Systems Metabolic Engineering to Elucidate and Enhance Intestinal Metabolic Activities of Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is one of the most widely used probiotics to treat gastrointestinal diseases. Recently, many studies have engineered EcN to release therapeutic proteins to treat specific diseases. However, because EcN exhibits intestinal metabolic activities, it is difficult to predict outcomes after administration. In silico and fermentation profiles revealed mucin metabolism of EcN. Multiomics revealed that fucose metabolism contributes to the intestinal colonization of EcN by enhancing the synthesis of flagella and nutrient uptake. The multiomics results also revealed that excessive intracellular trehalose synthesis in EcN, which is responsible for galactose metabolism, acts as a metabolic bottleneck, adversely affecting growth. To improve the ability of EcN to metabolize galactose, otsAB genes for trehalose synthesis were deleted, resulting in the ΔotsAB strain; the ΔotsAB strain exhibited a 1.47-fold increase in the growth rate and a 1.37-fold increase in the substrate consumption rate relative to wild-type EcN.

The Probiotic Enterococcus Lactis SF68 as a Potential Food Fermentation Microorganism for Safe Food Production.

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes. Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

Design of an agro-industrial by-products-based media for the production of probiotic bacteria for fish nutrition.

Probiotic production in commercial culture media is expensive, so, it is necessary to design culture media based on "low-cost" components like agro-industrial by-products. Therefore, this study aimed to design an agro-industrial by-product-based culture media using whey, sugarcane molasses, and palm kernel cake as components to produce Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10 isolated from Nile tilapia (Oreochromis niloticus) associated gut microbiota. Higher bacterial concentrations were achieved at high whey concentrations and low concentrations of sugarcane molasses and palm kernel cake (PKC) using agitation. The optimal conditions were whey, 3.84% w/v; sugarcane molasses, 7.39% w/v; PKC, 0.77% w/v; and agitation speed, 75 RPM. Bacterial growth under optimal conditions was compared to that in commercial Brain-Heart Infusion (BHI) broth. L. lactis A12 showed similar growth in the optimal media and BHI. The estimated cost of the culture media based on component prices was USD $ 3.01/L, which is 86.93% lower than BHI broth (USD $ 23.04/L). It was possible to design a "low-cost agro-industrial by-product-based culture media to produce L. lactis A12 and the two Priestia species under monoculture conditions.

In vitro digestive system simulation and anticancer activity of soymilk fermented by probiotics and synbiotics immobilised on agro-industrial residues.

In this study, a variety of probiotic strains, including Lactiplantibacillus plantarum, Lacticaseibacillus casei, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum, Limosilactobacillus reuteri, Lactobacillus delbrueckii subsp. bulgaricus, Lacticaseibacillus rhamnosus, and Bifidobacterium bifidum, were utilized for soymilk fermentation both as free cells and as synbiotics on agro-industrial residuals such as okara, whey protein, banana peels, apple pomace, sugarcane bagasse, orange peels, and lemon peels. Among these, Lacticaseibacillus rhamnosus emerged as the most significant strain for soymilk fermentation, exhibiting a viability of 10.47 log cfu/mL, a pH of 4.41, total acidity of 1.12%, and organic acid contents (lactic and acetic acid) of 11.20 and 7.50 g/L, respectively. As a synbiotic Lacticaseibacillus rhamnosus immobilised on okara, showed even more impressive results, with a viability of 12.98 log cfu/mL, a pH of 4.31, total acidity of 1.27%, and organic acid contents of 13.90 and 9.30 g/L, respectively. Over a 12-h fermentation period, cell viability values increased by 10.47-fold in free cells and 11.19-fold in synbiotics. Synbiotic supplementation of fermented soymilk proved more beneficial than free cells in terms of viability, acidity, and organic acid content. Furthermore, when synbiotic fermented soymilk was freeze-dried to simulate the digestive system in vitro, synbiotics and freeze-dried cells demonstrated superior gastrointestinal tract survival compared to free cells. Both the probiotic bacteria and the synbiotics exhibited cytotoxicity against colon and liver cancer cell lines, with half-maximal inhibitory concentrations ranging from 41.96 to 61.52 µL/well.

Exploiting tropical fruit processing coproducts as circular resources to promote the growth and maintain the culturability and functionality of probiotic lactobacilli.

This study evaluated the use of acerola (Malpighia glabra L., CACE), cashew (Anacardium occidentale L., CCAS), and guava (Psidium guayaba L., CGUA) fruit processing coproducts as substrates to promote the growth, metabolite production, and maintenance of the viability/metabolic activity of the probiotics Lactobacillus acidophilus LA-05 and Lacticaseibacillus paracasei L-10 during cultivation, freeze-drying, storage, and exposure to simulated gastrointestinal digestion. Probiotic lactobacilli presented high viable counts (≥8.8 log colony-forming units (CFU)/mL) and a short lag phase during 24 h of cultivation in CACE, CCAS, and CGUA. Cultivation of probiotic lactobacilli in fruit coproducts promoted sugar consumption, medium acidification, and production of organic acids over time, besides increasing the of several phenolic compounds and antioxidant activity. Probiotic lactobacilli cultivated in fruit coproducts had increased survival percentages after freeze-drying and during 120 days of refrigerated storage. Moreover, probiotic lactobacilli cultivated and freeze-dried in fruit coproducts had larger subpopulations of live and metabolically active cells when exposed to simulated gastrointestinal digestion. The results showed that fruit coproducts not only improved the growth and helped to maintain the viability and metabolic activity of probiotic strains but also enriched the final fermented products with bioactive compounds, being an innovative circular strategy for producing high-quality probiotic cultures.

Optimization of fermentation parameters to improve the biosynthesis of selenium nanoparticles by Bacillus licheniformis F1 and its comprehensive application.

BACKGROUND: Selenium nanoparticles (SeNPs) are increasingly gaining attention due to its characteristics of low toxicity, high activity, and stability. Additionally, Bacillus licheniformis, as a probiotic, has achieved remarkable research outcomes in diverse fields such as medicine, feed processing, and pesticides, attracting widespread attention. Consequently, evaluating the activity of probiotics and SeNPs is paramount. The utilization of probiotics to synthesize SeNPs, achieving large-scale industrialization, is a current hotspot in the field of SeNPs synthesis and is currently the most promising synthetic method. To minimize production costs and maximize yield of SeNPs, this study selected agricultural by-products that are nutrient-rich, cost-effective, and readily available as culture medium components. This approach not only fulfills industrial production requirements but also mitigates the impact on downstream processes. RESULTS: The experimental findings revealed that SeNPs synthesized by B. licheniformis F1 exhibited a spherical morphology with diameters ranging from 110 to 170 nm and demonstrating high stability. Both the secondary metabolites of B. licheniformis F1 and the synthesized SeNPs possessed significant free radical scavenging ability. To provide a more robust foundation for acquiring large quantities of SeNPs via fermentation with B. licheniformis F1, key factors were identified through single-factor experiments and response surface methodology (RSM) include a 2% seed liquid inoculum, a temperature of 37 ℃, and agitation at 180 rpm. Additionally, critical factors during the optimization process were corn powder (11.18 g/L), soybean meal (10.34 g/L), and NaCl (10.68 g/L). Upon validating the optimized conditions and culture medium, B. licheniformis F1 can synthesize nearly 100.00% SeNPs from 5 mmol/L sodium selenite. Subsequently, pilot-scale verification in a 5 L fermentor using the optimized medium resulted in a shortened fermentation time, significantly reducing production costs. CONCLUSION: In this study, the efficient production of SeNPs by the probiotic B. licheniformis F1 was successfully achieved, leading to a significant reduction in fermentation costs. The exploration of the practical applications of this strain holds significant potential and provides valuable guidance for facilitating the industrial-scale implementation of microbial synthesis of SeNPs.

LC-HRMS-based metabolomics and lipidomics analyses of a novel probiotic Akkermansia Muciniphila in response to different nutritional stimulations.

The mucin-degrading gut commensal Akkermansia muciniphila (A. muciniphila) negatively correlates with various diseases, including metabolic disorders, neurodegenerative disorders, and cancers, through interacting with host receptors by diverse molecules. Still, their exact metabolic capability within the nutrient-rich environment (such as in the human gut) is not fully characterized. Therefore, in the present study, we investigated the comprehensive metabolome and lipidome of A. muciniphila after supplementation of four major gut microbial nutrients: mucin, inorganic salts, bile salts, and short-chain fatty acids (SCFAs). Our results showed that mucin is the predominant driver of the different lipidomic and metabolomic profiles of A. muciniphila, and it promotes the overall growth of this bacteria. While the addition of inorganic salts, bile salts, and SCFAs was found to inhibit the growth of A. muciniphila. Interestingly, inorganic salts affected the purine metabolism in A. muciniphila cultures, while adding bile salts significantly increased the production of other bile acids and N-acyl amides. Lastly, SCFAs were identified to alter the A. muciniphila energy utilization of triglycerides, fatty acyls, and phosphatidylethanolamines. To our knowledge, this is the first study to examine the comprehensive lipidome and metabolome of A. muciniphila, which highlights the importance of nutritional impacts on the lipidome and metabolome of A. muciniphila and hence providing foundational knowledge to unveil the potential effects of A. muciniphila on host health.

Functional roles and engineering strategies to improve the industrial functionalities of lactic acid bacteria during food fermentation.

In order to improve the flavor profiles, food security, probiotic effects and shorten the fermentation period of traditional fermented foods, lactic acid bacteria (LAB) were often considered as the ideal candidate to participate in the fermentation process. In general, LAB strains possessed the ability to develop flavor compounds via carbohydrate metabolism, protein hydrolysis and amino acid metabolism, lipid hydrolysis and fatty acid metabolism. Based on the functional properties to inhibit spoilage microbes, foodborne pathogens and fungi, those species could improve the safety properties and prolong the shelf life of fermented products. Meanwhile, influence of LAB on texture and functionality of fermented food were also involved in this review. As for the adverse effect carried by environmental challenges during fermentation process, engineering strategies based on exogenous addition, cross protection, and metabolic engineering to improve the robustness and of LAB were also discussed in this review. Besides, this review also summarized the potential strategies including microbial co-culture and metabolic engineering for improvement of fermentation performance in LAB strains. The authors hope this review could contribute to provide an understanding and insight into improving the industrial functionalities of LAB.

Label-Free Monitoring of Coculture System Dynamics: Probing Probiotic and Cancer Cell Interactions via Infrared Spectroscopic Imaging.

Evaluating the dynamic interaction of microorganisms and mammalian cells is challenging due to the lack of suitable platforms for examining interspecies interactions in biologically relevant coculture conditions. In this work, we demonstrate the interaction between probiotic bacteria (Lactococcus lactis and Escherichia coli) and A498 human cancer cells in vitro, utilizing a hydrogel-based platform in a label-free manner by infrared spectroscopy. The L. lactis strain recapitulated in the compartment system secretes polypeptide molecules such as nisin, which has been reported to trigger cell apoptosis. We propose a mid-infrared (IR) spectroscopic imaging approach to monitor the variation of biological components utilizing kidney cells (A498) as a model system cocultured with bacteria. We characterized the biochemical composition (i.e., nucleic acids, protein secondary structures, and lipid conformations) label-free using an unbiased measurement. Several IR spectral features, including unsaturated fatty acids, ß-turns in protein, and nucleic acids, were utilized to predict cellular response. These features were then applied to establish a quantitative relationship through a multivariate regression model to predict cellular dynamics in the coculture system to assess the effect of nisin on A498 kidney cancer cells cocultured with bacteria. Overall, our study sheds light on the potential of using IR spectroscopic imaging as a label-free tool to monitor complex microbe-host cell interactions in biological systems. This integration will enable mechanistic studies of interspecies interactions with insights into their underlying physiological processes.

Whole-genome analysis, evaluation and regulation of in vitro and in vivo GABA production from Levilactobacillus brevis YSJ3.

Gamma-aminobutyric acid (GABA) produced by lactic acid bacteria (LAB) is safe and has several health benefits. Levilactobacillus brevis YSJ3 was selected from 110 LAB. It exhibited the highest in vitro GABA production level of 970.10 µg/mL. Whole-genome analysis revealed that L. brevis YSJ3 contained gadR, gadC, gadB and gadA. Furthermore, the Luedeking-Piret model was fitted, which indicated that GABA production was divided into three stages. The gadR 0079, gadC 0080, and gadB 0081 were confirmed to promote GABA synthesis. Moreover, 55 metabolites, particularly those involved in arginine metabolism, were significantly different at 6 and 20 h of cultivation. Notably, L. brevis YSJ3 significantly improved sleep in mice and increased GABA levels in the mice's gut compared with the control group. This suggests that the oral administration of L. brevis YSJ3 improves sleep quality, probably by increasing intestinal GABA levels. Overall, L. brevis YSJ3 was confirmed as a GABA-producing strain in vitro and in vivo, making it a promising probiotic candidate for its application in food and medicine.

Microbial community-scale metabolic modelling predicts personalized short-chain fatty acid production profiles in the human gut.

Microbially derived short-chain fatty acids (SCFAs) in the human gut are tightly coupled to host metabolism, immune regulation and integrity of the intestinal epithelium. However, the production of SCFAs can vary widely between individuals consuming the same diet, with lower levels often associated with disease. A systems-scale mechanistic understanding of this heterogeneity is lacking. Here we use a microbial community-scale metabolic modelling (MCMM) approach to predict individual-specific SCFA production profiles to assess the impact of different dietary, prebiotic and probiotic inputs. We evaluate the quantitative accuracy of our MCMMs using in vitro and ex vivo data, plus published human cohort data. We find that MCMM SCFA predictions are significantly associated with blood-derived clinical chemistries, including cardiometabolic and immunological health markers, across a large human cohort. Finally, we demonstrate how MCMMs can be leveraged to design personalized dietary, prebiotic and probiotic interventions aimed at optimizing SCFA production in the gut. Our model represents an approach to direct gut microbiome engineering for precision health and nutrition.

Exploring the interaction and impact of probiotic and commensal bacteria on vitamins, minerals and short chain fatty acids metabolism.

There is increasing evidence that probiotic and commensal bacteria play a role in substrate metabolism, energy harvesting and intestinal homeostasis, and may exert immunomodulatory activities on human health. In addition, recent research suggests that these microorganisms interact with vitamins and minerals, promoting intestinal and metabolic well-being while producing vital microbial metabolites such as short-chain fatty acids (SCFAs). In this regard, there is a flourishing field exploring the intricate dynamics between vitamins, minerals, SCFAs, and commensal/probiotic interactions. In this review, we summarize some of the major hypotheses beyond the mechanisms by which commensals/probiotics impact gut health and their additional effects on the absorption and metabolism of vitamins, minerals, and SCFAs. Our analysis includes comprehensive review of existing evidence from preclinical and clinical studies, with particular focus on the potential interaction between commensals/probiotics and micronutrients. Finally, we highlight knowledge gaps and outline directions for future research in this evolving field.

Production of CaCO3-single-coated probiotics and evaluation of their spectroscopic properties, morphological characteristics, viability, and intestinal delivery efficiency.

The intake of probiotics offers various health benefits; however, their efficacy depends on the maintenance of viability during industrial processing and digestion. Probiotic viability can be compromised during encapsulation, freeze-drying, storage, and digestion, necessitating multiple coatings. This complicates production and raises costs. In this study, CaCO3-single-coated probiotics (CSCPs) were prepared, an approach rarely reported before. Through instrumental analyses, the encapsulation of probiotics within CaCO3 was confirmed, ensuring their high viability. This proposed technology effectively preserves the viability of probiotics during the encapsulation and freeze-drying processes, resulting in minimal cell loss. Moreover, CSCPs demonstrated exceptional viability performance under simulated gastric and intestinal conditions. Notably, 100% of these microorganisms reached the intestines, delivering over 10 billion CFUs of probiotics in a viable state. This study highlights the potential of CSCPs as a feasible solution for overcoming probiotic encapsulation challenges and optimizing therapeutic benefits.

Process optimization, growth kinetics, and antioxidant activity of germinated buckwheat and amaranth-based yogurt mimic.

This study aimed to investigate the integration of cereal and germinated pseudocereals into set-type yogurt mimic, resulting in a novel and nutritious product. Four groups of yogurts mimic, namely CPY-1, CPY-2, CPY-3, and CPY-4, were prepared using different probiotic cultures, including L. acidophilus 21, L. plantarum 14, and L. rhamnosus 296 along with starter cultures. Notably, CPY-2 cultured with L. plantarum and L. rhamnosus and incubated for 12 h exhibited the most desirable attributes. The resulting yogurt demonstrated an acidity of 0.65%, pH of 4.37 and a probiotic count of 6.38 log CFU/mL. The logistic growth model fit revealed maximum growth rates (k, 1/h) and maximum bacterial counts (Nm log CFU/mL) for each CPY variant. The results revealed that CPY-2 significantly improved protein, dietary fiber, phenols and antioxidant capacities compared to the control. Scanning electron microscopy showed more structured and compact casein network in CPY-2, highlighting its superior textural characteristics. Overall, this study demonstrates the incorporation of cereal and germinated pseudocereals into set-type yogurt mimic offers health benefits through increased dietary fiber and ß-glucan.

Christensenella strain resources, genomic/metabolomic profiling, and association with host at species level.

The gut commensal bacteria Christensenellaceae species are negatively associated with many metabolic diseases, and have been seen as promising next-generation probiotics. However, the cultured Christensenellaceae strain resources were limited, and their beneficial mechanisms for improving metabolic diseases have yet to be explored. In this study, we developed a method that enabled the enrichment and cultivation of Christensenellaceae strains from fecal samples. Using this method, a collection of Christensenellaceae Gut Microbial Biobank (ChrisGMB) was established, composed of 87 strains and genomes that represent 14 species of 8 genera. Seven species were first described and the cultured Christensenellaceae resources have been significantly expanded at species and strain levels. Christensenella strains exerted different abilities in utilization of various complex polysaccharides and other carbon sources, exhibited host-adaptation capabilities such as acid tolerance and bile tolerance, produced a wide range of volatile probiotic metabolites and secondary bile acids. Cohort analyses demonstrated that Christensenellaceae and Christensenella were prevalent in various cohorts and the abundances were significantly reduced in T2D and OB cohorts. At species level, Christensenellaceae showed different changes among healthy and disease cohorts. C. faecalis, F. tenuis, L. tenuis, and Guo. tenuis significantly reduced in all the metabolic disease cohorts. The relative abundances of C. minuta, C. hongkongensis and C. massiliensis showed no significant change in NAFLD and ACVD. and C. tenuis and C. acetigenes showed no significant change in ACVD, and Q. tenuis and Geh. tenuis showed no significant change in NAFLD, when compared with the HC cohort. So far as we know, this is the largest collection of cultured resource and first exploration of Christensenellaceae prevalences and abundances at species level.

Effects of gut bacteria and their metabolites on gut health of animals.

The intestine tract is a vital site for the body to acquire nutrients, serving as the largest immune organ. Intestinal health is crucial for maintaining a normal physiological state. Abundant microorganisms reside in the intestine, colonized in a symbiotic manner. These microorganisms can generate various metabolites that influence host physiological activities. Microbial metabolites serve as signaling molecules or metabolic substrates in the intestine, and some intestinal microorganisms act as probiotics and promote intestinal health. Researches on host, probiotics, microbial metabolites and their interactions are ongoing. This study reviews the effects of gut bacteria and their metabolites on intestinal health to provide useful references for animal husbandry.

The roles of Saccharomyces cerevisiae on the bioaccessibility of phenolic compounds.

Phenolic compounds are a group of non-essential dietary compounds that are widely recognized for their beneficial health effects, primarily due to their bioactive properties. These compounds which found in a variety of plant-based foods, including fruits, vegetables, and grains are known to possess antimicrobial, antioxidant, anti-inflammatory, and anti-carcinogenic properties. However, the health effects of these compounds depend on their bioaccessibility and bioavailability. In recent years, there has been growing interest in the use of probiotics for promoting human health. Saccharomyces cerevisiae is a yeast with potential probiotic properties and beneficial health effects. Biosorption of phenolic compounds on Saccharomyces cerevisiae cell walls improves their bioaccessibility. This characteristic has also allowed the use of this yeast as a biosorbent in the biosorption process due to its low cost, safety, and easy availability. S. cerevisiae enhances the bioaccessibility of phenolic compounds as a delivery system under in vitro digestion conditions. The reason for this phenomenon is the protective effects of yeast on various phenolic compounds under digestion conditions. This article shows the role of S. cerevisiae yeast on the bioaccessibility of various phenolic compounds and contributes to our understanding of the potential impact of yeasts in human health.

Metabolomics analysis of potential functional metabolites in synbiotic ice cream made with probiotic Saccharomyces cerevisiae var. boulardii CNCM I-745 and prebiotic inulin.

Probiotic lactic acid bacteria have been widely studied, but much less was focused on probiotic yeasts in food systems. In this study, probiotic Saccharomyces cerevisiae var. boulardii CNCM I-745 was employed to prepare ice cream added with and without inulin (1%, w/v). Metabolomics analysis on the effect of inulin showed 84 and 147 differentially expressed metabolites identified in the ice cream samples from day 1 and day 30 of storage (-18 °C), respectively. Various potential functional metabolites were found, including citric acid, ornithine, D-glucuronic acid, sennoside A, stachyose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, cis-aconitic acid, gamma-aminobutyric acid, L-threonine, L-glutamic acid, tryptophan, benzoic acid, and trehalose. Higher expression of these metabolites suggested their possible roles through relevant metabolic pathways in improving survivability of the probiotic yeast and functionality of ice cream. This study provides further understanding on the metabolic characteristics of probiotic yeast that potentially affect the functionality of ice cream.

Effects of fructooligosaccharides and Saccharomyces boulardii on the compositional structure and metabolism of gut microbiota in students.

Fructooligosaccharides (FOS) are a common prebiotic widely used in functional foods. Meanwhile, Saccharomyces boulardii is a fungal probiotic frequenly used in the clinical treatment of diarrhea. Compared with single use, the combination of prebiotics and probiotics as symbiotics may be more effective in regulating gut microbiota as recently reported in the literature. The present study aimed to investigate the effects of FOS, S. boulardii and their combination on the structure and metabolism of the gut microbiota in healthy primary and secondary school students using an in vitro fermentation model. The results indicated that S. boulardii alone could not effectively regulate the community structure and metabolism of the microbiota. However, both FOS and the combination of FOS and S. boulardii could effectively regulate the microbiota, significantly inhibiting the growth of Escherichia-Shigella and Bacteroides, and controlling the production of the gases including H2S and NH3. In addition, both FOS and the combination could significantly promote the growth of Bifidobacteria and Lactobacillus, lower environmental pH, and enhance several physiological functions related to synthesis and metabolism. Nevertheless, the combination had more unique benefits as it promoted the growth of Lactobacillus, significantly increased CO2 production and enhanced the functional pathways of carbon metabolism and pyruvic acid metabolism. These findings provide guidance for clinical application and a theoretical basis for the development of synbiotic preparations.

A Novel Bacillus amyloliquefaciens Specifically Improving the Solubility and Antioxidant Activities of Edible Bird's Nest.

Edible bird's nest (EBN), a most highly priced and valuable foodstuff, contains high percentage of proteins and carbohydrates. However, proteins adhering to these carbohydrates make the EBN hard and tough, which need to be boiled as the bird's nest soup to make the Chinese cuisine. To overcome the hard and tough texture of EBN and improve the digestion degrees, the present study screened and identified a probiotic strain Bacillus amyloliquefaciens YZW02 from 5-year stored EBN sample completely solubilizing EBN for the first time. The 24-h B. amyloliquefaciens fermented EBN contained 20.30-21.48 mg/mL of the soluble protein contents with a recovery rate of 98-100%, DPPH radical scavenging rate of 84.76% and ABTS radical scavenging capacity of 41.05%. The mixed fermentation of B. amyloliquefaciens YZW02 and Bacillus natto BN1 were further applied to improve the low-MW peptide percentages and antioxidant activities. The mixed-fermentation of B. natto BN1 with 4-h cultured B. amyloliquefaciens YZW02 had the lowest percentage (82.23%) of >12-kDa proteins/peptides and highest percentages of 3-12 kDa, 1-3 kDa and 0.1-1 kDa peptides of 8.6% ± 0.08, 7.57% ± 0.09, 1.77% ± 0.05 and 0.73% ± 0.05, with the highest DPPH, ABTS and •OH scavenging capacity of 90.23%, 46.45% and 49.12%, respectively. These findings would provide an efficient strategy for improving the solubility and antioxidant activities of EBNs.