Characterization of the Staphylococcus xylosus methylome reveals a new variant of type I restriction modification system in staphylococci.

Restriction modification (RM) systems are known to provide a strong barrier to the exchange of DNA between and within bacterial species. Likewise, DNA methylation is known to have an important function in bacterial epigenetics regulating essential pathways such as DNA replication and the phase variable expression of prokaryotic phenotypes. To date, research on staphylococcal DNA methylation focused mainly on the two species Staphylococcus aureus and S. epidermidis. Less is known about other members of the genus such as S. xylosus, a coagulase-negative commensal of mammalian skin. The species is commonly used as starter organism in food fermentations but is also increasingly considered to have an as yet elusive function in bovine mastitis infections. We analyzed the methylomes of 14 S. xylosus strains using single-molecular, real-time (SMRT) sequencing. Subsequent in silico sequence analysis allowed identification of the RM systems and assignment of the respective enzymes to the discovered modification patterns. Hereby the presence of type I, II, III and IV RM systems in varying numbers and combinations among the different strains was revealed, clearly distinguishing the species from what is known for other members of the genus so far. In addition, the study characterizes a newly discovered type I RM system, encoded by S. xylosus but also by a variety of other staphylococcal species, with a hitherto unknown gene arrangement that involves two specificity units instead of one (hsdRSMS). Expression of different versions of the operon in E. coli showed proper base modification only when genes encoding both hsdS subunits were present. This study provides new insights into the general understanding of the versatility and function of RM systems as well as the distribution and variations in the genus Staphylococcus.

Transcriptomic analysis of the response of Photobacterium phosphoreum and Photobacterium carnosum to co-contaminants on chicken meat.

This study investigated the impact of Brochothrix (B.) thermosphacta and Pseudomonas (Ps.) fragi on the transcriptomes of Photobacterium (P.) phosphoreum and P. carnosum on chicken meat under modified atmosphere (MA) and air atmosphere (AA). P. phosphoreum TMW2.2103 responded to MA with a reduced transcript number related to cell division and an enhanced number related to oxidative stress. Concomitantly, the analysis revealed upregulation of fermentation and downregulation of respiration. It predicts enhanced substrate competition in presence of co-contaminants/MA. In contrast, the strain upregulated the respiration in AA, supposably due to improved substrate accessibility in this situation. For P. carnosum TMW2.2149 the respiration was downregulated, and the pyruvate metabolism upregulated under MA. MA/co-contaminant resulted in multiple upregulated metabolic routes. Conversely, AA/co-contaminant resulted only in minor regulations, showing inability to cope with fast growing competitors. Observations reveal different strategies of photobacteria to react to co-contaminants on meat.

Impact of Modified Atmospheres on Growth and Metabolism of Meat-Spoilage Relevant Photobacterium spp. as Predicted by Comparative Proteomics.

Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O2, 30% CO2, red and white meats) and oxygen-free MAP (70% N2, 30% CO2, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of Photobacterium carnosum and P. phosphoreum was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for P. carnosum. Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose (P. phosphoreum) or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro, and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

SxsA, a novel surface protein mediating cell aggregation and adhesive biofilm formation of Staphylococcus xylosus.

Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces, and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished and significantly reduced in TMW 2.1023. Macro- and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared with the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, Staphylococcus paraxylosus, and Staphylococcus nepalensis and additionally encoded in a small group of Staphylococcus cohnii and Staphylococcus saprophyticus strains. This study provides insights into protein-mediated biofilm formation of S. xylosus and identifies a new cell wall-associated protein influencing cell aggregation and biofilm formation.

Optimization of a cultivation procedure to selectively isolate lactic acid bacteria from insects.

AIMS: Natural niches and transmission routes of lactic acid bacteria (LAB) are highly versatile. Proposed routes of transmission to food fermentations are from plant material via insects or vice versa. This study aimed to establish a method for the selective isolation of LAB from insects. METHODS AND RESULTS: Varied parameters that influence growth and selectivity are temperature, type of carbohydrate and atmosphere. Additionally, the effects of antibiotics to suppress non-LAB species were evaluated. A model consortium consisting of 12 species representing different lifestyles was inoculated in a growth medium to identify conditions for the highest diversity and recovery rate. The method was applied to isolate LAB from Drosophila melanogaster, Sitotroga cerealella, Tribolium castaneum and Tenebrio molitor. Isolated species were Leuconostoc mesenteroides, Paucilactobacillus vaccinostercus and Lactiplantibacillus plantarum from D. melanogaster and L. mesenteroides, Pediococcus pentosaceus and Latilactobacillus curvatus from T. molitor. No LAB could be isolated from T. castaneum and S. cerealella. 16S rDNA amplicon sequencing of DNA obtained from insects corroborated part of our results. CONCLUSION: A combination of different enrichment conditions ensures a high probability to isolate LAB species from insects and can be helpful above already known non-cultivation methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel method allows to selectively isolate LAB from insects and the strategy of the method is of interest to study other niches.

Bap-Independent Biofilm Formation in Staphylococcus xylosus.

The biofilm associated protein (Bap) is recognised as the essential component for biofilm formation in Staphylococcus aureus V329 and has been predicted as important for other species as well. Although Bap orthologs are also present in most S. xylosus strains, their contribution to biofilm formation has not yet been demonstrated. In this study, different experimental approaches were used to elucidate the effect of Bap on biofilm formation in S. xylosus and the motif structure of two biofilm-forming S. xylosus strains TMW 2.1023 and TMW 2.1523 was compared to Bap of S. aureus V329. We found that despite an identical structural arrangement into four regions, Bap from S. xylosus differs in key factors to Bap of S. aureus, i.e., isoelectric point of aggregation prone Region B, protein homology and type of repeats. Disruption of bap had no effect on aggregation behavior of selected S. xylosus strains and biofilm formation was unaffected (TMW 2.1023) or at best slightly reduced under neutral conditions (TMW 2.1523). Further, we could not observe any typical characteristics of a S. aureus Bap-positive phenotype such as functional impairment by calcium addition and rough colony morphology on congo red agar (CRA). A dominating role of Bap in cell aggregation and biofilm formation as reported mainly for S. aureus V329 was not observed. In contrast, this work demonstrates that functions of S. aureus Bap cannot easily be extrapolated to S. xylosus Bap, which appears as non-essential for biofilm formation in this species. We therefore suggest that biofilm formation in S. xylosus follows different and multifactorial mechanisms.

Comparative genomics of Photobacterium species from terrestrial and marine habitats.

Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn't, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.

The diversity among the species Tetragenococcus halophilus including new isolates from a lupine seed fermentation.

BACKGROUND: Tetragenococcus (T.) halophilus can be isolated from a variety of fermented foods, such as soy sauce, different soy pastes, salted fish sauce and from cheese brine or degraded sugar beet thick juice. This species contributes by the formation of short chain acids to the flavor of the product. Recently, T. halophilus has been identified as a dominant species in a seasoning sauce fermentation based on koji made with lupine seeds. RESULTS: In this study we characterized six strains of T. halophilus isolated from lupine moromi fermentations in terms of their adaptation towards this fermentation environment, salt tolerance and production of biogenic amines. Phylogenic and genomic analysis revealed three distinctive lineages within the species T. halophilus with no relation to their isolation source, besides the lineage of T. halophilus subsp. flandriensis. All isolated strains from lupine moromi belong to one lineage in that any of the type strains are absent. The strains form lupine moromi could not convincingly be assigned to one of the current subspecies. Taken together with strain specific differences in the carbohydrate metabolism (arabinose, mannitol, melibiose, gluconate, galactonate) and amino acid degradation pathways such as arginine deiminase pathway (ADI) and the agmatine deiminase pathway (AgDI) the biodiversity in the species of T. halophilus is greater than expected. Among the new strains, some strains have a favorable combination of traits wanted in a starter culture. CONCLUSIONS: Our study characterized T. halophilus strains that were isolated from lupine fermentation. The lupine moromi environment appears to select strains with specific traits as all of the strains are phylogenetically closely related, which potentially can be used as a starter culture for lupine moromi. We also found that the strains can be clearly distinguished phylogenetically and phenotypically from the type strains of both subspecies T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis.

Microbiota dynamics and volatile compounds in lupine based Moromi fermented at different salt concentrations.

Fermented soy sauces are used as food seasonings in Eastern countries and all over the world. Depending on their cultural origins, their production differs in parameters such as wheat addition, temperature, and salt concentration. The fermentation of lupine seeds presents an alternative to the use of soybeans; however, the microbiota and influencing factors are currently unknown. In this study, we analyse the microbiota of lupine Moromi (mash) fermentations for a period of six months and determine the influence of different salt concentrations on the microbiota dynamics and the volatile compound composition. Cultured microorganisms were identified by protein profiling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene amplicon sequencing provided an overview of the microbiota including non-cultured bacteria. The volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS). At all salt concentrations, we found that Tetragenococcus halophilus (up to 1.4 × 109 colony forming units (CFU)/mL on day 21) and Chromohalobacter japonicus (1.9 × 109 CFU/mL, day 28) were the dominating bacteria during Moromi fermentation. Debaryomyces hansenii (3.6 × 108 CFU/mL, day 42) and Candida guilliermondii (2.2 × 108 CFU/mL, day 2) were found to be the most prevalent yeast species. Interestingly, Zygosaccharomyces rouxii and other yeasts described as typical for soy Moromi were not found. With increasing salinity, we found lower diversity in the microbiota, the prevalence-gain of typical species was delayed, and ratios differed depending on their halo- or acid tolerance. GC-MS analysis revealed aroma-active compounds, such as pyrazines, acids, and some furanones, which were mostly different from the aroma compounds found in soy sauce. The absence of wheat may have caused a change in yeast microbiota, and the use of lupine seeds may have led to the differing aromatic composition. Salt reduction resulted in a more complex microbiome, higher cell counts, and did not show any spoiling organisms. With these findings, we show that seasoning sauce that uses lupine seeds as the sole substrate is a suitable gluten-free, soy-free and salt reduced alternative to common soy sauces with a unique flavour.

Characterization of the influence of carbon sources on fum1 gene expression in the fumonisin producer Fusarium verticillioides using RT - LAMP assay.

Fusarium verticillioides is one of the major fumonisin producers. The ingestion of this mycotoxin represents a risk for both human and animal health. The development of F. verticillioides is associated with environmental conditions, especially carbon sources. We developed a reliable and fast reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and determined fum1 gene expression upon growth of two F. verticillioides strains isolated from maize and wheat in Czapek's medium containing four different sugars as sole carbon sources. Fumonisin B1 (FB1) production was determined by LC-MS/MS analysis. High growth and production of FB1 were observed in fructose-containing medium for the strain that originated from maize. Less production of FB1 occurred using maltose as sole carbon source for both strains. The fum1 gene expression started between 2 and 4 days of incubation, and positive signals were detected prior to the initial production of FB1. The RT-LAMP assay was effective in the detection of fum1 gene expression at very early stages of F. verticillioides growth and allowed the prediction of FB1 formation.

Contribution to the ongoing discussion on fluoride toxicity.

Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.

Influence of the packaging atmosphere and presence of co-contaminants on the growth of photobacteria on chicken meat.

Fresh meat is commonly packaged in modified atmosphere to decelerate spoilage processes. The applied gas mixture affects the growth of spoilage organisms and selectively shapes the spoilage community. In this study, we investigated the impact of O2 and CO2 on the growth of Photobacterium (P.) phosphoreum and P. carnosum strains in situ on chicken meat by packaging under different modified atmospheres (air, 70% O2/30% CO2, 70% N2/30% CO2, 100% N2). Combination of 70% O2 and 30% CO2 resulted in significant growth reduction of the analyzed strains, suggesting inhibitory effects of both gases in combination. In contrast, 30% CO2 alone had only a minor effect and photobacteria are supposed to have a growth advantage over other meat spoilers in this atmosphere. Additionally, single growth of the strains in the different atmospheres was compared when challenged with the presence of Pseudomonas (Ps.) fragi or Brochothrix (B.) thermosphacta as prominent co-contaminants in different ratios (10:1, 1:1, 1:10). Presence of co-contaminants resulted in increased cell numbers of P. carnosum TMW2.2149 but reduced or unchanged cell numbers of P. phosphoreum TMW2.2103 in most packaging atmospheres. The initial ratio of photobacteria and co-contaminants defined the relative abundance during storage but did not change the type of the interaction. Our results suggest either a commensalistic (P. carnosum) or competitive interaction (P. phosphoreum) of photobacteria and co-contaminants on modified atmosphere packaged chicken, respectively. Furthermore, in a mix comprising seven prominent spoilers, strains of both Photobacterium species prevailed as a constant part of the spoilage microbiome during 7 days of refrigerated storage on chicken meat packaged under O2/CO2 atmosphere.

Hydrostatic pressure- and halotolerance of Photobacterium phosphoreum and P. carnosum isolated from spoiled meat and salmon.

Photobacterium spp. occur frequently in marine environments but have been recently also found as common spoilers on chilled meats. The environmental conditions in these ecological niches differ especially regarding salinity and ambient pressure. Linking the occurrence of photobacteria in different niches may elucidate its ecology and bring insights for the food industry. We investigated tolerance of Photobacterium (P.) phosphoreum and P. carnosum strains to high hydrostatic pressure and salinity and aligned our observations with presence of relevant genes. The strains were isolated from packaged meats and salmon (or the sea) to identify adaptations to marine and terrestrial habitats. Growth of all P. carnosum strains was reduced by 40 MPa hydrostatic pressure and >3% sodium chloride, suggesting loss of traits associated with marine habitats. In contrast, P. phosphoreum strains were only slightly affected, suggesting general adaptation to marine habitats. In accordance, these strains had gene clusters associated with marine niches, e.g. flagellar and lux-operons, being incomplete in P. carnosum. Occurrence of P. carnosum strains on packaged salmon and P. phosphoreum strains on meats therefore likely results from cross-contamination in meat and fish processing. Still, these strains showed intermediate traits regarding pressure- and halotolerance, suggesting developing adaptation to their respective environment.

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety.

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data.

ß-Glucan Production by Levilactobacillus brevis and Pediococcus claussenii for In Situ Enriched Rye and Wheat Sourdough Breads.

Sourdough fermentation is a common practice spread across the globe due to quality and shelf life improvement of baked goods. Above the widely studied exopolysaccharide (EPS) formation, which is exploited for structural improvements of foods including baked goods, ß-glucan formation, by using lactic acid bacteria (LAB), offers additional values. Through renunciation of sucrose addition for bacterial ß-d-glucan formation, which is required for the production of other homopolysaccharides, residual sweetness of baked goods can be avoided, and predicted prebiotic properties can be exploited. As promising starter cultures Levilactobacillus (L.) brevis TMW (Technische Mikrobiologie Weihenstephan) 1.2112 and Pediococcus (P.) claussenii TMW 2.340 produce O2-substituted (1,3)-ß-d-glucan upon fermenting wheat and rye doughs. In this study, we have evaluated methods for bacterial ß-glucan quantification, identified parameters influencing the ß-glucan yield in fermented sourdoughs, and evaluated the sourdough breads by an untrained sensory panel. An immunological method for the specific detection of ß-glucan proved to be suitable for its quantification, and changes in the fermentation temperature were related to higher ß-glucan yields in sourdoughs. The sensory analysis resulted in an overall acceptance of the wheat and rye sourdough breads fermented by L.brevis and P.claussenii with a preference of the L. brevis fermented wheat sourdough bread and tart-flavored rye sourdough bread.

Comparative Proteomics Reveals the Anaerobic Lifestyle of Meat-Spoiling Pseudomonas Species.

The ability of certain Pseudomonas (P.) species to grow or persist in anoxic habitats by either denitrification, acetate fermentation, or arginine fermentation has been described in several studies as a special property. Previously, we had isolated strains belonging to the species P. lundensis, P. weihenstephanensis, and P. fragi from anoxic modified atmosphere packaged (MAP) minced beef and further proved their anaerobic growth in vitro on agar plates. This follow-up study investigated the anaerobic growth of two strains per respective species in situ on inoculated chicken breast filet under 100% N2 modified atmosphere. We were able to prove anaerobic growth of all six strains on chicken breast filet with cell division rates of 0.2-0.8/day. Furthermore, we characterized the anaerobic metabolic lifestyle of these Pseudomonas strains by comparative proteomics, upon their cultivation in meat simulation media, which were constantly gassed with either air or 100% N2 atmospheres. From these proteomic predictions, and respective complementation by physiological experiments, we conclude that the Pseudomonas strains P. fragi, P. weihenstephanensis, P. lundensis exhibit a similar anaerobic lifestyle and employ arginine fermentation via the arginine deiminase (ADI) pathway to grow anaerobically also on MAP meats. Furthermore, glucose fermentation to ethanol via the ED-pathway is predicted to enable long term survival but no true growth, while respiratory growth with nitrate as alternative electron acceptor or glucose fermentation to acetate could be excluded due to absence of essential genes. The citric acid cycle is partially bypassed by the glyoxylate shunt, functioning as the gluconeogenetic route without production of NADH2 under carbon limiting conditions as e.g., in packaged meats. Triggered by an altered redox balance, we also detected upregulation of enzymes involved in protein folding as well as disulfide bonds isomerization under anoxic conditions as a counteracting mechanism to reduce protein misfolding. Hence, this study reveals the mechanisms enabling anaerobic grow and persistence of common meat-spoiling Pseudomonas species, and further complements the hitherto limited knowledge of the anaerobic lifestyle of Pseudomonas species in general.

Influence of fermentation conditions on the secretion of seripauperin 5 (PAU5) by industrial sparkling wine strains of Saccharomyces cerevisiae.

The yeast mannoprotein seripauperin 5 (PAU5) from Saccharomyces cerevisiae is a negative gushing biomarker in sparkling wine with a direct gushing-reducing effect. The knowledge about the influence of the yeast strain and the fermentation conditions on the PAU5 content in the final product could reduce the gushing potential of sparkling wines and avoid economic losses for sparkling wine producers. The potential of 30 different commercially used (sparkling) wine S. cerevisiae strains to produce PAU5 was analyzed by RP-HPLC. The experiments revealed great differences between strains and identified three high-PAU5-producing yeast strains. The influence of different fermentation conditions was analyzed in two selected strains. Cultivation conditions of elevated temperature and lower inoculation density, as well as cultivation under diffuse daylight significantly enhanced PAU5 production by the tested S. cerevisiae strains as compared to standard conditions. However, cultivation as agitated cultures and co-cultivation with Metchnikowia pulcherrima or Torulaspora delbrueckii led to a reduction of PAU5 production as compared to standard conditions.

Studies on the gushing potential of Penicillium expansum.

Gushing describes the spontaneous excessive over-foaming of carbonated beverages leading to considerable economic losses and reputational damages to the beverage industry. Surface-active proteins produced by filamentous fungi are involved in the induction of gushing. In the current study, the role of Penicillium expansum in sparkling wine gushing was investigated. Almost 40 P. expansum strains were analyzed regarding their ability to secrete surface-active proteins and to induce gushing in carbonated water as a model system and in sparkling wine. The majority of the strains produced surface-active compounds and induced gushing. The severity of gushing depended on the volume of culture supernatant added to carbonated liquids. Moreover, sparkling wine showed more severe gushing than carbonated water. A protein with a molecular mass of 20 kDa was prominent in gushing-inducing P. expansum culture supernatants. It was identified as PEX2_044840 from P. expansum. This protein was heterologously expressed in Pichia pastoris (Komagataella phaffi). The purified recombinant protein induced gushing in sparkling wine after addition of at least 30 µg/mL of protein sample.

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.