RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

人工液体培养发菜与野生发菜营养成分比较

为了合理开发利用人工培养发菜,比较分析了人工液体培养发菜和野生发菜的营养成分。通过高效液相色谱(HPLC)、电感耦合等离子体发射光谱(ICP-OES)、苯酚硫酸、凯氏定氮和索氏抽提法测定了2种发菜的Ca、Mg、Cu、Zn、Sr、Fe、Se元素、氨基酸、粗多糖、蛋白质、脂肪的含量。结果表明:人工液体培养发菜中粗蛋白、脂肪含量分别为26.17%、0.23%,而野生发菜中为23.05%、0.31%;人工培养发菜的胞外多糖含量为36.95 mg/L,人工培养发菜与野生发菜的荚膜多糖含量分别为15.03 mg/kg和13.84 mg/kg,氨基酸总量分别为21.99 mg/g和21.20 mg/g,其中必需氨基酸含量分别为11.73 mg/g和7.97 mg/g。人工培养发菜的Mg、Cu、Zn、Se含量优于野生发菜,分别是野生发菜的2.9、3.4、3.8、1.6倍。综合分析认为,人工液体培养发菜能分离较多的胞外多糖;粗蛋白、脂肪、荚膜多糖、氨基酸和Ca、Sr、Fe元素含量与野生发菜相近。     

pH-dependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli in the presence of various acidulants

The effect of acidulant identity on the pH-dependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli was studied. Nine strains of E. coli (seven O157:H7, one O111:H-, and one biotype 1 reference strain) were cultured individually for 18 h at 37 degrees C in tryptic soy broth (TSB) plus 1% dextrose and in TSB without dextrose to yield acid resistance induced and noninduced stationary-phase cells, respectively. These cultures were then inoculated into brain heart infusion broth (BHI) supplemented with 0.5% citric, malic, lactic, or acetic acid and adjusted to pH 3.0 with HCl. The BHI tubes were incubated at 37 degrees C for up to 7 h and samples were removed after 0, 2, 5, and 7 h and plated for counting CFU on BHI agar and MacConkey agar (MA). The results were compared to data previously obtained with HCl only. Acid resistance varied substantially among the isolates, being dependent on the strain, the acidulant, and the induction of pH-dependent acid resistance. Hydrochloric acid was consistently the least damaging to cells; lactic acid was the most detrimental. The relative activity of the other acids was strain dependent. Inducing pH-dependent acid resistance increased the already substantial acid tolerance of stationary-phase E. coli. The extent of injury also varied with acid and strain, with as much as a 5-log-cycle differential between BHI agar and MA CFU counts. The accurate determination of the survival of enterohemorrhagic E. coli in acidic foods must take into account the biological variability of the microorganism with respect to its acid resistance and its ability to enhance survival through the induction of physiological stress responses.     

Surveillance and antimicrobial resistance of Salmonella strains isolated from slaughtered pigs in Spain

The prevalence of and the antibiotic resistance shown by Salmonella isolated from pigs in Andalusia (southern Spain) is reported. Salmonella enterica was recovered from 40 (33%) of 121 sampled herds, and a total of 65 isolates were serotyped. The most common Salmonella serotypes were Typhimurium and Rissen (30.7% each); others included Derby (9.2%), Brandenburg (9.2%), Newport (7.7%), Bredeney (4.6%), Anatum (3.0%), Hadar (1.5%), and Goldcoast (1.5%). One strain (1.5%) belonging to the monophasic variant of the Typhimurium serotype (Salmonella 4,5,12:i: -) was also detected. Definitive phage type (DT) 104b was the most common Typhimurium phage type isolated. These Salmonella strains were resistant to various antimicrobial agents, including tetracycline (84.6%), streptomycin (69.2%), neomycin (63.0%), sulfonamides (61.5%), ampicillin (53.8%), and amoxicillin (53.8%). All isolates were fully susceptible to ceftriaxone, ciprofloxacin, and colistin. Thirty-nine strains (64%) resistant to four or more antimicrobial agents were defined as multidrug resistant. Multidrug resistance profiles were observed in Salmonella serotypes Typhimurium, Rissen, Brandenburg, Bredeney, a monophasic variant, Gold-coast, Hadar, and Anatum, with serotypes Typhimurium and Brandenburg showing the most complicated resistance patterns (resistant to > or = 11 drugs).     

Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance

The effects of growth temperature (in the range 10-45 degrees C) and acidification up to pH 4.5 of the culture medium (Brain Heart Infusion, BHI) with different organic acids (acetic, citric and lactic) and hydrochloric acid on membrane fatty acid composition and heat resistance of Salmonella typhimurium CECT 443 were studied. The heat resistance was maximal in cells grown at 45 degrees C (cells grown in non-acidified BHI showed a D58-value of 0.90 min) and decreased with decreasing growth temperature up to 10 degrees C (D58-value of 0.09 min). The growth of cells in acidified media caused an increase in their heat resistance. In general, acid adapted cells showed D-values of between 1.5 and 2 times higher than the corresponding for non-acid adapted control cells. This cross-protection response, which has important implications in food processing, was not dependent on the pH value and the acid used to acidify the growth medium. A membrane adaptation corresponding to an increase in the unsaturated to saturated fatty acids ratio (UFA/SFA) and membrane fluidity was observed at low growth temperature. Moreover, the acidification of the growth medium caused a decrease in UFA/SFA ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in cyc19 relative concentration. Thus, acid adapted cells showed CFA levels 1.5 times higher than non-acid adapted control cells. A significant proportion of unsaturated fatty acids were converted to their cyclopropane derivatives during acid adaptation. These changes in membrane fatty acid composition result in cells with decreased membrane fluidity. A clear relation between membrane fatty acid composition and heat resistance was observed. In general, D-values were maximum for cells with low UFA/SFA ratio, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting acid adapted cells from heat inactivation. However, changes observed in membrane fatty acid composition are not enough to explain the great thermotolerance of cells grown at 45 degrees C. Thus, other mechanisms, such as the synthesis of Heat Shock Proteins, could be responsible for this increase in the bacterial heat resistance.     

纺织品 生物舒适性 稳态条件下热阻和湿阻的测定

本标准是修改采用ISO 11092：1993《纺织品生理舒适性稳态条件下热阻和湿阻的测定（蒸发热板法）》,对GB／T 11048—1989《纺织品保温性能的测定》的修订。本标准与ISO 11092：1993的主要差异如下：     

曲酸生产菌种的分离筛选与发酵条件研究

从土壤中分离出一株曲酸产量较高的米曲霉(Aspergillusoryzan)菌株,对该菌株的发酵条件进行了初步研究。结果表明,该菌株在淀粉8%、酵母膏0.2%、MgSO4·7H2O0.05%、K2HPO40.1%、pH6.0的培养基中,28℃恒温摇床振荡培养6d,摇瓶发酵液曲酸含量为41.2mg/mL。     

两株魏斯氏菌的益生特性比较

以分离自泡菜的2株魏斯氏菌为研究对象,对其耐酸、耐胆盐、耐人工肠液、抑菌、降解胆固醇和甘油三酯、表面特性的益生特性进行了比较研究。实验结果表明:绿色魏斯菌ZY-6的耐酸特性好于希腊魏斯菌WS-419,pH值为2时的存活率分别为45. 18%和24. 65%,pH为3和4时,存活率> 100%;在质量浓度3 g/L的胆盐浓度中存活率分别为75. 89%和72. 05%,都显示出较好的胆盐耐受性; 2株菌对人工肠液具有一定的抗性,3 h后的存活率在80%左右; ZY-6的抑菌活性稍好于WS-419,两者对大肠杆菌、金黄色葡萄球菌、沙门氏菌的抑菌圈直径分别为22. 61、27. 02、30. 53 mm和22. 59、25. 82、27. 96 mm; 2株魏斯菌降解胆固醇的能力相似,降解率分别为32. 79%和32. 13%; WS-419对甘油三酯的降解率好于ZY-6,两者分别为42. 80%和26. 70%; 2株菌的表面疏水性和表面凝集性皆大于60%,显示出较好的表面特性。绿色魏斯菌ZY-6和希腊魏斯菌WS-419的益生性能较好,可作为功能性食品开发的候选菌株。     

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.     

Survival of biofilm-forming Salmonella on stainless steel bolt threads under dry conditions

We examined the survival of two biofilm-forming strains and two biofilm-deficient strains of non typhoid Salmonella (NTS) on stainless steel bolt threads under dry conditions. Five μL of tryptone soya broth or egg yolke mulsion containing NTS strains at a concentration of 9 log cfu/mL was dropped onto the thread surfaces of hexagonal bolts. After inoculation, the bolts were screwed into the nuts, and then removed (Separate type) or not removed (Unit type). The two types of samples were kept in a dry environment (20.0-25.0°C, 2-15% humidity) and bacteria on the surfaces were periodically counted. Biofilm-forming strains were recovered from all samples after 336 days of incubation, but biofilm-deficient strains were isolated from only two of 8 samples after 336 days. This finding demonstrates that NTS can survive for approximately one year on bolt threads, providing direct evidence of the potential risk of constructions having crevices or uneven surfaces as possible contamination sources. The risk of cross-contamination may be higher for biofilm-forming strains than for biofilm-deficient strains.     

Influence of lactate and acetate salt adaptation on Salmonella Typhimurium acid and heat resistance

The aim of the present study was to determine the survival of Salmonella Typhimurium adapted with sodium lactate (NaL), potassium lactate/sodium acetate mixture (KL/NaA) or sodium acetate (NaA) in simulated gastric fluid (SGF) and during heat treatment. NaL-, KL/NaA- and NaA-adapted cells were prepared by incubating in tryptic soy broth (TSB) containing these salts at 5, 5 and 3% (w/v) concentration levels, respectively, for 24 h at 37 °C. The Baranyi model was used to compare the growth kinetic parameters of adapted cells. The acid and heat resistance of adapted cells were determined by incubating in SGF (pH 2.04) at 37 °C and in TSB at 55.8, 57.8 and 59.8 °C, respectively. Adapted cells had significantly (P < 0.05) longer lag phase duration (LPD) and slower maximum growth rate (MGR) than non-adapted cells. The acid resistance of KL/NaA-adapted cells was not significantly (P > 0.05) different from that of non-adapted cells. NaL-adapted cells were more susceptible to the low pH environment, whereas NaA-adapted cells showed enhanced acid resistance compared to non-adapted and other adapted cells. Unlike acid resistance, both NaL- and NaA-adapted cells showed enhanced heat resistance with increased D-values, regardless of treatment temperatures. Thus, this study indicates that adaptation of S. Typhimurium to 5% NaL or 3% NaA could enhance their ability to survive thermal processes or in the human stomach, possibly increasing the risk of Salmonella outbreaks.     

Changes in culturability and virulence of Salmonella typhimurium during long-term starvation under desiccating conditions

The survival of Salmonella typhimurium under desiccation and starvation conditions commonly associated with farm buildings was investigated in a desiccation model system: filtration onto polycarbonate membranes placed in a sealed desiccator with 0.0067 g/m³ absolute humidity. Heterogeneities within bacterial populations in relation to time of desiccation were investigated on a single-cell basis by epifluorescence microscopy coupled with an image analysis system in conjunction with fluorescent dyes Chemchrome V6 and DAPI. Changes in cellular states were compared to the results of plate counts (colony forming units, CFU) on selective (modified semi-solid Rappaport Vassiliadis (MSRV)) and non-selective (nutrient agar (NA) and R2A agar) media, and to the measurements of infectivity and virulence using two animal models (chicks and mice). During 9 weeks of experimental desiccation, total cell counts (DAPI) of starved S. typhimurium remained stable, as did esterase activity (Chemchrome V6), but DAPI fluorescence intensity decreased slowly. Bacterial cells entered gradually into non-culturable states (decrease of CFU counts on MSRV, NA and R2A agar media) and the total loss of culturability on NA (defined as probability of presence of 1 CFU on the membrane inferior to 10⁻⁶) was obtained after 9 weeks. Loss of chick infectivity and mice virulence in animal models occurred more rapidly, within three weeks of experimental desiccation.     

Antimicrobial resistance and phage and molecular typing of Salmonella strains isolated from food for human consumption in Spain

The aims of this study were to ascertain the population structure and antimicrobial susceptibility of Salmonella enterica serovars isolated in 2002 from food in 16 Spanish regions. Serovars were characterized by serotyping, phage typing, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) typing, and 264 nonrelated strains were selected for further analysis. The main sources were eggs and their derivatives (21.6% of strains), poultry and related products (16.6%), and seafood (16.3%). High serotype diversity was detected (51 serotypes); the most common were Enteritidis (n = 96, 36.3%) and Typhimurium (n = 53, 20.1%), followed by a miscellaneous group of 49 different serotypes (n = 115, 43.5%). A 15% increase in Salmonella Enteritidis isolation was observed. Common phage types for Salmonella Enteritidis were PT1 (41.6% of isolates), PT4 (9.4%), PT6 (9.4%), and PT6a (9.4%), and common types for Salmonella Typhimurium were DTU302 (18.8%), DT104 (15.1%), and DT104B (13.2%). Salmonella Enteritidis strains were categorized into eight PFGE types with a similarity of 81 to 96%, and 73.9% of the strains were grouped into just one cluster. Salmonella Typhimurium isolates were divided into 13 PFGE types with a similarity of 64 to 86%, and one predominant clone contained 41.5% of the strains. Resistance rates for Salmonella Enteritidis, Salmonella Typhimurium, and the miscellaneous group were, respectively, 8.3, 69.8, and 13.9% for ampicillin, 3.1, 52.8, and 59% for streptomycin, 40.6, 22.6, and 10.4% for nalidixic acid, 15.6, 71.7, and 31.1% for tetracycline, 7.3, 18.8, and 9.5% for trimethoprim-sulfamethoxazole, 0, 50.9, and 4.3% for chloramphenicol, and 6.2, 71.7, and 17.4% for multiple (at least four) antimicrobials. All the strains remained susceptible to other beta-lactams and fluoroquinolones. Surveillance of S. enterica isolated from food is strongly recommended to reduce community exposure to antimicrobial resistant strains.     

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.     

对米根霉在不同条件下产酸情况的研究

乳酸是目前世界上公认的3大有机酸之一,其广泛应用于食品、医药、化工、制革、纺织、环保和农业等诸多领域。用微生物发酵法生产L-乳酸可以达到较高的纯度要求。根霉属中的米根霉(Rhizopus oryzae)是目前发酵生产L-乳酸的重要菌株,本课题研究的是从自然界选取的米根霉在不同条件下的产酸情况。采用分析方法包括还原糖的测定,pH测定,NaOH滴定确定乳酸产量。通过对不同条件下产酸情况的分析,可以优化产酸条件,提高产酸率。最终确定出在葡萄糖浓度为130g/L,氯化铵作为氮源,其浓度在2.0g/L的条件下,实验室菌株的最佳产酸量是44.2g/L,葡萄糖的转化率是40.1%。     

Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions

The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D _121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores.     

Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions

 The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D _121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores. Received: 1 July 1997