Fermentation, Volume 10, Issue 1 (January 2024) – 71 articles

Jujubes (Ziziphus jujuba Mill), characterized by a rich profile of bioactive compounds, have been historically less exploited due to their unappealing sensory characteristics when dried, including delayed bitterness and a limited shelf life when fresh. Co-fermented jujube puree has emerged as a strategy for enhancing its functional food potential. This study examined the impact of 8-day bicultured Junzao jujube puree, employing both commercial and indigenous Chinese lactic and acetic acid bacteria. Our investigation encompassed an assessment of functionality (cellular profile, antioxidant properties, color, free amino acids, phenolic profiling, volatiles elucidation using headspace-solid phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC-MS), aroma analysis using electronic nose), and microstructural analysis using scanning electron microscopy (SEM). Viable counts of bicultured purees showed probiotic effects exceeding 6–7 log CFU/mL. Strong positive correlations were observed between phenolic compounds (chlorogenic acid, rutin, p-coumaric acid) and antioxidant capacities (ABTS-RSA and DPPH-RSA). The darker color of raw jujube puree was modified, exemplified by a significant (p < 0.05) negative correlation between overall color difference and cyanidin 3-O-rutinoside (R² = −0.768). Purees, particularly those containing bicultures of Lactobacillus helveticus Lh 43 and Acetobacter pasteurianus Ap-As.1.41 HuNiang 1.01 exhibited the highest potential free amino acid content (157.17 ± 1.12 mg/100 g FW) compared to the control (184.03 ± 1.16 mg/100 g FW) with a distinctive formation of L-methionine in biculture of Lactiplantibacillus plantarum Lp 28 and A. pasteurianus Ap-As.1.41 HuNiang 1.01. The phenolic profile of Lacticaseibacillus casei Lc 122 and A. pasteurianus Ap-As.1.41 HuNiang 1.01 increased by 22.79% above the control (48.34 mg/100 g FW) while biculture: L. helveticus Lh 43 and A. pasteurianus Ap-As.1.41 HuNiang 1.01 were enhanced by 4.37%, with the lowest profile in Lp. plantarum Lp 28 and A. pasteurianus Ap-As.1.41 HuNiang 1.01 (46.85 mg/100 g FW). The electronic nose revealed the predominant presence of sulfur, terpenes, and alcohol sensor bioactives in the fermented purees. HS-SPME-GC-MS analysis identified 80 volatile compounds in the bicultured purees, with esters constituting the major group (42%). Furthermore, SEM analysis unveiled massive microscopic alterations in the bicultured purees compared to the unfermented puree. These results collectively demonstrated that lactic–acetic acid co-fermentation serves to biovalorize Junzao jujube puree, enhancing its organoleptic appeal and extending its shelf life. Full article

Aerobic composting is one of the methods for the resource utilization of submerged plant residues. This study investigated the effects of biochar, wetland sediments and microbial agents added individually or combined on the humification process, lignocellulose degradation and microbial communities during Ceratophyllum demersum and Potamogeton wrightii composting. The results showed that the addition of wetland sediment and biochar was found to significantly elevate the composting temperature and humification of compost products. The average content of lignin in wetland sediment and/or biochar treatments was 12.2–13.5%, which was higher than the control group (10.9–11.45%). Compared with the organic matter (19.4%) and total nitrogen concentration (35.3%) of compost treated with complex microbial agent treatments, the homemade microbial agents significantly increased the values by 22.1% and 41.0%, respectively. By comparing the differences in microbial communities among different treatments, the sediments and homemade agents demonstrated greater increases in activity and diversity of lignocellulose degradation-related microbes, especially for Truepera and Actinomarinale. Humus component and temperature were the most critical parameters influencing the changes in the bacterial community. Based on these results, a combination of biochar and homemade agents was a promising additive for an effective composting strategy, and sediment was identified as a potential control of bacterial diversity in wetland plant compost. Full article

In recent years, magnetic fields have emerged as a non-thermophysical treatment with a significant impact on microbial fermentation processes. Brassica trispora is a microorganism known for its industrial-scale production of lycopene and high yield of single cells. This study aimed to investigate the impact of low-frequency magnetic fields on lycopene synthesis by Brassica trispora and elucidate the underlying mechanism for enhancing lycopene yield. The results indicate that both the intensity and duration of the magnetic field treatment influenced the cells. Exposing the cells to a 0.5 mT magnetic field for 48 h on the second day of fermentation resulted in a lycopene yield of 25.36 mg/g, representing a remarkable increase of 244.6% compared to the control group. Transcriptome analysis revealed that the alternating magnetic field significantly upregulated genes related to ROS and the cell membrane structure, leading to a substantial increase in lycopene production. Scanning electron microscopy revealed that the magnetic field treatment resulted in a rough, loose, and wrinkled surface morphology of the mycelium, along with a few micropores, thereby altering the cell membrane permeability to some extent. Moreover, there was a significant increase in intracellular ROS content, cell membrane permeability, key enzyme activity involved in lycopene metabolism, and ROS-related enzyme activity. In conclusion, the alternating frequency magnetic field can activate a self-protective mechanism that enhances lycopene synthesis by modulating intracellular ROS content and the cell membrane structure. These findings not only deepen our understanding of the impact of magnetic fields on microbial growth and metabolism but also provide valuable insights for developing innovative approaches to enhance carotenoid fermentation. Full article

Non-conventional yeasts (NCYs) (i.e., non-Saccharomyces) are used as alternative starters to promote aroma complexity of fermented foods (e.g., bakery products). A total of 66 yeasts isolated from artisanal food matrices (bread and pizza sourdoughs and milk whey) from different geographical areas of the Campania region (Italy) were screened for physiological and technological characteristics such as leavening ability, resistance to NaCl and pH, exopolysaccharide and phytase activity production, and carbohydrate assimilation. Selected and isolated microorganisms were also used to study the leavening kinetics in experimental doughs as mixed inocula of two different strains. Volatile organic compounds (VOCs) of the inoculated doughs were analyzed with solid-phase microextraction/gas chromatography–mass spectrometry (SPME/GC-MS). Most of the strains belonged to non-Saccharomyces species (Pichia kudriavzevii, Kluyveromyces marxianus) and Saccharomyces (S. cerevisiae). Several strains produced exopolysaccharides (EPSs), that are important for dough rheological properties. Moreover, yeasts isolated from whey showed extracellular phytase activity. The mixed starter culture of the S. cerevisiae and NCY strains showed a synergic effect that enhanced the doughs’ aroma complexity. The use of non-conventional yeasts mixed with S. cerevisiae strains can be advantageous in the bakery industry because they improve the bread aroma profiles and nutritional properties by bioactive molecule production. Full article

Glycolate plays an important role as a platform chemical in both polymeric material and cosmetic industries. However, the microbial production of glycolate often encounters challenges associated with unbalanced metabolic flux, leading to a notably low titer. Additionally, the use of expensive inducers, such as IPTG, contributes to an increase in the overall production cost. To address these issues, the key enzymes involved in the glycolate biosynthetic route, including citrate synthase (gltA), isocitrate lyase (aceA), isocitrate dehydrogenase kinase/phosphatase (aceK) and glyoxylate reductase (ycdW), were overexpressed in E. coli under the control of inducible promoters with varying strengths in order to determine the optimal combination. Subsequently, the glycolate pathway was further modulated by replacing inducible promoters with various constitutive synthetic promoters. Through this systematic optimization, the best strain, named Mgly4T1562, produced 3.02 g/L glycolate with 97.32% theoretical yield in shake-flask cultivation. The titer further increased to 15.53 g/L in a fed-batch experiment. Notably, this study marks the first successful utilization of synthetic promoters in tuning the glycolate biosynthetic pathway for glycolate biosynthesis. The strategy presented in this research holds significant promise for facilitating the cost-effective and industrially viable production of glycolate without the need for expensive inducers. Full article

Koumiss, a traditional fermented beverage made from mare’s milk, is typically consumed by nomads. Industrialized production of koumiss has been increasingly applied recently due to the increased demand for the beverage and awareness of its potential health benefits. However, it is unknown whether industrial koumiss is comparable to the traditional koumiss in terms of quality. In this study, we compared the microbiological and physicochemical properties in the industrial and traditional koumiss fermentation processes synchronously using culture-dependent and culture-independent approaches. Although Lactobacillus and Kazachstania species were similarly dominant in the bacterial and fungal communities, respectively, in both processes, the microbial counts and diversity in the traditional koumiss were significantly higher than those in the industrial koumiss. Furthermore, the traditional koumiss fermentation consumed more lactose, produced more flavor substances including acetic acid, lactic acid, ethanol, and free amino acids, and reached a lower pH value at the final stage. The physicochemical characters of traditional koumiss were mainly associated with Lactobacillus and Kazachstania species, which, in turn, were positively correlated with each other but negatively correlated with other non-dominant microbes. The starter was the major source of the microbial community of industrial koumiss, whereas both the starter and environment were the major sources of traditional koumiss. Random forest analysis recognized 11 significantly important genera as microbial indicators to distinguish industrial from traditional koumiss. Overall, this study shows that the microbial and physicochemical dynamics during the traditional and industrial fermentation of koumiss differ significantly, and the results obtained are valuable for improving the quality of industrial koumiss. Full article

ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative mainly produced by the aerobic, filamentous bacterium Streptomyces albulus. Therefore, it is crucial to breed superior ε-PL-producing strains to enhance fermentation efficiency to reduce production costs. Metabolic engineering is an effective measure for strain modification, but there are few reports on key genes for ε-PL biosynthesis. In this study, metabolic flux analysis was employed to identify potential key genes in ε-PL biosynthesis in S. albulus WG-608. A total of six potential key genes were identified. Three effective key genes (ppc, pyc and pls) were identified for the first time in ε-PL biosynthesis through overexpression experiments. It also presents the first demonstration of the promoting effects of ppc and pyc on ε-PL biosynthesis. Three genes were then co-expressed in S. albulus WG-608 to obtain OE-ppc-pyc-pls, which exhibited an 11.4% increase in ε-PL production compared to S. albulus WG-608, with a 25.5% increase in specific ε-PL production. Finally, the metabolic flux analysis of OE-ppc-pyc-pls compared to S. albulus WG-608 demonstrated that OE-ppc-pyc-pls successfully altered the metabolic flux as expected. This study not only provides a theoretical basis for the metabolic engineering of ε-PL-producing strains but also provides an effective approach for the metabolic engineering of other metabolites. Full article

Plasmids are commonly used tools in microbiology and molecular biology and have important and wide-ranging applications in the study of gene function, protein expression, and compound synthesis. The complex relationship between necessary antibiotic addition, compatibility between multiple plasmids, and the growth burden of host bacteria has plagued the wider use of compatibility plasmids. In this study, we constructed the terminal polymerization pathway of PSA by exogenously expressing the neuA, neuD, and neuS genes after the knockdown of Eschesrichia coli BL21 (DE3). Duet series vectors were utilized to regulate the expression level of neuA, neuD, and neuS genes to study the gene expression level, plasmid copy number growth burden, pressure of antibiotic addition, stability of compatible plasmids, and the level of expression stability of exogenous genes, as well as the effect on the biological reaction process. The results showed that the three genes, neuA, neuD, and neuS, were enhanced in the recombinant strain E. coli NA-05, with low copy, medium copy, and high copy, respectively. The effect of PSA synthesis under standard antibiotic pressure was remarkable. The results of this thesis suggest the use of a Duet series of compatible expression vectors to achieve the stable existence and co-expression of multiple genes in recombinant bacteria, which is a good reason for further research. Full article

Anaerobic digestion of sewage sludge is limited at the hydrolysis stage of the process. The goal of this study was to assess the effects of sludge retention times and ultrasound pretreatment on the ammonium concentration and organic matter transformation in anaerobic digesters treating sewage sludge. To achieve this, two laboratory-scale semicontinuous anaerobic digesters were operated for a period of over 70 d, including a control reactor and another fed by pretreated sludge. Both anaerobic systems were fed with mixed sludge (50%/50% primary/secondary treatment) in mesophilic conditions (37 °C), with solid retention times (SRT) of 7.5 d (Phase I) and 3 d (Phase II). The performance of the anaerobic digestion process was assessed in terms of the methane yield and the total and soluble chemical organic demand, total solids, and volatile solids removal. The results showed that the ultrasound pretreatment caused an increase of around 22.2% in CODt removal for an SRT of 7.5 d. Meanwhile, an SRT of 3 d resulted in a decrease of up to 92.4% in CODt removal. The performance in terms of biogas production and organic matter removal was significantly affected by the SRT reduction to 3 d, showing that the process is not viable in these conditions. Full article

This experiment was aimed at comparing how a chemical additive or an inoculant would affect the dry matter (DM) losses, fermentation pattern, yeast and mold counts, and aerobic stability (AS) of the ensiled high-moisture barley grain (675 g kg⁻¹ DM). Crimped barley grain was ensiled with or without chemical additive AIV Ässä Na and an inoculant SiloSolve FC, totaling three treatments (1 × 3 factorial scheme) for the fermentation periods lasting 7, 14, 28, 60, and 90 days. The application of a chemical additive showed higher pH levels, retained water-soluble carbohydrates (WSCs) better, lowered DM loss, and reduced concentrations of fermentation products. Barley grain treated with chemicals initially showed a marked reduction in yeast and fungal growth and a higher AS up to day 60 of storage but became less effective in later stages of storage, leading to decreased AS. Barley grain inoculated with homo- and heterofermentative strains had decreased silage pH; its WSC content was similar to control and had higher content of weak acids and 1,2 propanediol, reducing mold and yeast counts. With fermentation duration from 28 days and beyond (i.e., 90 days), the inoculant treatment achieved the longest AS and the lowest increase in pH and weight loss during the period of air exposure. Full article

The sesquiterpene β-caryophyllene (BCP) is a major component of various plant essential oils, to which it confers a unique spicy aroma. It is mainly used as a fragrance additive in the food, cosmetic and perfume industries, with an annual consumption ranging between 100 and 1000 metric tons worldwide. Recently, BCP has attracted attention as a promising precursor for the production of high-density fuels and for its various biological activities and pharmacological effects. These include antioxidant, anti-inflammatory, anticancer, immune–modulatory, and many other activities. Due to its underlying mechanisms, β-caryophyllene interacts with various human receptors, including CB2 of the endocannabinoid system, which defines it as a phytocannabinoid with therapeutic potential for certain serious conditions. Due to β-caryophyllene’s high utility, various green and sustainable strategies for its production in microorganisms have been developed. This article provides an update on the state-of-the-art in this field to identify directions for further development to extend the compound’s potential. Full article

Catalase (CAT) is an enzyme involved in the first line of cellular antioxidant defense. It plays a key role in the protection of a wide range of Antarctic organisms against cold stress. Extracellular catalase is very rare and data on it are extremely scarce. The aim of the present study was to select an efficient producer of extracellular catalase from amongst Antarctic filamentous fungi. Sixty-two Antarctic filamentous fungal strains were investigated for their potential ability to synthesize intracellular and extracellular CAT. The Antarctic strain Penicillium rubens III11-2 was selected as the best producer of extracellular catalase. New information on the involvement of the extracellular antioxidant enzymes superoxide dismutase and CAT in the response of filamentous fungi against low-temperature stress was obtained. An efficient scheme for the purification of CAT from culture fluid was developed. An enzyme preparation with high specific activity (513 U/mg protein) was obtained with a yield of 19.97% and a purification rate of 98.4-fold. The purified enzyme exhibited maximal enzymatic activity in the temperature range of 5–40 °C and temperature stability between 0 and 30 °C, therefore being characterized as temperature sensitive. To our knowledge, this is the first purified extracellular cold active catalase preparation from Antarctic filamentous fungi. Full article

Fermented beverages have been a constant companion of humans throughout their history. A wide range of products have been developed with time, depending on the availability of raw materials and ambient conditions. Their differentiation was based on the specific characteristics of each product, resulting from the cultivation of different varieties and the variability of environmental conditions and agricultural practices, collectively described by the term ‘terroir’ that was developed in winemaking. The health benefits that have been associated with their consumption, which include the control of blood pressure and glycemic control, along with immunomodulatory, hypocholesterolemic, hepatoprotective, and antiproliferative activities, directed their re-discovery that occurred over the last few decades. Thus, the dynamics of the microbial communities of fermented beverages during fermentation and storage have been thoroughly assessed. The functional potential of fermented beverages has been attributed to the chemical composition of the raw materials and the bioconversions that take place during fermentation and storage, due to the metabolic capacity of the driving microbiota. Thus, the proper combination of raw materials with certain microorganisms may allow for the modulation of the organoleptic properties, as well as enrichment with specific functional ingredients, enabling targeted nutritional interventions. This plasticity of fermented beverages is their great advantage that offers limitless capabilities. The present article aims to critically summarize and present the current knowledge on the microbiota and functional potential of fermented beverages and highlight the great potential of these products. Full article

Amino alcohols are important compounds that are widely used in the polymer and pharmaceutical industry, particularly when used as chiral scaffolds in organic synthesis. The hydroxylation of polyamide polymers may allow crosslinking between molecular chains through the esterification reactions of hydroxyl and carboxyl groups. Therefore, this may alter the functional properties of polyamide polymers. 2-hydroxycadaverine (2HyC), as a new type of chiral amino alcohol, has potential applications in the pharmaceutical, chemical, and polymer industries. Currently, 2HyC production has only been realized via pure enzyme catalysis or two-stage whole-cell biocatalysis, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2HyC in industrial applications. Here, we designed and constructed a promising artificial pathway in Escherichia coli for producing 2HyC from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs two enzymes to sequentially convert lysine into 2HyC. However, the catalytic activity of wild-type pyridoxal phosphate-dependent decarboxylase from Chitinophage pinensis (DC_Cp) toward 3-hydroxylysine is lower, resulting in the lower production of 2HyC. Thus, the higher catalytic activity of DC_Cp is desired for low-cost and expanded industrial applications of 2HyC. To improve the catalytic activity of DC_Cp, a mutant library of DC_Cp was first built using a semi-rational design. The K_cat/K_m of mutant DC_Cp (R53D/V94I) increased by 63%. A titer of 359 mg/L 2HyC was produced in shake flasks, with a 2HyC titer increase of 54% compared to control strain ML101. The results show that the production of 2HyC was effectively increased through a semi-rational design strategy. These findings lay the foundation for the development and utilization of renewable resources to produce 2HyC in microorganisms via an efficient, green, and sustainable biosynthetic strategy for further industrial application. Full article

Soy leghemoglobin (LegH) has been gaining interest over the last years as an efficient flavor and aroma compound in plant-based meat substitutes. Hence, in the following article, we demonstrate the methods for LegH production using a recombinant Komagataella phaffii strain. Multiple fed-batch fermentation with an alternative to a BSM medium, where glucose was used as the main carbon source, was implemented and the growth kinetics, e.g., a maximal specific biomass growth of 0.239 g·g⁻¹·h⁻¹, a biomass yield from the substrate of 0.298 g·g⁻¹, and a maximal specific substrate consumption rate of 0.81 g·g⁻¹·h⁻¹ were identified. Leghemoglobin production resulted in a yield of 0.513 mg·gDCW⁻¹, while the highest biomass density achieved in this study was 121.80 gDCW·L⁻¹. The applied medium that showed potential for additional optimization studies, which, in contrast to BSM, made it possible to separate pH control from nitrogen supply, does not affect medium turbidity measurements and does not induce metabolite synthesis during yeast biomass growth. Full article

Brewer’s spent yeast (BSY) is one of the brewing industry’s most plentiful side-streams. Abundant, low-cost and high in nutrients, it has great potential for application in food technology and human nutrition. With the ever-increasing interest in sustainability, waste reduction and circular food systems, the use of BSY as a novel food ingredient may be the route to add exponential value while reducing the environmental impact. However, negative flavour characteristics and high amounts of alcohol severely limit the current applications of BSY. This study explores the use of processing involving lactic acid bacteria (LAB) fermentation technology as a means of improving BSY quality characteristics and examines the effects of this process on the chemical, nutritional and sensory characteristics of BSY. The results reveal that BSY is a suitable substrate for LAB fermentation, successfully supporting the growth of Lactobacillus amylovorus FST 2.11. Compared to the unfermented BSY (CBSY), fermentation significantly reduced the perceptible bitterness of the BSY as detected by a sensory panel, from 6.0 ± 2.8 units to 0.9 ± 0.7 units, respectively. Fermented BSY (PBSY) had enhanced sour and fruity flavours, and a variety of other volatile compounds and metabolites were determined. Protein profiles showed significant protein degradation, and free amino acid levels were greatly increased following fermentation, from 2.8 ± 0.2 g/100 g to 10.5 ± 0.4 g/100 g, respectively. Protein quality was high, with the CBSY and PBSY providing well over the required level (>100%) of essential amino acids per gram protein, with the exception of sulphur amino acids (98%). Major physical differences were observed using scanning electron microscopy. This study concludes that LAB fermentation positively affects the sensory and nutritional characteristics of BSY and can aid in the incorporation of brewer’s spent yeast into foods for human consumption. Full article

Fermented foods such as table olives are produced through a spontaneous process that has been improved over the years, ensuring the safety and quality of the final product. The aim of the present work was to study the action of starter cultures of lactic acid bacteria (Lacticaseibacillus rhamnosus GG ATCC53103, Levilactobacillus brevis ATCC8287, and Lactiplantibacillus plantarum ATCC14917) which were previously shown to have probiotic and antioxidant potential during the fermentation of natural Greek-style black olives (Kalamata) in brine containing 6% (w/v) NaCl at a temperature of 20 °C for a period of 150 days. At a molecular level, the main metabolites in every fermentation process were identified using an HPLC method. The results showed that the concentration of the metabolites increased gradually, developing a stable pattern after the 90th day of fermentation. In addition, the DL-p-hydroxyphenyllactic acid (OH-PLA) was identified as the phenolic acid with the highest concentration, independently of the selected starter culture. Microbial genomic DNA was also extracted from the olives’ surface at the final stages of fermentation (150 days) and was subjected to 16S rRNA sequencing using the Nanopore MinION™ NGS tool, enabling a comprehensive analysis of the microbial community. According to the findings, the most abundant genera were Lactobacillus and Leuconostoc. To the best of our knowledge, this is the first study exploring these particular starters for olive fermentation. Full article

Malolactic fermentation (MLF) in wine is driven by the lactic acid bacterium Oenococcus oeni in most cases. Although this bacterium is resistant to wine stress conditions, it often faces difficulties completing MLF. Previous studies indicate that yeast mannoproteins may improve O. oeni growth and survival in wine. However, very little is known about this topic. This study evaluated the effect of the addition of mannoprotein extracts to culture media on O. oeni growth and its survival to stress conditions and MLF performance. Three commercial mannoprotein extracts were characterized in terms of polysaccharide and protein richness and were used for O. oeni culture media supplementation. The addition of mannoprotein extracts improved the survival of the two evaluated O. oeni strains, PSU-1 and VP41, after acid shock (pH 3.2) in comparison to that of the control. The transcriptional response of four genes involved in mannose metabolism was different depending on the strain, indicating the complexity of sugar metabolism in O. oeni. PSU-1 cells grown with two of the mannoprotein extracts performed faster MLF compared with the control condition, indicating that mannoprotein addition may improve the performance of O. oeni starter cultures, although this effect depends on the strain. Full article

Saccharomyces cerevisiae var. boulardii (Sb) is currently receiving significant attention as a synthetic probiotic platform due to its ease of manipulation and inherent effectiveness in promoting digestive health. A comprehensive exploration of Sb and other S. cerevisiae strains (Sc) would shed light on the refinement and expansion of their therapeutic applications. This review aims to provide a thorough overview of Saccharomyces yeasts from their native health benefits to recent breakthroughs in the engineering of Saccharomyces yeasts as synthetic therapeutic platforms. Molecular typing and phenotypic assessments have uncovered notable distinctions, including the superior thermotolerance and acid tolerance exhibited by Sb, which are crucial attributes for probiotic functions. Moreover, parabiotic and prebiotic functionalities originating from yeast cell wall oligosaccharides have emerged as pivotal factors influencing the health benefits associated with Sb and Sc. Consequently, it has become imperative to select an appropriate yeast strain based on a comprehensive understanding of its actual action in the gastrointestinal tract and the origins of the targeted advantages. Overall, this review underscores the significance of unbiased and detailed comparative studies for the judicious selection of strains. Full article

Representatives of the genus Bacillus have been established as one of the most important industrial microorganisms in the last few decades. Genetically modified B. subtilis and, to a lesser extent, B. licheniformis, B. amyloliquefaciens, and B. megaterium have been used for the heterologous expression of numerous proteins (enzymes, vaccine components, growth factors), platform chemicals, and other organic compounds of industrial importance. Vectors designed to work in Bacillus spp. have dramatically increased in number and complexity. Today, they provide opportunities for genetic manipulation on every level, from point mutations to systems biology, that were impossible even ten years ago. The present review aims to describe concisely the latest developments in the shuttle, integrative, and CRISPR-Cas9 vectors in Bacillus spp. as well as their application for large-scale bioengineering with the prospect of producing valuable compounds on an industrial scale. Genetic manipulations of promoters and vectors, together with their impact on secretory and metabolic pathways, are discussed in detail. Full article

In recent years, probiotics have received considerable attention for improving the health of aquaculture organisms, such as fish and shrimp, by stimulating immune activity and increasing growth rates. Oplegnathus fasciatus is a common and economically important cultured fish species in Asia. In this study, we aimed to investigate the potential of lactic acid bacteria (LAB; Limosilactobacillus reuteri)-fermented feed to promote growth and enhance immune function in O. fasciatus. The feed contained the highest proportion of LAB after L. reuteri fermentation for 3 days in anaerobic conditions. Oplegnathus fasciatus was fed LAB-fermented feed for 30 days. The administration of LAB-fermented feed (live bacteria > 10⁹ CFU/g) significantly increased the growth rate (weight gain = 174.8%; FCR = 4.23) and intestinal probiotic levels of O. fasciatus. After LAB-fermented feeding, the immunity index was evaluated by superoxide anion production, the phagocytic activity of leukocytes, and bactericidal and lysozyme activities in the serum of O. fasciatus. We found that LAB-fermented feed treatment potentially elevated the proportions of intestinal Bifidobacterium, Blautia, and Dorea species and reduced pathogenic bacterial growth (Acinetobacter, Escherichia_Shigella, and Megasphaera) in O. fasciatus. This study demonstrated that LAB-fermented feed containing extracellular vesicles improves growth performance and the inhibition of pathogenic Acinetobacter baumannii. Full article

Lactic acid bacteria effectively utilize the nutrients and active compounds in plant-based materials via their powerful metabolic pathways and enzyme systems, achieving a combination of nutrition, functionality, and deliciousness. Currently, the majority of review articles predominantly concentrate on summarizing the fermentation of fruits and vegetables by lactic acid bacteria, devoting comparatively less attention to researching other plant species varieties and plant-based by-products. Furthermore, the summary of the research on the active substances and functional properties lacks sufficient depth. This review provides a comprehensive overview of the status of and technological progress in lactic acid bacteria fermentation of various plant species and plant-based by-products, and the effects of lactic acid bacteria on the active substances and functional properties are emphasized. In addition, this review emphasizes that active substances give products more functionality. The aim of this review is to emphasize the significant contribution of lactic acid bacteria to the active substances and functional properties of plant-based products, which will assist researchers in better comprehending the application value of lactic acid bacteria in the plant-based domain and direct attention towards the interaction mechanisms between active substances and product functionality. Concurrently, this review provides a certain theoretical foundation and reference for the application of fermented functional products in promoting health and preventing diseases. Full article

Improper disposal of vegetable waste can cause serious environmental pollution, but because they contain huge water content and organic matter, they are not suitable for disposal by methods such as incineration and landfill. However, vegetable waste contains a large amount of nutrients and have some complementary effects with rice straw in terms of physical structure, nutrients, and moisture. In this experiment, the plant feed (corn husk, peanut shells and sorghum shells) was used as the control group (CON group), and the mixed silage of Chinese cabbage waste and rice straw (mixed silage) was used as the experiment group (TRE group), and its safety performance was evaluated by testing its toxin content, pesticide residues, vitamin contents and feeding experiment of Hu sheep. In the animal experiment, 16 healthy Hu sheep (5.5 months, 39.11 ± 4.16 kg) were randomly divided into two groups of 8 each. The results of the safety performance evaluation showed that the content of mycotoxins, heavy metals, and nitrites as well as pesticide residues in the crude feeds of both groups were within the range of Chinese feed hygiene standards. In addition, the levels of deoxynivalenol (DON) and aflatoxin (AFT) in the CON group were lower, while the content of ochratoxin (OTA) and zearalenone were higher than those in the TRE group (p < 0.05). The levels of plumbum(Pb), chromium (Cr), cadmium (Cd), and nitrite in the CON group were lower than the mixed silage, while the levels of As were higher than the mixed silage (p < 0.05). It is worth noticing that the content of vitamin B₂ (VB₂) and vitamin C (VC) in the TRE group was higher than the CON group (p < 0.05). The results of the feeding experiment showed that the mixed silage did not affect the growth performance, nutrient digestibility, organ index, and intestinal index of Hu sheep (p > 0.05). In addition, the mixed silage reduced the weight of omasum, the proportion of omasum to live weight before slaughter, the amount of compound stomach, and the proportion of compound stomach to live weight before slaughter, which were higher than those in the TRE group (p < 0.05). The thickness of the basal layer of the rumen abdominal sac, the red blood cell count, the content of IL-10, and TNF-α in the blood, and TNF-α content in the rumen of the Hu sheep in the TRE group were higher than the CON group (p < 0.05). In conclusion, the feed safety index content of the mixed silage did not exceed the Chinese feed hygiene and safety standards and did not cause adverse effects on the growth performance of the Hu sheep, and it improved the immune performance of the body and digestive tract of the sheep to a certain extent and promoted the healthy development of the sheep. Full article

Morchella esculenta is a precious edible and medicinal fungus rich in protein, polysaccharides, polyphenols, amino acids, triterpenes, and other active components. In this study, MS-1 was isolated from the fruiting body of M. esculenta. Through conducting single-factor experiments and the response surface analysis of the culture conditions, the optimal culture components of an M. esculenta fermentation broth for extracellular polysaccharide production were determined, namely, 3.7% glucose, 2% yeast extract, and 0.15% sodium chloride. The polysaccharides MSF and MSL were extracted from the fruiting body of M. esculenta and the fermentation broth, respectively, and analyzed with gel permeation chromatography (GPC), monosaccharide composition, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and in vivo and in vitro antioxidant and anti-inflammatory activity analyses. The research results show that the calculated MW of MSF is 2.34 × 10⁵ Da, and the calculated MW of MSL is 1.40 × 10⁵ Da. MSF is composed of three monosaccharides: D-galactose, D-glucose, and D-mannose (molar ratio of 4.34:90.22:5.45). MSL consists of five monosaccharides: D-arabinose, D-galactose, D-glucose, D-mannose, and glucuronic acid (molar ratio of 0.31:14.71:13.03:71.43:0.53). The in vitro antioxidant test results show that MSF and MSL both have significant antioxidant activities. Activity experiments on MSF and MSL in zebrafish showed that MSF and MSL have significant repair effects on the oxidative damage caused by metronidazole in zebrafish embryos, and there were significant changes in the transcriptional activity levels of the oxidative stress-related genes SOD, Keap1, and Nrf2. Therefore, the polysaccharides MSF and MSL from MS-1 can be used as important raw materials for functional foods and drugs. Full article

The feeding value of five varieties of whole-plant cassava (SC5, SC7, SC9, SC14, and SC205) was assessed through analysis of the nutritional composition and in situ ruminal degradability. The results showed abundant nutrients in whole-plant cassava, and the means of starch and crude protein (CP) were 267.7 and 176.8 (g kg⁻¹), and ranged from 223.7 g kg⁻¹ (SC9) to 296.4 g kg⁻¹ (SC14) and from 142.4 g kg⁻¹ (SC5) to 195.8 g kg⁻¹ (SC9) (p < 0.05), respectively. Meanwhile, the moderate neutral detergent fiber (NDF) of whole-plant cassava was also observed and ranged from 266.2 g kg⁻¹ in SC9 to 286.6 g kg⁻¹ in SC14 (p < 0.05). In addition, the trace elements, such as Fe, Mn, Cu, and Zn, in whole-plant cassava were relatively enriched, and their mean concentrations were 135.8, 1225.2, 5.8, and 105.3 mg kg⁻¹ (p < 0.05), respectively. Both the highest essential amino acid and total amino acid concentrations were obtained in SC7 (p < 0.01). The hydrogen cyanide (HCN) content of fresh and dried whole-plant cassava ranged from 76.5 to 131.6 and from 36.0 to 56.7 mg kg⁻¹ (p < 0.05), respectively. The in situ dry matter ruminal degradability and metabolizable energy (ME) varied significantly (p < 0.05) and were consistently lowest and highest in SC9 and SC14, ranging from 50.9% to 80.0% and from 7.5 to 12.3 MJ kg⁻¹, respectively. Collectively, all varieties of whole-plant cassava had a high feeding value, as reflected by abundant starch, minerals, amino acid, and water-soluble carbohydrates, while having a low fiber content and HCN toxicity, as well as excellent ruminal digestibility characteristics, and they could be used as a potential feed resource for ruminants. Full article

Due to the increasing costs of livestock farming, it is important to find cost-effective alternatives of feed stuffs. This study investigated the effects of high-moisture ear corn (HMEC) feeding on the production performance, serum antioxidant capacity, immunity, and ruminal fermentation and microbiome of dairy cows. Thirty pluriparous Chinese Holstein cows were randomly allocated to two groups: steam-flaked corn (SFC) and HMEC (replacement of 2 kg equal dry matter SFC) and fed for a 60 day trial. The results showed replacing SFC with HMEC significantly increased dry matter intake, milk yield, and 4% fat-corrected milk yield (p < 0.05). Serum levels of superoxide dismutase, glutathione peroxidase, and immunoglobulins G, M, and A were significantly higher, and those of creatinine and cholesterol were significantly lower, in the HMEC group than in the SFC group (p < 0.05). HMEC also significantly increased total volatile fatty acid and acetate (p < 0.05) concentrations. In both groups, the dominant phyla of ruminal bacteria were Bacteroidetes, Firmicutes, and Actinobacteria, and the dominant genera were Prevotella, NK4A214-group, and Succiniclasticum. Mogibacterium, Eubacterium nodatum group, norank-f-Lachnospiraceae, and Eubacterium brachy group were significantly enriched in the ruminal fluid of HMEC-group cows (p < 0.05). In conclusion, replacing SFC with HMEC improved production performance, antioxidant capacity, and immunity, while regulating both ruminal fermentation and the composition of the ruminal microbiome in dairy cows. Full article

The purpose of the present study was to investigate the effect of additives on the fermentation properties of ensiled mixed alfalfa and perennial ryegrass silage in the karst terrain of Southwest China. A mixture of alfalfa and perennial ryegrass was ensiled at a ratio of 3:7 using three experimental treatments: (1) CK (without additives) and distilled water (5 mL kg⁻¹ fresh weight (FW)); (2) FA and formic acid (88%) (5 mL kg⁻¹ FW); and (3) LAB combined with the application of Lactiplantibacillus plantarum and Lentilactobacillus buchneri (2 × 10⁷ cfu/g FW). All samples were packed manually into polyethylene bags, and three polyethylene bags from each treatment were sampled on days 7, 15, and 45. The findings demonstrated that the pH values of all the mixed silages gradually decreased during ensiling. The lactic acid (LA) and acetic acid (AA) contents increased gradually with ensiling time and peaked after 45 days of ensiling. After 45 days of ensiling, the FA and LAB groups effectively preserved the nutrient content of the mixed silage, which presented a reduced neutral detergent fiber and acid detergent fiber content (p < 0.05) and higher water-soluble carbohydrate content (p < 0.05) than the CK group. The fermentation quality of the mixed silages in the FA and LAB groups improved, as indicated by higher (p < 0.05) LA contents and lower (p < 0.05) pH and ammoniacal nitrogen contents after 45 days of ensiling compared to those in the CK group. As fermentation progressed, the abundance of harmful microorganisms (Hafnia obesumbacterium, Enterobacteriaceae, and Sphingomonas) and beneficial microorganisms (Lactiplantibacillus and Lentilactobacillus) decreased and increased, respectively. In addition, compared to those in the CK group, the FA group had higher abundances of “lipid metabolism” and “biosynthesis of antibiotics” and lower abundances of “membrane transport”. Briefly, the results of this study suggest that the incorporation of FA and LAB additives could improve the quality of fermented mixed silage, and that FA is better than LAB. This information is useful for combining forage resources to satisfy the requirements for high-protein feed and for manufacturing ruminant feed annually. Full article

The objective of this study was to evaluate the effect of anaerobic fermentation on the in vitro ruminal production of total gas (TG), methane (CH₄), carbon monoxide (CO) and hydrogen sulfide (H₂S), as well as on the characteristics of ruminal fermentation and CH₄ conversion efficiency of whole-plant maize (WPM) from four native maize genotypes (Amarillo, Olotillo, Tampiqueño and Tuxpeño) from Mexico, and a commercial hybrid was used as a control. In all genotypes, the fermented WPM produced the lowest amounts (p ≤ 0.0236) of TG and CH₄ from degraded dry matter (DM), and Tampiqueño and Tuxpeño presented the highest production of these gases. In addition, Tuxpeño also presented the highest proportion of CH₄ (mL 100 mL⁻¹ TG), and Olotillo presented the lowest proportion of both gases. Something similar occurred in H₂S, where the fermented WPM produced the lowest (p ≤ 0.0077) amount per DM degraded, and Amarillo and Tampiqueño presented the highest and lowest production, respectively. However, the fermented WPM presented the highest (p = 0.0128) CO production from degraded DM, and Tuxpeño and Olotillo presented the highest and lowest production, while the rumen pH was lower (p < 0.0001) in the fermented WPM, and Tuxpeño and Olotillo presented the highest and lowest pH, respectively. Furthermore, the fermented plant presented the greatest (p ≤ 0.0055) DM degradation, and the Amarillo and hybrid genotypes presented the highest percentages, while Olotillo presented the lowest. The short-chain fatty acid (SCFA) content and metabolizable energy (ME) did not differ (p ≥ 0.0899) between genotypes and were higher (p = 0.0009) in the fresh WPM. Despite the above, the fermented WPM was more efficient (p ≤ 0.0249), and the Amarillo and hybrid genotypes produced less CH₄ per unit of SCFAs, ME and organic matter. In conclusion, the Amarillo genotype equaled the hybrid one, and although the production of CO increased, anaerobic fermentation showed the potential to reduce the rumen production of TG, CH₄ and H₂S, as well as to improve DM degradability and CH₄ conversion efficiency. Full article