Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.     

Discrimination of dairy industry isolates of the Lactobacillus casei group

Lactobacilli are a major part of the microflora of the gut and of many fermented dairy products, and are found in a variety of environments. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus zeae form a closely related taxonomic group within the facultatively heterofermentative lactobacilli. The classification and nomenclature of these bacteria are controversial. In this study, relationships between these species were investigated using type strains and dairy industry isolates examined with DNA-based techniques and conventional carbohydrate use tests. Carbohydrate use patterns gave poor discrimination of some species, but DNA PCR using specific primers targeted to sequences of the 16S rRNA gene discriminated 4 types consistent with the currently recognized species. Pulsed-field agarose gel electrophoresis of chromosomal NotI restriction fragments identified 18 different band patterns from 21 independent Lactobacillus isolates and confirmed the identity of L. casei strains from 2 culture collections (CSCC 5203 and ASCC 290), both representing the type strain of L. casei. Some isolates were reclassified as L. rhamnosus, suggesting that the prevalence of L. rhamnosus as a natural component of the microflora of dairy foods and dairy environments has previously been underestimated. These methods can provide a practical basis for discrimination of the species and identification of individual industrial strains.     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

副干酪乳杆菌诱导Caco-2细胞凋亡及其发酵乳贮藏品质的研究

以新疆马奶酒中自行分离得到的副干酪乳杆菌（Lactobacillus paracasei）M5L为对象,研究该菌株对结肠癌Caco-2细胞的抑制作用并分析发酵乳贮藏品质变化。结果表明,副干酪乳杆菌M5L可以在一定的盐浓度、胆盐浓度及酸性环境中较好的生长。当菌体以100∶1的感染系数作用于Caco-2细胞72 h后抑制率最佳;显微镜下观察活菌处理后的细胞发生明显的凋亡现象;流式细胞仪检测到副干酪乳杆菌M5L可以诱导Caco-2细胞内活性氧显著增加,并将细胞周期阻滞在合成期,且通过增大Caspase-3和Bax基因表达量,降低Bcl-2基因表达量来促进肿瘤细胞的凋亡。其发酵酸乳贮藏5 d时品质较佳,长时间的贮存会导致酸乳品质的劣变。     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

Nucleotide sequencing,purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.     

Antialcoholic liver activity of whey fermented by Lactobacillus casei isolated from koumiss

Whey fermented liquid (WFL) was studied for its hepatoprotective effects by using chronic alcohol-induced mice. Whey fermented liquid, prepared by inoculating whey with 4% (vol/vol) Lactobacillus casei and then incubating at 41°C for 8 h, was used to orally treat alcohol-induced mice at 3 dosages for 5 wk. Ethanol consumption significantly reduced the activity of superoxide dismutase and glutathione peroxidase, while lowering glutathione content and increasing levels of aspartate aminotransferase, alanine aminotransferase, total triglyceride, malondialdehyde, and cytochrome P450 2E1. Treatment with WFL significantly attenuated the increased levels of alanine aminotransferase, aspartate aminotransferase, triglyceride, and cytochrome P450 2E1, while decreasing superoxide dismutase, glutathione peroxidase, malondialdehyde, and glutathione levels. Pathological changes in the livers of mice who had ingested alcohol were improved by the administration of WFL. These results suggest that WFL may exert a protective effect against alcoholic liver disease by increasing antioxidant activity, which supports the use of WFL as an antialcoholic liver disease treatment.     

Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: A multivariate screening study

Sixty-three strains of the taxonomically related species Lactobacillus plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum and L. pentosus isolated from sourdoughs and other food and non-food sources and 14 strains of other members of the genus Lactobacillus were screened for their tolerance of acid, alkaline, heat, oxidative, osmotic, detergent and starvation stresses in order to evaluate the diversity of stress response. Most strains of the L. plantarum group were highly tolerant of acid, alkaline and osmotic stress and highly sensitive to detergent stress, while a larger diversity was found for other stress. Multivariate analysis allowed grouping the strains in clusters with similar response patterns. Stress response patterns in the L. plantarum group were similar to those of species of the L. casei/L. paracasei group but clearly different from those of other mesophilic Lactobacillus. No relationship was found between grouping obtained on the basis of stress response patterns and by genotypic fingerprinting (rep-PCR), nor with the taxonomic position or isolation source of the strains. Further experiments with selected strains showed that exponential phase cells were generally but not always more sensitive than stationary phase cells. The ability to grow under stressful conditions showed a slightly better correlation with the ecological conditions prevailing in the isolation niches of the strains.This study will be the basis for further investigations to identify and exploit the basis of diversity in the stress response of lactic acid bacteria.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

副干酪乳杆菌N1115发酵乳对小鼠便秘的改善作用

本研究基于盐酸咯哌丁胺建模制造小鼠便秘模型,考察副干酪乳杆菌 N1115发酵乳对治疗小鼠便秘的 作用。低、中、高剂量副干酪乳杆菌N1115发酵乳（活菌含量分别为1×107、1×108、1×109 CFU/mL）连续灌胃 15 d后,与对照组相比小鼠的排便量及粪便含水量均显著升高（P＜0.05）;肠道推进率分别为57.3%、67.3%和 71.3%,显著高于对照组（P＜0.05）。气相色谱仪分析小鼠粪便中的短链脂肪酸发现,灌胃副干酪乳杆菌N1115 发酵乳后,小鼠粪便中的乙酸、丙酸、丁酸、戊酸含量显著升高（P＜0.05）,且短链脂肪酸种类较模型组更为丰 富。对小鼠远端结肠的组织形态观察发现,灌胃副干酪乳杆菌N1115发酵乳的小鼠结肠黏膜完整,细胞排列整齐, 而对照组的肠黏膜厚度降低,凹窝变浅。通过c-kit抗体对小鼠结肠中的肠间质细胞（interstitial cells of Cajal,ICC）进 行免疫组化分析,结果显示,灌胃副干酪乳杆菌N1115发酵乳后小鼠的ICC含量显著增加,表明副干酪乳杆菌N1115发 酵乳通过提高ICC活性,促进肠道蠕动。综上,副干酪乳杆菌N1115发酵乳能够有效缓解小鼠的便秘症状。     

Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces

Lactobacillus casei cells were immobilized on fruit (apple and pear) pieces and the immobilized biocatalysts were used separately as adjuncts in probiotic cheese making. In parallel, cheese with free L. casei cells and cheese only from renneted milk were prepared. The produced cheeses were ripened at 4 to 6°C and the effect of salting and ripening time on lactose, lactic acid, ethanol concentration, pH, and lactic acid bacteria viable counts were investigated. Fat, protein, and moisture contents were in the range of usual levels of commercial cheeses. Reactivation in whey of L. casei cells immobilized on fruit pieces after 7 mo of ripening showed a higher rate of pH decrease and lower final pH value compared with reactivation of samples withdrawn from the remaining mass of the cheese without fruit pieces, from cheese with free L. casei, and rennet cheese. Preliminary sensory evaluation revealed the fruity taste of the cheeses containing immobilized L. casei cells on fruit pieces. Commercial Feta cheese was characterized by a more sour taste, whereas no significant differences concerning cheese flavor were reported by the panel between cheese containing free L. casei and rennet cheese. Salted cheeses scored similar values to commercial Feta cheese, whereas unsalted cheese scores were significantly lower, but still acceptable to the sensory panelists.     

Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage

Lactobacillus casei Zhang is a novel strain that was screened out of koumiss collected in Inner Mongolia, and our previous research showed that L. casei Zhang has health benefits such as cholesterol-reducing and immunomodulating effects. The fermentation characteristics of L. casei Zhang in soymilk and bovine milk and the transit tolerance of L. casei Zhang in fermented milk products during refrigerated storage for 28 d were assessed. A faster decrease in pH and faster growth of L. casei Zhang during fermentation were observed in soymilk compared with bovine milk at various inoculation rates, probably because of the low pH buffering capacity of soymilk. The fermented bovine milk samples had much higher final titratable acidity (TA) values (between 0.80 and 0.93%) than the soymilk samples (between 0.40 and 0.46%). Dramatic increases in TA values in the fermented soymilk samples during storage were observed, and the TA values of the fermented soymilk samples changed from <0.56% to values between 0.86 and 0.98%. On the other hand, only slight increases in TA were observed in the bovine milk samples during the 28 d of storage. The survival rates of freshly prepared cultures of L. casei Zhang in simulated gastric juice at pH 2.0 and 2.5 were 31 and 69%, respectively, and the delivery of L. casei Zhang through fermented soymilk and bovine milk significantly improved the viability of L. casei Zhang in simulated gastric transit. Lactobacillus casei Zhang showed good tolerance to simulated gastric juice and intestinal juice in the fermented soymilk and bovine milk samples, and maintained high viability (>10⁸ cfu/g) during storage at 4°C for 28 d. Our results indicated that both soymilk and bovine milk could serve as vehicles for delivery of probiotic L. casei Zhang, and further research is needed to elucidate the mechanism of the change in pH and TA of L. casei Zhang in fermented milk samples during fermentation and storage and to understand the difference between soy- and milk-based systems.     

A novel lactic acid bacterium for the production of high purity L-lactic acid, Lactobacillus paracasei subsp. paracasei CHB2121

Fermentation-derived lactic acid has several potential industrial uses as an intermediate carbon chemical and a raw material for biodegradable polymer. We therefore undertook the identification of a novel bacterial strain that is capable of producing high concentrations of lactic acid and has potential commercial applications. A novel L(+)-lactic acid producing bacterium, Lactobacillus paracasei subsp. paracasei CHB2121 was isolated from soil obtained near an ethanol production factory and identified by 16S rRNA gene sequence analysis and characterization using an API 50 CHL kit. L. paracasei subsp. paracasei CHB2121 efficiently produced 192 g/L lactic acid from medium containing 200 g/L of glucose, with 3.99 g/(L·h) productivity, and 0.96 g/g yield. In addition, the optical purity of the produced lactic acid was estimated to be 96.6% L(+)-lactic acid. The newly identified L. paracasei subsp. paracasei CHB2121 efficiently produces high concentrations of lactic acid, and may be suitable for use in the industrial production of lactic acid.     

Effects of a probiotic fermented milk beverage containing Lactobacillus casei strain Shirota on defecation frequency,intestinal microbiota,and the intestinal environment of healthy individuals with soft stools

The effects of drinking a fermented milk beverage that contains Lactobacillus casei strain Shirota (LcS) at 40 billion bacterial cells/bottle for 4 weeks (probiotics, 1 bottle/day) on defecation frequency, intestinal microbiota and the intestinal environment of healthy individuals with soft stools were evaluated. Thirty-four healthy adults who had soft stools were randomised into 2 groups, and the effects of a regular 4-week intake of probiotics were evaluated by a placebo-controlled, double-blind, parallel-group comparative design. Defecation frequency significantly decreased after the 4-week intake period compared with before the probiotic treatment. The stool quality significantly improved (hardened) compared to the placebo. Also, the water content of the stools was lower in the probiotic group than in the placebo group. Live LcS was recovered at 6.9 ± 1.3 and 7.2 ± 0.8 log₁₀ CFU per 1 g of stool after 2 and 4 weeks, respectively, of probiotic treatment. The number of bifidobacteria in the stools also increased significantly compared with the level before starting the probiotics. The organic acid levels (total, acetic acid, propionic acid, and butyric acid) significantly increased compared with the level before intake in both the probiotic and placebo groups, but they returned to the original levels after the end of the intake period. These results suggest that probiotic fermented milk beverage has an intestine-conditioning effect by improving the frequency of defecation and stool quality and increasing the intrinsic bifidobacteria in healthy individuals with soft stool.     

Species-specific identification of commercial probiotic strains

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.