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.

Proof of concept: predicting the onset of meat spoilage by an integrated oxygen sensor spot in MAP packages.

During storage of modified atmosphere packaged (MAP) meat, the initial microbiota grows to high cell numbers, resulting in perceptible spoilage after exceeding a specific threshold level. This study analyses, whether elevated oxygen consumption in the headspace of MA-packages would enable a prediction method for meat spoilage. We monitored the growth of single spoiling species inoculated on high-oxygen MAP beef and poultry, performed sensorial analysis and determined oxygen concentrations of the headspace via a non-invasive sensor spot technology. We detected microbial headspace oxygen consumption occurring prior to perceptible meat spoilage for certain species inoculated on beef steaks. However, headspace oxygen consumption and cell counts at the onset of spoilage were highly species-dependent, which resulted in a strong (Brochothrix thermosphacta) and moderate (Leuconostoc gelidum subspecies) decrease of the headspace oxygen content. No linear decrease of the headspace oxygen could be observed for Carnobacterium divergens and Carnobacterium maltaromaticum inoculated on poultry meat. We demonstrate the applicability of an incorporated oxygen sensor spot technology in MAP meat packages for detection of spoilage in individual packages prior to its perceptible onset. This enables individual package evaluation and sorting within retail, and consequently reduces meat disposal as waste.

Diversity and anaerobic growth of Pseudomonas spp. isolated from modified atmosphere packaged minced beef.

AIMS: This study aimed to monitor development of spoilage-associated microbiota on high-oxygen modified atmosphere packaged (MAP) minced beef, assess diversity of Pseudomonas sp. therein employing a polyphasic approach and probe their ability to grow anaerobically in the presence of carbon dioxide. METHODS AND RESULTS: Headspace atmosphere and total viable count of MAP minced beef were monitored, and spoilage-associated microbiota was identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Pseudomonas spp. represented a major part of the spoilage-associated microbiota throughout the spoilage process and were characterized with a polyphasic approach including MALDI-TOF, randomly amplified polymorphic DNA biotyping, 16S rDNA and rpoD sequence analysis, and carA multiplex polymerase chain reaction. Pseudomonas isolates displayed high diversity and varying assertiveness under conditions employed in MAP minced beef with P. fragi, P. lundensis and P. weihenstephanensis as dominant species. CONCLUSIONS: The polyphasic approach enabled high-throughput characterization of Pseudomonas sp. Their adapted capability to grow anaerobically and resistance to high levels of CO2 is suggested to be a general feature within the genus, which is hitherto underexplored. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that diverse Pseudomonas generally regarded as strict aerobes and CO2 -sensitive appear well adapted to grow under MAP conditions, leading to high cell counts in minced beef and ultimately contribute to spoilage of the product.

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.

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.

An adapted isolation procedure reveals Photobacterium spp. as common spoilers on modified atmosphere packaged meats.

The genus Photobacterium comprises species of marine bacteria, commonly found in open-ocean and deep-sea environments. Some species (e.g. Photobacterium phosphoreum) are associated with fish spoilage. Recently, culture-independent studies have drawn attention to the presence of photobacteria on meat. This study employed a comparative isolation approach of Photobacterium spp. and aimed to develop an adapted isolation procedure for recovery from food samples, as demonstrated for different meats: Marine broth is used for resuspending and dilution of food samples, followed by aerobic cultivation on marine broth agar supplemented with meat extract and vancomycin at 15°C for 72 h. Identification of spoilage-associated microbiota was carried out via Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry using a database supplemented with additional mass spectrometry profiles of Photobacterium spp. This study provides evidence for the common abundance of multiple Photobacterium species in relevant quantities on various modified atmosphere packaged meats. Photobacterium carnosum was predominant on beef and chicken, while Photobacterium iliopiscarium represented the major species on pork and Photobacterium phosphoreum on salmon, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates highly frequent isolation of multiple photobacteria (Photobacterium carnosum, Photobacterium phosphoreum, and Photobacterium iliopiscarium) from different modified-atmosphere packaged spoiled and unspoiled meats using an adapted isolation procedure. The abundance of photobacteria in high numbers provides evidence for the hitherto neglected importance and relevance of Photobacterium spp. to meat spoilage.

Assertiveness of meat-borne Lactococcus piscium strains and their potential for competitive exclusion of spoilage bacteria in situ and in vitro.

AIMS: This study aimed to investigate intraspecies assertiveness of meat-borne Lactococcus piscium isolates, inhibitory effects on unwanted and harmful meat spoilers, and the prevalence on beef deliberately inoculated with Lc. piscium. METHODS AND RESULTS: Co-inoculation of Lc. piscium isolates and spoilers (Brochothrix thermosphacta, Leuconostoc gelidum subsp. gasicomitatum, Carnobacterium divergens, Pseudomonas weihenstephanensis, Serratia liquefaciens, Hafnia alvei) were conducted in sterile meat simulation medium. Differentiation of Lc. piscium strains was carried out with colony-based RAPD-PCR. Selective cultivation was used to differentiate spoilers from Lc. piscium. Intraspecies assertiveness revealed Lc. piscium TMW2.1614 as most assertive strain. Co-inoculation of selected Lc. piscium strains caused substantial growth reduction of spoilers while the extent was strain- and spoiler dependent. Monitoring the microbiota on beef steaks deliberately inoculated with Lc. piscium revealed prevalence over the endogenous microbiota while maintaining a ripened sensory impression without undesired alterations. CONCLUSIONS: This study reveals Lc. piscium strains TMW2.1612/2.1614/2.1615 as highly competitive against spoilers in vitro while beef deliberately inoculated with these strains maintained acceptable organoleptics. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected Lc. piscium strains exhibit high potential for application as bioprotective cultures for competitive exclusion on beef in order to extend minimum shelf life and enhance product safety of meat.

Monitoring of spoilage-associated microbiota on modified atmosphere packaged beef and differentiation of psychrophilic and psychrotrophic strains.

AIMS: This study aimed to explore the discriminatory power of MALDI-TOF MS as a high-throughput method to monitor growth dynamics of meat-spoiling bacteria on modified atmosphere packaged (MAP) beef and to differentiate psychrophilic and psychrotrophic spoilage-associated strains. METHODS AND RESULTS: MAP beef steaks were incubated for 21 days at constant 4 and 10°C. Development of headspace gas composition, pH, CFU and spoilage-associated microbial composition were monitored. MALDI-TOF MS exhibited high discriminatory power for reliable, high-throughput identification of spoilage-associated, psychrotrophic microbiota. Microbiota development was highly dependent on initial abundance of specific species. Organoleptic onset of spoilage was concomitant with an alteration of headspace atmosphere and pH. Screening for psychrophiles at 4°C on beef revealed the abundance of Leuconostoc gelidum subsp. gelidum TMW2·1998 with characteristic psychrophilic growth behaviour. CONCLUSIONS: We suggest that control of initial contaminants is crucial to predict the onset of spoilage and that headspace atmosphere and pH are important parameters with spoilage-indicative potential. SIGNIFICANCE AND IMPACT OF THE STUDY: MALDI-TOF MS proved suitable for high-resolution monitoring of psychrotrophic and psychrophilic spoilage-associated microbiota and enables improved insights in the spoilage progress. The presence of psychrophilic strains on beef is the likely causative for unexplained spoilage events.

Evidence of gushing induction by Penicillium oxalicum proteins.

AIMS: The aim of this study was to elucidate whether grape-associated fungi exert an influence on gushing by their production of surface-active compounds. METHODS AND RESULTS: In preliminary experiments, 58 grape-associated isolates of species within Penicillium and Aspergillus genera were tested for their ability to modify the surface activity of culture supernatants. As the genus Penicillium had a higher potential to change surface activity, further research focused on that genus. Subsequently, supernatants of 36 Penicillium isolates were assessed for their potential to induce gushing in a model system. Isolates of Penicillium oxalicum had the highest potential. Different external factors were investigated for their influence on the intensity of gushing. By using reversed-phase high performance liquid chromatography and subsequent MALDI-TOF MS, SDS-PAGE and nano-ESI-LC-MS/MS analysis, two proteins in the exoproteome of P. oxalicum were identified, which could be linked to the induction of gushing. CONCLUSIONS: Our results suggest that infection of grapes by P. oxalicum may contribute to gushing in sparkling wine. SIGNIFICANCE AND IMPACT OF THE STUDY: In contrast to gushing of beer, the reason for its development in sparkling wine is widely unexplored. Nonetheless, sparkling wine producers have also been affected by this economically damaging phenomenon. This study has first suggested about the occurrence of primary gushing in sparkling wine.

Thermal treatment of lupin-based milk alternatives - Impact on lupin proteins and the network of respective lupin-based yogurt alternatives.

In this study lupin-based milk alternative (LBMA), obtained from protein isolate of Lupinus angustifolius cv. Boregine, was homogenized and was subjected to different heat treatments (unheated; pasteurized: 80°C, 60s; ultra-high temperature heating (UHT): 140°C, 10s). Upon thermal treatment, lupin proteins, namely ß-conglutin and α-conglutin, were stepwise denatured, which was accompanied by the exposure of masked sulfhydryl groups, the decrease of free sulfhydryl groups and reoxidation to disulfide bridges. Moreover, quaternary structure of lupin proteins was influenced upon heat treatment displayed with molecular weight distribution (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Further, particle size distribution showed that heat treatment promoted the formation of smaller particles. Differently heated LBMA were additionally fermented to lupin-based yogurt alternatives (LBYA) with different lactic acid bacteria (Lactobacillus plantarum TMW 1.460 and 1.1468, Pediococcus pentosaceus BGT B34 and Lactobacillus brevis BGT L150) and microstructural observations (Confocal Laser Scanning Microscopy, cryo-Scanning Electron Microscopy) were performed. Micrographs showed that harsher thermal treatments of LBMA (UHT heating) led to more close-meshed microstructures of respective LBYA. Further, the choice of microorganisms seemed to play a decisive role in microstructural appearance of respective LBYA as the composition of the networks were fundamental different.

Fermentation performance of lactic acid bacteria in different lupin substrates-influence and degradation ability of antinutritives and secondary plant metabolites.

AIMS: The main objectives were to determine the influence of secondary plant metabolites and antinutritives in lupin seeds on the fermentation performance of lactic acid bacteria and to study their ability to degrade these substances. The suitability of lupin raw materials as fermentation substrates was examined. METHODS AND RESULTS: To evaluate the fermentation performance, microbial growth, metabolite formation and substrate uptake in three different lupin substrates was monitored. On the one hand, a lupin protein isolate, which contained only trace amounts of phytochemicals was used in the study. On the other hand, the flour of Lupinus angustifolius cv. Boregine and the flour of the alkaloid rich lupin Lupinus angustifolius cv. Azuro were inoculated with Bifidobacterium animalis subsp. lactis, Pediococcus pentosaceus, Lactobacillus plantarum and Lactococcus lactis subsp. lactis. The micro-organisms showed no significant differences in the fermentation performance on the different lupin flours. Similarly, the growth of most strains on lupin protein isolate was comparable to that on the lupin flours. The fermentation with Bifidobacterium animalis subsp. lactis led to a significant decrease in flatulence causing oligosaccharides. During fermentation with Lactobacillus plantarum the phytic acid content was partially degraded. CONCLUSIONS: Neither the secondary plant metabolites nor the antinutritives of lupin flour inhibited the growth or metabolic activity of the tested micro-organisms. Therefore, lupin flour is suitable for lactic fermentation. Some strains showed the ability to degrade oligosaccharides or phytic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the fundamental knowledge of the metabolism of lactic acid bacteria during fermentation of lupin substrates. Fermentation of lupin raw materials could be used to improve the nutritional value of the substrates due to the reduction of antinutritives.

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.

Glutamine deamidation by cereal-associated lactic acid bacteria.

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.

Characterization of AfpA, an alkaline foam protein from cultures of Fusarium culmorum and its identification in infected malt.

AIMS: The objective of this study was to evaluate the capability of Fusarium culmorum to produce non-hydrophobin surface-active proteins in vitro, to isolate and characterize such proteins from liquid cultures, to analyse their effect on overfoaming (gushing) of beer and to elucidate their prevalence in pure cultures and infected malt. METHODS AND RESULTS: A 20 kDa protein was isolated from liquid cultures of F. culmorum BBA 62182 upon enrichment by foaming. BLAST search with N-terminal and internal sequences of the protein revealed high homology with a hypothetical protein predicted within the F. graminearum PH1 genome sequence. Oligonucleotide primers designed to bind 30 nt upstream and downstream of the predicted gene were used to amplify a 695 nt PCR fragment from genomic DNA of F. culmorum BBA 62182. Cloning and sequencing of the product revealed a 635 nt open reading frame which had 98% homology to the predicted F. graminearium PH1 gene code. Removal of a 59 nt intron and translation resulted in a 191 amino acid protein of 20.754 kDa with a calculated pI of 9.1. Amino acids obtained by Edman sequencing of fragments within the 20 kDa protein were 100% homologous with the sequence deduced from the DNA sequence. According to its properties, the new protein was termed alkaline foam protein A (AfpA). Sequence comparison revealed some homologies with proteins in Emericella nidulans, which are involved in phialide development and response to antifungal agents. Homologies with other hypothetical fungal proteins suggest a new group of proteins, for which we suggest the name fungispumins. Addition of AfpA to beer showed that overfoaming (gushing) is not induced in stable beer but can significantly enhance this effect in beer showing moderate gushing. Use of a polyclonal anti-AfpA antibody in a Western blot revealed that the protein is produced by various F. culmorum strains and also by F. graminearum, but not by other Fusarium spp. tested. PCR testing of 69 species of Fusarium and Trichoderma reesei with a gene specific primer pair revealed that the gene may be present exclusively in F. culmorum, F. graminearum, F. cerealis, F. lunulosporum and F. oxysporum f. sp . dianthi. Immunochemical detection of AfpA in malts artificially inoculated with F. culmorum and F. graminearum showed that the protein was present in gushing inducing malts (gushing test) but absent in malts which were negative in a gushing test. CONCLUSIONS: AfpA is a member of a new protein class, fugispumins, and can be isolated from pure liquid cultures of F. culmorum. A homologous protein is synthesised by F. graminearum. The protein is produced in contaminated malt and enhances gushing of beer. The gene coding for AfpA is restricted to Fusarium species presumably involved in the induction of beer gushing. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a new class of proteins, fungispumins, the natural function of which remains to be elucidated. Findings add useful information to research on the mechanisms involved in foam stability of beer. AfpA may be useful as a marker for gushing in future quality control applications for the brewing industry.

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis.

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Differential inactivation of glucose- and glutamate dependent acid resistance of Escherichia coli TMW 2.497 by high-pressure treatments.

The inactivation by 200-400 MPa and post-pressure survival at acid conditions of E. coli TMW 2.497 was characterized by the measurement of intracellular pH (pHin), viable cell counts, glutamate (Glu) and arginine (Arg) consumption, and the influence of mild adaptation to mild acid stress prior to pressure treatment. Glutamate and arginine did not affect viable cell counts or the pHin during pressure application but improved the ability to maintain a high pHin after pressure treatment. In pH 4.0 buffer without arg and glu, a 3 log reduction of cell counts occurred after 24 h of incubation, whereas little or no loss of viability was observed after 24 h incubation in the presence of glu and arg. During post-pressure incubation at pH 4.0, 10 mM glutamate were metabolized but only 2 mM arginine were used, indicating that glutamate rather than arginine was responsible for the protective effect on pHin and survival. In conclusion, the pressure induced, irreversible loss of the transmembrane deltapH correlates to cell death and glu stabilizes the pHin of E. coli during post-pressure incubation.

Extracellular homopolysaccharides and oligosaccharides from intestinal lactobacilli.

AIMS: To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. METHODS AND RESULTS: Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and >25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. CONCLUSIONS: A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level.

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.

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 percent 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.

Molecular diagnosis of ochratoxinogenicAspergillus species.

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.