Partitioning and in vitro bioaccessibility of apple polyphenols during mechanical and physiological extraction: A hierarchical clustering analysis with LC-ESI-QTOF-MS/MS.

Polyphenol partitioning during mechanical (cold-pressing) and physiological (digestion) extraction at the individual polyphenol and subclass level was investigated. UHPLC-ESI-QTOF-MS/MS analysis yielded a comprehensive identification of 45 polyphenols whose semi-quantification revealed a hierarchical clustering strongly determined by polyphenol structure and their location within the apple tissue. For instance, pomace retained most flavonols and flavanols (degree of polymerization DP 5-7), which were highly hydrophobic, hydroxylated, or large (>434 Da), and more abundant in peel. In vitro digestion UHPLC-ESI-QTOF-MS/MS analysis of whole apple (and its corresponding matrix-free extract) clustered polyphenols into five main groups according to their interaction with plant cell walls (PCWs) during each digestion phase. This grouping was not reproduced in pomace, which exhibited a greater matrix effect than whole apple during oral and gastric digestion. Nevertheless, the interaction between most polyphenol groups, including dihydrochalcones, flavanols (DP 1-4) and hydroxycinnamic acid derivatives, and pomace PCWs was lost during intestinal digestion.

Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose.

AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Fucose modifies short chain fatty acid and H2S formation through alterations of microbial cross-feeding activities.

Algae are a rich but unexplored source of fibers with the potential to contribute to the next generation of prebiotics. The sulfated brown algae polysaccharide, fucoidan, is mainly composed of the deoxy-hexose L-fucose, which can be metabolized to 1,2-propanediol (1,2-PD) or lactate by gut microbes as precursors of propionate and butyrate. It was the aim of this study to investigate the impact of fucoidan on the fermentation capacity of the fecal microbiota and to compare to fucose. In batch fermentations of fecal microbiota collected from 17 donor samples, fucose promoted the production of propionate while no consistent effect was observed for commercial fucoidan and Fucus vesiculosus extract prepared in this study containing laminarin and fucoidan. H2S production was detected under all tested conditions, and levels were significantly lower in the presence of fucose in a dose-dependent manner. The addition of high fucose levels led to higher relative abundance of microbial 1,2-PD and lactate cross-feeders. Our results highlight that fucose and not fucoidan addition impacted fermentation capacity and increased the proportions of propionate and butyrate, which allows for precise modulation of intestinal microbiota activity.

The quest for the perfect loaf of sourdough bread continues: Novel developments for selection of sourdough starter cultures.

Sourdough fermentation, one of the oldest unit operations in food production, is currently experiencing a revival in bread production at the household, artisanal, and the industrial level. The expanding use of sourdough fermentation in bread production and the adaptation of fermentation to large scale industrial bread production also necessitate the development of novel starter cultures. Developments in the last years also have expanded the tools that are used to assess the metabolic potential of specific strains, species or genera of the Lactobacillaceae and have identified multiple ecological and metabolic traits as clade-specific. This review aims to provide an overview on the clade-specific metabolic potential of members of the Lactobacillaceae for use in sourdough baking, and the impact of these clade-specific traits on bread quality. Emphasis is placed on carbohydrate metabolism, including the conversion of sucrose and starch to soluble polysaccharides, conversion of amino acids, and the metabolism of organic acids. The current state of knowledge to compose multi-strain starter cultures (synthetic microbial communities) that are suitable for back-slopping will also be discussed. Taken together, the communication outlines the current tools for selection of microbes for use in sourdough baking.

Comparative genomic analysis of Periweissella and the characterization of novel motile species.

The genus Periweissella was proposed as a novel genus in the Lactobacillaceae in 2022. However, the phylogenetic relationship between Periweissella and other heterofermentative lactobacilli, and the genetic and physiological properties of this genus remain unclear. This study aimed to determine the phylogenetic relationship between Periweissella and the two closest genera, Weissella and Furfurilactobacillus, by the phylogenetic analysis and calculation of (core gene) pairwise average amino acid identity. Targeted genomic analysis showed that fructose bisphosphate aldolase was only present in the genome of Pw. cryptocerci. Mannitol dehydrogenase was found in genomes of Pw. beninensis, Pw. fabaria, and Pw. fabalis. Untargeted genomic analysis identified the presence of flagellar genes in Periweissella but not in other closely related genera. Phenotypes related to carbohydrate fermentation and motility matched the genotypes. Motility genes were organized in a single operon and the proteins shared a high amino acid similarity in the genus Periweissella. The relatively low similarity of motility operons between Periweissella and other motile lactobacilli indicated the acquisition of motility by the ancestral species. Our findings facilitate the phylogenetic, genetic, and phenotypic understanding of the genus Periweissella.ImportanceThe genus Periweissella is a heterofermentative genus in the Lactobacillaceae which includes predominantly isolates from cocoa fermentations in tropical climates. Despite the relevance of the genus in food fermentations, genetic and physiological properties of the genus are poorly characterized and genome sequences became available only after 2020. This study characterized strains of the genus by functional genomic analysis, and by determination of metabolic and physiological traits. Phylogenetic analysis revealed that Periweissella is the evolutionary link between rod-shaped heterofermentative lactobacilli and the coccoid Leuconostoc clade with the genera Weissella and Furfurilactobacillus as closest relatives. Periweissella is the only heterofermentative genus in the Lactobacillaceae which comprises predominantly motile strains. The genomic, physiological, and metabolic characterization of Periweissella may facilitate the potential use of strains of the genus as starter culture in traditional or novel food fermentations.

The C-Terminal Domain of Liquorilactobacillus nagelii Dextransucrase Mediates the Production of Larger Dextrans Compared to Liquorilactobacillus hordei.

Dextransucrases released by certain lactic acid bacteria form glucose polymers with predominantly α-1,6-linkages and may be exploited biotechnologically for the tailored production of polysaccharides with application potential. Despite releasing two closely related dextransucrases, previous studies showed that water kefir borne Liquorilactobacillus (L.) hordei TMW 1.1822 and L. nagelii TMW 1.1827 produce different amounts of polysaccharides with distinct particle sizes (molecular weight and radius of gyration) and molecular architectures. To investigate where these differences originate and thus to provide deeper insights into the functionally diverse nature of polysaccharide formation during water kefir fermentation, we constructed two variants of the L. nagelii dextransucrase-a full-length enzyme and a truncated variant, devoid of a C-terminal glucan-binding domain that reflects the domain architecture of the L. hordei dextransucrase-and applied them at various enzyme concentrations to form dextran over 24 h. The full-length enzyme exhibited a high activity, forming constant amounts of dextran until a four-fold dilution, whereas the truncated variant showed a gradual decrease in activity and dextran formation at an increasing dilution. The application of the full-length enzyme resulted in higher average particle sizes compared to the truncated variant. However, the dilution of the enzyme extracts also led to a slight increase in the average particle size in both enzymes. Neither the domain architecture nor the enzyme concentration had an impact on the structural architecture of the dextrans. The presented results thus suggest that the comparatively higher processivity of the L. nagelii dextransucrase is predominantly caused by the additional C-terminal glucan-binding domain, which is absent in the L. hordei dextransucrase. The average particle size may be influenced, to some extent, by the applied reaction conditions, whereas the structural architecture of the dextrans is most likely caused by differences in the amino acid sequence of the catalytic domain.

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose.

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Living the Sweet Life: How Liquorilactobacillus hordei TMW 1.1822 Changes Its Behavior in the Presence of Sucrose in Comparison to Glucose.

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests that L. hordei TMW 1.1822 favors fructose over other sugars. The dextransucrase was expressed irrespectively of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix but is also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for its use as a starter culture in plant-based food fermentations with in situ dextran formation.

A systematic approach to study the pH-dependent release, productivity and product specificity of dextransucrases.

BACKGROUND: Dextransucrases are extracellular enzymes, which catalyze the formation of α-1→6-linked glucose polymers from sucrose. These enzymes are exclusively expressed by lactic acid bacteria, which commonly acidify the extracellular environment due to their physiology. Dextransucrases are thus confronted with steadily changing reaction conditions in regards to the environmental pH, which can further affect the amount of released dextransucrases. In this work, we studied the effect of the environmental pH on the release, the productivity and the product specificity of the dextransucrase expressed by Lactobacillus (L.) hordei TMW 1.1822. Dextransucrases were recovered as crude extracts at pH 3.5-pH 6.5 and then again used to produce dextrans at these pH values. The respectively produced dextran amounts and sizes were determined and the obtained results finally systematically correlated. RESULTS: Maximum dextran amounts were produced at pH 4.0 and pH 4.5, while the productivity of the dextransucrases significantly decreased at pH 3.5 and pH 6.5. The distribution of dextran amounts produced at different pH most likely reflects the pH dependent activity of the dextransucrases released by L. hordei, since different transglycosylation rates were determined at different pH using the same dextransucrase amounts. Moreover, similar hydrolysis activities were detected at all tested conditions despite significant losses of transglycosylation activities indicating initial hydrolysis prior to transglycosylation reactions. The molar masses and rms radii of dextrans increased up to pH 5.5 independently of the stability of the enzyme. The gelling properties of dextrans produced at pH 4.0 and pH 5.5 were different. CONCLUSIONS: The presented methodological approach allows the controlled production of dextrans with varying properties and could be transferred and adapted to other microbes for systematic studies on the release and functionality of native sucrases or other extracellular enzymes.

Identification and comparison of two closely related dextransucrases released by water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α-1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827 were identified and comparatively investigated. Differences between both proteins mainly arise from an additional C-terminal glucan-binding domain and the presence of a signal motif in the L. nagelii TMW 1.1827 dextransucrase. L. hordei TMW 1.1822 released the enzyme only in the presence of its substrate sucrose in contrast to L. nagelii TMW 1.1827, while both strains functionally expressed the dextransucrases independently of sucrose. Both enzymes could be recovered as crude protein extracts in culture supernatants, as they are not covalently bound to the cell surface. This enabled the formation of dextrans at equal reaction conditions as well as their subsequent structural analysis in terms of molecular structure and molecular weight. The volumetric transglycosylation and hydrolysis activities were distinctly different for both enzymes, which produced O3-branched dextrans with a comparable degree of branching. Moreover, identical oligosaccharides were obtained for both dextrans upon endo-dextranase digestion, while some differences in the polysaccharide fine structures could be identified from the varying portions of certain oligosaccharides. Dextrans synthesized by the dextransucrase released by L. nagelii exhibited an averaged molecular weight (Mw) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an Mw of 6.1×107 Da. Moreover, glycosylation of glucansucrases by LAB was identified for the first time for the released dextransucrase of L. nagelii TMW 1.1827. Our study therefore reveals new molecular insights into how dextransucrases released by water kefir borne L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 contribute to the complex formation of the traditional beverage water kefir.

Detailed Structural Characterization of Glucans Produced by Glucansucrases from Leuconostoc citreum TMW 2.1194.

The water kefir organism Leuconostoc citreum TMW 2.1194 forms highly branched dextrans with O3- and O4-bound side chains. To obtain detailed information on the enzymatic synthesis of these polymers, the four glucansucrases encoded by Leuconostoc citreum TMW 2.1194 were cloned, heterologously expressed, and used for polysaccharide production. Molecular and macromolecular structure of the synthesized glucans were analyzed by methylation analysis, two-dimensional NMR spectroscopy, oligosaccharide analysis after partial hydrolysis, and asymmetric flow field-flow fractionation. It was demonstrated that two glucansucrases form insoluble glucans with variously branched dextran sections and varying portions of consecutive, 1,3-linked glucose units. In contrast, the other two glucansucrases synthesized O3- (Lc6255) and O4-branched (Lc1785) soluble dextrans. Analysis, isolation, and characterization of enzymatically liberated oligosaccharides showed that monomeric and elongated side chains are abundant in both polysaccharides. From the structures and size distributions it was concluded that Lc1785 is mainly responsible for synthesis of fermentatively produced soluble dextrans.

Proteomic Analysis of Lactobacillus nagelii in the Presence of Saccharomyces cerevisiae Isolated From Water Kefir and Comparison With Lactobacillus hordei.

Water kefir is a slightly alcoholic and traditionally fermented beverage, which is prepared from sucrose, water, kefir grains, and dried or fresh fruits (e.g., figs). Lactobacillus (L.) nagelii, L. hordei, and Saccharomyces (S.) cerevisiae are predominant and stable lactic acid bacteria and yeasts, respectively, isolated from water kefir consortia. The growth of L. nagelii and L. hordei are improved in the presence of S. cerevisiae. In this work we demonstrate that quantitative comparative proteomics enables the investigation of interactions between LAB and yeast to predict real-time metabolic exchange in water kefir. It revealed 73 differentially expressed (DE) in L. nagelii TMW 1.1827 in the presence of S. cerevisiae. The presence of the yeast induced changes in the changes in the carbohydrate metabolism of L. nagelii and affected reactions involved in NAD+/NADH homeostasis. Furthermore, the DE enzymes involved in amino acid biosynthesis or catabolism predict that S. cerevisiae releases glutamine, histidine, methionine, and arginine, which are subsequently used by L. nagelii to ensure its survival in the water kefir consortium. In co-culture with S. cerevisiae, L. nagelii profits from riboflavin, most likely secreted by the yeast. The reaction of L. nagelii to the presence of S. cerevisiae differs from that one of the previously studied L. hordei, which displays 233 differentially expressed proteins, changes in citrate metabolism and an antidromic strategy for NAD+/NADH homeostasis. So far, aggregation promotion factors, i.e., formation of a specific glucan and bifunctional enzymes were only detected in L. hordei.

Label-free quantitative proteomic analysis reveals the lifestyle of Lactobacillus hordei in the presence of Sacchromyces cerevisiae.

Water kefir is a fermented beverage, which is traditionally prepared from sucrose, kefir grains, dried or fresh fruits, and water. L. hordei and S. cerevisiae are isolated as predominant and stable species of lactic acid bacteria and yeasts, respectively. In this study we demonstrate that label free quantitative proteomics is useful to study microbial interaction along the response of co-cultivated L. hordei TMW 1.1822 in the presence of S. cerevisiae TMW 3.221 as compared with their single cultures in a water kefir model. It is shown and L. hordei responds to S. cerevisiae in many respects revealing a mutualistic relationship. The data suggest that L. hordei responds to the presence of S. cerevisiae with adjustment of intracellular redox reactions controlled of proteins, which are part of Rex regulons and proteins involved in the glycolytic pathway and energy fermentation. An NADH, H+-driven metabolic switch to preferential production of butanediol instead of acetate or lactate, and up-regulation of arginine deiminase, alleviated acid stress and concomitantly protected S. cerevisiae against an acidic environment, which L. hordei generated in single culture. Moreover, the data suggest that the presence of S. cerevisiae in the nitrogen and fatty acids limited environment of the water kefir facilitated and improved the growth of L. hordei by delivering gluconate, fructose, amino acids, fatty acids or substrates for their biosynthesis. Up-regulation of the OppABCDF peptide transport and enzymes involved in amino acid metabolism indicates enhanced peptide uptake, as well as cross-feeding of L. hordei by glutamine, glutamate, histidine, tryptophan, methionine, proline, tryptophan delivered by S. cerevisiae.

Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic, proteomic and physiological characterization.

Water kefir is a traditional fermented beverage made from sucrose, water, kefir granules, dried or fresh fruits. In our water kefir granules, Lactobacillus (L.) hordei is one of the predominant lactic acid bacteria (LAB) species of this presumed symbiotic consortium. It faces abundant sucrose versus limitation of amino- and fatty acids in an acidic environment. Sequencing of the genome of L. hordei TMW 1.1822 revealed one chromosome plus three plasmids. The size of the chromosome was 2.42 Mbp with a GC content of 35% GC and 2461 predicted coding sequences. Furthermore, we identified 1474 proteins upon growth on water kefir medium. Metabolic prediction revealed all enzymes required for the glycolytic Embden-Meyerhof (EMP) and phosphoketolase (PKP) pathways. Genes encoding all enzymes involved in citrate, pyruvate and mannitol metabolism are present. Moreover, it was confirmed that L. hordei is prototrophic for 11 amino acids and auxotrophic for 6 amino acids when combining putative biosynthesis pathways for amino acids with physiological characterization. Still, for glycine, serine and methionine no sure auxotype could be determined. The OppABCDF peptide transport system is complete, and 13 genes encoding peptidases are present. The arginine deiminase system, was predicted to be complete except for carbamate kinase, thus enabling neutralization reactions via ammonium formation but no additional energy generation. Taken together our findings enable prediction of the L. hordei lifestyle in water kefir: Abundant sucrose is consumed directly via parallel EMP and PK pathways and is also extracellularly converted to dextran and fructose by a glucansucrase, leaving fructose as additional carbon source. Essential amino acids (in the form of peptides) and citrate are acquired from fruits. In the lack of FabB unsaturated fatty acids are synthesized by predicted alternative enzymes. Formation of acetoin and diacetyl as well as arginine conversion reactions enable acidification limitation. Other members of the water kefir consortium (yeasts, acetic acid bacteria) likely facilitate or support growth of L. hordei by delivering gluconate, mannitol, amino- and fatty acids and vitamins.

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry.