Fermentation, Volume 9, Issue 5 (May 2023) – 90 articles

This study shows the impact of three different commercial inactive yeast derivatives (IYDs) (Opti Less™, Noblesse™, Optimum White™, Lallemand, Canada and Oenolees MP™ Lafort, USA) during the 6-month aging period on the volatile profile, sensory attributes and antiradical activity, including polyphenols and the total free sulfhydryl (-SH groups) content, of Grašac wines made in sequential fermentation with native Hanseniaspora uvarum S-2 and Saccharomyces cerevisiae QA23. The addition of IYDs helped in maintaining the constant values of antiradical activity during aging by increasing polyphenolic values and mitigating the decrease in -SH groups. HS-SPME-GC-MS analysis showed that esters were the major volatile compounds, with ethyl-acetate and 2-phenyl-ethyl-acetate being the most abundant among all the samples, followed by ethyl-dodecaonate, ethyl-decanoate and 3-methyl-butyl-octanoate, all of them contributing to fruity and floral aromas in wine. As the concentration of IYDs increased, a corresponding rise in the levels of certain volatiles, such as 2-methyl-1-propanol, phenyl-ethyl-alcohol and ethyl-octanoate, was observed. Sensory analysis showed that the addition of IYDs generally improved the taste and odor profile of the wine by reducing astringency and increasing fullness and complexity, regardless of the IYD type. The results demonstrated that different IYDs may have varying effects on wine, with each product having its specific purposes, providing the tools for winemakers to carefully regulate and obtain the desired sensory profile of the wine. Full article

The content of 2,3-butanediol ((R,R) and meso isomers) and 1,2-propanediol in grape berries and “liquid samples” (all non-berry extracts) from the Tokaj wine region of Hungary was investigated. Our aim was to find out how the activity of Botrytis cinerea influences the concentrations of these compounds compared with healthy grapes. Based on the measured concentrations, we can make a distinction between healthy berries and noble, rotted, so-called aszú berries. We also investigated if there is a difference between finished aszú wines and liquids intended for aszú production. We wanted to investigate the amount and distribution of the stereoisomers of 2,3-butanediol and their proportions. The results of the HS-SPME-GC-FID analysis of the samples showed significant differences in the 2,3-butanediol content between healthy and botrytised, aszú berries and between liquid samples for aszú production and aszú wines. In the berry samples, meso-2,3-butanediol could not be detected, whereas in the liquid samples, we found good amounts of this isomer. This may be due to the fact that the appearance of the meso form of 2,3-butanediol is a consequence of alcoholic fermentation. Significant differences were found between wines from healthy grapes and wines from botrytised grapes in terms of the levo-2,3-butanediol content, so that from an analytical point of view, a difference can be made between wines from healthy and botrytised grapes. No significant differences were found between berry and liquid samples in terms of 1,2-propanediol concentrations during our tests. Full article

In this study, the effect of solid-state fermentation with Bacillus subtilis GYB6, Saccharomyces cerevisiae NJ1, and Bacillus amyloliquefaciens Y8 on the anti-nutritional factors, nutritional components, bioactive compounds, antioxidant activity, functional properties, and structure of rapeseed meal (RSM) were investigated. Results showed that the action of three strains in the fermentation of RSM caused a significant decline in glucosinolates, phytic acid, crude fiber, and tannins by 99.18%, 42.41%, 27.21%, and 34.17%, respectively. The amount of crude protein, amino acids, and peptides of RSM increased significantly after fermentation. The SDS-PAGE results showed that 12S globulin and 2S albumin protein were almost entirely degraded. Fermentation considerably increased the concentration of total phenolics and flavonoids, and activated antioxidant activity and functional properties. Furthermore, the structural variation was observed by scanning electron microscopy and FTIR spectroscopy. Thus, these results indicated that the solid-state fermentation process in this study was a promising approach to enhance both the nutritional value and bioactivity of RSM, which could be used as value-added functional animal food ingredients. Full article

Green fractionation and a comprehensive overview of lignin molecular structures during the DES (deep eutectic solvent) pretreatment are very important for lignin valorization and the whole biorefinery process. Herein, intractable woody biomass (poplar wood) was pretreated with five types of carboxylic-acid-based DESs (acetamide served as an HBA (hydrogen bond acceptor), propanedioic acid, tartaric acid, malic acid, glutaric acid, and succinic acid served as HBDs (hydrogen bond donors)) under the optimized pretreatment conditions. Results showed that the optimal delignification ratio was achieved for tartaric-acid-based DES at 140 °C for 20 min under microwave-assisted heating. Two-dimensional HSQC NMR data demonstrated that the isolated poplar DES lignin consisted mainly of β-β, β-O-4 (normal and acylated forms), β-5, and esterified p-hydroxybenzoates (PBs) in different contents. Especially, the contents of β-O-4 in the isolated DES lignin fractions varied based on the pretreatment temperature and different chemical compositions of the DES. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH analysis), microstructure (scanning electron microscope, SEM), and molecular weights (gel permeation chromatography, GPC) of the DES lignin fractions demonstrated that the DES delignification promoted the rapid assembly of lignin nanoparticles (LNPs) and could yield homogeneous lignin (1.23 < PDI < 1.58) with controlled nanometer size (30–170 nm) and good antioxidant activity. This study will improve the knowledge of structural changes of lignin during the different carboxylic-acid-based DES pretreatments and maximize the lignin valorization. Full article

One of the earliest biotechnological processes is brewing, which uses conventional raw materials like barley malt and, to a lesser extent, wheat malt. Today, adjuncts are used in the brewing of 85–90% of the world’s beer, with significant regional differences. The results of this study’s brewing were compared to those of beer made only from malted barley. Malted and unmalted sorghum were suggested for use in this study’s brewing. In order to improve the technical mashing operation and raise output yield, commercial enzymes were introduced. The following physicochemical analyses of the finished beer were carried out in accordance with regulatory requirements: original extract (% m/m), apparent extract (% m/m), alcohol content (% v/v, % m/m), density (g/cm³), turbidity (EBC), pH, color (EBC), bitterness value (IBU), oxygen content (mg/L), carbon dioxide content (g/L). A nine-point hedonic scale was used to conduct the sensory evaluation of the beer samples. Sorghum was easily included into the technological process to create a finished product that, in many ways, resembled traditional beer, making sorghum appropriate for typical beer drinkers. The laboratory brewing formula that produced the highest-quality results of all the tested variants included 60% sorghum malt and 40% unmalted sorghum: original extract 11.26% m/m, apparent extract 4.59% m/m, alcohol content 4.12% v/v, turbidity 0.74 EBC, CO₂ content 5.10 g/L. The resulting sorghum beer typically has low alcohol content, a complex, aromatic, slightly sour flavor, a mild bitter or astringent sensation, and less stable foam. Full article

In the present study, a wild-type Lacticaseibacillus paracasei SP5 (L. paracasei SP5) potential probiotic strain (previously isolated from kefir grains) was applied for the 1-day fermentation of an apple–orange–carrot mixed juice. After the fermentation, the mixed juice was refrigerated in cold storage at 4 °C, and the microbiological stability, characterization of volatiles, physicochemical properties (pH, total titratable acidity (TTA), residual sugar content and organic acids content), the sensorial validation (aroma, taste and overall acceptability) of the juice, and the viability of the potential probiotic strain were analyzed. The stored juice exhibited zero colonies of yeasts/fungi and simultaneously the viability of L. paracasei SP5 was retained to 8.28 Log CFU/mL, even after the 4th week of cold storage. The pH values ranged from 3.80 to 3.35 and the TTA values ranged from 0.1344% to 0.1844% lactic acid for the unfermented juice up until the 4th week of cold storage. Furthermore, the organic acids content consisted mostly of lactic acid (4.6 to 9.1 g/L), while malic acid (3.7 to 1.5 g/L), acetic acid (0.6 g/L) and propionic acid (0.3 g/L) were detected only after the 4th week of cold storage. Residual sugar content ranged from the initial value of 122.2 g/L and 106.6 g/L at the end of cold storage. As far as the volatiles’ characterization is concerned: 9 esters, 2 organic acids, 12 alcohols, 3 aldehydes, 1 ketone, 6 terpenes and 4 sesquiterpenes (37 in total) were detected in the unfermented mixed juice and 33 compounds in the fermented juice after 4 weeks of cold storage. The sensorial properties (aroma, taste and overall acceptability) of the fermented mixed juice samples were positively influenced. Consequently, L. paracasei SP5 potential probiotic strain may be applied for the production of probiotic mixed juices, with satisfying viability, volatile profile and organoleptic results. Full article

2,3-Butanediol, acetoin and tetramethylpyrazine have a wide range of applications as important chemicals in the chemical, food and pharmaceutical fields. Bacillus subtilis has a very wide application potential in many industries as a food-safe grade strain and has a good performance as a potential strain for tetramethylpyrazine production. In this study, we constructed a recombinant plasmid with dual promoter to overexpress 2,3-butanediol dehydrogenase and introduced it into Bacillus subtilis BS2. The constructed strain (BS-ppb11) was then optimized for fermentation conditions, and the maximum concentration of 2,3-butanediol produced was 96.5 g/L, which was 36.4% higher than that of the original strain, in which the (R,R)-2,3-butanediol had a chiral purity of 94.7%. Meanwhile, BS-ppb11 produced a maximum concentration of 82.2 g/L acetoin, which was 36.7% higher than that of the original strain. Subsequently, through optimization of metabolic conditions, BS-ppb11 produced 34.8 g/L of tetramethylpyrazine in staged batch replenishment fermentation, which was 95.5% higher than the original strain and was the highest ferritin production reported to date for Bacillus subtilis. In addition, we introduced a photocatalytic coenzyme regeneration system in BS-ppb11 to further improve the metabolic yield of 2,3-butanediol by regulating cofactor homeostasis, which laid the foundation for the subsequent in-depth study of the related mechanism. Full article

The generation of royalties on the use of foreign-made inoculum strains or starters as a result of the implementation of the ‘Nagoya Protocol’ has led to efforts and healthy competition to secure useful biological resources in each country. In this study, we recognized the necessity and importance of securing useful strains in Korea and produced starters using five strains of fungi (Aspergillus oryzae and Aspergillus niger). Specifically, the quality characteristics exhibited by different strain inoculum concentrations (1, 3, 5% (v/w)) and drying conditions (low-temperature air drying at 35 °C for 24 h and high-temperature hot air drying at 45 °C for 18 h) during the starter manufacturing process were analyzed. Parameters such as enzyme activity and free amino acid and volatile flavor compound content, which may vary based on production conditions, are expected to produce basic data for the production of Korean-type starters. Full article

A positive effect of flaxseed mucilage (FSM) addition (at concentrations of 0.1, 0.2, and 0.4%) to MRS and milk whey nutrient medium on the survival, auto-aggregation, hydrophobicity, adhesive, and antioxidant properties of L. bulgaricus, L. fermentum AG8, and L. plantarum AG9 was shown. It was found that the AG 8 strain became less sensitive to 7% NaCl concentrations (the cell survival rate in the experiment with 0.4% flaxseed mucilage increased by 10% compared to the control). Cultivation in the presence of FSM led to an increase in auto-aggregation, especially in the case of AG8 (from 60 to 85%) and AG9 (from 50 to 80%) strains, and an increase in hydrophobicity was seen: for L. fermentum AG8, it was from 30% to 62–72%, for L. fermentum AG9 from 30% to 35–42%, and for L. bulgaricus from 20% to 30%. The adhesive properties of the L. fermentum AG8 and L. plantarum AG9 cells increased from 0.472 to 1 nN (nanonewton) and from 0.630 to 2.5 nN, respectively. The presence of flaxseed mucilage increased the total phenolic content in cell-free supernatants after 48 h of cultivation. The concentration of 0.1–0.2% FSM increased the OH-scavenging activity of milk whey nutrient medium cell-free supernatants of strains AG8 and AG9 by 7–10%. Flaxseed mucilage can serve as a promising bioactive additive that elevates antioxidant activity, increases the resistance and survival of Lactobacillus cells in the gastrointestinal tract, and leads to the synthesis of lipase and α-glucosidase inhibitors. The co-culture of these lactic acid bacteria in the presence of FSM and milk components in the form of whey leads to the synthesis of lipase and α-glucosidase inhibitors more than the culturing on de Man, Rogosa, and Sharpe broth. Full article

Indigestible glucans (IGs) are dietary fibers that can promote human health via fermentation by the gut microbiota, where their physico-chemical properties play a crucial role. This effect remains to be fully explored. The aim of the current study was to comprehensively investigate and compare the fermentation characteristics of IGs with various structural properties, as well as their effects on the gut microbiota. Barley β-glucan (BG), laminarin (L), yeast β-glucan (BY), pachyman (PAC), resistant starch (R), and litesse (Lit) were anaerobically batch-fermented using the human fecal microbiota for 48 h. All the IGs were utilized by the gut microbiota at different rates, and 2% of L, 14% of BG, 23% of BY and PAC, and 35% of R and Lit were unfermented at the 48^th hour. During fermentation, mono-, di-, or trisaccharides were released from BG, L, and Lit, and the pH of broth was greatly lowered by IGs, especially BG and L, along with the production of short-chain fatty acids. Interestingly, PAC favored butyric acid production, while BG, L, and BY preferred propionic acid. Moreover, lactic acid, but not succinic acid, was detected in considerable amounts, but only with BG and L after 5 h. 16S rDNA analysis showed different microbial structures and the selective promotion of bacteria with different IGs, while only PAC did not decrease microbial α-diversity. Further qPCR analysis confirmed that BG was more potent at proliferating Faecalibacterium prausnitzii; BY preferred total bacteria, Prevotella, and Lactobacillus; and R favored Bifidobacterium. The IG-induced changes in the gut microbiota were strongly correlated with carboxylic acid production. In conclusion, the six IGs differed in fermentation characteristics and gut microbiota regulation capacity, and each one could have specific applications in human health promotion. Full article

Low fertilizer quality and remediation ability are considered the major factors hampering the land application of anaerobic digestate. Therefore, the role of Myrothecium verrucaria treatment in enhancing the fertilizer quality and remediation ability of digestate for land application was explored. Higher content of humic acid (7.5 g/L) with a higher degree of humification index and oxygen-containing functional groups was observed in the digestate receiving Myrothecium verrucaria treatment. Likewise, humic acid formed from Myrothecium verrucaria treatment had a higher capacity of heavy metal binding. Moreover, the viable and culturable count of S. faecalis, S. typhi, C. perfringens, and E. coli pathogens in the digestate decreased to approximately 12.50%, 41.70%, 18.87%, and 50.00% and 25.97%, 64.44%, 37.51%, and 75.27%, respectively, after treatment with Myrothecium verrucaria. This study provided a novel strategy to enhance the fertilizer quality, remediation ability, and biological safety of anaerobic digestate for land application. Full article

Saccharomyces cerevisiae is the most important industrial microorganism used to fuel ethanol production worldwide. Herein, we obtained a mutant S. cerevisiae strain with improved capacity for ethanol fermentation, from 13.72% (v/v for the wild-type strain) to 16.13% (v/v for the mutant strain), and analyzed its genomic structure and gene expression changes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that the changed genes were mainly enriched in the pathways of carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and lipid metabolism. The gene expression trends of the two strains were recorded during fermentation to create a timeline. Venn diagram analysis revealed exclusive genes in the mutant strain. KEGG enrichment of these genes showed upregulation of genes involved in sugar metabolism, mitogen-activated protein kinase pathway, fatty acid and amino acid degradation, and downregulation of genes involved in oxidative phosphorylation, ribosome, fatty acid and amino acid biogenesis. Protein interaction analysis of these genes showed that glucose-6-phosphate isomerase 1, signal peptidase complex subunit 3, 6-phosphofructokinase 2, and trifunctional aldehyde reductase were the major hub genes in the network, linking pathways together. These findings provide new insights into the adaptive metabolism of S. cerevisiae for ethanol production and a framework for the construction of engineered strains of S. cerevisiae with excellent ethanol fermentation capacity. Full article

Bacillus pumilus is widely used as a biocontrol agent. To further develop the biological control potential of B. pumilus LYMC-3 against pine blight, a statistical experimental design was used to optimize a liquid medium using low-cost substrates to improve its antagonistic activity against Sphaeropsis sapinea. Through the plate antagonism test and greenhouse control effect test, this study determines the antifungal effect of strain LYMC-3 against S. sapinea and pine blight. Moreover, response surface optimization methodologies were used to systematically optimize the medium composition and culture conditions of the LYMC-3 strain. The plate antagonism test showed that the inhibition rate of LYMC-3 fermentation filtrate (diluted 5-fold) was 66.09%. The greenhouse control effect test showed that the control effect of its fermentation filtrate on shoot blight reached 89.99%. The response surface optimization test ultimately determined that a higher inhibition rate can be achieved under these conditions: the optimal medium components were 7.2 g/L glucose, 15 g/L peptone, and 7.1 g/L magnesium sulfate; the optimal culture conditions were 52% liquid volume, 28 °C culture temperature, an initial pH of 7, and 1% bacterial inoculation volume. Under the optimized system, the five-fold diluted LYMC-3 fermentation filtrate inhibition rate against S. sapinea was 81.23%, which was 15.84% higher than that before optimization. Meanwhile, optimize the selection of lower-cost and more commonly used glucose instead of beef paste as the carbon source for the culture medium, and choose cheaper magnesium sulfate instead of sodium chloride as the nitrogen source. Full article

When plant biomass is anaerobically digested, seeds may survive the energy production process and contaminate the digestate. Hard-seeded (HS), i.e., physically dormant, species were found to be difficult to inactivate. Here, we aimed to verify this finding from lab-scale experimental reactors (ERs) in a full-scale commercial reactor (CR). In addition, we tested seed survival in a pH-buffered water bath (WB). Seeds were exposed to CR, ER and WB treatments at 42 °C for a maximum of 36 days. The viability of seeds was checked by measuring germination and response to tetrazolium staining and modeled as a function of exposure time using a dose–response approach. CR killed seeds more effectively than ER and WB treatments. The non-HS reference species, Chenopodium album, was completely inactivated by all treatments. Responses of the HS species ranged from complete inactivation to complete insensitivity. The most resistant was Malva sylvestris. The least resistant species were inactivated mainly by temperature, while additional mortality factors were effective in the more resistant species. We concluded that mesophilic AD in CRs can reduce the risk of seed contamination in the digestate for non-HS but not for HS species. Moreover, WB treatments seem suitable to estimate the minimum mortality of non-HS species in CR. Full article

The application of fermented total mixed ration (FTMR) is an effective method to prolong the use time of feed, but the understanding of the interaction mechanism between fungal microorganisms and silage quality and aerobic stability in FTMR is still limited. This study aimed to evaluate the effects of alfalfa (Medicago sativa L.) hay to oat (Avena sativa L.) hay ratios on chemical composition, fermentation characteristics, and fungal communities during aerobic exposure of fermented total mixed ration (FTMR). The supplement levels of oat were as follows: 200 g/kg oat hay (LO), 300 g/kg oat hay (MO), and 400 g/kg oat hay (HO). The water content of the three treatments was adjusted to 50% using a sprayer. After 60 days of ensiling, the bags were opened, and the chemical composition, fermentation characteristics, and fungal communities were measured after 3, 6, 9, and 12 days of aerobic exposure. The results suggested that the LO treatment significantly (p < 0.05) increased the aerobic stability than that in other treatments. The crude protein and lactic acid content in the three treatments were significantly decreased with the extension of the aerobic exposure period. Additionally, there was a remarkable (p < 0.05) higher lactic acid content observed in the LO treatment than that in the HO treatment during the aerobic stage. The PCoA showed that the compositions of the fungal community in the HO treatment were distinctly separated from the other two treatments. Compared with HO and LO treatments, the MO treatment observed relatively higher OTU, Shannon, and Chao1 indexes. Compared with LO and MO treatments, the abundances of the genes Saccharomyces and Wallemia were greater increased and decreased in the HO treatment, respectively. Integrated correlation analysis also underscores a possible link between the fermentation characteristics, aerobic stability, and significantly altered fungal community. This study suggested that the use of FTMR in production might prolong aerobic storage time when alfalfa was fermented in a mixture with ≤30% oat. Full article

Rice bran (RB) is a promising food ingredient that can improve biological function. In this study, we investigated the effects of RB, both unfermented (RB30) and fermented (RBF30), with five different microorganisms on the neurobehavioral activity in zebrafish larvae. Analytical methods such as LC–UV and LC–MS were performed for the analysis of RB30 and RBF30 extracts. Interestingly, niacin content, which is known to improve brain functions such as cognition and emotion, was found to be higher in RBF30 than in RB30. Furthermore, niacin content was highly increased in the RBF30-exposed fish, compared to those in the control fish. Therefore, we profiled behavioral patterns and various neurochemistry in zebrafish larvae following supplementation with RBF30 as well as performed calcium imaging on Tg (huC:GAL4-VP16); (UAS:GCaMP7a) zebrafish larvae to determine the correlation of neural activity. RBF30 revealed greater stimulation of locomotor activity without negatively affecting decision-making behavior in zebrafish larvae, as compared to RB30 or niacin. Its behavioral activation is mainly linked with the elevations of neural activity and several neurochemicals such as serotonergic and dopaminergic systems that are implicated in the control of anxiety and stress. Taken together, these results suggest that RBF30 could be a food material that improves the behavioral health by modulating neural activity and brain neurochemistry. Full article

Sucrose is an abundant, cheap, and renewable carbohydrate which makes it an attractive feedstock for the biotechnological production of chemicals. Escherichia coli W, one of the few safe E. coli strains able to metabolize sucrose, was examined for the production of pyruvate. The repressor for the csc regulon was deleted in E. coli W strains expressing a variant E1 component of the pyruvate dehydrogenase complex, and these strains were screened in a shake flask culture for pyruvate formation from sucrose. The pyruvate accumulated at yields of 0.23–0.57 g pyruvate/g sucrose, and the conversion also was accompanied by the accumulation of some fructose and/or glucose. Selected strains were examined in 1.25 L controlled batch processes with 40 g/L sucrose to obtain time–course formation of pyruvate and monosaccharides. Pyruvate re-assimilation was observed in several strains, which demonstrates a difference in the metabolic capabilities of glucose- and sucrose-grown E. coli cultures. An engineered strain expressing AceE[H106M;E401A] generated 50.6 g/L pyruvate at an overall volumetric productivity of 1.6 g pyruvate/L·h and yield of 0.68 g pyruvate/g sucrose. The results demonstrate that pyruvate production from sucrose is feasible with comparable volumetric productivity and yield to glucose-based processes. Full article

Fermentation technology has a long history and low-temperature fermentation has now become the focus of research. This paper reviews the mechanism and application of low-temperature fermentation and the optimization of relevant strains. Low-temperature fermentation leads to a differential expression of growth in metabolism genes (PSD1, OPI3, ERG3, LCB3 and NTH1). Low-temperature fermentation can be applied to foods and has various advantages, such as increasing changes in volatile flavor compounds and other corresponding metabolic substances of the strain, and inhibited growth of spurious bacteria. The focus of low-temperature fermentation in the long run lies in strain optimization, which is to protect and optimize the strains through a variety of methods. Low-temperature fermentation can greatly improve product quality. At present, the most effective methods to promote low-temperature fermentation are gene knockout and probiotic microencapsulation. Full article

Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture Chlorella vulgaris and Rhodotorula glutinis for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m²/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of Chlorella vulgaris and Rhodotorula glutinis in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production. Full article

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis. Full article

The purpose of this study was to reveal the changes in total phenolic content and antioxidant capacity of broccoli, and an untargeted metabolomics approach was developed to investigate the effect of lactic acid bacteria fermentation on the metabolome of broccoli florets. The results showed that the total phenolic content and antioxidant capacity significantly increased after fermentation. The untargeted metabolite profile showed that the main chemical components of fermented and unfermented broccoli are lipids and lipid-like molecules, organic acids and derivatives and organoheterocyclic compounds. Univariate and multivariate statistical analyses of the identified metabolites showed some metabolites such as sorbitol are upregulated after fermentation, and that other metabolites such as l-malic acid are downregulated after fermentation. Moreover, metabolite pathway analyses were used to study the identification of subtle but significant changes among groups of related metabolites that cannot be observed with conventional approaches. KEGG pathway analysis showed that metabolites are mainly enriched in the glucagon signaling pathway, pyruvate metabolism, glycolysis/gluconeogenesis and fructose and mannose metabolism after fermentation, compared with raw broccoli. The results of this study can help to further our understanding of the impact of LAB fermentation on bioactivity changes in and the metabolites profile of fermented broccoli, and the application of fermented broccoli in health foods and special dietary foods. Full article

Kombucha is a fermented tea beverage containing bioactive compounds from tea and vital compounds such as acetic acid, D-saccharic acid-1,4-lactone, and glucuronic and gluconic acids produced from the metabolic activities of bacteria and yeasts, which benefit human health. Kombucha contains a symbiotic culture of bacteria and yeast (SCOBY), which actively ferments sugar. Kombucha microbial compositions vary due to environmental conditions and the starter culture. Saccharomyces sp., Schizosaccharomyces pombe, Schizosaccharomyces sp., and Brettanomyces sp. (yeasts) and Acetobacter aceti, Komagataeibacter xylinum (formerly known as Gluconacetobacter xylinum), Gluconobacter oxydans, and Acetobacter pasteurianus (acetic acid-producing bacteria) are commonly found in kombucha. This review focused on the microbial compositions of kombucha and their functionality. Aspects discussed include: (i). developments in kombucha, (ii). microbial compositions of kombucha, (ii). microbial production of kombucha cellulose, (iv). factors influencing kombucha microbial compositions, (v). tea type and kombucha bioactive compounds, (vi). kombucha health benefits, and (v). potential risk factors of kombucha consumption. Current gaps, recommendations, and prospects were also discussed. Kombucha production using rooibos as the tea base is recommended, as rooibos is caffeine-free. Upcycling kombucha wastes, mainly SCOBY, for producing cellulose filters, improving food flavors and as a substrate in food fermentations is touched on. Full article

A demonstrator plant of a recently patented process for improved sludge degradation has been implemented on a municipal scale. In a 1500 m³ sewage sludge digester, an intermediary stage with aerobic sewage sludge reactivation was implemented. This oxic activation increased the biogas yield by up to 55% with a 25% reduction of the remaining fermentation residue volume. Furthermore, this process allowed an NH₄-N removal of over 90%. Additionally, 16S rRNA gene amplicon high-throughput sequencing of the reactivated digestate showed a reduced number of methane-forming archaea compared to the main digester. Multiple ammonium-oxidizing bacteria were detected. This includes multiple genera belonging to the family Chitinophagaceae (the highest values reached 18.8% of the DNA sequences) as well as a small amount of the genus Candidatus nitrosoglobus (<0.3%). In summary, the process described here provides an economically viable method to eliminate nitrogen from sewage sludge while achieving higher biogas yields and fewer potential pathogens in the residuals. Full article

In sheep, temperament is known to affect animal welfare and the quality of animal products. While the composition of the gut microbiota is different between depressed patients and healthy human patients, in sheep, the influence of temperament on ruminal microbial species and abundance remains unknown. This study investigated the effects of temperament on parameters of rumen fermentation and microbial composition of rumen contents of Hu ram lambs. Using the pen score test, 6 lambs that scored 2 points or below (calm) and 6 lambs that scored 4 points or more (nervous) were selected from 100 ram lambs. The sheep were fed a standard diet for 60 days and rumen samples were collected at slaughter. The concentrations of propionic acid, isovaleric acid, valeric acid, and the ammonia nitrogen concentration were different between the calm and the nervous groups (p < 0.05). At the phylum level, there were significant differences in Bacteroidetes, Tenericutes, and Spirochetes (p < 0.05); and at the genus level, there were significant differences in the Christensenellaceae R-7 group, Treponema 2, Fibrobacter, and Ruminococcaceae UCG-003 (p < 0.05). The present study suggests that differences in the rumen microbiota between the calm group and the nervous group could have an impact on the metabolism of carbohydrates and polysaccharides and explain why Calm Hu sheep have a higher energy utilization efficiency than nervous Hu sheep. More studies are needed to further understand the effect of temperament on specific pathways of the rumen microbiota. Full article

Fermented foods have gained immense popularity in recent years due to their distinct flavor profile. Given the increasing demand, there is a growing focus on optimizing their nutritional quality while also reducing their costs. In this study, using a novel approach, hemp seed meal was utilized as a solid fermentation substrate to produce nattokinase (NK). Using a combination of one-factor-at-a-time experiments, Plackett–Burman design, and Box–Behnken design, the optimal fermentation conditions of Bacillus subtilis 13932 (NK-producing strain) were determined. The initial ratio of HSM (hemp seed meal) to water was 1:2.0 (v:w), the thickness of the substrate was 2.9 cm, the bacterial inoculum volume was 10% (v:w), the relative humidity was 75.2%, the temperature was set at 35 °C, and the fermentation time was 20 h. The NK activity under these conditions was measured to be 7067.12 IU/g. During fermentation, 15.15% of soluble peptides were produced, which exhibited hydroxyl radical removal ability and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical removal ability of 14.85%, down from 32.96%. Furthermore, trypsin inhibitor and urease in HSM decreased by 42.6% and 73.6%, respectively, improving the nutritional quality of HSM. Sensory evaluations indicated that HSM is expected to be a popular food, highlighting the potential of using HSM as a solid fermentation substrate for NK production. Full article

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD_{600 nm} dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes. Full article

Anaerobic methanogenesis plays an important role in the sustainable management of high concentration organic wastewater and bioenergy recovery. Interspecies electron transfer (IET) is a new type of mutualistic symbiosis that can accelerate microbial metabolism and overcome thermodynamic barriers in the metabolic process, thus facilitating anaerobic methanogenesis. IET is classified into Direct Interspecies Electron Transfer (DIET) and Mediated Interspecies Electron Transfer (MIET) according to the different electron transfer methods. This paper summarizes the recent research progress related to interspecies microbial electron transfer in anaerobic methanogenic system, describes the possible specific mechanisms of DIET and MIET, and analyzes the differences between DIET and MIET methods in terms of methanogenic performance, thermodynamics, kinetics, and the microbial communities involved in them. Finally, it was found that, through DIET, microorganisms in the process of anaerobic methanogenesis could not only strengthen the extracellular electron transfer of microorganisms and alleviate the inhibition of high organic loading rate, organic acids, and toxic substances, they could also help ferment bacteria and allow methanogenesis to break through the thermodynamic barriers and efficiently degrade complex organic matter. This can overcome several problems, such as low efficiency of electron transfer and acidification of traditional anaerobic digestion. Full article

This study aimed to explore the dynamic variations in fermentation characteristics, bacterial diversity and community composition at two preservation temperatures as preservation time extended. Six rumen fluid samples collected from high-grain feeding sheep were stored at −20 °C or −80 °C for 0 day, 7 days, 14 days, 30 days, 60 days, 120 days, and 240 days. The results showed that the current preservation temperature did not alter the fermentation characteristics, bacterial diversity and community composition (p > 0.05). The concentrations of ammonia, microbial crude protein, acetate, propionate, butyrate, valerate, and total volatile fatty acids were higher when stored at 60 days (p < 0.05). Preservation time had no influence on bacterial richness and evenness (p > 0.05), whilst the relative abundances of Bacteroidota and Prevotella were numerically higher when stored at 30 days, and the opposite results were observed regarding Firmicutes. Both principal co-ordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) showed clusters among treatments in terms of preservation time and preservation temperature. Analysis of similarities (ANOSIM) also revealed similarities between treatments (p > 0.05). This study indicates that most fermentation characteristics in rumen fluid were altered after a 60-day preservation, whilst the preservation time for rumen bacterial community profile alteration was 30 days. It is recommended to finish the sample determination of rumen fluid within 30 days. This study may assist decision-making regarding the practicable time for rumen fluid determination, as well as viable preservation conditions for inoculum used for in vitro fermentation testing. Full article

Delaying flavour staling has been one of the greatest and most significant challenges for brewers. The choice of suitable raw materials, particularly malting barley, is the critical starting point to delay the risk of beer staling. Malting barley and the malting process can have an impact on beer instability due to the presence of pro-oxidant and antioxidant activities. Malt contains various compounds originating from barley or formed during the malting process, which can play a significant role in the fundamental processes of brewing through their antioxidant properties. This review explores the relationship between malt quality, in terms of antioxidant and pro-oxidant activities, and the flavour stability of beer. Full article