Analysis of exopolysaccharide production by Lactobacillus casei CG11, isolated from cheese.

Exopolysaccharide-producing Lactobacillus casei CG11 was isolated from soft, white, homemade cheese. In basal minimal medium, it produces a neutral heteropolysaccharide consisting predominantly of glucose (about 75%) and rhamnose (about 15%). Plasmid curing experiments revealed that exopolysaccharide production by strain CG11 is linked to a plasmid approximately 30 kb in size.     

Analysis of exopolysaccharide production by Lactobacillus casei CG11, isolated from cheese.

Exopolysaccharide-producing Lactobacillus casei CG11 was isolated from soft, white, homemade cheese. In basal minimal medium, it produces a neutral heteropolysaccharide consisting predominantly of glucose (about 75%) and rhamnose (about 15%). Plasmid curing experiments revealed that exopolysaccharide production by strain CG11 is linked to a plasmid approximately 30 kb in size.     

Diacetyl and Acetoin Production by Lactobacillus casei

Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.     

Exopolysaccharide and Kestose Production by Lactobacillus sanfranciscensis LTH2590

The effect was investigated of sucrose concentration on sucrose metabolism and on the formation of exopolysaccharide (EPS) by Lactobacillus sanfranciscensis LTH2590 in pH-controlled fermentations with sucrose concentrations ranging from 20 to 160 g liter⁻¹. The EPS production increased and the relative sucrose hydrolysis activity decreased by increasing the sucrose concentration in the medium. The carbon recovery decreased from 95% at a sucrose concentration of 30 g liter⁻¹ to 58% at a sucrose concentration of 160 g liter⁻¹ because of the production of an unknown metabolite by L. sanfranciscensis. This metabolite was characterized as a fructo-oligosaccharide. The oligosaccharide produced by L. sanfranciscensis was purified and characterized as a trisaccharide with a glucose/fructose ratio of 1:2. The comparison of the retention time of this oligosaccharide and that of pure oligosaccharide standards using two different chromatography methods revealed that the oligosaccharide produced by L. sanfranciscensis LTH2590 is 1-kestose. Kestose production increased concomitantly with the initial sucrose concentration in the medium.     

Production and Regeneration of Lactobacillus casei Protoplasts

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCl₂, 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl₂, CaCl₂, gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.     

Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11.     

基因组改组提高干酪乳杆菌耐酸性生产L-乳酸

首先采用紫外线与亚硝基胍两种传统微生物诱变方法对干酪乳杆菌进行诱变,经低pH平板、碳酸钙平板和摇瓶试验获得了5株耐酸性提高的突变菌株.以获得的突变菌株为出发菌株,应用灭活双亲原生质体融合后致死损伤得到互补获得活性融合子的方法,对其进行基因组改组,经过低pH平板、碳酸钙平板和摇瓶筛选,获得4株可以在pH3.8平板上旺盛生长且产酸量较高的改组菌株.将改组菌株与原始菌株分别于pH 3.8和3.4的YE液体培养基中培养,改组菌株能够在原始菌株无法生存的pH条件(pH 3.4)下生长.在pH 3.8的条件下,对改组菌株与原始菌株的发酵特征进行比较,37℃发酵48小时后,改组菌株产酸量为原始菌株的2.4倍,表明基因组改组技术能有效提高多基因调控表型的进化.     

Production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei

In order to make full use of soybean stalk produced in large quantity annually in China, a process is proposed for production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei. Experiments were conducted using the proposed process and experimental results indicate that the potential of 242 mg (g stalk)⁻¹ fermentable sugar is released from hydrolysate through enzymatic saccharication with a saccharication of 51%. The main sugar released from pretreated soybean stalk through enzymatic hydrolysis was a mixture of glucose, xylose and cellobiose at a ratio of 3.9:1.7:1. Fermentation of soybean stalk hydrolysate by L. sake and L. casei yielded the lactic acid conversion of 48% and 56%, respectively, however, lactic acid conversion increased to 71% by co-inoculation of both strains. L. sake and L. casei were able to degrade glucose, but unable to completely assimilate xylose and cellobiose. The proposed process can be used to produce lactic acid from soybean stalk hydrolysate.     

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS⁻) and EPS-producing (EPS⁺) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS⁺ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS⁺ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.     

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.     

Optimization of the fermentation of whey by Lactobacillus casei

Optimization studies have been carried out for the production of L-lactic acid from the fermentation of whey by Lactobacillus casei. Two Statistical Designs, a full 2⁴ Factorial Design and a full 3² Factorial Design, have been used to determine the most suitable values of the operational variables for obtaining the maximum lactic acid production rate. The variables selected for study were temperature, pH, lactose concentration, and inoculum concentration. Among them only temperature and pH were found to affect the response significantly. A second-order polynomial empirical model relating both the response (lactic acid production rate) and the variables was formulated. The maximum acid production rate was found when the values of pH and temperature were 5.4 and 38°C, respectively. All conclusions are restricted to the experimental range studied.     

Enhancement of Host Resistance against Listeria Infection by Lactobacillus casei: Activation of Liver Macrophages and Peritoneal Macrophages by Lactobacillus casei

The functions of liver macrophages and peritoneal macrophages obtained after injection of Lactobacillus casei were examined. Listericidal activity in vivo was enhanced in liver macrophages 13 days after L. casei injection but was somewhat suppressed in the macrophages 2 days after the injection. The listericidal activity in vitro was enhanced in peritoneal macrophages obtained 13 days after L. casei injection but was suppressed in cells obtained 2 days later. The PMA-triggered respiratory burst in the liver macrophages elicited by L. casei was higher than that of resident macrophages. Alkaline phosphodiesterase activity in the liver macrophages was decreased by L. casei injection, as was also the case with peritoneal macrophages. These observations indicate that L. casei augmented cellular functions of both liver and peritoneal macrophages.     

Binding and transport of thiamine by Lactobacillus casei.

The relationship between thiamine transport and a membrane-associated thiamine-binding activity has been investigated in Lactobacillus casei. Thiamine transport proceeds via a system whose general properties are typical of active uptake processes; entry of the vitamin into the cells requires energy, is temperature dependent, exhibits saturation kinetics, and is inhibited by substrate analogs. A considerable concentration gradient of unchanged thiamine can be achieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. casei also contains a high-affinity, thiamine-binding component which could be measured by incubation of intact cells with labeled substrate at 4 degrees C (conditions under which transport is negligible). Binding was insensitive to iodoacetate, occurred at a level (0.5 nmol per 10(10) cells) nearly 20-fold higher than could be accounted for by facilitated diffusion, and was found to reside in a component of the cell membrane. Participation of this binder in thiamine transport is supported by the observations that the processes of binding and transport showed similarities in their (i) regulation by the concentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) susceptibility to inhibition by thiamine analogs.     

Optimization of Exopolysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus RR Grown in a Semidefined Medium

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by Lactobacillus delbrueckii subsp. bulgaricus RR in a semidefined medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and five replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of L. delbrueckii subsp. bulgaricus RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% confidence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specific production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.