Rapid identification of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii species using species-specific primers

Based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR), an identification tool for rapid differentiation of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii, species isolated recently from French sourdough was developed. The DNA fragments containing ISRs were amplified with primers pairs 16S/p2 and 23S/p7. Clone libraries of the PCR-amplified rDNA with these primers were constructed using a pCR2.1 TA cloning kit and sequenced. The DNA sequences obtained were analyzed and species-specific primers were designed from these sequences. Two PCR amplicons, which were designated small ISR (S-ISR) and large ISR (L-ISR), were obtained for all Lactobacillus species studied. The L-ISR sequence reveale2d the presence of two tRNA genes, tRNAAla and tRNAIle. Species-specific primers designed allowed rapid identification of these species. The specificity of these primers was positively demonstrated as no response was obtained for more than 200 other species tested.     

Changes within Lactobacillus and Bifidobacterium species

Laboratory rats with a gut flora unambiguous free from Bifidobacterium revealed three days after an application of a lactose-rich food a dominating Lactobacillus plantarum flora. Up from this date, Bifidobacterium could be detected for the first time. At the beginning, there was to be observed merely B. adolescentis and B. pseudolongum, at the 5th to the 10th day B. bifidum supervened. Finally this species together with B. infantis was dominating. With in vitro experiments, by a systematic modifying of the medium changes from Bifidobacterium species typical to faeces from infants to such species only to meet in faeces from adults could be observed only after a long time of cultivation.     

泡菜中生物转化共轭脂肪酸的植物乳杆菌

共轭脂肪酸是指一类含共轭双键的多不饱和脂肪酸的总称,包括共轭亚油酸(conjugated linoleic acid,CLA),共轭亚麻酸(conjugated linolenic acid,CLNA)和共轭十八碳四烯酸(conjugated stearidonic acid,CSA)。CLA和CLNA分别是由亚油酸和亚麻酸衍生所得的多种位置与几何异构体的总称,具有多种生理功能,如抗癌、抗动脉粥样硬化、提高机体免疫力和减肥等,已成为功能性脂质的研究热点。采用GC-MS(gas chromatographymass spectrometry)法分析了筛选自泡菜的22株植物乳杆菌转化共轭脂肪酸的能力。结果显示,CCFM47和CCFM232两株菌具有极高的共轭亚油酸和共轭亚麻酸转化能力,其中亚油酸的转化率均超过30%,发酵液中CLA总浓度分别达到0.166 4 mg/ L和0.151 4 mg/mL,而对α-亚麻酸的转化率高达60%,发酵液中CLNA总浓度分别达到0.196 4 mg/mL和0.172 1 mg/mL。     

Production of organic acids by Lactobacillus strains in three different media

Ten strains of Lactobacillus (Lb). casei, Lb. rhamnosus, Lb. plantarum, Lb. paracasei and Lb. curvatus species were chosen to determine the production of organic acids after cultivation in skimmed milk, MRS broth and Jerusalem artichoke (JA) medium. The highest acidity was obtained in MRS broth and the weakest acidification was found in skimmed milk. Lb. casei Shirota produced the highest amount and Lb. rhamnosus VT1 the lowest amount of substances being estimated as titratable acidity. All strains produced lactic acid in the investigated broth and most of the strains produced acetic acid in MRS broth except Lb. curvatus 2768 and Lb. casei Shirota, in JA broth except Lb. paracasei SF1 and in skimmed milk except Lb. casei 2750, Lb. curvatus 2768, Lb. curvatus 2775 and Lb. casei Shirota. All strains, except Lb. plantarum 01, produced butyric acid in MRS broth. Beside the lactic and acetic acids, formic, citric, succinic and glutamic acids were also produced in MRS broth; formic and succinic acids were produced in skimmed milk and succinic acid in JA broth. Some strains showed change in their fermentation profile from homofermentative to mix-acid fermentation in milk. The antifungal efficiency of the lactic and acetic acid in the amount produced by lactobacilli was investigated. None of the investigated aspergilli were inhibited. The inhibitory effect of acids against Fusarium increased unequivocally with the increasing concentration. The study pointed at the dissimilarity of organic acid production of Lactobacillus strains, which was considerably influenced by the media.     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

氨酸对嗜酸乳杆菌生长及冻干的影响

探讨了氨基酸对嗜酸乳杆菌生长的影响.采用单因子试验研究了16种氨基酸对嗜酸乳杆菌增菌效果和抗冷冻效应,结果表明,谷氨酸、精氨酸、亮氨酸、赖氨酸、甲硫氨酸、脯氨酸、苯丙氨酸显著促进嗜酸乳杆菌生长,丙氨酸、异亮氨酸、半胱氨酸、丝氨酸、苯丙氨酸、大冬氨酸、甘氨酸、脯氨酸能提高嗜酸乳杆菌抗冷冻能力.研究发现,向发酵液中添加异亮氨酸0.03g/L、半胱氨酸0.03g/L、天冬氨酸0.03g/L、丝氨酸0.03g/L、甘氨酸0.03g/L组成的复合氨基酸或半胱氨酸0.03g/L、天冬氨酸0.03g/L、丙氨酸0.03g/L、甘氨酸0.03g/L组成的复合氨基酸,均可使嗜酸乳杆菌冻干存活率达到95%以上,而对照组只有42.95%.     

New insights in antibiotic resistance of Lactobacillus species from fermented foods

Bacteria belonging to the genus Lactobacillus are used as starter cultures or that develop naturally as fermenting microbiota in the production of various foods. On the detrimental side, lactobacilli may act as reservoir of antibiotic resistance genes, which can spread to commensal bacteria in humans or animals, or to food-associated pathogens. In the last decade, advances in molecular biology and in genome sequencing have provided more information on antibiotic resistances in foodborne bacteria. The aim of this review was to consider and provide an up-to-date status on phenotypic and genotypic antibiotic resistance profiles in Lactobacillus species from fermented foods and also to highlight new information on the distribution of glycopeptide and chloramphenicol resistance genes in Lactobacillus genomes. In silico screening of vanZ (glycopeptide resistance) and cat (chloramphenicol resistance)-like sequences in Lactobacillus species isolated from fermented foods revealed for the first time the occurrence of vanZ and cat genes in Lactobacillus species being highly conserved genes in the chromosome of each species, presumably non-transferable. Further studies involving genome sequences of Lactobacillus isolated from fermented foods, especially those relying on spontaneous fermentation, is crucial to increase knowledge on the potential presence and spread of antibiotic resistance genes via the food route.     

Monitoring the growth of Lactobacillus species during a rye flour fermentation

The natural microbial community conducting an industrial sourdough fermentation was investigated by molecular biological methods using the following strategy: strains were isolated and subjected to RAPD (randomly-amplified polymorphic DNA) PCR. After computer-supported pattern analysis and clustering of the strains the 16S rDNA of members of each distinct cluster were partially (530 bp) or completely (1570 bp) sequenced and identified by comparative sequence analysis. The predominant strains of this fermentation could be allotted to the species Lactobacillus amylovorus, Lactobacillus pontis and a species, which phylogenetically takes an intermediate position between L. pontis and L. panis. Sporadically, strains were identified as L. reuteri. In a second step the effect of external factors was investigated under the controlled conditions of a lab-scale process. Fermentations were carried out at 34°C, 40°C and 46°C. The development of the flora was consistent in independent fermentations as proved by RAPD typing of randomly-picked colonies. The microbial community in these fermentations was identical to those found in an industrial scale. The qualitative composition of the flora was not affected by the temperature. L. amylovorus was the dominant species. With increasing fermentation time, a shift toward the predominance of heterofermentative lactobacilli was observed. This finding was underlined by metabolic studies and stoichiometric calculations of the metabolic pathways. With increasing temperature the percentage of homofermentative organisms was reduced. Furthermore, the growth rate and the metabolic activity increased, followed by an immediate decrease of the growth rate at 46°C and lower terminal values of lactate, acetate and ethanol, respectively.     

Bioactive fatty acids of three commercial scallop species

The fat content and fatty acid profile of commercially important scallops Flexopecten glaber, Mimachlamys varia, and Pecten jacobaeus were investigated in samples of adductor muscle, gonad, mantle, and viscera. The viscera showed the highest lipid content in all species examined. Signi?cant differences were found in the fatty acid composition among tissues and among scallops. All pectinids exhibited high levels of eicosapentaenoic and docosahexaenoic acids in the adductor muscle, with a maximum value of 211 mg/100 g tissue and 252 mg/100 g tissue in the viscera of F. glaber. Highest n3/n6 ratios were recorded in F. glaber gonad and viscera, in P. jacobaeus muscle, and in the gonad of M. varia. M. varia adductor muscle had the lowest values of atherogenicity and thrombogenicity indices used as indicators of beneficial health effects. These data contribute to the overall evaluation of the nutritional quality of scallops and suggest that their consumption may provide health benefits.     

Lactobacillus casei, dominant species in naturally fermented Sicilian green olives

This study investigated the phenotypic and genotypic characteristics of lactic acid bacteria in naturally fermented green olives, collected from different areas of Sicily. Both classical biochemical tests and PCR/Restriction Fragments Length Polymorphism (RFLP) of 16S rDNA were used to characterize the isolates. The identity of the isolates was obtained by the partial sequencing analysis of the 16S rDNA. The BioMerieux software assigned the 13 heterofermentative strains to the Lactobacillus brevis species; 24 homofermentative strains were classified as Lactobacillus casei and the remaining 11 homofermentative lactobacilli were identified as Lactobacillus plantarum. The rapid ID 32 STREP test identified coccal-shaped strains as Enterococcus faecium species. The PCR/RFLP analysis showed a remarkable bacterial heterogeneity within the isolates. The 16S rDNA partial sequencing did not confirm biochemical identification, revealing a strong dominance of isolates belonging to the L. casei species. It is noteworthy that this species has never been reported as dominant species in fermented vegetables.A combination of molecular and biochemical analysis allowed the identification of species involved in natural food fermentations.     

The inhibitory effects of Lactobacillus casei and Lactobacillus helveticus on Bacillus species isolated from raw milk in various salt concentrations

Nineteen strains of Bacillus were isolated and identified from 111 samples of raw milk. The inhibitory effects of Lactobacillus casei and Lactobacillus helveticus on strains of Bacillus were studied. The inhibitory effects were evaluated (using the well diffusion method) on nutrient agar, with NaCl added at concentrations of 40, 45, 50, 55, 60 and 65 g/L. Lactobacillus casei inhibited 16 strains of Bacillus on nutrient agar without salt, and 18 strains in the presence of salt. When the salt concentration was 6.5%, the inhibitory effect decreased, and L. casei inhibited only eight strains of Bacillus ( Bacillus cereus , Bacillus sphaericus , Bacillus licheniformis , Bacillus macerans , Bacillus firmus , Bacillus coagulans , Bacillus polymyxa and Bacillus stearothermophilus ). Lactobacillus helveticus formed the inhibitory zones against five strains of Bacillus on nutrient agar without salt, but with 4% NaCl concentration, it showed an inhibitory effect on 13 strains; a further two strains were inhibited in the presence of 4.5 and 5.0% salt. In 5.5% NaCl, the same 15 strains were inhibited by L. helveticus, whilst in 6 and 6.5% salt, the inhibitory effect decreased and only six and seven strains were affected, respectively. The maximum inhibitory effect with both L. casei and L. helveticus was observed in the presence of 4.5–5.5% salt.     

Polyphasic approach for quantitative analysis of obligately heterofermentative Lactobacillus species in cheese

Obligately heterofermentative lactobacilli (OHL) present in cheese during ripening can influence the flavour and texture of the final product. In order to better evaluate, follow and control this population, there is a current need for easy-to-use tools. In this study, a culture-dependent quantitative method (ABEV medium) was set up for direct and selective enumeration of total OHL from cheese, and a culture-independent method based on specific real time PCR (qPCR) assays was developed to target Lactobacillus fermentum and Lactobacillus parabuchneri individual species. These tools were applied for OHL quantification in manufactured Emmental and Tomme cheeses. The ABEV medium was well adapted for specific enumeration and isolation of OHL species present in milk-derived samples, even in the presence of background microbiota. qPCR assays showed 100% specificity and could accurately quantify the targeted species in various types of cheese. Culture-dependent and -independent techniques evaluated in manufactured cheese samples generated similar bacterial counts. The behaviour of L. fermentum and L. parabuchneri was characterized from milk samples to the end of ripening. In addition, PCR-TTGE was used to confirm the presence of inoculated species and to globally analyze the composition of naturally present species. This polyphasic approach illustrates the complementarity of the different methods.     

1株植物乳杆菌发酵豆粕产有机酸的研究

从发酵豆粕中筛选得到1株生长迅速产酸量大的乳酸菌,经鉴定为植物乳杆菌,命名为JUN-DY-6,利用该菌对豆粕进行固态发酵。通过高效液相色谱法(HPLC)对发酵豆粕中的有机酸组成进行了定性定量分析。结果显示:用JNU-DY-6生产的发酵豆粕中主要有6种有机酸,其中以乳酸、乙酸、丙酸和柠檬酸为主。且发酵豆粕工艺中糖蜜的添加与否对发酵豆粕中丙酸含量影响较大。碱性蛋白酶添加量0.5%,糖蜜添加量为3%的条件下发酵豆粕,产生有机酸含量峰值为14.646 g/L,比仅使用乳酸菌发酵的情况下提高了23.1%。SDS-PAGE凝胶电泳结果显示,发酵豆粕中的大豆蛋白基乎完全降解为小分子多肽。     

乳酸菌代谢保护剂中糖产酸对冷冻保护的影响

真空冷冻干燥是乳酸菌菌粉制备的关键技术手段,在冻干前须将菌泥与保护剂混匀并进行预冻。文中系统研究了菌泥与保护剂混匀过程中的代谢产酸情况,及产酸引起的pH值降低对预冻的影响。测定了不同菌泥添加保护剂后在室温和冷藏温度下的产酸速率,以及不同pH值下冷冻后菌株的存活率,分析了不同pH值条件下冷冻对菌株ATP酶、β-半乳糖苷酶、乳酸脱氢酶及细胞膜完整性和流动性的影响。结果表明,菌泥与保护剂混合后,室温环境下的产酸速率很快,可在短时间内将菌悬液的pH值降至4.0。而pH值降低确实会对冷冻菌株的存活产生影响,pH值在降至4.0时,菌株冷冻存活率显著降低。低pH值条件下的冷冻,会损伤ATP酶、β-半乳糖苷酶及降低细胞膜的流动性,是导致冷冻存活率降低的原因。因此,为提高真空冷冻干燥的菌株存活率,首先须控制菌泥与保护剂混合时的温度和时间,确保预冻阶段乳酸菌无损失。     

"冷冻-培养"循环冷冻刺激对植物乳杆菌细胞膜脂肪酸组成的影响

研究-20℃的“冷冻-培养”循环冷冻刺激对冷敏感性植物乳杆菌细胞膜脂肪酸组成及反复冻融后存活率的影响。采用气相-质谱分析方法研究细胞膜脂肪酸组成和平板菌落计数法计数反复冻融后存活的菌数,从而计算存活率。首先比较了4株乳杆菌对-20℃低温的敏感性．发现植物乳杆菌B1在-20℃备件下冷冻24h后存活的活菌数最低,在4株乳杆菌中对低温的敏感性最高;其次,发现经过10次冷冻-培养循环冷冻刺激后存活的菌,其细胞膜的不饱和脂肪酸与饱和脂肪酸的比值（U／S）较出发菌株提高了60．391％,并且经循环冷冻刺激后存活菌的C16：0和C18：0占总脂肪酸的比例均下降,相反C18：2的比例则升高。循环冷冻刺激后的存活菌,经过4次反复冻融后的存活菌数较出发菌株提高一个数量级。结果表明,循环冷冻刺激可以改变植物乳杆菌细胞膜的脂肪酸组成,提示循环冷冻刺激后存活菌对反复冻融抗性的增强可能与细胞膜中U／S比例提高有关。     

短乳杆菌发酵苹果汁工艺优化及有机酸变化

该研究利用短乳杆菌发酵苹果汁,生产能够调节人体肠道功能的乳酸菌苹果汁。通过单因素实验确定最适碳源、氮源以及缓冲因子;并以总酸、还原糖的变化为依据,进行响应面优化,确定最适发酵工艺条件,采用高效液相色谱法测定苹果汁发酵过程中有机酸组成及含量变化。结果表明,优化后发酵苹果汁的最佳培养基条件为:葡萄糖质量浓度50 g/L,蛋白胨质量浓度9 g/L,K₂HPO₄质量浓度4 g/L;菌种接种量为8%,发酵温度为36℃,发酵时间为6 d。在该条件下测得苹果汁的总酸为2. 61 g/100 g、还原糖为5. 027 g/100 g;经短乳杆菌发酵后苹果汁的品质明显优于发酵前。发酵后乳酸、富马酸、乙酸、奎宁酸含量显著增多;苹果酸、琥珀酸含量均持续减少,酒石酸含量无明显变化。     

风波平息再话核酸

<正> 核酸分为脱氧核糖核酸(DNA)和核糖核酸(RNA)两大类,由碱基、核糖和磷酸组成。核酸是高分子化合物,是细胞的核心,存在于人体60亿万个细胞之中,细胞分裂时需要核酸来提高营养。一般细胞的生命周期为200天、因此每天都有大量的细胞产生。核酸在机体的生长、发育和遗传变异中起着根本性的作用,应该说核酸是生命之本,因为人体的所有功能最终要通过细胞来实现,而支配细胞活动的是核酸,它是细胞赋活不可缺少的营养素,没有核酸,人体根本无法生存。     

Fermentation of Vigna sinensis var. carilla flours by natural microflora and Lactobacillus species

Natural fermentation and an inoculum containing 10% (vol/vol) Lactobacillus fermentum or Lactobacillus plantarum were used to obtain fermented flours from Vigna sinensis L. var. carilla seeds that had been washed with distilled water and dried at 55 degrees C for 24 h. To optimize the fermentation parameters (lactic acid bacterium level, bean flour concentration, and fermentation time), several small-scale fermentation processes were carried out. On the basis of the results obtained, fermentor-scale bean fermentation by microorganisms present on the seeds (natural fermentation [NF]) or by inoculation with L. plantarum (PF) was carried out at 37 degrees C for 48 h with a concentration of 300 g of bean flour per liter. The fermented flours (NF and PF) were also autoclaved. The levels of alpha-galactosides, inositol phosphates, trypsin inhibitor activity (TIA), soluble carbohydrates, starch (total and available), total available carbohydrates, thiamin, and riboflavin were determined for the processed cowpea flours, and microbiological studies were also carried out. The beans' levels of alpha-galactosides, TIA, and inositol hexaphosphate decreased by 95, 50, and 85%, respectively, for the NF flour and by 87, 27, and 85%, respectively, for the PF flour, while inositol pentaphosphate and inositol tetraphosphate were present in both fermented flours. The sucrose content decreased, and glucose, fructose, and galactose appeared as a result of fermentation. The levels of total available sugars and thiamin decreased by 2 and 12% and by 69 and 43%, respectively, while the riboflavin content increased by 106 and 94% for NF and PF flours, respectively. When NF and PF cowpea flours were heated in an autoclave for 20 min, TIA decreased further (by 80 and 56%, respectively). According to the chemical and microbiological results obtained in this study, fermentation with L. plantarum and autoclaving is an excellent process by which to produce a new functional food from the seed of a cheap legume (Vigna sinensis L. var. carilla).     

Edible table (bio)spread containing potentially probiotic Lactobacillus and Bifidobacterium species

A prototype of a reduced fat (60% w/w) edible table biospread, with an added viable, nongrowing, mixed-strain and potentially probiotic culture was developed. Conventional commercial aqueous-phase ingredient and reduced fat spread processing technologies were modified to achieve acceptable strain viability ( ≥   10⁵ cfu/mL) during scraped-surface heat exchange emulsion processing and biospread shelf life. The modifications consisted of:

• 1

   spilt-stream pasteurization of the aqueous and lipid phases (thereby obviating the need for in-line pasteurization of the water-in-oil emulsion during processing);