Monitoring of assertive Lactobacillus sakei and Lactobacillus curvatus strains using an industrial ring trial experiment.

AIMS: In a previous study, we used a 5-day fermenting sausage model to characterize assertiveness of Lactobacillus curvatus and Lactobacillus sakei starter strains towards employ autochthonous contaminants. In this work, we probed those findings and their transferability to real sausage fermentation including the drying process in an industrial ring trial experiment. METHODS AND RESULTS: Raw fermented sausages ('salami') were produced with three L. curvatus and four L. sakei strains as starter cultures in cooperation with three manufacturers from Germany. We monitored pH, water activity and microbiota dynamics at strain level over a total fermentation and ripening time of 21 days by MALDI-TOF-MS identification of isolates. The principal behaviour of the strains in real sausage fermentations was the same as that one observed in the 5-day model system delineating single strain assertiveness of a bacteriocin producer from co-dominance of strains. CONCLUSIONS: The water activity decrease, which is concomitant with the sausage ripening process has only limited impact on the assertiveness and survival of the starter strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of a 5-day model can provide insight in the assertiveness of a specific starter strain in sausage fermentation.

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

AIMS: Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. METHODS AND RESULTS: A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. CONCLUSION: This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

Comparative phylobiomic analysis of the bacterial community of water kefir by 16S rRNA gene amplicon sequencing and ARDRA analysis.

AIMS: The aim of this study was to analyse the bacterial microbiota of water kefir using culture-independent methods. METHODS AND RESULTS: We compared four water kefirs of different origins using 16S rDNA amplicon sequencing and ARDRA. The microbiota consisted of different proportions of the genera Lactobacillus (Lact.), Leuconostoc (Leuc.), Acetobacter (Acet.) and Gluconobacter. Surprisingly, varying but consistently high numbers of sequences representing members of the genus Bifidobacterium (Bif.) were found in all kefirs. Whereas part of the bifidobacterial sequences could be assigned to Bifidobacterium psychraerophilum, a majority of sequences identical to each other could not be assigned to any known species. A nearly full-length sequence of the latter exhibited a beyond-species similarity (96.4%) with the sequence from the closest relative species Bif. psychraerophilum. A Bifidobacterium-specific ARDRA analysis reflected the abundance of the novel Bifidobacterium species by revealing its unique MboI restriction profile. Attempts to isolate the bifidobacteria were successful for Bif. psychraerophilum only. CONCLUSIONS: The complexity of the water kefir microbiota has been underestimated in previously studies. The occurrence of bifidobacteria as part of the consortium is novel. SIGNIFICANCE AND IMPACT OF THE STUDY: These data give new insights into the understanding of the complexity of food fermentations and underline the need for approaches detecting noncultivable organisms.

Impact of different sugars and glycosyltransferases on the assertiveness of Latilactobacillus sakei in raw sausage fermentations.

Latilactobacillus sakei comprises a biodiversity of strains, which display different assertiveness upon their application as starter cultures in raw sausage fermentation. While the assertiveness of winning partner strains has been referred to competitive exclusion based on genomic settings enabling occupation of multiple niches of the sausage habitat, single strain assertiveness of L. sakei remained unexplained. In this study we assessed the impact of the expression of a glycosyltransferase enabling the production of a glucan from sucrose to the assertiveness of L. sakei TMW 1.411, which expresses a plasmid-encoded glycosyltransferase (gtf). In a sausage fermentation model wild type L. sakei TMW 1.411 and its plasmid-cured mutant were employed in competition with each other and with other Latilactobacillus sakei strains. To differentiate any effects resulting from general sugar utilization from those of glucan formation, the experiments were carried out with glucose, fructose, and sucrose, respectively. It was shown that the type of sugar affects the individual strains behaviour, and that the wild type was more competitive than the mutant in the presence of any of these sugars. In direct competition between wild type and mutant, a clear competitive advantage could also be demonstrated for the strain possessing the plasmid with the glycosyltransferase. Since this competitive advantage was observed with all sugars, not just sucrose, and Gtf expression has been shown as independent of the employed sugar, it is suggested that possession of the gtf gene-carrying plasmid confers a competitive advantage. It appears that the Gtf contributes to competitive exclusion and the establishment of colonization resistance, to a larger extent by an adhesive functionality of the Gtf on the cellular surface than by the production of glucan. Hence, gtf genes can be used as a possible additional marker for the selection of assertive L. sakei starter strains in sausage fermentation.

Fermentation profile, cholesterol-reducing properties and chemopreventive potential of ß-glucans from Levilactobacillus brevis and Pediococcus claussenii - a comparative study with ß-glucans from different sources.

The aim of the present study was to investigate whether ß-glucans obtained from the lactic acid bacteria (LAB) Levilactobacillus (L.) brevis and Pediococcus (P.) claussenii exhibit similar physiological effects such as cholesterol-binding capacity (CBC) as the structurally different ß-glucans from oat, barley, and yeast as well as curdlan. After in vitro fermentation, fermentation supernatants (FSs) and/or -pellets (FPs) were analyzed regarding the concentrations of short-chain fatty acids (SCFAs), ammonia, bile acids, the relative abundance of bacterial taxa and chemopreventive effects (growth inhibition, apoptosis, genotoxicity) in LT97 colon adenoma cells. Compared to other glucans, the highest CBC was determined for oat ß-glucan (65.9 ± 8.8 mg g-1, p < 0.05). Concentrations of SCFA were increased in FSs of all ß-glucans (up to 2.7-fold). The lowest concentrations of ammonia (down to 0.8 ± 0.3 mmol L-1) and bile acids (2.5-5.2 µg mL-1) were detected in FSs of the ß-glucans from oat, barley, yeast, and curdlan. The various ß-glucans differentially modulated the relative abundance of bacteria families and reduced the Firmicutes/Bacteroidetes ratio. Treatment of LT97 cells with the FSs led to a significant dose-dependent growth reduction and increase in caspase-3 activity without exhibiting genotoxic effects. Though the different ß-glucans show different fermentation profiles as well as cholesterol- and bile acid-reducing properties, they exhibit comparable chemopreventive effects.

A gene cluster encoding plantaricin 1.25beta and other bacteriocin-like peptides in Lactobacillus plantarum TMW1.25.

Plantaricin 1.25beta is a thermostable class two bacteriocin produced by Lactobacillus plantarum TMW1.25 isolated from sausage fermentation. It is co-produced with several other bacteriocin-like peptides. Using oligonucleotides derived from previously determined peptide sequences, a 3.8 kb DNA fragment could be amplified. A neighboring 1.8 kb fragment was amplified using ligation-anchored single-specific-primer PCR. Sequencing of the complete 5.6 kb stretch revealed that the structural gene for plantaricin 1.25beta, plnB, was located downstream of another bacteriocin gene, plnC. Seven other open reading frames were detected, including plnK encoding a bacteriocin-like peptide, but not including any putative immunity genes. Interestingly, the gene cluster contained an IS30-like insertion sequence, designated IS125, as well as an ISS1 homolog.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis.

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Maltose metabolism of Lactobacillus sanfranciscensis: cloning and heterologous expression of the key enzymes, maltose phosphorylase and phosphoglucomutase.

The maltose degradation operon containing genes encoding maltose phosphorylase mapA and phosphoglucomutase pgmA from Lactobacillus sanfranciscensis DSM20451T were cloned and expressed in Escherichia coli. These genes represent the first genetic data available for this species beyond taxonomic classification. MapA encodes a 754-amino acid polypeptide representing maltose phosphorylase, MapA, with a calculated molecular mass of 85.7 kDa. Comparative sequence analysis showed that mapA is of a new type distinct from other alpha-glucosidase genes sequenced so far. Putatively, pyridoxal 5'-phosphate is required as cofactor. The deduced amino acid sequence of pgmA shows an overall similarity of 39% to the phosphoglucomutase of Lactococcus lactis. pgmA is separated by a single nucleotide from the preceding mapA gene indicating effective translation by translational coupling. Upon subcloning mapA was heterologously expressed in E. coli. Additionally, upstream of the maltose-degrading operon ORF1 and ORF2 are located in the opposite direction. These genes show homology to fabZ and accB from E. coli and Bacillus subtilis, respectively, both involved in fatty acids biosynthesis.

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation.

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.

Diversity of lactic acid bacteria associated with ducks.

The diversity of lactic acid bacteria occurring in the crop and intestinal content of ducks of different ages (5 to 34 days) was evaluated using the RAPD-PCR technique and comparative sequence analysis of 16S rRNA. Cluster analysis derived from isolates grown on Rogosa agar used for the enrichment of lactic acid bacteria, revealed at least twenty six different groups of organisms representing species, subspecies and strains belonging to Streptococcus, Enterococcus, Pediococcus, Weissella and Lactobacillus. Homofermentative and heterofermentative lactobacilli were identified, belonging to the phylogenetically defined Lb. acidophilus, Lb. salivarius and Lb. reuteri groups.

Polyphasic identification of wild yeast strains isolated from Greek sourdoughs.

A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs. Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR). The latter methods confirmed the classical identification. According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica.

Cloning, expression, and characterization of acetate kinase from Lactobacillus sanfranciscensis.

In the metabolism of Lactobacillus sanfranciscensis, the acetate kinase (AK) is a key enzyme and responsible for dephosphorylation of acetyl phosphate with the concomitant production of acetate and ATP. The L. sanfranciscensis ack gene was identified by PCR methods. It encodes a 397 amino acid protein sharing 56% similarity with Bacillus subtilis AK. Whereas cotranscription of ack and pta (phosphotransacetylase) is reported in previously characterised organisms, the L. sanfranciscensis ack gene is not located in direct neighbourhood to the encoding gene. AK was heterologously expressed in E. coli and characterised by its v(max) and Km values and by the dependence of enzyme activity on temperature and pH. Based on this data the in vivo role of the enzyme is discussed.

Lactobacillus frumenti sp. nov., a new lactic acid bacterium isolated from rye-bran fermentations with a long fermentation period.

Within the framework of the characterization of the microflora of an industrial sourdough fermentation, strains of Lactobacillus amylovorus, Lactobacillus pontis and two other strains were isolated which could not be associated with a valid species. These latter strains were Gram-positive, catalase-negative, non-spore-forming, non-motile rods that could be clearly differentiated from known species by 16S rDNA sequence analysis. For further characterization, the morphological, physiological (sugar fermentation, formation of DL-lactate, hydrolysis of arginine, growth temperature, CO2 production) and chemotaxonomic (G+C content, cell wall composition, SDS-PAGE of whole-cell proteins) properties were determined. Fitting of the complete 16S rDNA sequence into alignments of such sequences, together with the subsequent phylogenetic calculations, allowed the reconstruction of a phylogenetic tree. These data showed that the two strains were phylogenetically related but formed an independent cluster distinct from their closest neighbours, L. pontis, Lactobacillus panis, Lactobacillus oris, Lactobacillus vaginalis and Lactobacillus reuteri. The results of DNA-DNA hybridization experiments indicated that the two isolates represent a new Lactobacillus species, for which the name Lactobacillus frumenti is proposed; the type strain of this species is DSM 13145T (= LMG 19473T).

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation.

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

Cloning of the phosphotransacetylase gene from Lactobacillus sanfranciscensis and characterization of its gene product.

The phosphotransacetylase (PTA) (EC 2.3.1.8) catalyzes a key branch point reaction in the carbohydrate pathway of Lactobacillus sanfranciscensis. In this report, we describe the cloning of the pta gene. The DNA sequence analysis revealed a 987 bp open reading frame encoding a protein with a molecular mass of 35.5 kD. These are the first studies on a PTA of an organism representative for the heterofermentative lactic acid bacteria. Unlike in most other bacteria analysed so far, in L. sanfranciscensis the pta gene is not adjacent located to the gene encoding acetate-kinase. The PTA was heterologously expressed as a biotinylated fusion protein in E. coli and purified to homogeneity. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for acetylphosphate and CoA (forward reaction) were 1.3 and 0.1 mM, respectively. The apparent Vmax was 194 U/mg. The enzyme also catalyzed in vitro the reverse reaction with apparent Km values for acetylCoA and phosphate of 0.6 and 6.7 mM, respectively (Vmax of 38 U/mg). The PTA showed a wide range of temperature for optimal activity (49 degrees C to 58 degrees C). It was inactivated after 15 min at 60 degrees C. Its activity was not affected by addition of MgCl2 (10 mM) or KCl (100 mM).

In vitro stability and expression of green fluorescent protein under high pressure conditions.

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry.

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.

Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T.

AIMS: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451T under various sublethal stress conditions. METHODS AND RESULTS: Stress conditions which reduce the growth rate of L. sanfranciscensis DSM 20451T to 10% of its maximum were determined. These conditions were met at 43, 12.5 degrees C, a pH value of 3.7, 1.9% NaCl, or 80 MPa respectively. In contrast to heat preincubation, other prestresses, including salt, cold and pressure led to an increase of barotolerance by hydrostatic pressure of 300 MPa for 30 min. Stationary-phase cells also showed an increased barotolerance. Sublethal pressure leads to enhanced heat tolerance. CONCLUSIONS: Stress response to salt, low temperature and acidic pH as well as starvation overlap with that one to high pressure by inducing barotolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation of bacteria by high pressure treatment is influenced by their history which modulates barotolerance. Mechanisms of barotolerance appear different from heat shock defence.

Carnimonas nigrificans gen. nov., sp. nov., a bacterial causative agent for black spot formation on cured meat products.

Nine different strains, CTCBS1T or CTCBS9, were identified to be the causative agents of black spots on the surface of raw cured meat products. The formation of black spots under aerobic conditions is reproducible upon reinoculation of meat products with any of these strains, indicating that they are the causative agent. The strains were Gram-negative, catalase-positive and obligately aerobic rods. The G+C content of DNA of strain CTCBS1T is 56.0 +/- 0.3 mol%. The content of non-polar main fatty acids were 16:0, 16:1, 18:1 and 19:0 cyc. Its phylogenetic position was elucidated by comparative sequence analysis of the 16S rRNA gene. Overall sequence similarity to other bacteria does not exceed 93.3%. Isolate CTCBS1T clustered phylogenetically within the gamma-subclass of the Proteobacteria and is closely related to members of Halomonas (90:5-91.9%) and to Zymobacter palmae (93.3%). A genetic homogeneity of the nine strains was demonstrated by M13 random amplified polymorphic DNA-PCR, whereas differentiation from other genera, e.g. Zymobacter and Pseudomonas, could easily be achieved by their chemotaxonomic characteristics. Taxonomic data revealed the status of a separate genus for which the name Carnimonas gen. nov., sp., nov. is proposed. Despite chemotaxonomic and physiological similarities, the new genus is at present not a member of the family Halomonadacease because of the lack of two out of 15 descriptive 16S rRNA signature sequences. The first member of the new genus is Carnimonas nigrificans. The use of a specific, 16S rRNA-targeted oligonucleotide primer allowed the identification of all nine strains of C. nigrificans in a PCR assay. Toxicological studies showed no pathogenic potential for C. nigrificans strain CTCBS1T (CECT 4437T).

Characterization of the bacterial flora of Sudanese sorghum flour and sorghum sourdough.

The microflora of a Sudanese sorghum flour, a spontaneously fermented sourdough and a long-term sourdough produced in a Sudanese household by consecutive re-inoculations, was studied. The dominant contaminants of sorghum flour were Gram-negative, catalase-positive, rod-shaped bacteria with counts of about 10(5) cfu g-1. The spontaneously fermented sorghum sourdough showed a bacterial succession from Gram-negative, catalase-positive contaminants to Enterococcus faecalis, Lactococcus lactis, Lactobacillus fermentum and Lact. reuteri. The total bacterial count reached about 10(10) cfu g-1 and the pH dropped from 6.4 to 3.35 in about 42 h. In this phase, only the Latter two species remained dominant in a ratio of 1:1. From the Sudanese long-term dough, seven strains of Lactobacillus were isolated, representing the dominant flora. Sequence comparison of partial 16S rRNA gene sequences were used to clarify their phylogenetic positions. Five strains were classified as Lact. vaginalis and could be regarded as heterogeneous biovars of this species. The other two strains could be assigned to Lact. helveticus. RAPD-PCR and sugar fermentation patterns were useful in differentiation of these strains.