Detection of histamine-producing bacteria using polymerase chain reaction techniques and DNA probes

The polymerase chain reaction technique was used to enable the detection of DNA specific amplicons, corresponding to gene fragments coding for histidine decarboxylase present in the histidine decarboxylating bacteria frequently found in canned fish. The level of histamine in several fish product samples was quantified by HPLC and the DNA of the samples containing histamine was isolated and amplified with hdcA histidine-decarboxylase gene-specific primers. The primer sets used, CL1/CL2, JV16HC/JV17HC and CL1/JV17HC, amplified 150 bp, 370 bp and 500 bp products respectively. Non-expected fragments (100 bp, 150 bp and 250 bp) were also amplified by JV16HC/JV17HC. Some amplified fragments were sequenced automatically, presenting high homologies with the Clostridium perfringens hdcA gene. A DNA probe from a C. perfringens pure strain was hybridized with the DNA fragments amplified from the contaminated samples. The hybridization blots were then detected by colorimetry, revealing that the samples were contaminated by C. perfringens.     

A comparative study of different PCR-based DNA fingerprinting techniques for typing of lactic acid bacteria

A set of 92 lactic acid bacteria strains and 10 type/reference strains was analysed using three molecular techniques (randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), enterobacterial repetitive intergenic consensus (ERIC-PCR) and the polytrinucleotide (GTG)₅-PCR), in order to compare their discriminatory ability for typing of these bacteria. The results indicated that RAPD-PCR generated patterns with the largest number of bands, adequately clustering the isolates belonging to the same genus. Likewise, it showed the highest discriminatory capacity, while no discrimination between isolates of different genera was possible with ERIC-PCR and (GTG)₅-PCR. Therefore, the use of RAPD-PCR has turned out to be the most suitable procedure for typing LAB isolates belonging to different genera and species obtained from some fermented foods such as goat and Manchego cheeses, Almagro eggplant fermentations and Tempranillo wines.     

Production of pyroglutamic acid by thermophilic lactic acid bacteria in hard-cooked mini-cheeses

Pyroglutamic acid is present in high amounts (0.5g/ 100g) in many cheese varieties-and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano. An in vivo model system for cooked mini-cheese production and ripening acceleration was set up to demonstrate the ability of thermophilic lactic acid bacteria, used as a starter, to produce pyroglutamic acid (pGlu). In mini-cheeses stored at 38 and 30 degrees C for up to 45 d, all starters tested produced different amounts of pGlu. In descending order of pGlu production, the bacteria analyzed were: Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. lactis. Evidence for the presence of glutamine to pGlu cyclase activity in lactic acid bacteria was provided. Cell lysates obtained from cultures of L. helveticus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and S. thermophilus showed the ability to cyclize glutamine to pGlu, resulting in processing yields from 1.4 to 30.3%, depending on the subspecies. Formation of pGlu from free glutamine appeared to be similar to that observed using a glutamine-glutamine dipeptide substrate. Under the experimental conditions applied, pGlu aminopeptidase activity was only detected in L. helveticus. Thus, pGlu formation in long-ripened cooked cheese may depend on the activity of thermophilic lactic acid bacteria.     

Identification of spoilage yeasts in a food-production chain by microsatellite polymerase chain reaction fingerprinting

A survey of yeast strains present in the production chain of mayonnaise and salad dressings was carried out over a period of 14 months. Attempts were made to identify the isolated yeasts with the API system, but identification of all species involved was not possible. In the investigation the performance of the microsatellite polymerase chain reaction (PCR) fingerprinting analysis with the microsatellite oligonucleotide primers (GAC)₅and (GTG)₅appeared to be superior. Several yeast species were encountered in the production lines but only the speciesZygosaccharomyces bailiiandZygosaccharomyces bisporuswere present in the final products. Only microsatellite PCR fingerprinting analysis allowed discrimination between species of theZygosaccharomycesgenus. In addition, the PCR-based technique allowed the discrimination of different types within theZ. bailiispecies.Z. bailiistrains isolated from spoiled products displayed PCR-fingerprinting types that appeared to be identical to some of those generated by strains isolated from one specific production line. This suggested that microsatellite PCR fingerprinting is useful in tracing back the origin of spoilage outbreaks, and that it can be applied in microbiological quality assurance monitoring systems in industrial environments.     

Fast detection and quantification of four dairy propionic acid bacteria in milk samples using real-time quantitative polymerase chain reaction

Propionibacterium freudenreichii is added to vat milk to create the characteristic eyes and typical nutty flavour of Emmentaler Protected Designation of Origin (PDO) cheese, but leads to serious quality defects in other raw milk cheeses from Switzerland. To trace propionic acid bacteria (PAB) in raw milk, we developed and validated a fast quantitative polymerase chain reaction (qPCR)-based method for P. freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii, and Propionibacterium acidipropionici. qPCR-standard curves were linear over five log units down to 10¹ copies per reaction (R ≥ 0.997); efficiencies ranged from 0.83 to 0.97. In spiking experiments, the lower limits of quantification were 10¹–10² cfu mL⁻¹ raw milk. Fifty one vat milk samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method gave faster and species specific determination of four dairy PAB in milk and yielded comparable quantitative results.     

Autolysis detection and evaluation of some lactic acid bacteria by renaturing sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and polymerase chain reaction assays

Eleven lactic acid bacterial strains were tested for autolysis ability and the presence of autolytic enzymes by renaturing sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Polymerase chain reaction (PCR) assays for the detection of lysogenic strains were performed. Autolysis in a buffer system was observed in Lactobacillus delbrueckii subsp. bulgaricus DSM 20080, L. delbrueckii subsp. bulgaricus DSM 20081, Bifidobacterium longum subsp. infantis DSM 20088, B. angulatum DSM 20098 and Streptococcus thermophillus DSM 20617. Mitomycin C induction of prophage was demonstrated in B. longum subsp. infantis DSM 20088, B. angulatum DSM 20098, Lactobacillus acidophilus DSM 20242 and S. thermophillus DSM 20617. The presence of genes encoding known bacteriophage lysins was demonstrated by a PCR assay and correlated well with the autolytic phenotypes of the strains, indicating that PCR screening is useful in the rapid identification of autolytic strains.     

Technological properties of milks fermented with thermophilic lactic acid bacteria at suboptimal temperature

In the present work the synergistic relationship between different strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was studied at optimal (44 degrees C) and suboptimal temperatures (30 degrees C). Acidification, viscosity, whey syneresis, and bacterial concentration of the final product were evaluated on single-strain and mixed cultures after 24 h at 30 degrees C and 6 h at 44 degrees C. Three pairs of strains (LBB + CP2, LBP + CP2, and LBR + CP2) showed synergistic effect, which was reflected by the viscosity and syneresis of the coagulum. These results were more significant when cultures were incubated at 30 degrees C, reaching apparent viscosity values of 19 to 28 mPa x s. On the other hand, lactobacilli cultures enhanced the growth of two streptococci strains (CP2 and CP4). These results were confirmed by cultures of streptococci supplemented with supernatants of culture of lactobacilli. Those supernatants stimulate the viscosity produced by CP2 and CP4 strains and reduce the syneresis of all cultures of streptococci. Neither the increase of viscosity nor reduction of syneresis could be attributed to a decrease of pH.     

Detection of peanut using real-time polymerase chain reaction

Preliminary results are presented on a sensitive and robust assay for the identification of peanut in commercial products using real-time PCR technology. Peanut specific primers and probe, designed using the Arah 2 gene, were optimised for real-time PCR using an ABI PRISM 7700. Commercial extraction kits employing different technological strategies were assessed for the extraction of PCR quality peanut DNA template. The specificity of the primer and probe set was determined using a wide range of food items and the limit of detection and quantification calculated using dilutions of peanut DNA. The assay was used to detect spiked or trace level of peanut in commercial samples and was finally used to detect peanut in a biscuit prepared with 2 ppm of lightly roasted peanut powder.An erratum to this article can be found at     

Detection of horse DNA in food and feedstuffs using a polymerase chain reaction assay

BACKGROUND: Development of accurate methods for rapid identification of animal materials in food and feedstuffs is essential to protect consumers and also to enforce feed bans. The aim of this study was to develop a polymerase chain reaction (PCR) assay for the specific detection of horse DNA in food and feedstuffs. RESULTS: The primers designed amplified a horse‐specific fragment of 114 bp of the mitochondrial 12S ribosomal RNA gene. The specificity of the primers was verified by PCR analysis of DNA from 32 non‐target species including mammals, birds, fish and plant species. The PCR assay developed allowed the detection of raw and heated horse tissues in muscle/oats mixtures even when the concentration of horse‐derived materials was reduced to 1 g kg^?1. CONCLUSION: The performance of the method was not affected by prolonged heat treatment (up to 133 °C for 20 min at 300 kPa), and consequently it could be very useful in verifying the origin of raw materials in food and feedstuffs submitted to denaturing technologies, for which other methods cannot be applied. Copyright © 2009 Society of Chemical Industry     

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.     

Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.     

Identification of yeast strains using the polymerase chain reaction

Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.     

发酵型大米乳酸菌饮料加工工艺研究

以大米为主要原料,经过粉碎、糊化、糖化、过滤等处理后得到米浆原液,加入一定量蔗糖、脱脂乳,接入两种乳酸菌进行发酵,制备发酵型大米饮料。通过预实验确定了最佳米/水比为1∶16,并且对乳酸菌进行了定向驯化。通过正交实验确定了产品最佳制备工艺,即蔗糖添加量4%、脱脂乳添加量5%、鼠李糖乳杆菌接种量3%、保加利亚乳杆菌接种量为4%,在37℃下发酵8 h。     

Enzymatic hydrolysis of soybean protein using lactic acid bacteria

The proteolytic activity of 12 lactic acid bacteria (LAB) strains, assayed on soy protein extract at a temperature of 37 °C for 6 h, was evaluated by SDS–PAGE, reverse-phase HPLC and free-amino acid analyses. The results indicated that α- and α′-subunits of β-conglycinin were the preferred substrates for the majority of the LAB. Only a few strains exerted some action against the basic polypeptides of glycinin, this fraction was the least degraded of all soy protein fractions. Whole-cell suspensions of LAB used in this study generated hydrophilic and hydrophobic peptides from mainly soy protein fractions. RP-HPLC analyses indicated differences in the profiles of the hydrolysates, with several peaks decreasing in size and new peaks being formed. Three of the selected strains assayed increased the level of total free amino acids in the soy protein extract (SPE) and hydrolyzed principally essential amino acids and flavour precursor amino acids.     

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount ofA. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships (R2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 x 10(9) copies/microl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 x 10(8) and 2.7 x 10(8) copies/microl for woodland and grassland, respectively), high DNA yield (6.4 microg/g and 1.8 microg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.     

Effects of exopolysaccharide-producing strains of thermophilic lactic acid bacteria on the texture of stirred yoghurt

Summary Rheological and sensory properties of yoghurts prepared from commercially available cultures that had ropy and non-ropy characteristics were compared. Our results from both instrumental and sensory data suggest that it may not be the amount of polysaccharide that is important to rheological properties, but the type of exopolysaccharide (EPS)-producing strains and consequently the interaction of the polymer with the milk proteins during the fermentation. Data also suggest that the interaction and co-operative growth that occurs in mixed cultures also influences the yield of EPS production in the fermentation, as combining two ropy strains does not lead to an increase in total polysaccharide, although the viscosity can be improved. Texture measurements for viscosity correlated with sensory evaluation of viscosity and with slipperiness.     

味全乳酸菌

乳品是消费者每天都需要摄入的东西,但因消费者自身原因会在摄入后产生不适的状况．加之最近频繁爆出乳品安全问题,让消费者对乳品的选择有了一些担忧．所以纯奶制品的销售额一直不太稳定,这给纯奶企业与经销商的投资带来了不利因素。但如果不是纯奶制品的乳品．例如果奶、花生奶等,却一直没有在销售业绩上出现下滑征兆。