Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum

Lipoteichoic acid is a major cell wall virulence factor of gram-positive bacteria. LTAs from various bacteria have differential immunostimulatory potentials due to heterogeneity in their structures. Although recent studies have demonstrated that LTA isolated from Lactobacillus plantarum (pLTA) has anti-inflammatory properties and is less inflammatory than LTAs from pathogenic bacteria, little is known about the structure of pLTA. In this study, high-field NMR spectra of the pLTA were compared with those of LTA from pathogenic bacterium, Staphylococcus aureus (aLTA). The 2D NMR results demonstrated that pLTA possesses α-linked hexose sugar substituents on the poly-glycerophosphate backbone instead of N-acetylglucosamine substituents, and unsaturated fatty acids in its glycolipids. The sugar substituents were revealed as an approximately 29:1 molar ratio of the glucose to galactose by HPAEC-PAD analysis. MALDI-TOF/TOF MS analyses identified the presence of unsaturated fatty acids in the glycolipid moieties of pLTA. In addition, the glycolipid structure was found to be composed of trihexosyl-diacyl- and/or trihexosyl-triacyl-glycerol ceramide units by means of unique fragment ions of the glycolipids. These results enabled us to elucidate the pLTA structure, which is distinctively different from canonical LTA structure, and suggest that the unique immunological property of pLTA might be caused by the pLTA structure.     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test

Soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea inhibited the mutagenicity of the directly-acting mutagens: 4-nitro-o-phenylenediamine on Salmonella typhimurium strain TA 98 and NaN₃ on S. typhimurium strain TA 100; and indirectly-acting mutagens, 2-aminofluorene using strain TA 98 and benzo[a]pyrene using strain TA 100, in the presence of a supernatant solution from mammalian hepatic microsomes.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza

Bacteriocins ST414BZ and ST664BZ, produced by Lactobacillus plantarum, inhibited the growth of a number of lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. Optimal production of bacteriocin ST664BZ (12 800 AU/mL) was recorded in MRS broth with an initial pH of 6.0 and 6.5. Bacteriocin ST414BZ was produced in MRS broth at lower pH values, ranging from 6.5 to 5.0. Low levels of bacteriocin activity were produced in BHI, M17, 10% (w/v) soy flour and 10% (w/v) molasses, suggesting that specific nutrients are required for optimal production. Bacteriocin ST414BZ production doubled (from 12 800 to 25 600 AU/mL) in MRS broth with tryptone as sole nitrogen source, or when glucose was replaced with maltose. Bacteriocin ST664BZ production, on the other hand, was less influenced by changes in nitrogen content, but increased two-fold (to 25 600 AU/mL) when glucose was replaced with sucrose, maltose or mannose, or when MRS broth was supplemented with 2.0 g/L KH₂ PO₄. Enrichment of MRS broth with vitamins B₁₂, B₁ or C did not stimulate production of the two bacteriocins. Growth in the presence of DL-6,8-thioctic acid increased bacteriocin ST664BZ production to 25 600 AU/mL. Concluded from these results, optimal levels of bacteriocins ST414BZ and ST664BZ will be produced in boza enriched with tryptone and maltose.     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

In vitro importance of probiotic Lactobacillus plantarum related to medical field

Lactobacillus plantarum is a Gram positive lactic acid bacterium commonly found in fermented food and in the gastro intestinal tract and is commonly used in the food industry as a potential starter probiotic. Recently, the consumption of food together with probiotics has tremendously increased. Among the lactic acid bacteria, L. plantarum attracted many researchers because of its wide applications in the medical field with antioxidant, anticancer, anti-inflammatory, antiproliferative, anti-obesity and antidiabetic properties. The present study aimed to investigate the in vitro importance of L. plantarum toward medical applications. Moreover, this report short listed various reports related to the applications of this promising strain. In conclusion, this study would attract the researchers in commercializing this strain toward the welfare of humans related to medical needs.     

Fermentation of brined turnip roots using Lactobacillus plantarum and Leuconostoc mesenteroides starter cultures

The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures led to earlier acid production and earlier and more pronounced inhibition of Enterobacteriaceae than with uninoculated (natural) fermentation. Unlike the natural fermentation, the controlled fermentations did not show a yeast secondary fermentation and also had a better colour. Due to its ability to produce higher amounts of acid, the use of Lact. plantarum is more desirable than of Leuc. mesenteroides.     

Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium

The growth rate of Lactobacillus plantarum in a complex medium with 55.6 mM glucose decreased during aerobic incubation (relative to anaerobic incubation). The decrease occurred much earlier than an increase in the rate of oxygen utilization by the culture which led to H₂O₂ accumulation. The concentration of H₂O₂ accumulated in the medium was easily tolerated by the culture and elimination of the H₂O₂ did not prevent the decrease in growth rate. Increased O₂ utilization was accompanied by a switch in metabolism which resulted in acetate rather than lactate accumulation in aerobic cultures.Abbreviation MRSG Man, Rogosa and Sharpe (1960). Medium modified as in Materials and methods with glucose as fermentation substrate     

Utilization of high lysine-producing strains of Lactobacillus plantarum as starter culture for nutritional improvement of ogi

A high lysine-producing mutant of Lactobacillus plantarum (OG 261-5) derived from a wild type strain (OG 261) previously isolated from fermenting ogi was evaluated for nutritional improvement of ogi in a modified fermentation process. Results indicate that ogi obtained by fermentation with the pure culture of the mutant compared to traditional ogi increased in concentration of available lysine from 228.5 ± 12.0 mg/100 g to 525.1 ± 25.8 mg/100 g, tryptophan from 58.6 ± 8.0 mg/100 g to 114.3 ± 11.0 mg/100 g and tyrosine from 408.5 ± 13.7 mg/100 g to 4636.5 ± 11.3 mg/100 g. However, the contents of valine, leucine, isoleucine and phenylalanine were substantially reduced which may affect the protein quality of the modified ogi. The modified process shortened the period of traditional ogi production from five days to one by reducing the two-stage fermentation (i.e. soaking and souring stages) to a one-stage process and the total protein recovery was better compared to traditional ogi processing. There was no significant difference (P > 0.05) in organoleptic quality attributes of colour, flavour, and overall acceptability of ogi produced by the modified process and the traditional ogi.     

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.     

Combined effects of pH, yeast extract, carbohydrates and di-ammonium hydrogen citrate on the biomass production and acidifying ability of a probiotic Lactobacillus plantarum strain, isolated from table olives, in a batch system

A four variables-five levels Central Composite Design (CCD) was developed to model the individual and interactive effects of carbohydrates (lactose or maltose), yeast extract, di-ammonium hydrogen citrate and pH on the biomass production (Abs_600 nm), viable and cultivable cell number and acidifying ability of a probiotic strain of Lactobacillus plantarum, isolated from table olives “Bella di Cerignola”. pH values were modeled through a negative Gompertz equation, in order to obtain the parameter α (metabolic adaptation time). This value and the biomass were submitted to a stepwise procedure and second order polynomial equations were derived. The parameter α was affected by the initial pH and lactose; the effect of the maltose, however, was not significant. The biomass production increased with increasing of yeast extract, di-ammonium hydrogen citrate and maltose concentrations and was maximum at pH 6.0 and 20 g l⁻¹ of lactose.     

Use of starter culture of Lactobacillus plantarum BP04 in the preservation of dining-hall food waste

In this work, Lactobacillus plantarum BP04 was employed as starter culture in dining-hall food waste storage with different inoculant levels at 0, 2 and 10% (v/w) to suppress the outgrowth of pathogenic and spoilage bacteria. Inoculation by Lactobacillus plantarum BP04 was effective in accelerating pH drop and reducing the growth period of enterobacteria to 9, 7 and 2 days, corresponding to inoculant levels at 0, 2 and 10% (v/w). Increasing inoculum levels were found to inhibit the growth of Lactobacillus brevis and Leuconostoc lactis. HPLC analysis revealed that lactic acid was the predominant organic acid during the treatment of dining-hall food waste. Its concentration varied among the fermented processes reflecting variations of microbial activity in the fermented media.     

Enhancing Nutritional Quality of Silage by Fermentation with Lactobacillus plantarum

The present study was aimed to investigate the nutritive profiles, microbial counts and fermentation metabolites in rye, Italian rye-grass (IRG) and barley supplemented with Lactobacillus plantarum under the field condition, and its probiotic properties. After preparation of silage, the content of crude protein (CP), crude ash, acid detergent fiber (ADF), and neutral detergent fiber (NDF), microbes such as lactic acid bacteria (LAB), yeast and fungi counts, and fermentation metabolites lactic acid, acetic acid and butyric acid was assessed. Results indicated that the content of ADF and NDF were significantly varied between rye, IRG and barley mediated silages. The content of CP was increased in L. plantarum supplemented with IRG, but slightly decreased in rye and barley mediated silages. The maximum LAB count was recorded at 53.10 × 10⁷ cfu/g in rye, 16.18 × 10⁷ cfu/g in IRG and 2.63 × 10⁷ cfu/g in barley silages respectively. A considerable number of the yeasts were observed in the IRG silages than the rye silages (P < 0.05). The amount of lactic acid production is higher in L. plantarum supplemented silages as compared with control samples (P < 0.05). It was confirmed that higher amount of lactic acid produced only due to more number of LAB found in the silages. L. plantarum was able to survive at low pH and bile salt and the duodenum passage with the highest percentage of hydrophobicity. Furthermore, the strain was sensitive towards the antibiotics commonly used to maintain the microbes in food industrial setups. In conclusion, supplementation of L. plantarum is most beneficial in rye, IRG and barley silage preparations and probiotic characteristics of L. plantarum was an intrinsic feature for the application in the preparation of animal feeds and functional foods.     

Rapid identification of Lactobacillus plantarum group using the SNaPshot minisequencing assay

This study used SNaPshot minisequencing for species identification within the Lactobacillus plantarum group. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 35 strains of L. plantarum group species. The results showed that the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus fabifermentans. In conclusion, a rapid, accurate and cost-effective assay was successfully developed for species identification of the members of the L. plantarum group.     

Effects of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet

The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2, an isolate from Chinese traditional Tibet kefir, on cholesterol-lowering and microflora of rat in vivo. Rats were fed on cholesterol-enriched experimental diet, supplemented with lyophilized L. plantarum MA2 powder, with a dose of 10¹¹ cells/day per mice. The results showed that L. plantarum MA2 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides level, while there was no change in high-density lipoprotein cholesterol. In addition, liver total cholesterol and triglycerides was also decreased. However, fecal cholesterol and triglycerides was increased significantly (P < 0.05) in comparison with the control. Also, L. plantarum MA2 increased the population of lactic acid bacteria and bifidobacteria in the fecal, but it did not change the number of Escherichia coli as compared to control. Moreover, pH, moisture, and organic acids in the fecal were also measured. The present results indicate the probiotic potential of the L. plantarum MA2 strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.     

Complete nucleotide sequence and structural organization of pPB1, a small Lactobacillus plantarum cryptic plasmid that originated by modular exchange

A small cryptic plasmid designated pPB1 was isolated from Lactobacillus plantarum BIFI-38 and its complete 2899 bp nucleotide sequence was determined. Sequence analysis revealed four putative open reading frames. Based on sequence analysis two modules could be identified. First, the replication module consisted of a sequence coding for a replication protein (RepB) and its corresponding target site, and two putative repressor proteins (RepA and RepC). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate RepB production. A putative lagging-strand initiation site was also found, suggesting that pPB1 replicates via a rolling circle mechanism. The second module of pPB1 consisted of a sequence coding for a putative mobilization protein and its corresponding oriT site. Since the nucleotide sequence of the replication module showed 94.5% identity to the similar region on the Leuconostoc lactis plasmid pCI411, and the nucleotide sequence of the mobilization module had 97.5% identity to L. plantarum plasmid pLB4, it is concluded that pPB1 originated by modular exchange between two such plasmids by homologous recombination. Putative recombination sites where crossover might have taken place were also identified.