Bacteriocin properties of Lactobacillus fermenti, Lactobacillus brevis and Lactobacillus buchneri

Four bacteriocins of L. fermenti, 3 bacteriocins of L. brevis and 1 bacteriocin of L. buchneri were studied with respect to morphology of the inhibition growth zones of the indicator strains, capacity for diffusion through cellophane, sensitivity to high temperature, bacterial proteases, trypsin, chymotrypsin, pepsin, papain, nucleases and lysozyme. According to the differences in their properties the bacteriocins were classified as belonging to 8 types, including 4 types of L. fermenti bacteriocins and 3 types of L. brevis bacteriocins.     

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

Lactose metabolism in Lactobacillus curvatus and Lactobacillus sake

Abstract The lactose metabolism was investigated in five strains of Lactobacillus curvatus and 14 strains of L. sake isolated from meat or meat-derived products. Strains with the ability to ferment lactose were found in both species. They exhibited either phospho-β-galactosidase (P-β-gal) or β-galactosidase (β-gal) activity, or both. P-β-gal activity of L. curvatus and L. sake was induced and detected only in the presence of lactose or galactose. Furthermore, catabolite repression by glucose was demonstrated. The immunological properties of the P-β-gal enzymes of these organisms resemble those of Lactococcus lactis . Several strains of L. sake but none of L. curvatus exhibited β-gal activity which was constitutive. In hybridisation experiments, the β-gal genes of L. sake and L. casei ATCC393 showed over 60% DNA-homology. The presence of β-gal genes in L. sake was demonstrated in both β-gal-producing and non-producing strains. This observations is consistent with a genetic potential of lactic acid bacteria exceeding their physiological capabilities.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype

Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.     

Lactobacillus protoplast transformation

A method for the transformation of Lactobacillus protoplasts by plasmid DNA is reported. The procedure involves polyethylene glycol treatment of protoplasts to induce DNA uptake. A transformation efficiency ranging from 5 to 1000 transformants per microgram of DNA is achieved; the efficiency of protoplast regeneration ranged from 10 to 20%.     

Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.     

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus

AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.     

鼠李糖乳杆菌-格氏乳杆菌联用对阴道致病菌的抑制作用

目的本文通过提取细菌基因组进行16S rDNA PCR扩增和测序,分析菌株的进化树分支,鉴定一株乳酸菌菌株RD-0060并检测RD-0060与已有菌株RD-0046联用的抑菌能力和细胞粘附能力。方法结合现有菌株RD-0046(格氏乳杆菌,Lactobacillus gasseri),采用牛津杯法研究RD-0060单菌、RD-0060和RD-0046联用抑制致病菌的能力。通过共培养细菌和阴道上皮细胞VK2/E6E7,研究RD-0060单菌和RD-0060/RD-0046二联菌粘附能力。结果 RD-0060为鼠李糖乳杆菌(Lactobacillus rhamnosus),具有抑制阿托波菌、阴道加德纳菌和常见好氧型病菌的功能,对阴道上皮细胞也有较强的粘附能力;RD-0060和RD-0046二联菌的抑菌效果和细胞粘附能力比单菌株更强。结论鼠李糖乳杆菌和格氏乳杆菌联用能显著抑制阴道致病菌生长,并且能够大量粘附阴道细胞,而且两菌株联用有协同效果,具有良好的临床应用和开发前景。     

Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages

AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.