Fruit preparations mainly serve as pasteurized intermediate products for the dairy industry to incorporate fruits into products like yoghurts, with strawberry being the most common type. Floating or sedimentation of fruit pieces is usually prevented by added hydrocolloids. In this study, the suitability of structure-forming pear fermentates as an alternative to conventional stabilizers was explored. Pear puree was fermented with β-D-glucan-forming LAB strains, either Levilactobacillus brevis TMW 1.2112 or Pediococcus parvulus LTH 1110. Fermentation outcomes in pear puree were evaluated based on the analysis of carbohydrates, organic acids, amino acids, and minerals. Pasteurization of the fermented purees, lyophilization and grinding yielded pear fermentates, which were incorporated as stabilizers into strawberry model fruit preparations. The latter underwent a comprehensive rheological assessment of flow properties, viscoelastic behaviour, thixotropy, and shear stability to investigate the structure-forming potential of the pear fermentates relative to control model products, which were stabilized using uninoculated pear fermentate blank. Increasing the fermentate dose from 10 to 15% resulted in a significant 2.6-fold increase in viscosity. However, at each fermentate dose, only insignificant differences were found between control fruit preparations and those with any one of the two LAB strains. Therefore, no fermentatively induced rheological effect was observed. Although no fermentatively induced structure formation was rheologically measurable for all pear fermentate variants, the rheological properties of resultant model fruit preparations were comparable to those of conventionally stabilized food products.

Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens causing serious human diseases such as bloody diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS). In recent years, the consumption of minimally processed products, such as fresh produce, has been increasingly associated with outbreaks of E. coli O157:H7 involving the development of HUS. Alternatives to the classical preservatives for prevention of EHEC transmission to humans, such as the use of strictly lytic phages are considered as powerful tools for the biocontrol of EHEC. In the current study, the two tailed bacteriophages vB_EcoS_MM-1 (MM-1) and vB_EcoS_MM-2 (MM-2) were isolated from a local sewage water plant. Genome analysis revealed that both phages belong to the genus Tequintavirus and contain a double-stranded DNA genome of approximately 118,000 bp. Phenotypic characterization revealed that both phages infect important clinical EHEC, food-borne STEC and Salmonella enterica serotypes. MM-1 and MM-2 were infectious at a broad temperature (4°C to 60 °C) and pH range (pH 3-12). Both phages combine a short latent period of appr. 15-20 min with a burst size of appr. 100 PFU mL-1. Growth of E. coli O157:H7 strain EDL933 was inhibited effectively by both phages. The analysis of Shiga toxin 2a gene expression using different MOIs did not reveal significant upregulation after phage infection compared to the non-infected control, even in the presence of subinhibitory concentrations of the SOS response-inducing antibiotic norfloxacin. This study highlights that vB_EcoS_MM-1 and vB_EcoS_MM-2 are promising and effective biocontrol agents against EHEC for further use in food safety applications and phage therapy.

In this study, 11 Streptococcus salivarius strains, one Streptococcus raffinosi strain isolated from the human oral cavity and a gastric lavage sample, as well as one S. salivarius reference strain, were whole-genome sequenced to investigate their genomic heterogeneity. Bioinformatic analysis indicated two major clades.

Non-nutritive sweeteners (NNS) are widely employed in foodstuffs. However, it has become increasingly evident that their consumption is associated with bacterial dysbiosis, which, in turn, is linked to several health conditions, including a higher risk of type 2 diabetes and cancer. Among the NNS, stevia, whose main component is rebaudioside A (rebA), is gaining popularity in the organic food market segment. While the effect of NNS on bacteria has been established, the impact of these sweeteners on bacterial viruses (phages) has been neglected, even though phages are crucial elements in maintaining microbial eubiosis. The present study sought to provide a proof-of-concept of the impact of NNS on phage infectivity by assessing the binding of rebA to phage proteins involved in the infection process of enteropathogenic bacteria, namely the fiber protein gp17 of Yersinia enterocolitica phage φYeO3-12 and the tubular baseplate protein gp31 of Klebsiella pneumoniae phage 32. We employed docking analysis and a panel of in vitro confirmatory tests (microscale thermophoresis, RedStarch™ depolymerization, adsorption, and lysis rates). Docking analysis indicated that NNS can bind to both fiber and baseplate proteins. Confirmatory assays demonstrated that rebA can bind gp31 and that such binding increased the protein’s enzymatic activity. Moreover, the binding of rebA to gp17 resulted in a decrease in the adsorption rate of the recombinant protein to its host but increased the Yersinia bacteriolysis caused by the whole phage compared to unexposed controls. These results support the hypothesis that NNS can impair phage infectivity, albeit the resulting effect on the microbiome remains to be elucidated.

The upper gastrointestinal (uGI) microbiota has been implicated in infectious, metabolic, and immunological conditions, yet remains poorly characterized due to invasive sampling and low microbial biomass. We developed and validated a contamination-controlled 16S rRNA gene and transcript-based protocol to profile the murine and human uGI microbiota from low-biomass samples. We applied this protocol to murine esophageal, gastric, and duodenal tissues, and to human saliva, gastric, and duodenal aspirates from patients undergoing endoscopy for suspected food-related, mild GI symptoms. Our objective was to identify conserved compositional and structural uGI microbiota patterns and assess their clinical relevance in relation to pathogen burden and inflammation. In mice, we found evidence for transcriptionally inactive and active intestinal taxa along the uGI tract, supporting horizontal microbiota transfer. In humans, we identified two distinct, inversely correlated salivary microbiota types - one dominated by the Prevotella 7 genus - which were conserved in the duodenum. The Prevotella 7-dominated uGI microbiota type was associated with lower relative abundances of gastrointestinal and extraintestinal opportunistic pathogens. These patterns were reproducible in an independent cohort and associated with lower systemic TNF-α levels. Our findings suggest that noninvasive salivary microbiota profiling can stratify individuals based on uGI microbiota composition and inflammation-associated risk traits, offering new opportunities for clinical applications and translational studies.

Lactic acid bacteria (LAB) play a vital role in the production of fermented foods, with certain strains being capable of producing exopolysaccharides (EPS) that may enhance the texture and functionality of fermented food products. Among these, bacterial β-glucan produced by LAB - a specific type of EPS - offers favorable rheological properties and potential health benefits, making it particularly valuable for food applications. In this study, a combined phenotypic and genotypic screening strategy was used to identify LAB strains potentially capable of producing β-glucan and suitable for use in food fermentations, with an emphasis on fruit-based products. From a collection of 246 LAB isolates, six EPS-producing strains harboring the gtf-2 gene, which encodes β-glucan synthase, were selected for further characterization. Notably, Lacticaseibacillus paracasei LTH 2407, isolated from Gouda cheese, is the first known member of its genus carrying a gtf-2 gene. Genomic and metabolic analyses revealed strain-specific profiles related to sugar transport, carbohydrate metabolism, and EPS biosynthesis, indicating their potential applicability in diverse food matrices, including fruit fermentations. A genome-based safety assessment showed no evidence of toxin production, antimicrobial resistance genes, or the genetic potential for producing highly toxic biogenic amines such as histamine or tyramine. These findings support the potential suitability of the selected strains for safe use as starter cultures in food fermentations. The study provides a solid basis for further analysis of the fermentation performance, EPS production capacity, and industrial relevance of these β-glucan-producing LAB strains.

Bacillus species are beneficial fermentation microbes that exhibit useful technological traits including the expression of extracellular amylolytic and proteolytic enzymes and antimicrobial lipopeptides. In this study, strains of Bacillus spp. were screened through genome analysis and the effect of fermentation of plant-based cheese analogues with the acidification cultures Lactococcus lactis, Lactococcus cremoris and Leuconostoc mesenteroides, and the adjunct culture Lentilactobacillus. buchneri plus Bacillus spp. was investigated. Based on genome analyses of 9 strains of Bacillus spp., B. velezensis FUA2155, B. amyloliquefaciens FUA2153, and B. subtilis FUA2114 that harbor genes encoding for amylases and proteases and lipopeptide synthases were selected for fermentation of plant cheese. Bacillus strains exhibited metabolic activity during bean germination but were inactive after acidification of the cheese matrix. The strains prolonged the mould-free storage time of plant-based cheese analogues and enhanced proteolysis. Of the three strains, only B. velezensis FUA2155 contributed to accumulation of taste-active glutamate. Lt. buchneri accelerated the inactivation of Ln. mesenteroides and enhanced the prevention against fungal contaminants in plant-based cheese analogues with bacilli at the ripening condition rH of 0.78. Taken together, this study provides evidence that the use of proteolytic strains of Bacillus in combination with the acidification cultures Lc. lactis and Lc. cremoris associated with Ln. mesenteroides and adjunct culture Lt. buchneri improved the quality of fermented plant-based cheese analogues.

Plant-based milk alternatives can be produced from a variety of raw materials. The microbial load of the used raw materials can vary greatly, affecting the heating parameters necessary for reducing the microbial load. In this study, plant-based raw materials for producing oat-, almond-, pea-, and rice-based drinks were examined for their microbial load. In this context, flours, flakes, protein isolates, and syrups were tested. The microbiological tests included i) the mesophilic viable cell count (mVCC), ii) the thermophilic viable cell count (tVCC), iii) the mesophilic spore count (mSC), and iv) the thermophilic spore count (tSC). Pure cultures were isolated from each sample, and bacterial species were identified using 16S rRNA gene analysis. The plant-based raw materials (oat, almond, pea and rice) showed wide variations in the viable cell and spore count, ranging from 1 to 8.5 log10 CFU/g. Most of the raw materials contained a high proportion of spores in the viable cell count. Despite previous ultra-high temperature treatment (UHT treatment), the oat and rice syrups showed spore levels of 1 to 4 log10 CFU/g. In total, 435 bacterial isolates were classified with the most frequent species belonging to the genus Bacillus. Among these, B. licheniformis, B. subtilis, and B. tequilensis were the most prevalent. However, other species such as B. cereus, B. amyloliquefaciens, P. etheri, and G. stearothermophilus were also present. Based on the initial spore load of the raw materials, the required effect of the heat treatment B* can be calculated to ensure a commercially sterile plant-based drink. For an average mesophilic bacterial load of oat flours with 5 log10 CFU/g, a 12 log10 reduction is required and for a higher contamination with 9 log10 CFU/g a 16 log10 reduction is already necessary.

The common microbiota of strawberry fruits was characterized by sequencing with a special focus on spoilage organisms. A comprehensive understanding of the microbiota of strawberries might reduce food loss by spoilage and facilitate the manufacture of safe products. Altogether, 19 samples of fresh strawberries from Spain, Germany, and Austria were collected and analyzed by Illumina amplicon sequencing. A shared microbiota of the investigated strawberries is represented by 16 bacterial and 14 fungal genera. Potential spoilage organisms including the genera Bacillus, Cladosporium, and Botrytis (in 95% of samples) were identified as part of this common microbiota of the analyzed strawberries. These spoilage organisms might pose a particular risk to pasteurized strawberry products by their ability to form spores. In this context, Cladosporium herbarum and Botrytis caroliniana could be of particular importance. Human pathogens already known to be associated with foodborne illness caused by strawberries were not identified in the microbiota of investigated strawberries. The present study underlines the importance of understanding the microbiota of strawberry fruits to produce safe and shelf-stable food from raw strawberries.

Fruit preparations are intermediate food products that are primarily used in the dairy industry for the production of fruit yogurt or frozen desserts. Typically, they are stabilized by added hydrocolloids like pectins. The objective of this study was to investigate the potential replacement of conventional stabilizers by structure-forming fermentates produced by exopolysaccharides (EPS)-forming lactic acid bacteria (LAB). Peach puree was selected as fermentation matrix. Prior to 72 h of incubation, it was inoculated with either the heterofermentative LAB strain Levilactobacillus brevis TMW 1.2112 or the homofermentative LAB strain Pediococcus parvulus strain LTH 1110, both being known to produce EPS in form of β-D-glucan. The lyophilized fermentates were applied as stabilizers to produce strawberry fruit preparations. Flow curves, viscoelastic behaviour and shear stability were measured to investigate the effect of fermentate incorporation on the rheological properties of the products. A fermentatively induced effect was observed in terms of a 1.3-fold increase in viscosity of strawberry model fruit preparations with 10 % fermentate of Lv. brevis TMW 1.2112 compared to the addition of the same dose of fermentate blank. Further, increasing the fermentate blank dose from 10 % to 15 % resulted in a 2.4-fold viscosity increase of the model fruit preparations. High shear stability was found in all model strawberry fruit preparations. However, fermentation had no clear benefit in terms of viscoelastic behaviour and shear stability of the fruit preparations. Although the fermentatively induced thickening potential was limited, production of viscosity-increasing peach fermentate with minor changes in the sugar and amino acid profiles of the fruit proved to be feasible.

Shiga toxins (Stx) produced by Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) are ribosome-inactivating AB5 proteins that consist of one enzymatic active A-subunit (StxA) and a pentamer of non-covalently linked B-subunits (StxB). The description of Stx as an AB5 protein and the observation that A-subunits without their corresponding B-subunits also intoxicate eukaryotic cells, led to the question whether A- and B-subunits are produced in the bacteria in a 1:5 ratio or whether the A-subunit of the clinically most prominent subtype Stx2a is transcribed in excess revealing free A-subunits released in the bacterial environment. The aim of this study was therefore, to investigate the genetic and protein-based background for this observation in six Stx2a-encoding STEC and EHEC wildtype strains. For this purpose, transcriptional analysis of the Stx2a subunit genes, stxA2a and stxB2a, was performed by quantitative real-time PCR in one foodborne O113:H21 STEC isolate (strain TS18/08) and five HUS-associated EHEC strains with the serotypes O157:H7/H- (HUSEC003, HUSEC004), O103:H- (HUSEC008), O26:H11 (HUSEC018), and O104:H4 (LB226692). Contrary to the hypothesis that the A- and B-subunit genes are expressed in a ratio of 1:5 comparable to the holotoxin structure or in a ratio of 1:1 based on the operon structure, the results showed that stxA2a was expressed 1.90 ± 0.55-times stronger than the gene encoding the B-subunit, possibly indicating the presence of free A-subunits. In addition, strain-specific differences regarding the mRNA fold-changes of the A-subunit gene were observed. By use of native polyacrylamide gel electrophoresis and subsequent Western blot analysis, those single A-subunits were indeed detected in the culture supernatants of all six strains. To investigate whether the transcription ratios between A- and B-subunits observed are in a similar range as the amount of subunit proteins present after translation, a quantitative ELISA specific for StxA2a and StxB2a was established. Quantification of the subunits on protein level by use of ELISA revealed that the subunit ratio of StxA2a:StxB2a is 1.10 ± 0.20 for the strains HUSEC003, HUSEC004 and HUSEC008, but 4.63 ± 0.31 for the strains TS18/08, LB226692, and HUSEC018. The results of this study demonstrated that on both, the transcriptional and the translational level, the established 1:5 subunit ratio is not present in all investigated strains. In addition, the ratios observed after translation indicate that in some strains StxA2a subunits are even produced in higher amounts than B-subunits.

A core genome phylogenomic analysis of two lactic acid bacteria isolates from spoiled beer, Furfurilactobacillus sp. LTH 5742 and LTH 5750, one isolate from sourdough, Furfurilactobacillus sp. C5, and the type strains of five Furfurilactobacillus species were performed. Average nucleotide identity and digital DNA-DNA hybridization analyses revealed two distinct phylogenetic lineages, both clearly separated from the five previously described Furfurilactobacillus species. Based on these data, two novel species were proposed: Furfurilactobacillus cerevisiae sp. nov., represented by strains LTH 5742T (=DSM 120514T=LMG 34151T) and LTH 5750, and Furfurilactobacillus cerealis sp. nov., represented by strain C5T (=DSM 120515T=LMG 34152T). Comparative genomic analyses highlighted distinct metabolic signatures concerning the 1,2-propanediol and hydroxycinnamic acid metabolism. The gene aldA encoding lactaldehyde dehydrogenase, contributing to 1,2-propanediol production from lactate, is present in Ff. spp. LTH 5742T and LTH 5750, Ff. spp. C5T and Furfurilactobacillus rossiae but not in Furfurilactobacillus milii. Genes for 1,2-propanediol conversion to propionate and propanol are present in Ff. rossiae but not in one of the other species. The glutathione reductase gene, gshR, was uniquely detected in Ff. cerealis, Ff. rossiae and 'Furfurilactobacillus entadae'. These findings expand the diversity of the genus Furfurilactobacillus and provide insight into their ecological adaptation in cereal-based fermentations.

Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the “beany” off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.

The objective of this study was to identify a suitable surrogate for E. coli O157:H7 strain 19685/91 and O113:H21 strain TS18/08, by assessing their thermal resistance at temperatures of 60°C, 65°C, and 72°C in strawberry nectar. The influence of the matrix and the research methodology on the decimal reduction time (D-value) was investigated. Thermal kinetics and safety assessment demonstrated that E. coli ATCC 8739 is a suitable surrogate. The study demonstrated that the presence of fruit particles in the nectar increased thermal resistance of the tested strains. Variations in D-values were observed depending on the research method employed, with D-values in glass capillaries were up to 6.6 times lower compared to larger sample volumes. Encapsulation of E. coli ATCC 8739 exhibited high efficiency of 90.25±0.26% and maintained stable viable counts after 26 days of storage in strawberry nectar at 4°C. There were no significant differences in thermal resistance between surrogates directly inoculated into strawberry nectar and those encapsulated in alginate beads. Additionally, the encapsulated strains did not migrate outside the beads. Therefore, encapsulated E. coli ATCC 8739 in alginate beads can be effectively utilized in industrial settings to validate thermal treatments as a reliable and safe method.

We report the complete genome sequence of the novel Kozakia baliensis strain LTH 7215, isolated from grape must. This strain exhibits a mucoid phenotype on solid medium, associated with exopolysaccharide production. The high-quality and full-length genome sequence was obtained using PacBio Sequencing technology, refined with Illumina sequencing.

Heat resistance, D- and z-values of five Salmonella enterica strains, namely S. Senftenberg LTH 5703, S. Typhimurium ATCC 13311 and ATCC 14028, S. Saintpaul LTH 6494, and S. Enteritidis ATCC 13076, as well as of a Salmonella cocktail and three potential surrogate strains were investigated in phosphate buffered saline and strawberry nectar (12° Brix). Thermal inactivation was performed at 60°C, 65°C, and 72°C. Moreover, the influence of pre-incubation under stressful environmental conditions on the heat resistance was tested. The results identified Escherichia coli ATCC 11229 as a suitable surrogate candidate for Salmonella strains, as its calculated D-values in both matrices were higher or statistically the same than those of the examined pathogens. The safety analysis showed no virulence factors that could classify E. coli ATCC 11229 as a pathogen. Furthermore, the results show significant differences in D-values of all strains in different matrices, which indicates a clear influence of the environment on heat resistance of bacteria. The pre-incubation under stress conditions had no significant effect on the heat resistance of E. coli ATCC 11229. However, further research is needed to explore the influence of stress conditions and duration on bacterial heat resistance.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications. Genome sequence mining and computational analyses lead to the discovery and functional characterization of conserved bacterial toxins with activity against bacteria and fungi.