Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses

AIMS: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. METHODS AND RESULTS: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. CONCLUSIONS: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.     

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.     

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation

AIMS: To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. METHODS AND RESULTS: A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. CONCLUSIONS: In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.     

乳酸菌对微囊藻毒素的清除

【目的】研究唾液乳杆菌(Lactobacillus salivarius BBE09-18)、发酵乳杆菌(L.fermentum BBE09-29)以及干酪乳杆菌(L.casei Zhang)对藻毒素的清除能力以及影响乳酸菌清除藻毒素的主要因素,以期为进一步解析乳酸菌清除藻毒素的作用机制提供理论基础,并为去除食品体系中的藻毒素提供新的思路。【方法】研究乳酸菌细胞的不同生理状态(活细胞与死细胞)对藻毒素清除能力的影响,考察菌浓差异、起始藻毒素浓度差异、葡萄糖的供给等对乳酸菌清除藻毒素效能的影响。【结果】3株实验乳酸菌均具有清除藻毒素的能力,其中,L.casei Zhang的藻毒素清除能力最强,当藻毒素初始浓度为150μg/L时,24h后残留藻毒素浓度为85.5μg/L,清除率可达43%。此外,研究还发现乳酸菌活细胞的清除能力显著高于热失活后的死细胞。添加外源物质葡萄糖能显著提高实验菌株清除藻毒素的效率,在初始浓度为1800μg/L的藻毒素溶液中,当添加5%(w/v)葡萄糖后,L.casei Zhang经24h可清除92%的藻毒素。【结论】3株乳酸菌均具有藻毒素清除能力;同时,菌体浓度以及菌体自身的生理状态对藻毒素的清除效率具有重要影响,乳酸菌对藻毒素的清除可能与菌体的代谢活性相关。     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

两种乳酸菌胞外多糖对人体粪便菌群的影响

目的 在体外利用7名志愿者的新鲜粪便作为来源,选择菊粉作为对照,研究干酪乳杆菌LC2W与鼠李糖乳杆菌Y37胞外多糖对人体肠道菌群的影响.方法 利用PCR-DGGE(变性梯度凝胶电泳)方法分析样品中菌群组成,高效液相色谱分析培养前后胞外多糖组分变化.结果 与菊粉相比,粪便菌群在添加乳杆菌胞外多糖培养后,Parabacteroides类群细菌明显增加,4名志愿者粪便样品中出现明显的双歧杆菌条带,两种乳杆菌胞外多糖对粪便菌群的影响差异无统计学意义;而添加菊粉培养后,Bacteroides类群细菌的条带明显增加.两种乳杆菌胞外多糖中主要是大分子量的部分被利用.结论 干酪乳杆菌LC2W与鼠李糖乳杆菌Y37的胞外多糖在体外有调节粪便菌群的作用.     

Food-grade gene expression in lactic acid bacteria

In the 1990s, significant efforts were invested in the research and development of food-grade expression systems in lactic acid bacteria (LAB). At this time, Lactococcus lactis in particular was demonstrated to be an ideal cell factory for the food-grade production of recombinant proteins. Steady progress has since been made in research on LAB, including Lactococcus, Lactobacillus and Streptococcus, in the areas of recombinant enzyme production, industrial food fermentation, and gene and metabolic pathway regulation. Over the past decade, this work has also led to new approaches on chromosomal integration vectors and host/vector systems. These newly constructed food-grade gene expression systems were designed with specific attention to self-cloning strategies, food-grade selection markers, plasmid replication and chromosomal gene replacements. In this review, we discuss some well-characterized chromosomal integration and food-grade host/vector systems used in LAB, with a special focus on sustainability, stability and overall safety, and give some attractive examples of protein expression that are based on these systems.     

乳酸菌合成叶酸的研究进展

叶酸普遍存在于各类食物中,但由于叶酸的不稳定性以及饮食习惯的差异性,各国叶酸缺乏现象普遍存在。叶酸是参与核酸合成及细胞分裂分化的重要物质,叶酸缺乏引起机体功能的混乱,由此引发癌症等一系列的疾病。大部分乳酸菌是叶酸缺陷型菌株,但越来越多的研究表明其很多种类都具有叶酸合成能力。本文主要概述产叶酸乳酸菌的分类,乳酸菌生物合成叶酸的机制,以及利用乳酸菌进行的叶酸基因工程研究进展。     

4株乳酸菌对14种抗生素的敏感性试验

实验模拟饲料中益生菌与抗生素接触环境,对益生菌的存活比率进行量化表示,对用于饲料添加剂的4株乳酸菌进行抗生素敏感性试验。结果表明除极个别乳酸菌和抗生素的组合外,乳酸菌均在此种实验方法下对抗生素有很好的耐受性,实验结果对微生物饲料添加剂的研制及其在畜禽生产中的应用具有指导意义。     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Enhanced lactic acid bacteria viability with yeast coincubation under acidic conditions

ABSTRACT

The enhancing effects of yeasts on the viability of lactic acid bacteria (LAB) under acidic conditions were investigated. Meyerozyma guilliermondii, coaggregative with both LAB strains under acidic conditions, significantly enhanced the viability of Lactobacillus pentosus and L. paracasei in pH 3.0 lactic acid (LA) buffer at 10°C (p < 0.05). Non-coaggregative yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Cyberlindnera saturnus) also significantly enhanced the LAB viability (p < 0.05), and physical contact between LAB and yeasts was not essential for the viability-enhancing effect, indicating that the coaggregation had no relation to the enhancing mechanism. Although yeast metabolites and LA assimilation had no enhancing effect, hydrogen peroxide (H₂O₂) decreased after yeast coincubation, and H₂O₂ elimination improved L. pentosus viability. H₂O₂ elimination alone did not sufficiently improve L. paracasei viability, but the addition of antioxidants was effective. These results suggest that the antioxidant activity of yeast increased the LAB viability under acidic conditions.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

The impact of lactic acid bacteria on cheese flavor

Abstract Chesse flavor is a manifestation of complex interactions of volatile and non-volatile flavor-active compounds plus tactual perception. Numerous agents, including lactic acid bacteria, procece the flavou sensations. The effect of lactic acid bacteria is more dominant in cheese varieties with limited growth of secondary flora. This review describes the indirect and direct impacts of lactic acid bacteria in cheese with emphasis on carbohydrate fermentation, changes in oxidation-reduction potential, interactions with non-starter bacteria, autolysis, proteolytic and peptidolytic activities, transport of metabolites and flavor production.     

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.     

Development of detection medium for hard-to-culture beer-spoilage lactic acid bacteria

Aims:  To develop a detection medium for hard-to-culture beer-spoilage lactic acid bacteria (LAB). Methods and Results:  Four hard-to-culture beer-spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild-type strains in beer. To develop a countermeasure against these hard-to-culture beer-spoilage LAB, a beer-based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard-to-culture beer-spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer-based medium. Further comparative study was performed with five other media widely used for the detection of beer-spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer-spoiler detection (ABD) medium, possessed superior sensitivity for hard-to-culture beer-spoilage LAB and comparable sensitivity with easy-to-culture beer-spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro-organisms, and thereby allow the selective growth of beer-spoilage LAB. Conclusions:  Advanced beer-spoiler detection medium is considered as an effective tool for comprehensive detection of beer-spoilage LAB in breweries. Significance and Impact of the Study:  The detection by ABD medium can be used as an indicator for differentiating the beer-spoilage ability of LAB without further confirmatory tests in breweries.     

Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of amino acids

Co-culture of Lactobacillus brevis subsp. lindneri or L. plantarum with Saccharomyces cerevisiae or S. exiguus from sourdough did not modify the yield of the yeasts but gave higher growth rates and final yields of both lactic acid bacteria (LAB) than in their respective mono-cultures. Co-cultures of L. brevis subsp. lindneri with S. cerevisiae or S. exiguus in a medium without valine or leucine, which are essential for growth of the LAB, led to growth of the LAB due to excretion of these amino acids by the yeasts.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, Via S. Costanzo, 06100 Perugia, Italy     

Immunomodulatory function of lactic acid bacteria

Using mice, we found that oral administration of Bifidobacterium breve YIT4064 (B. breve) activated the humoral immune system, augmented anti-rotavirus IgA production or anti-influenza virus (IFV) IgG production and protected against rotavirus infection or influenza infection, respectively. Furthermore, when the B. breve was given to infants from an infant home, there was a significant reduction of the frequency of rotavirus shedding in stool samples during the administration of the bacteria. It was also found, again using mice, that oral administration of Lactobacillus casei strain Shirota (LcS) stimulated type 1 helper T (Th1) cells, activated the cellular immune system and inhibited incidence of tumors and IgE production. These results demonstrated that these two strains of lactic acid bacteria modulated the different immune systems each in its own way and prevented against various diseases.     

Changes in lactic acid bacteria and components of Awa-bancha by anaerobic fermentation

ABSTRACT

Awa-bancha is a post-fermented tea produced in Naka and Kamikatsu, Tokushima, Japan. We investigated the lactic acid bacteria in each stage of production of Awa-bancha and evaluated the relationships with the components. Lactic acid bacteria were isolated from tea leaves cultured with de Man, Rogosa, and Sharpe (MRS) agar plates, and the species were identified by homology of the 16 S rRNA gene and multiplex polymerase chain reaction (PCR) of the recA gene to distinguish the Lactobacillus plantarum group. As a result, a variety of species were isolated from the raw tea leaves, and Lactobacillus pentosus was isolated most frequently after anaerobic fermentation. Regarding the tea leaf components, organic acids, such as lactic acid, increased, free amino acids decreased, and catechins changed owing to anaerobic fermentation. Our results suggest that the microbial flora mainly composed of L. pentosus is important in the anaerobic fermentation process for flavor formation of Awa-bancha.