The growing demand for non-alcoholic beers (NAB) requires innovative strategies to enhance diversity and overcome common shortcomings such as excessive sweetness, wort-like flavour, and poor mouthfeel. This study assessed pilot-scale wort production for fermentation with maltose-negative yeasts, with 40% substitution of barley malt by alternative cereals and pseudocereals (rye malt, oat flakes, buckwheat, khorasan, einkorn). Adjunct use reduced wort maltose (26.89 g/L to 22.11-24.84 g/L) and slightly increased oligosaccharides compared with the reference wort from 100% barley malt. Buckwheat wort showed 90% higher glucose, while oat flakes and khorasan lowered glucose by 14-21%. Unmalted adjuncts increased soluble high-molecular-weight proteins by 36-67% and decreased amino acids by 30-37%. Rye malt raised viscosity by 56%, likely improving mouthfeel but reducing processability. Off-flavour aldehydes and dimethyl sulphide remained low. Overall, these results demonstrate the feasibility of adjunct incorporation and highlight their potential to diversify NAB styles with reduced sweetness and enhanced mouthfeel.

Although recent research focuses on the search for maltose- and/or maltotriose-negative yeasts to produce non- and low-alcohol beers (NABLAB), an in-depth evaluation of commercial options is currently lacking. Therefore, this study aimed to compare the physicochemical and sensorial characteristics of NABLAB made with nine different commercial yeasts. Overall, NABLAB were successfully made with SafBrew™LA-01, SMARTBEV™ NEER®, NEER®Poly, and NEER®Punch, WLP603, LalBrew®London, and LalBrew®Windsor. Pichia kluyveri strains NEER®, NEER®Poly, and NEER®Punch assimilated little FAN, and produced a lot of esters, yielding fruity/sweetish beers. SafBrew™LA-01 was characterized by phenolic flavors. There was a strong reduction of wort aldehydes by these yeasts. Still, worty flavors were noticeable, especially in the WLP603 beers. The maltotriose-negative Saccharomyces cerevisiae strains LalBrew®London™ and LalBrew®Windsor™ produced more higher alcohols and these beers were perceived as more bitter due to less residual sugars. This research facilitates informed decision-making for industrial stakeholders or academics in yeast selection for NABLAB production.

This study aimed to evaluate the effects of citrus pectins obtained from lemon (LPP), orange (OPP), and bigarade (BPP) peels, at concentrations of 0.1 % and 0.5 %, on the fermentation and quality of fat-free set yogurt. The addition of pectin significantly accelerated fermentation. Notably, incorporating 0.5 % BPP reduced fermentation time from 12 to 4 h, attributed to rapid lactose breakdown (4.83 mg·mL-1·h-1), increased lactic acid production (3.12 mg·mL-1·h-1), and enhanced proliferation of lactic acid bacteria. Regarding yogurt quality, pectin addition markedly improved textural properties and reduced syneresis, particularly at higher concentrations. Yogurts enriched with 0.5 % BPP and OPP showed the lowest syneresis ratio (2.06 %) and wheying-off (0.62 %), respectively, along with increased firmness, consistency, and adhesiveness. Cryo-SEM imaging confirmed improved structural stability during storage. These findings suggest that citrus pectins, especially BPP, are promising natural additives for enhancing the fat-free set yogurt fermentation process, texture, and shelf life.

Strain LMG 33000T was isolated from a Bombus lapidarius gut sample. It shared the highest percentage 16S rRNA sequence identity, average amino acid identity, and amino acid identity of conserved genes with Convivina intestini LMG 28291T (95.86 %, 69.9 and 76.2 %, respectively), and the highest percentage OrthoANIu value with Fructobacillus fructosus DSM 20349T (71.4 %). Phylogenomic analyses by means of 107 or 120 conserved genes consistently revealed Convivina as nearest neighbour genus. The draft genome of strain LMG 33000T was 1.44 Mbp in size and had a DNA G+C content of 46.1 mol%. Genomic and physiological analyses revealed that strain LMG 33000T was a typical obligately fructophilic lactic acid bacterium that lacked the adhE and aldh genes and that did not produce ethanol during glucose or fructose metabolism. In contrast, Convivina species have the adhE and aldh genes in their genomes and produced ethanol from glucose and fructose metabolism, which is typical for heterofermentative lactic acid bacteria. Moreover, strain LMG 33000T exhibited catalase activity, an unusual characteristic among lactic acid bacteria, that is not shared with Convivina species. Given its position in the phylogenomic trees, and the difference in genomic percentage G+C content and in physiological and metabolic characteristics between strain LMG 33000T and Convivina species, we considered it most appropriate to classify strain LMG 33000T into a novel genus and species within the Lactobacillaceae family for which we propose the name Eupransor demetentiae gen. nov., sp. nov., with LMG 33000T (=CECT 30958T) as the type strain.

The increase in studies on bee microbiomes is prompted by concerns over global pollinator declines. Bumble bees host core and non-core microbiota which may contribute to increased lifetime fitness. The presence of Fructobacillus in the gut microbiomes of bumble bee workers, or the replacement of core symbionts with Fructobacillus bacteria, has been considered a marker of dysbiosis. A phylogenomic analysis and functional genomic characterization of the genomes of 21 Fructobacillus isolates from bumble bees demonstrated that they represented four species, i.e. Fructobacillus cardui, Fructobacillus fructosus, Fructobacillus tropaeoli, and the novel species Fructobacillus evanidus sp. Nov. Our results confirmed and substantiated the presence of two phylogenetically and functionally distinct Fructobacillus species clades that differ in genome size, percentage G + C content, the number of coding DNA sequences and metabolic characteristics. Clade 1 and clade 2 species differed in amino acid and, to a lesser extent, in carbohydrate metabolism, with F. evanidus and F. tropaeoli genomes featuring a higher number of complete metabolic pathways. While Fructobacillus genomes encoded genes that allow adhesion, biofilm formation, antibacterial activity and detoxification, other bacteria isolated from the bumble bee gut appeared better equipped to co-exist with the bumble bee host. The isolation and identification of multiple Fructobacillus species from several bumble bee gut samples in the present study also argued against a specific partnership between Fructobacillus species and their bumble bee hosts.

We provide a culturomics analysis of the cultivable bacterial communities of the crop, midgut and hindgut compartments, as well as the ovaries, of the invasive insect Vespa velutina, along with a cultivation-independent analysis of samples of the same nest through 16S rRNA amplicon sequencing. The Vespa velutina bacterial symbiont community was dominated by the genera Convivina, Fructobacillus, Lactiplantibacillus, Lactococcus, Sphingomonas and Spiroplasma. Lactococcus lactis and Lactiplantibacillus plantarum represented generalist core lactic acid bacteria (LAB) symbionts, while Convivina species and Fructobacillus fructosus represented highly specialised core LAB symbionts with strongly reduced genome sizes. Sphingomonas and Spiroplasma were the only non-LAB core symbionts but were not isolated. Convivina bacteria were particularly enriched in the hornet crop and included Convivina intestini, a species adapted towards amino acid metabolism, and Convivina praedatoris sp. nov. which was adapted towards carbohydrate metabolism.

Consumers are more than ever in search of novel and exciting beer choices, and brewers are, therefore, continuously experimenting to adapt their product portfolio. One interesting way to naturally incorporate novel flavors and tastes is by using alternative adjuncts, but this is not always an easy and straightforward process. In this study, a 40% unmalted alternative adjunct (einkorn, emmer, spelt, khorasan, quinoa, amaranth, buckwheat, sorghum, teff, and tritordeum) or reference (barley malt, unmalted barley, and unmalted wheat) was added to 60% barley malt, after which three different laboratory mashing processes (Congress mash, Congress mash with pre-gelatinization of the adjunct, and Evans mash) were performed, and their behavior during mashing and the resulting wort characteristics were investigated in detail. Overall, the extraction process of all 10 unmalted alternative adjuncts was not complete for all three laboratory mashing processes, whereby Congress mashing resulted in the highest extract and fastest filtration, whereas Evans mashing resulted in the lowest extract and slowest filtration. Pre-gelatinization of the unmalted was generally only beneficial for adjuncts with high onset starch gelatinization temperatures. This process also inactivated endogenous enzymes in the unmalted adjuncts, which had an adverse effect on the mashing process.

Wort was prepared according to the Congress mash protocol with the addition of 40% unmalted barley, tritordeum, or quinoa. Mashes with quinoa filtered significantly slower than mashes with barley. In contrast, tritordeum filtered similarly as barley, even though tritordeum does not possess a husk. The lack of husk may have contributed to a higher extract yield of tritordeum compared to barley. The final 65 degrees C mash protocol resulted in shorter saccharification times, but slower wort filtration compared to the Congress mash protocol. When unmalted adjuncts were pregelatinized by heating in water for 20min at 95 degrees C, the filtration following the Congress mash protocol was slower and the filtration following the final 65 degrees C mash protocol was faster than the same mashes prepared without pregelatinization. Pregelatinization also inactivated the endogenous enzymes in the (pseudo)cereals used. This was especially noticeable for quinoa, resulting in wort with markedly lower glucose concentrations, most likely due to the inactivation of endogenous amyloglucosidases. Wort filtration improved for both mashing protocols when an exogenous enzyme mix (Brewers Compass (R)) was added to each of the three different (pseudo)cereals. Furthermore, addition of the enzyme mix increased the extract yield of the final 65 degrees C mashes and the FAN levels of the Congress mashes. These findings confirmed the benefit of using the Brewers Compass (R) enzyme mix during mashing processes with high percentages of (up to 40%) unmalted (pseudo)cereals.

Acetic acid bacteria (family Acetobacteraceae) are found in the gut of most insects. Two clades are currently recognized: Commensalibacter-Entomobacter and Bombella-Oecophyllibacter. The latter group is only found in hymenopteran insects and the described species have been isolated from bees and ants. In this study, two new strains DDB2-T1T (=KACC 21507T=LMG 31759T) and DM15PD (=CCM 9165=DSM 112731=KACC 22353=LMG 32454) were isolated from wasps collected in the Republic of Korea and Germany, respectively. Molecular and phenotypic analysis revealed that the strains are closely related, with 16S rRNA gene sequences showing 100 % identity and genomic average nucleotide identity (ANI) values ≥99 %. The closest related species based on type strain 16S rRNA gene sequences are Swingsia samuiensis, Acetobacter peroxydans, Bombella favorum and Bombella intestini (94.8-94.7% identity), whereas the closest related species based on type strain genome analysis are Saccharibacter floricola and Bombella intestini (ANI values of 68.8 and 68.2 %, respectively). The reconstruction of a phylogenomic tree based on 107 core proteins revealed that the branch leading to DDB2-T1T and DM15PD is localized between Oecophyllibacter and Saccharibacter-Bombella. Further genomic distance metrics such as ANI, percentage of conserved proteins and alignment fraction values were consistent with these strains belonging to a new genus. The key phenotypic characteristics were one MALDI-TOF-MS peak (m/z=4601.9±2.0) and the ability to produce acid from d-arabinose. Based on this polyphasic approach, including phylogenetics, phylogenomics, genome distance calculations, ecology and phenotypic characteristics, we propose to name the novel strains Aristophania vespae gen. nov., sp. nov., with the type strain DDB2-T1T (=KACC 21507T=LMG 31759T).

Strain C17-3T was isolated from blueberry fruits collected from a farmland located in Damyang-gun, Jeollanam-do, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain C17-3T to the genus Acetobacter, where it occupied a rather isolated line of descent with Acetobacter ghanensis 430AT and Acetobacter lambici LMG 27439T as the nearest neighbours (98.9 % sequence similarity to both species). The highest average nucleotide identity and digital DNA-DNA hybridization values were 76.3 % and 21.7 % with Acetobacter garciniae TBRC 12339T; both values were well below the cutoff values for species delineation. Cells are strictly aerobic, Gram-stain-negative rods, catalase-positive and oxidase-negative. The DNA G+C content calculated from the genome sequence was 59.2 %. Major fatty acids were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C19 : 0cyclo ω8c. The major isoprenoid quinone was ubiquinone 9. On the basis of the results of phylogenetic analyses, phenotypic features and genomic comparisons, it is proposed that strain C17-3T represents a novel species of the genus Acetobacter and the name Acetobacter vaccinii sp. nov. is proposed. The type strain is C17-3T (= KACC 21233T = LMG 31758T).

To meet consumer demand and remain competitive, brewers attempt to broaden their product range with new, innovative flavors. One way to achieve this is by (partially) replacing barley malt with less common adjuncts, such as the ancient wheat varieties einkorn, emmer, spelt, and khorasan; the pseudocereals quinoa, amaranth, and buckwheat; or the alternative cereals sorghum, teff, and tritordeum. The physical (grain size and thousand kernel weight), chemical (starch, protein, fat, beta-glucan, and water content), and physicochemical (gelatinization temperature, amylase content) properties of these alternative cereals and pseudocereals were determined, as well as their aldehyde concentrations and flavor profiles. The starch content ranged from 45.2 +/- 1.2 %dm for einkorn to 75.5 +/- 0.6 %dm for teff. The protein content ranged from 10.6 +/- 0.0 %dm for barley malt to 18.4 +/- 0.6 %dm for khorasan. Major differences were found for the diastatic power, with quinoa having the lowest (6 +/- 4 degrees WK) and spelt the highest (277 +/- 19 degrees WK) value. Most alternative cereals and pseudocereals had an onset gelatinization temperature <63 degrees C, making them suitable for common brewing practices. This was not the case for quinoa, amaranth, teff, and sorghum, which had a gelatinization temperature >= 64 degrees C. Aldehyde concentrations were significantly lower in alternative cereals and pseudocereals, compared to malted barley.

For eight backslopping steps, eight series of water kefir fermentation processes differing in backslopping time and rinsing of the grains during each backslopping step and eight series of fermentation processes differing in incubation temperature and backslopping time were followed. Short backslopping times resulted in high relative abundances of Liquorilactobacillus nagelii and Saccharomyces cerevisiae , intermediate backslopping times in high relative abundances of Leuconostoc pseudomesenteroides , and long backslopping times in high relative abundances of Oenococcus sicerae and Dekkera bruxellensis . When the grains were rinsed during each backslopping step, the relative abundances of Lentilactobacillus hilgardii and Leuc . pseudomesenteroides increased and those of D . bruxellensis and Liql . nagelii decreased. Furthermore, rinsing of the grains during each backslopping step resulted in a slightly higher water kefir grain growth and lower metabolite concentrations. The relative abundances of Liquorilactobacillus mali were highest at 17°C, those of Leuc . pseudomesenteroides at 21 and 25°C, and those of Liql . nagelii at 29°C. With a kinetic modeling approach, the impact of the temperature and rinsing of the grains during the backslopping step on the volumetric production rates of the metabolites was determined.

Eleven series of water kefir fermentation processes differing in the presence of oxygen and the type and concentration of inoculum and substrate, were followed as a function of time to quantify the impact of these parameters on the kinetics of this process via a modeling approach. Increasing concentrations of the water kefir grain inoculum increased the water kefir fermentation rate, so that the metabolic activity during water kefir fermentation was mainly associated with the grains. Water kefir liquor could also be used as an alternative means of inoculation, but the resulting fermentation process progressed slower than the one inoculated with water kefir grains, and the production of water kefir grain mass was absent. Substitution of sucrose with glucose and/or fructose reduced the water kefir grain growth, whereby glucose was fermented faster than fructose. Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei), Lentilactobacillus hilgardii (formerly known as Lactobacillus hilgardii), Liquorilactobacillus nagelii (formerly known as Lactobacillus nagelii), Saccharomyces cerevisiae, and Dekkera bruxellensis were the main microorganisms present. Acetic acid bacteria were present in low abundances under anaerobic conditions and only proliferated under aerobic conditions. Visualization of the water kefir grains through scanning electron microscopy revealed that the majority of the microorganisms was attached onto their surface. Lactic acid bacteria and yeasts were predominantly associated with the grains, whereas acetic acid bacteria were predominantly associated with the liquor.

Kombucha tea is made by aerobically fermenting a sweetened tea infusion with a kombucha culture, a symbiotic culture of bacteria and yeasts. The resulting beverage is usually non-alcoholic, sour and refreshing, but not naturally sparkling. Many consumers are drinking kombucha because of its alleged health effects and as kombucha tea is increasing in popularity, consumers are increasingly demanding kombucha beverages of better quality. To better adapt their products to these consumer demands, brewers are looking to the scientific knowledge about kombucha for answers. However, the scientific exploration of this complex naturally fermented beverage is still limited. This review paper presents the current state-of-the-art. To further deepen the understanding of the kombucha fermentation process and to help brewers in their search for higher-quality beverages, the microbial species diversity and its dynamics during the fermentation process should be investigated in detail, as well as the kinetics of the substrate consumption and metabolite production, and the relationship between the substrate and metabolite concentrations, and the consumer preferences for the resulting beverage.

Eight water kefir fermentation series differing in buffer capacity and calcium concentration of the water used for fermentation were studied during eight backslopping steps. High buffer capacities resulted in high pH values and high calcium concentrations resulted in low pH values at the end of each backslopping step. When the water buffer capacity and/or calcium concentration were below certain minima, the water kefir grain growth decreased gradually over multiple backsloppings. High water buffer capacities resulted in high concentrations of residual total carbohydrate concentrations and low metabolite concentrations. Further, high water buffer capacities resulted in high ratios of lactic acid bacteria to yeasts, which was reflected in high molar ratios of the concentrations of lactic acid to ethanol and acetic acid to ethanol. The most prevalent microorganisms of the water kefir grain inoculum and grains of all fermentation series at the end of the eighth backslopping step were Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus paracasei, Bifidobacterium aquikefiri, Saccharomyces cerevisiae, and Dekkera bruxellensis. These microbial communities were influenced by the water buffer capacity and had an impact on the substrate consumption and metabolite production during water kefir fermentation.

Eight water kefir fermentation series differing in the presence of oxygen, the nutrient concentration, and the nutrient source were studied during eight consecutive backslopping steps. The presence of oxygen allowed the proliferation of acetic acid bacteria, resulting in high concentrations of acetic acid, and decreased the relative abundance of Bifidobacterium aquikefiri. Low nutrient concentrations resulted in slow water kefir fermentation and high pH values, which allowed the growth of Comamonas testosteroni/thiooxydans. Further, low nutrient concentrations favored the growth of Lactobacillus hilgardii and Dekkera bruxellensis, whereas high nutrient concentrations favored the growth of Lactobacillus nagelii and Saccharomyces cerevisiae. Dried figs, dried apricots, and raisins resulted in stable water kefir fermentation. Water kefir fermentation with dried apricots resulted in the highest pH and water kefir grain growth, whereas that with raisins resulted in the lowest pH and water kefir grain growth. Further, water kefir fermentation with raisins resembled fermentations with low nutrient concentrations, that with dried apricots resembled fermentations with normal nutrient concentrations, and that with fresh figs or a mixture of yeast extract and peptone resembled fermentations with high nutrient concentrations.

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

Aims To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process.Methods and Results Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these micro-organisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii and Saccharomyces cerevisiae were always present and may be the key micro-organisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of micro-organisms, fast fermentation and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria.Conclusions The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key micro-organisms was always present during fermentation.Significance and Impact of the Study This study allows a rational selection of a water kefir grain inoculum.