the synthesis of these small chiral molecules and their molecular properties is presented. The occurrence, taste, and bioactivity of all simple naturally occurring 2,5-DKPs to date have been reviewed and those found in food from yeasts, fungi, and bacteria that have been used in food preparation or contamination, as well as metabolites of sweeteners and antibiotics added to food are also reviewed.     

Histamine Formation in Fish Sauce Production

ABSTRACT: The formation of histamine in fish sauce made from Stolephorus sp was studied. In the first experiment, fish were iced on board and mixed with salt at the factory, while in the second experiment fish were mixed with salt on board collection vessels. Eight batches were fermented for 12 mo. Histamine levels increased during fermentation to 22 to 159 and 589 to 686 ppm in the first and second experiments respectively. Good correlation between histamine levels in raw material and final products was found. It was concluded that histamine was formed both in the raw material and during fermentation. It was speculated that histidine decarboxylase enzymes formed prior to fermentation produced histamine during fermentation.