Intraspecific diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides associated with vacuum-packed meat product spoilage analyzed by randomly amplified polymorphic DNA PCR

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA-restriction fragment length polymorphism profiles. The RAPD-M13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6 in L. sakei, 12 in L. plantarum, and 6 in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H'), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.     

Comparison of the anti‐obesity and hypocholesterolaemic effects of single Lactobacillus casei strain Shirota and probiotic cocktail

This study compared the efficacy of Lactobacillus casei strain Shirota (LAB13) and a probiotic cocktail for their anti‐obesity and other lipid profile modulating effects. Diet‐induced obese rats were supplemented with two different probiotics which are LAB13 (1 × 10⁹ CFU day^?1 per rat) and cocktail of five bacterial strains (1 × 10⁹ CFU day^?1 per rat) for 12 weeks. Comparative data on weight gain, energy intake, liver weight, subcutaneous fat, total fat weights, total cholesterol and leptin levels in both treatment groups showed significant reduction in probiotic‐treated groups compared to the obese control group. Both probiotics have the anti‐obesity and hypocholesterolaemic effects and are able to reduce body weight and fats via reduction in energy intake. Only LAB13 was able to reduce the level of triglyceride significantly. Therefore, the LAB13 is equally effective compared to the probiotic cocktail in weight reduction. LAB13 is more effective in improving lipid profile which is a common medical complication of obesity.     

Different stress tolerance of spray and freeze dried Lactobacillus casei Shirota microcapsules with different encapsulating agents

Food Science and Biotechnology - In this study, the effects of encapsulation with maltodextrin and reconstituted skim milk (RSM) and their binary and ternary blends with gum arabic (GA) by spray...     

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies.

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.     

Transit of radical scavenging activity of milk products prepared by Maillard reaction and Lactobacillus casei strain Shirota fermentation through the hamster intestine

Oxidative stress in the colon is associated with the incidence of colon cancer. In situ, the suppression of oxidative stress in the colon would be an effective form of prevention of the cancer. In this study we investigated the transit of the radical scavenging activity of milk products through the hamster intestinal tract. Two types of skim milk products were prepared by Maillard reaction and then lactic acid fermentation. Heat treatment enhanced the radical scavenging activity for 2,2-diphenyl-1-picrylhydrazyl radical of skim milk. The activity was further increased by fermentation with Lactobacillus casei strain Shirota. Normal hamsters were fed these milk products for 14 d. For potential radical scavenging activity per unit dry weight of feces and cecal content, the groups ranked in the order of fermented product-fed hamsters > heated product-fed hamsters > control hamsters, reflecting the order of the potential of the corresponding diets. Approximately 12% of the activity of the heated and the fermented product diets passed through the gastrointestinal tract. These results suggest that some of the radical scavenging activity generated by food processing reached the colon in nonabsorbable products.     

自然发酵肉制品中干酪乳杆菌的分离和鉴定

从自然发酵香肠和自制腌肉中分离得到68株乳酸菌,通过对菌株发酵适应性的研究,筛选出2株性能优良,适用于肉制品发酵的乳酸菌C67和R6,初步生化鉴定表明,2株均为干酪乳杆菌。文中还对所获得的菌株的相关发酵性能进行了测试。     

Characterization of spontaneous phage-resistant variants of Streptococcus thermophilus by randomly amplified polymorphic DNA analysis and identification of phage-resistance mechanisms

A total of 100 spontaneous phage-resistant mutants isolated from nine commercial Streptococcus thermophilus strains were characterized preliminarily by randomly amplified polymorphic DNA (RAPD) and the nature of their phage-resistance mechanisms was investigated. Only for mutants isolated from one strain, free phages were detected in their culture supernatants when these were titrated on the sensitive strain, suggesting that the mutants could have acquired the resistance phenotype by integrating the phage in their genomes (lysogeny). Adsorption interference was observed in the derivatives isolated from two strains. For mutants isolated from two other strains, restriction–modification (R–M) type systems were detected. In one of these cases, R–M was probably combined with another intracellular anti-phage system. In most cases, the molecular profiles (RAPD fingerprints) obtained with four arbitrary primers showed a high similarity among parent strains and their respective phage-resistant mutants. Some of these mutants were identified as potentially improved strains for industrial use.     

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR.

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.     

Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique

Strain-specific PCR primers for Lactococcus lactis subsp. cremoris FC were developed using the randomly amplified polymorphic DNA (RAPD) technique. RAPD was used to generate strain-specific markers. A 1164-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific for L. lactis subsp. cremoris FC was designed. The specificity of this primer pair was tested with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and was found to be strain-specific. Subsequently, this primer pair was subjected to the quantification of L. lactis subsp. cremoris FC in the feces of subjects fed fermented milk containing this strain. After administration, L. lactis subsp. cremoris FC was detected in the feces of all 7 subjects, with the maximum number being between 10(5) and 10(9) cells g(-1) of feces. Furthermore, this strain was detected in only one feces sample 2 weeks after administration was stopped. These results suggest that L. lactis subsp. cremoris FC can survive passage through the gastrointestinal tract.     

Viability of probiotic micro-organisms (Lactobacillus casei Shirota and Bifidobacterium animalis subspp. lactis) in a milk-based dessert with cranberry sauce

The objective of this study was to evaluate the survival of two probiotic micro-organisms, Lactobacillus casei Shirota and Bifidobacterium animalis subspp . lactis , in a milk-based dessert (2.7% fat) with cranberry sauce added. B. lactis had a final concentration of 1.99 × 10⁶ cfu/g after 21 days of study, with a logarithmic decrease of 8.87%. On other hand, L. casei Shirota had a final concentration of 2.05 × 10⁷ cfu/g at the end of the same period, a logarithmic decrease of 8.41%. Statistical analysis showed that significant differences existed between both micro-organisms and over various storage times, the more viable micro-organism being L. casei Shirota , which decreased less than one logarithmic cycle after 21 days.     

Concentrations and tracking of listeria monocytogenes strains in a seafood-processing environment using a most-probable-number enrichment procedure and randomly amplified polymorphic DNA analysis

Concentrations of environmental microflora and Listeria monocytogenes were monitored at multiple environmental locations within a seafood-processing facility over the course of 6 months. Concentrations of L. monocytogenes were determined using a most-probable-number (MPN) enrichment procedure. Two floor drains had persistent low concentrations of L. monocytogenes (0.03 to >1,100 MPN/cm2). In comparison, concentrations of the other organisms in the drain were much higher (heterotrophic plate count range of 10(5) to 10(8) CFU/cm2). Concentrations of environmental organisms (heterotrophic aerobic plate counts and counts of pseudomonads, Shewanella spp., Aeromonas hydrophila, and coliforms) were not correlated with concentrations of L. monocytogenes. The 178 confirmed L. monocytogenes isolates from the MPN procedure were further characterized by randomly amplified polymorphic DNA analysis. Sixteen different banding patterns were identified, and nine of the patterns were identified from samples collected on two or more collection dates. From all locations, banding type A was observed in 98 confirmed isolates (55%). Although present, L. monocytogenes was a relatively minor component in the ecosystem of the floor drains in this seafood-processing facility.     

Contamination routes of Gram-negative spoilage bacteria in the production of pasteurised milk,evaluated by randomly amplified polymorphic DNA (RAPD)

The sources of contamination for Gram-negative psychrotrophic bacteria (GNP), have been traced in three dairies by using the PCR-based method randomly amplified polymorphic DNA (RAPD). GNP isolated from raw and pasteurised milk, air, water and empty packages were RAPD-typed. Pasteurised milk was found to be recontaminated by a wide spectrum of different RAPD-types. Several of the milk contaminating RAPD-types were also found in condensed water on the filling nozzles, in waste-water at the bottom of the filling machine, and in the air in the immediate surroundings of the machine. Most of the RAPD-typed bacteria were categorised as pseudomonads. The majority of these belonged to Pseudomonas fluorescens, but also Pseudomonas putida, Pseudomonas corrugata, and Janthinobacterium lividum were identified. Indications of specific RAPD-types associated with the same filling machine over a time period of 6–15 months were found in two of the dairies.     

Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair

In this study, Lactobacillus salivarius CECT 5713 was originally isolated from feces of a one-month-old breast-fed infant. Since it has been suggested that the gut microbiota of breast-fed infants reflects that of the maternal breast milk, we investigated if this specific strain was present in breast milk of the respective mother. RAPD and PFGE analysis revealed the presence of the strain L. salivarius CECT 5713 in this biological fluid. To our knowledge, this is the first report of a L. salivarius strain isolated from breast milk. L. salivarius CECT 5713 produced l-lactate, acetate and hydrogen peroxide, which may be responsible for its antimicrobial activity against most of the indicator organisms used in this study; in addition, this strain showed a high survival rate after exposition to conditions simulating those found in the gastrointestinal tract. Finally, it was strongly adhesive to Caco-2 and HT-29 cells did not produce biogenic amines and were unable to degrade gastric mucin in vitro.     

Comparison between automatic ribotyping and random amplified polymorphic DNA analysis of Bacillus cereus isolates from the dairy industry.

Discrimination by automatic ribotyping and random amplified polymorphic DNA PCR, RAPD, was compared for 40 different B. cereus dairy isolates, 4 different B. mycoides isolates and 6 culture collection strains. RAPD-PCR has previously shown to be useful for tracing contamination routes of B. cereus to milk. Automatic ribotyping using EcoRI and PvuII separated the B. cereus and B. mycoides isolates/strains into 36 different ribotypes. RAPD-typing with primers generated 40 different RAPD-profiles. However, 17 isolates clustered into eight groups, irrespective of the primer and restriction enzyme used, and in all but one case, the isolates with the same pattern were isolated from the same dairy. Automatic ribotyping proved to be a useful, standardized and quick method to discriminate between B. cereus strains, only slightly less discriminatory than RAPD-typing.     

Proteolysis in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ŁOCK 0900 probiotic strain

Selected parameters related to proteolysis were evaluated in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ?OCK 0900 probiotic strain. Dehydration, pH, water activity and total nitrogen content were not affected by ultrasound treatment. Neither sonication nor inoculation has an influence on L* and a* colour parameters. Sonication significantly influenced the pattern of proteolysis providing higher nonprotein nitrogen content (NPN) and proteolysis index (PI). The highest intensity of proteolysis, as assessed by NPN and PI, occurred in an inoculated sample. Neither sonication nor inoculation with L. casei had a significant effect on total viable counts. Loins inoculated with L. casei had the highest lactic acid bacteria count, followed by the sonicated and nonsonicated control. This research shows that sonication followed by inoculation with L. casei ?OCK 0900 could be used as an effective measure to speed up the proteolytic changes in dry‐aged meat cuts.     

甜叶菊随机扩增多态性DNA技术优化及亲缘关系研究

以甜叶菊为试材,对影响其随机扩增多态性DNA标记反应体系的7个因子进行优化,同时对来自国内外的12个品种进行亲缘关系分析。结果表明,20μL的优化体系包括:双蒸水13.6μL,10×Buffer(含15mmol/LMgCl2)溶液3μL,2.5mmol/L的dNTPS1.2μL,10μmol/L的引物1μL,20ng的模板DNA1μL,1UTaq聚合酶;热循环程序为:94℃预变性4min,94℃变性30s,35℃退火30s,72℃延伸1min,40个循环,最后72℃延伸7min。聚类图结果表明,品种间的遗传距离变异范围为0.12～0.88,在遗传距离为0.37时,8个引物可将12个品种分为四大类,来自日本的2个品种与国内品种关系较远。     

分离自人体肠道的副干酪乳杆菌K56和ET-22的菌株鉴定

以婴儿和成人肠道来源的菌株K56和ET-22为研究对象,选取8株其他来源菌株作为参考,采用形态学、生理生化、基质辅助激光解析电离飞行时间质谱、全基因组测序、多位点序列分型分析和单核苷酸多态性(single nucleotide polymophism, SNP)分析,建立了适用于菌株K56和ET-22的鉴定方法。结果表明,菌株K56和ET-22及参考菌株的基质辅助激光解吸/电离飞行时间鉴定比对值大于2,平均核苷酸一致性值(average nucleotide identity, ANI)达到95%以上,在种水平均鉴定为副干酪乳杆菌(Lactobacillus paracasei);对副干酪乳杆菌K56、ET-22和参考菌株的1 701个共有核心基因开展基于全基因组测序的多基因序列位点分析,菌株K56和ET-22可与其他参考菌株有效区分,SNP分析表明不同培养代数间副干酪乳杆菌K56的SNP差异为103、ET-22的SNP差异为49,系统发育分析表明该方法可以有效区分副干酪乳杆菌K56、ET-22与其他参考菌株。益生菌的安全性和功能性均在菌株水平具有特异性,已成为业内新的科学共识。该研究...     

Use of highly variable gene (yycH) as DNA marker to resolve interspecific relationships within the Lactobacillus casei group and a target for developing novel species-specific PCR primers

Identifying Lactobacillus species group using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques yields inaccurate results. In this study, the partial yycH gene sequence was used for species discrimination by direct DNA sequencing and as target in a species-specific PCR assay. All examined Lactobacillus strains were clearly distinguished from the closely related species by comparative sequence analysis of the yycH gene. The average sequence similarity for the yycH gene (78.5 %) among type strains was significantly lower than that of the 16S rRNA sequence (99.0 %). Therefore, the yycH gene can be proposed as an additional molecular marker for Lactobacillus casei group that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the yycH gene sequences, which were then employed for PCR using the template DNA of Lactobacter strains. The PCR primer pairs were shown to be specific for Lactobacillus paracasei and Lactobacillus rhamnosus. Our data indicate that the phylogenetic relationships in the L. casei group are easily resolved by direct sequencing of the yycH gene and combined with species-specific PCR assays.     

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.     

Role of microbial strain and storage temperatures in the degradation of isoflavone phytoestrogens in fermented soymilk with selected β-glucosidase producing Lactobacillus casei strains

Soymilk fermented with 2 Lactobacillus casei strains were stored at various temperatures (80 °C, 4 °C, 24.8 °C and 37 °C) for 8 weeks and isoflavone concentration analysed at weekly intervals using RP-HPLC. The degradation of each isoflavone compound at each storage temperature was found to fit first order kinetic model. Aglycone as well as glucosides generally appeared to be stable during storage (P < 0.01) at the 4 storage temperatures. Aglycone forms had smaller degradation constants compared to glucosides at all storage temperature and in the presence of both microorganisms. Specifically, aglycones showed a unique trend of smaller degradation at lower storage temperatures (−80 °C and 4 °C) than at higher temperatures (24.8 °C and 37 °C). Glucoside genistin was least stable at all storage temperatures compared to other isoflavones in the fermented soymilk with each strain while aglycone daidzein was the most stable. L. casei 2607 in fermented soymilk stored at 4 °C after 8 weeks gave the least degradation for daidzein of a mere 3.78% loss from 9.53 to 9.17 ng/μL. L. casei 2607 showed greater hydrolytic potential than L. casei ASCC 290 as denoted by higher degradation of isoflavone glucosides in fermented soymilk at lower storage temperatures.