Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations.

Lactobacillus helveticus is a well characterized lactobacillus for dairy fermentations that is also found in malt whisky fermentations. The two environments contain considerable differences related to microbial growth, including the presence of different growth inhibitors and nutrients. The present study characterized L. helveticus strains originating from dairy fermentations (called milk strains hereafter) and malt whisky fermentations (called whisky strains hereafter) by in vitro phenotypic tests and comparative genomics. The whisky strains can tolerate ethanol more than the milk strains, whereas the milk strains can tolerate lysozyme and lactoferrin more than the whisky strains. Several plant-origin carbohydrates, including cellobiose, maltose, sucrose, fructooligosaccharide and salicin, were generally metabolized only by the whisky strains, whereas milk-derived carbohydrates, i.e. lactose and galactose, were metabolized only by the milk strains. Milk fermentation properties also distinguished the two groups. The general genomic characteristics, including genomic size, number of coding sequences and average nucleotide identity values, differentiated the two groups. The observed differences in carbohydrate metabolic properties between the two groups correlated with the presence of intact specific enzymes in glycoside hydrolase (GH) families GH1, GH4, GH13, GH32 and GH65. Several GHs in the milk strains were inactive due to the presence of stop codon(s) in genes encoding the GHs, and the inactivation patterns of the genes encoding specific enzymes assigned to GH1 in the milk strains suggested a possible diversification manner of L. helveticus strains. The present study has demonstrated how L. helveticus strains have adapted to their habitats.

Biodiversity of Lactobacillus helveticus isolates from dairy and cereal fermentations reveals habitat-adapted biotypes.

For the present study, we collected 22 Lactobacillus helveticus strains from different dairy (n = 10) and cereal (n  = 12) fermentations to investigate their biodiversity and to uncover habitat-specific traits. Biodiversity was assessed by comparison of genetic fingerprints, low-molecular-weight subproteomes, metabolic and enzymatic activities, growth characteristics and acidification kinetics in food matrices. A clear distinction between the dairy and cereal strains was observed in almost all examined features suggesting that the different habitats are domiciled by different L. helveticus biotypes that are adapted to the specific environmental conditions. Analysis of the low-molecular-weight subproteome divided the cereal isolates into two clusters, while the dairy isolates formed a separate homogeneous cluster. Differences regarding carbohydrate utilization were observed for lactose, galactose, sucrose and cellobiose as well as for plant-derived glucosides. Enzymatic differences were observed mainly for ß-galactosidase and ß-glucosidase activities. Further, growth temperature was optimal in the range from 33 to 37°C for the cereal strains, whereas the dairy strains showed optimal growth at 40°C. Taken together, adaptation of the various biotypes results in a growth benefit in the particular environment. Acidification and growth tests using either sterile skim milk or a wheat flour extract confirmed these results. Differentiation of these biotypes and their physiological characteristics enables knowledge-based starter culture development for cereal versus dairy products within one species.

Development of an S-layer gene-based PCR-DGGE assay for monitoring dominant Lactobacillus helveticus strains in natural whey starters of Grana Padano cheese.

Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.

In vitro and in vivo evaluation of an exopolysaccharide produced by Lactobacillus helveticus KLDS1.8701 for the alleviative effect on oxidative stress.

Correlations between oxidative stress and degenerative diseases have been gaining increasing attention. A number of studies affirm that exopolysaccharide (EPS) produced by lactic acid bacteria (LAB) can alleviate oxidative stress and further prevent the related diseases. In our previous study, Lactobacillus helveticus KLDS1.8701 has been shown to possess high antioxidant capacity in vitro. The aim of this study was to evaluate the ameliorative effects of EPS produced by L. helveticus KLDS1.8701 on oxidative stress. Firstly, EPS was isolated from the culture of L. helveticus KLDS1.8701 and purified using DEAE-Sepharose Fast Flow chromatography. Secondly, the antioxidant capacities of EPS fractions were evaluated using in vitro methods. Thirdly, an in vivo study was performed to investigate the possible protective effects of EPS on d-galactose (d-gal)-induced liver damage and gut microbiota disorder. In vitro antioxidant activity results suggested that EPS-1 exhibited strong scavenging properties on 2,2-diphenyl-1-picrylhydrazyl radical, superoxide radical, hydroxyl radical, and chelating activity on ferrous ion. In vivo, EPS-1 supplementation significantly attenuated oxidative status such as decreased organic index, liver injury and liver oxidative stress. EPS-1 supplementation shifted the gut microbiota composition to that of the control group. In addition, the analysis of Spearman's rank correlation suggested that the protective effects of EPS correlated with manipulating the gut microbiota composition in d-gal-induced mice. These results implied that EPS-1 supplementation could mitigate hepatic oxidative stress via manipulating the gut microbiota composition and be used as a potential candidate to attenuate oxidative damage.

Characterization of volatile compounds produced by Lactobacillus helveticus strains in a hard cheese model.

Starter cultures of Lactobacillus helveticus used in hard cooked cheeses play an important role in flavor development. In this work, we studied the capacity of three strains of L. helveticus, two autochthonous (Lh138 and Lh209) and one commercial (LhB02), to grow and to produce volatile compounds in a hard cheese extract. Bacterial counts, pH, profiles of organic acids, carbohydrates, and volatile compounds were analyzed during incubation of extracts for 14 days at 37 ℃. Lactobacilli populations were maintained at 106 CFU ml-1 for Lh138, while decreases of approx. 2 log orders were found for LhB02 and Lh209. Both Lh209 and LhB02 slightly increased the acetic acid content whereas mild increase in lactic acid was produced by Lh138. The patterns of volatiles were dependent on the strain which reflect their distinct enzymatic machineries: LhB02 and Lh209 produced a greater diversity of compounds, while Lh138 was the least producer strain. Extracts inoculated with LhB02 and Lh 209 were characterized by ketones, esters, alcohols, aldehydes, and acids, whereas in the extracts with Lh138 the main compounds belonged to aromatic, aldehydes, and ketones groups. Therefore, Lh209 and LhB02 could represent the best cheese starters to improve and intensify the flavor, and even a starter composed by combinations of LhB02 or Lh209 with Lh138 could also be a strategy to diversify cheese flavor.

In vitro assessment of the antimicrobial potentials of Lactobacillus helveticus strains isolated from traditional cheese in Sinkiang China against food-borne pathogens.

Lactobacillus helveticus, an obligatory hetero-fermentative LAB, is Generally Recognized as Safe (GRAS) and is gaining popularity for application in dairy products. Lactic acid bacteria (LAB) play a remarkable role in inhibiting the growth of pathogenic bacteria in food products, without disturbing the sensory attributes of the food. In this study, the screening of the antimicrobial potential of Lactobacillus helveticus KLDS 1.8701 against four food-borne pathogens including Listeria monocytogenes ATCC 19115, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, and Escherichia coli O157:H7 ATCC 43889 in vitro was inspected using the Oxford cup method and mixed culture inhibition assays. The organic acid production and antimicrobial potential of the cell-free supernatants (CFS) have been evaluated via different treatments and analysis using high performance liquid chromatography (HPLC). The analysis results revealed that KLDS 1.8701 exhibited the highest antimicrobial potential compared to other antimicrobial strains. The antimicrobial activity of KLDS 1.8701 resulted from the organic acids in the culture and CFS. From the study, it was found that carbon sources, as well as organic acid production, accelerate the antimicrobial activity of KLDS 1.8701 and the fructooligosaccharides (FOS) were considered the best for improving the proliferation of KLDS 1.8701 and supporting its antimicrobial action. Results of the mixed culture inhibition assays showed that part of the antimicrobial activity resulted from the inhibitory action of the bacteria itself in culture, and this action required cellular contact between the food-borne pathogens and KLDS 1.8701. Conversely, the results of the antimicrobial spectrum assay revealed that some Lactobacilli remained unaffected by KLDS 1.8701. KLDS 1.8701 might also be favorable for use as a supplementary starter in fermented dairy productions. Furthermore, KLDS 1.8701 could survive well under GI tract conditions. Further studies on in vivo inhibition assays and the probiotic effects are recommended.

Population structure of Lactobacillus helveticus isolates from naturally fermented dairy products based on multilocus sequence typing.

Lactobacillus helveticus is an economically important lactic acid bacterium used in industrial dairy fermentation. In the present study, the population structure of 245 isolates of L. helveticus from different naturally fermented dairy products in China and Mongolia were investigated using an multilocus sequence typing scheme with 11 housekeeping genes. A total of 108 sequence types were detected, which formed 8 clonal complexes and 27 singletons. Results from Structure, SplitsTree, and ClonalFrame software analyses demonstrated the presence of 3 subpopulations in the L. helveticus isolates used in our study, namely koumiss, kurut-tarag, and panmictic lineages. Most L. helveticus isolates from particular ecological origins had specific population structures.

Comparative genome-scale analysis of niche-based stress-responsive genes in Lactobacillus helveticus strains.

Next generation sequencing technologies with advanced bioinformatic tools present a unique opportunity to compare genomes from diverse niches. The identification of niche-specific stress-responsive genes can help in characterizing robust strains for multiple applications. In this study, we attempted to compare the stress-responsive genes of a potential probiotic strain, Lactobacillus helveticus MTCC 5463, and a cheese starter strain, Lactobacillus helveticus DPC 4571, from a gut and dairy niche, respectively. Sequencing of MTCC 5463 was done using 454 GS FLX, and contigs were assembled using GS Assembler software. Genome analysis was done using BLAST hits and the prokaryotic annotation server RAST. The MTCC 5463 genome carried multiple orthologs of genes governing stress responses, whereas the DPC 4571 genome lacked in the number of major stress-response proteins. The absence of the bile salt hydrolase gene in DPC 4571 and its presence in MTCC 5463 clearly indicated niche adaptation. Further, MTCC 5463 carried higher copy numbers of genes contributing towards heat, cold, osmotic, and oxidative stress resistance as compared with DPC 4571. Through comparative genomics, we could thus identify stress-responsive gene sets required to adapt to gut and dairy niches.

Differential effects of lactobacilli on activation and maturation of mouse dendritic cells.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.

Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed Lactobacillus helveticus as the dominant species.

AIMS: To assess the structure and stability of a dominant lactic acid bacteria (LAB) population during the propagation of rye sourdough in an industrial semi-fluid production over a period of 7 months. METHODS AND RESULTS: The sourdough was started from a 6-year-old freeze-dried sourdough originating from the same bakery. A unique microbial consortium consisting mainly of bacteria belonging to species Lactobacillus helveticus, Lactobacillus panis and Lactobacillus pontis was identified based on culture-dependent (Rep-PCR) and culture-independent [denaturing gradient gel electrophoresis (DGGE)] methods. Three of the isolated Lact. helveticus strains showed remarkable adaptation to the sourdough conditions. They differed from the type strain by the ability to ferment compounds specific to plant material, like salicin, cellobiose and sucrose, but did not ferment lactose. CONCLUSION: We showed remarkable stability of a LAB consortium in rye sourdough started from lyophilized sourdough and propagated in a large bakery for 7 months. Lactobacillus helveticus was detected as the dominant species in the consortium and was shown to be metabolically adapted to the sourdough environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an established and adapted microbial consortium as a starter is a good alternative to commercial starter strains.

Complete genome sequence of Lactobacillus helveticus R0052, a commercial probiotic strain.

Lactobacillus helveticus R0052 is a commercially available strain that is widely used in probiotic preparations. The genome sequence consisted of 2,129,425 bases. Comparative analysis showed that it was unique among L. helveticus strains in that it contained genes encoding mucus-binding proteins similar to those found in Lactobacillus acidophilus.

The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed.

Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain.

Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92.

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.

Characterisation of Lactobacillus helveticus strains producing antihypertensive peptides by RAPD and inverse-PCR of IS elements.

Lactobacillus helveticus is used for the manufacture of cheeses and milk-based products. Although it is not considered a probiotic microorganism, some strains demonstrated beneficial effects through the production of antihypertensive peptides from the hydrolysis of casein during milk fermentation. Strain-specificity of bioactive peptide production by L. helveticus makes the availability of reliable typing methods essential for both legal and good manufacturing processes. Accordingly, RAPD and inverse-PCR of five insertion sequence elements were comparatively evaluated for the molecular characterisation of four L. helveticus dairy cultures producing antihypertensive peptides and fourteen reference strains. Calculation of discriminatory indices and cluster analysis of the DNA fingerprints confirmed the suitability of both approaches for acceptable strain differentiation. Although RAPD was more discriminating, for a few test strains a neat discrimination was only achieved through multiplex inverse-PCR, thus suggesting the suitability of a combined analytical approach for a finer strain discrimination.

Exploitation of the diverse insertion sequence element content of dairy Lactobacillus helveticus starters as a rapid method to identify different strains.

The species Lactobacillus helveticus is a commonly used thermophilic starter and/or adjunct culture for Swiss and Cheddar cheese manufacture. Its use is normally associated with flavour improvement which is known to be associated with culture traits such as rapid autolysis and high proteolytic activity. The genome of the commercial strain, DPC4571, was recently sequenced and found to have an abundance of IS sequences in terms of both abundance (213 intact) and diversity (21 types). Given this unique diversity for a lactic acid bacterium, we investigated whether PCR-based IS fingerprinting could be used as a discriminatory tool to distinguish between different strains of Lb. helveticus. A set of ten primers targeting five of the most numerous groups (ISL1201, ISLhe65, ISLhe2, ISLhe15 and ISL2) of IS elements was designed. Multiplex-PCR with all primers resulted in 1-12 discreet amplicons for each strain tested. The resultant fingerprints (in the 0.5 kb-3 kb range) were found to be strain specific and reproducible. This approach thus provides a valuable method to distinguish between Lb. helveticus strains while giving some indication of the relative abundance of IS sequences in each strain.

Evaluation of microbial diversity during the manufacture of Fior di Latte di Agerola, a traditional raw milk pasta-filata cheese of the Naples area.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980).

Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.

Heterogeneity of putative surface layer proteins in Lactobacillus helveticus.

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.