食品中腐败酵母的实时荧光PCR鉴定

为研究食品中腐败酵母实时荧光PCR快速鉴定方法,设计和筛选出了可用于腐败酵母鉴定的多条探针和引物,并建立了针对酿酒酵母、鲁氏接合酵母、斯巴达克毕赤酵母和布鲁塞尔德克酵母等4种腐败酵母菌的实时荧光PCR鉴定方法。用文中建立的方法对从糕点、蜂蜜、饮料等市售食品中分离出的60余株酵母菌进行了鉴定,发现其中4株为酿酒酵母,21株为鲁氏酵母,其他为与上述4种不同的酵母。以实时荧光PCR方法鉴定酵母,全过程仅需约3 h,与常用的生化鉴定方法相比,简化了鉴定步骤,提高了鉴定准确性,缩短了鉴定时间。     

Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States

Minimum inhibitory concentrations were determined for 811 strains of Staphylococcus aureus isolated from cases of bovine mastitis in 11 countries. The countries and number of isolates included Denmark (105), England (92), Finland (95), Germany (103), Iceland (22), Ireland (42), Norway (101), Sweden (123), Switzerland (69), United States (53), and Zimbabwe (6). The antimicrobial agents tested were penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, amoxicillin + clavulanate, penicillin + novobiocin, enrofloxacin, premafloxacin, erythromycin, clindamycin, lincomycin, pirlimycin, neomycin, lincomycin + neomycin, and sulfamethazine. The MIC90 for these antimicrobial agents for all strains were 0.5, 1.0, 1.0, 0.5, 1.0, < or =0.06, 0.125, 0.125, < or =0.0078, 0.5, 1.0, 16.0, 1.0, 2.0, 0.5, and 4.0 microg/ml, respectively. Overall, only small variations between countries were seen in the MIC90 for the majority of compounds tested. Of the strains tested, 35.6% were positive for beta-lactamase production on initial testing, with an additional 21.3% positive after induction by penicillin. In conclusion, the overall level of resistance was generally low for all antimicrobial agents tested regardless of country. Given the differences in antimicrobial use in various countries, the widespread adoption of mastitis control programs to prevent infections limits the exposure of S. aureus infected animals to antimicrobial drugs.     

Comparison of lactococcal bacteriophage isolated in the United States and Argentina

Bacteriophage of Lactococcus lactis ssp. lactis and ssp. cremoris, isolated in the United States and Argentina, were compared with respect to host range, adsorption, latent period, burst size and immunological cross-reactivity. Only 1 out of 13 U.S. culture isolates was sensitive to Argentinian phage. Argentinian L. lactis ssp. lactis C2 mutants were resistant to 13 U.S. phage isolates (4 prolate and 9 isometric). While Argentinian phage Stl-3 multiplied on U.S. culture isolate 59-1, low adsorption (38%) and insignificant burst size and latent period data were evident. Antisera prepared against U.S. phage D59-1 (prolate) and F4-1 (isometric) neutralized the lytic activities of all Argentinian prolate phage although the F4-1 antiserum was less effective. The data suggest homology especially between U.S. phage D59-1 and the Argentinian phage.     

Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation

Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America.     

Occurrence of non-lactic acid bacteria populations involved in protein hydrolysis of cold-stored high moisture Mozzarella cheese

The aim of this study was to analyse non-lactic acid bacteria populations (NLABPs) and evaluate their role in proteolysis of cold-stored high moisture (HM) Mozzarella cheese. NLABPs reached values close to 8 log cfu mL⁻¹ after seven days of cold storage. Sequencing of 16 rDNA and rpoB genes and molecular biotyping allowed to identify 66 bacterial strains belonging to 25 species from 15 genera, mainly represented by Pseudomonas, Acinetobacter, and Rahnella. Fifteen strains showed proteolytic activity values higher than 1000.00 μg Gly mL⁻¹ after 24 h of growth in skimmed milk. Moreover, as shown by Urea-PAGE, 11 proteolytic strains caused partial or total disappearance of at least one of the caseins. Their proteolytic behaviour was assessed even when they grew inside the governing liquid together with HM Mozzarella cheese at 4 °C for 12 days. This is the first report that throws light on the complexity of NLABPs in HM Mozzarella cheese, demonstrating that some strains caused the partial hydrolysis of α, β, and γ caseins on its outer surface where a concomitant wrinkling and successive exfoliation became visible without significant changes in texture characteristics.     

Carotenoids of yeasts isolated from the Brazilian ecosystem

The carotenoid composition of pigmented yeasts isolated in Brazil was studied. The yeasts were cultured in yeast malt broth at 200 rpm, 25 °C, for 5 days, without illumination. Open column, thin layer and high performance liquid chromatography were used to separate, identify and quantify the carotenoids. The major pigments found in these yeasts were torulene and β-carotene. β-Carotene predominated in Rhodotorula graminis-125, Rhodotorula glutinis and Sporobolomyces roseus, while torulene was the principal carotenoid in Rhodotorula mucilaginosa. The yeast R. glutinis had the highest total carotenoid production (881 μg/l), followed by R. graminis (594 μg/l), Rhodotorula mucilaginosa-137 (590 μg/l), Rhodotorula mucilaginosa-108 (562 μg/l) and Rhodotorula mucilaginosa-135 (545 μg/l). Rhodotorula minuta and S. roseus had the lowest carotenoid contents (168 and 237 μg/l, respectively). In μg/g of dry cells, R. glutinis had a total carotenoid concentration of 132 μg/g.     

Incidence, concentration and effectiveness of malathion residues in wheat and maize (corn) exported from the United States

The incidence, concentration, and effectiveness of malathion treatments were measured in wheat and maize samples obtained from 79 port terminals in the United States. The presence of biologically active residue levels of malathion was inferred by bioassay assessment in 28% of 2058 wheat samples and in 8.4% of 2383 maize samples examined during the 2-year period, January 1977–December 1978. Malathion occurred most often in wheat from port terminals in the Pacific and Gulf regions where treatments were frequently part of the grain sale agreement and where grain was often treated immediately prior to sampling. At terminals where the grain was not treated just prior to sampling, the incidence of malathion on grain arriving at the ports was only 11.6% for wheat and 7.7% for maize.

Average malathion residues were 2.50 ppm in 210 samples of wheat and 1.03 ppm in 139 samples of maize and ranged from 2.71 ppm in Gulf wheat to 0.66 ppm in Pacific maize. Residues on wheat treated just prior to sampling, show inconsistent and generally inadequate malathion deposits resulting from port terminal treatments. No consistent relationship was evident between grain handling capacities and malathion residues.     

Comparison of the microflora isolated from spoiled cold-smoked salmon from three smokehouses

The microflora on spoiled, sliced and vacuum packed, cold-smoked salmon from three smokehouses was quantified and characterized in two independent experiments. Large variations in the microflora were observed both within (i.e. among vacuum packs from the same batch) and among the smokehouses. Lactic acid bacteria dominated the microflora, which reached 10⁷ cfu g⁻¹. Total viable counts of microorganisms alone were not related to quality, though spoilage characteristics were typical for microbiological spoilage. Among the lactic acid bacteria, Lactobacillus curvatus (ca 52–55%) was the most common species in both experiments with Lactobacillus saké, Lactobacillus plantarum, Carnobacterium spp. and Leuconostoc spp. present in smaller numbers. In some cases, large numbers of Enterobacteriaceae were also present and identified species were Serratia liquefaciens, Enterobacter agglomerans and Hafnia alvei. The microflora on cold-smoked salmon appeared to be related to the source of contamination i.e. the raw material and/or the smokehouse rather than being specific for the product, thus rendering the identification of the specific spoilage organisms difficult.     

Selection response for milk production in conventional production systems in Mexico, using genetic evaluations of Holstein sires from Canada and the United States

Polynomial regression models of the first, second, and third order were used to fit milk production deviations of daughters in Mexico on Canadian and US predicted transmitting ability values for 305-d mature-equivalent milk production (kg). For the pairs Canada-Mexico and Mexico-United States, 40 and 73 bulls with a minimum reliability of 0.75 were analyzed, respectively. Genetic correlations between pairs of countries were also estimated. The parameters were evaluated for all data, and for sires grouped according to the mean of the average phenotypic milk production (high and low) of their daughters’ herd mates. Quadratic and cubic effects were not significant in any analysis. From linear regression models, slopes of Mexican daughter deviations on US and Canadian predicted transmitting abilities were 1.01 and 0.93, respectively. Slopes were greater but intercepts were smaller for the high versus low level of production of the sires’ herd mates in Mexico. A greater difference between the genetic correlations was found for the high versus low environmental level than for the low level (0.79 vs. 0.70) for Mexico-US data compared with Canada-Mexico data (0.81 vs. 0.78). Genetic correlations between Mexico and the United States (0.74), and between Mexico and Canada (0.77), were smaller than the genetic correlation between the same Canadian and US sires (0.92), suggesting the presence of a moderate degree of genotype-environment interaction for milk production between Canada and the United States, and Mexico.     

浓香型白酒酵母对发酵糟醅中乙醇及主要酸、酯生成的影响

将分离自浓香型白酒酿造诸环节的20株酵母菌分别接种于装有入窖粮糟与黄水的玻璃坛中,并设立空白对照与窖内对照,遮光发酵50 d后,比较各发酵体系中主要酸、醇、酯的含量。结果显示,所有窖外发酵体系中乙酸、丁酸、乙醇、乳酯(文中均使用简称,下同)含量均高于窖内对照,己酯、乙酯、丁酯低于窖内对照;发酵糟醅中乙酸与乙酯、丁酸与丁酯有显著的线性正相关性,但己酸与己酯则无明显线性关系;供试的20株酵母中分别有16和12株对应的发酵糟醅中乙醇(最高达9.70 m L/100 g)和总酯(最高达11.52 g/L)高于空白对照;菌株Z8Y-15(Pichia、来自窖房空气)在浓香型白酒酿造中促进糟醅乙醇及己酯生成上具有较好的应用前景。     

Phenotypic and genetic diversity of Saccharomyces contaminants isolated from lager breweries and their phylogenetic relationship with brewing yeasts

A taxonomic study was carried out for isolates of Saccharomyces spp. identified as contaminants ("wild yeast") in 24 different lager breweries. With reference to the current taxonomy all isolates were found to belong to the Saccharomyces sensu stricto complex and 58% of the isolates were further identified as S. cerevisiae, 26% as S. pastorianus and 3% as S. bayanus. The remaining isolates (13%) could not be identified to the species level based on their phenotypic characteristics. However, some of these isolates were identified as S. cerevisiae by HaeIII restriction digest of PCR-amplified intergenic transcribed spacer (ITS) regions. Chromosome length polymorphism (CLP) was evident among the Saccharomyces brewing contaminants with chromosome profiles typical of Saccharomyces sensu stricto. Based upon cluster analysis of their chromosome profiles the majority of the brewing contaminants could be grouped as either S. cerevisiae or S. pastorianus/S. bayanus. Further, the technique was able to differentiate between almost all brewing contaminants and to separate them from any specific lager brewing yeast. The diversity of the Saccharomyces brewing contaminants clearly demonstrated by their CLP was further reflected by MAL genotyping. For the majority of the isolates more than two MAL loci were found with MAL1, MAL2 MAL3, MAL4 and MAL11, MAL31, MAL41 as the dominant genotypes. For all isolates MAL11 and MAL31 were found whereas MAL61 only was found for one isolate. The high number of MAL loci found in the SaccharomYces brewing contaminants indicate their adaptation to a maltose-enriched environment.     

Chemical and physical characteristics of corn silages and their effects on in vitro disappearance

Estimating the available energy in corn silage provides a unique challenge because the silage contains variable proportions of grain and stover, each of which can differ in availability due to chemical composition and physical form. The objectives of this study were to investigate relationships among chemical components and their relationships with in vitro disappearance of ground and unground dried silages, and to quantify minimally fragmented starch in corn silage and investigate its impact and that of mean particle size (MPS) on in vitro disappearance of unground silages. Thirty-two corn silages were selected to provide diversity in dry matter, protein, fiber, and MPS. Detergent fibers were highly correlated with each other and with nonfiber carbohydrates, and were used to develop prediction equations between these constituents. Sieves with apertures ≥4.75 mm were used to isolate intact kernels and large kernel fragments, which were collected and analyzed to measure minimally fragmented starch (Starch_>4.75). Dividing Starch_>4.75 by total starch defined the proportion of minimally fragmented starch (Starch_>4.75/Total), which ranged from 9 to 100% with a mean of 52%. Starch_>4.75/Total was positively correlated with MPS (r = 0.46). The inverse of Starch_>4.75/Total is an index of kernel fragmentation. Silages were prepared as whole material or ground to pass through a 4- or 1-mm screen of a cutter mill. In vitro dry matter disappearance (IVDMD) was greater for ground than for whole samples (71.7 and 61.2%, respectively). Increased IVDMD for ground samples was attributed to greater in vitro neutral detergent fiber (NDF) and neutral detergent solubles (NDS) disappearances. The IVDMD of ground samples was related to NDF and acid detergent lignin (R² = 0.80). The IVDMD of whole corn silage was related to acid detergent lignin, Starch_>4.75, MPS, and dry matter. When IVDMD was partitioned into in vitro digestible NDS (IVdNDS) and in vitro digestible NDF, the IVdNDS of whole was not uniform or completely fermented. The difference in IVdNDS between ground and whole was related to Starch_>4.75/Total. In conclusion, the proportion of minimally fragmented starch provides a corn silage fragmentation index that is related to the in vitro digestion of whole silages that, if validated by in vivo trials, may be a useful quantitative substitute for the qualitative processing adjustment factor that is used currently in summative equations for estimating the total digestible nutrients of corn silages.     

Virulence and stress susceptibility of clinical and environmental strains of Vibrio vulnificus isolated from samples from Taiwan and the United States

Vibrio vulnificus is an estuarine bacterium that causes severe wound infection and septicemia with high mortality. It also can be transmitted through the consumption of raw contaminated seafood and is an important foodborne pathogen. A total of 40 environmental and clinical V. vulnificus strains isolated from the United States and Taiwan were analyzed for virulence in animals, the presence of virulence-associated factors, and susceptibility to environmental stresses. Virulence in mice was exhibited by 85% of the environmental strains and 95% of the clinical strains. Strains from environmental or clinical sources were similar in virulence-associated phenotypes (protease activity, utilization of transferrin-bound iron, hemolysis, and inactivation in serum) and susceptibility to various stresses (4 and 52 degrees C, 0.1 and 10% NaCl, and pH 3.2), except freeze-thaw treatment. The clinical strains killed experimental animals after a shorter incubation time than did the environmental strains. Most of the 15 virulence-associated genes examined were present in most of the strains, regardless of their sources or virulence, with the exception of vvh, flgF, and purH. vvh was significantly more common in clinical strains than in environmental strains, and vvh, flgF, and purH were more common in virulent strains than in nonvirulent strains. These data may be helpful in devising strategies to manage or reduce the presence of V. vulnificus in foods.     

The effect of Lactobacillus buchneri 40788 on the fermentation and aerobic stability of high moisture corn in laboratory silos

The production of antifungal compounds during fermentation could be a useful mechanism to improve the aerobic stability of fermented feeds when they are exposed to air. High moisture corn (26% moisture) was ground and inoculated with various amounts of Lactobacillus buchneri 40788, a heterolactic acid bacteria, and ensiled in laboratory silos. Inoculation with L. buchneri 40788 from 1 x 10(5) to 1 x 10(6) cfu/g of corn had minor effects on the end products of fermentation during the early stage of ensiling (< or = 14 d). However, after 49, 92, and 166 d of ensiling, increasing the application rate of L. buchneri 40788 applied to corn increased the concentration of acetic acid when compared to untreated corn. Addition of L. buchneri 40788 had few other effects on the end products of fermentation. Dry matter recovery and aerobic stability were measured after 92 and 166 d of ensiling. At these times, dry matter recovery was not different among treatments, and numbers of yeasts and molds tended to decrease as the application rate of L. buchneri 40788 increased. Aerobic stability (number of h prior to a 2 degrees C rise in temperature after exposure to air) was markedly improved by the addition > or = 5 x 10(5) cfu/g of L. buchneri 40788. Combining L. buchneri 40788 with L. plantarum did not impart better aerobic stability than when L. buchneri 40788 was applied alone to corn. Addition of L. buchneri 40788 did not affect the rate of fermentation in high moisture corn, but after prolonged storage higher application rates increased production of acetic acid and markedly improved aerobic stability.     

Hexabromocyclododecanes in indoor dust from Canada, the United Kingdom, and the United States

alpha-, beta-, and gamma-hexabromocyclododecane diastereomers (HBCDs) were measured in house dust from Birmingham, U.K. (n=31, median concentration=730 ng sigmaHBCDs g(-1)); Amarillo/Austin, TX (n=13, 390 ng g(-1)); and Toronto, Canada (n=8, 640 ng g(-1)). Concentrations in dust (n=6, 650 ng g(-1)) from U.K. offices were within the range for UK homes. Concentrations from each country were statistically indistinguishable. In one UK house dust sample, 110,000 ng g(-1) was recorded-the highest recorded in indoor dust to date. While upper bound average U.K. dietary exposures for adults and toddlers, respectively, are 413 and 240 ng sigmaHBCDs day(-1), U.K. adults and toddlers daily ingesting, respectively, 50 and 200 mg of dust contaminated at the 95th percentile concentration are exposed, respectively, to 1100 and 4400 ng sigmaHBCDs day(-1). Normalized to body weight, this high-end exposure scenario estimate for toddlers is within the range reported elsewhere for occupationally exposed adults. While in commercial formulations gamma-HBCD predominates (>80%), alpha-HBCD in dust constitutes 14-67% of sigmaHBCDs (average 32%). Hence the predominance of the alpha-diastereomer in humans may arise partly from dust ingestion, and not solely to in vivo metabolism (when alpha-HBCD is formed from bioisomerization of other diastereomers), or dietary exposure (where alpha-HBCD predominates in most foodstuffs).     

Feeding value and nutrient preservation of high moisture corn ensiled in conventional silos forlactating dairy cows.

Sixty-one Holstein cows were used at varying stages of lactation to evaluate the feeding value of high moisture shelled corn ensiled in conventional silos. Control rations consisted of comparable amounts of conventional shelled corn dried in commercial drying facilities. Cows fed high moisture corn produced milk of higher fat content (3.09 versus 2.64%) and greater quantities of fat (.73 versus .64 kg) while they consumed less concentrate (9.28 versus 9.84 kg), resulting in the crude fiber content of the dry matter being 14.60% compared to 13.82% for control cows. Covariate adjustment of treatment means to equal intake of crude fiber eliminated differences above. Milk, 4% fat corrected milk, silage, total dry matter intake, and rumen volatile fatty acids were not significantly different between groups. Dry matter recovery following the ensiling process was 96.0 plus or minus 1.9% with 3.0 plus or minus 1.1% classified as spoiled corn, resulting in a total of 7% loss. Recoveries of all proximate constituents were similar to dry matter with the exception of ash (82.4%). Costs of the two systems were evaluated for varying corn prices and moisture.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

Effect of lactic acid bacteria inoculants on in vitro digestibility of wheat and corn silages

The aim of the study was to determine the effect of 10 sources of lactic acid bacteria (LAB) on dry matter digestibility (DM-D) and neutral detergent fiber digestibility (NDF-D), in various combinations with starch, in vitro. The soluble starch represented a concentrate feed, whereas silage represented feeding only roughage. The DM-D and NDF-D were determined after 24 and 48 h of incubation to represent effective (24 h) and potential (48 h) digestibility. Addition of LAB was both by direct application of the inoculants to rumen fluid (directly fed microbials) and by the use of preinoculated silages. For each feed combination, tubes without added LAB served as controls. The results indicate that, overall, some LAB inoculants applied at ensiling or added directly to the rumen fluid had the potential to increase the DM-D and NDF-D. The major significant inoculant effect on NDF-D was obtained after 24 h of incubation, whereas the effect after 48 h was mainly nonsignificant. The effective inoculants seemed to minimize the inhibitory effect of the starch on NDF-D within 24 h, perhaps by competition with lactate-producing rumen microorganisms.     

Genotypic diversity of bacteria and yeasts isolated from Tibetan kefir

In this study, we have investigated the intraspecies genotypic diversity of bacteria and yeasts isolated from Tibetan kefir via the (GTG)₅‐REP‐PCR technology. According to the cluster analysis of generated fingerprints, forty‐nine bacterial isolates were grouped into nine clusters and formed thirteen genotypes, and twenty‐two yeast isolates were grouped into two clusters and formed two genotypes. It's worth noting that isolates of Lactobacillus kefiranofaciens, Streptococcus thermophilus, Lactobacillus kefiri and Lactobacillus paracasei exhibited the intraspecies genotypic diversity, whereas isolates of Saccharomyces cerevisiae and Kazachstania unispora did not exhibit. These results confirmed that bacterial isolates possessed higher level of intraspecies genotypic diversity than yeast isolates studied in this research. These findings revealed that (GTG)₅‐REP‐PCR was an efficient and sensitive molecular typing tool for revealing the intraspecies genotypic diversity among the bacterial isolates originated from Tibetan kefir.