Effect and potential mechanism of nitrite reductase B on nitrite degradation by Limosilactobacillus fermentum RC4.

Nitrite has the potential risk of hypoxic poisoning or cancer in pickled food. In our previous study, Limosilactobacillus fermentum (L. fermentum) RC4 is effective in nitrite degradation by producing nitrite reductase B (NirB). To investigate the detailed mechanism from the genome, response, and regulation of NirB, the whole-genome sequence of L. fermentum RC4 was analyzed, the L. fermentum-EGFP-nirB with enhanced green fluorescent protein (EGFP) labeled the nitrite reductase large subunit nirB, and the recombined L. fermentum-NirB with overexpression NirB strain was conducted. The key genes within the dominant metabolism pathways may be involved in stress tolerance to regulate the degrading process. The green fluorescence density of EGFP indicated that NirB activity has a threshold and peaked under 300 mg/L nitrite concentration. NirB overexpressed in L. fermentum RC4 boosted the enzyme activity by 39.6% and the degradation rate by 10.5%, when fermented in 300 mg/L for 40 h, compared to the control group. RNA-seq detected 248 differential genes mainly enriched in carbohydrate, amino acid, and energy metabolism. The ackA gene for pyruvate metabolism and the mtnN gene for cysteine metabolism were up-regulated. NirB regulates these genes to produce acid and improve stress resistance for L. fermentum RC4 to accelerate nitrite degradation.

Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.

The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.

Elimination of Pathogen Biofilms via Postbiotics from Lactic Acid Bacteria: A Promising Method in Food and Biomedicine.

Pathogenic biofilms provide a naturally favorable barrier for microbial growth and are closely related to the virulence of pathogens. Postbiotics from lactic acid bacteria (LAB) are secondary metabolites and cellular components obtained by inactivation of fermentation broth; they have a certain inhibitory effect on all stages of pathogen biofilms. Postbiotics from LAB have drawn attention because of their high stability, safety dose parameters, and long storage period, which give them a broad application prospect in the fields of food and medicine. The mechanisms of eliminating pathogen biofilms via postbiotics from LAB mainly affect the surface adhesion, self-aggregation, virulence, and QS of pathogens influencing interspecific and intraspecific communication. However, there are some factors (preparation process and lack of target) which can limit the antibiofilm impact of postbiotics. Therefore, by using a delivery carrier and optimizing process parameters, the effect of interfering factors can be eliminated. This review summarizes the concept and characteristics of postbiotics from LAB, focusing on their preparation technology and antibiofilm effect, and the applications and limitations of postbiotics in food processing and clinical treatment are also discussed.

Legume protein fermented by lactic acid bacteria: Specific enzymatic hydrolysis, protein composition, structure, and functional properties.

In recent years, with increasing emphasis on healthy, green, and sustainable consumption concepts, plant-based foods have gained popularity among consumers. As widely sourced plant-based raw materials, legume proteins are considered sustainable and renewable alternatives to animal proteins. However, legume proteins have limited functional properties, which hinder their application in food products. LAB fermentation is a relatively natural processing method that is safer than chemical/physical modification methods and can enrich the functional properties of legume proteins through biodegradation and modification. Therefore, changes in legume protein composition, structure, and functional properties and their related mechanisms during LAB fermentation are described. In addition, the specific enzymatic hydrolysis mechanisms of different LAB proteolytic systems on legume proteins are also focused in this review. The unique proteolytic systems of different LAB induce specific enzymatic hydrolysis of legume proteins, resulting in the production of hydrolysates with diverse functional properties, including solubility, emulsibility, gelability, and foamability, which are determined by the composition (peptide/amino acid) and structure (secondary/tertiary) of legume proteins after LAB fermentation. The correlation between LAB-specific enzymatic hydrolysis, protein composition and structure, and protein functional properties will assist in selecting legume protein raw materials and LAB strains for legume plant-based food products and expand the application of legume proteins in the food industry.

Preparation of modified chitosan-based nano-TiO2-nisin composite packaging film and preservation mechanism applied to chilled pork.

Here, we developed a nano-TiO2-nisin-modified chitosan composite packaging film and investigated its properties and antibacterial activity, as well as its effect on chilled pork preservation time. The results indicated that the preservation time of chilled pork coated with a nano-TiO2-nisin-modified chitosan film (including 0.7 g/L nano-TiO2, irradiated with ultraviolet light for 40 min, and dried for 6 h) followed by modified atmosphere packaging (50% CO2 + 50% N2) increased from 7 to 20 days at 4 °C. Both nano-TiO2 and nisin enhanced the mechanical strength of the chitosan film, and nisin promoted nano-TiO2 dispersion and compatibility in chitosan. Treatment with 0.4 g/L nano-TiO2 for 60 min considerably inhibited spoilage bacteria, particularly Acinetobacter johnnii XBB1 (A. johnnii XBB1). As nano-TiO2 concentration and photocatalytic time increased, K+, Ca2+, and Mg2+ leakage in A. johnnii XBB1 increased but Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities decreased. In A. johnnii XBB1, TiO2 significantly downregulated the expression of putrefaction-related genes such as cysM and inhibited cell self-regulation and membrane wall system repair. Therefore, our nano-TiO2-nisin-modified chitosan film could extend the shelf life without the addition of any chemical preservatives, demonstrating great potential for application in food preservation.

Phosphate type dependent phosphorylation on the interfacial and emulsion stabilizing behaviors of goose liver protein: Perspective of protein charging.

Elucidation on the emulsifying behaviors of goose liver protein (GLP) from interfacial perspective was scarce when protein charging was altered. This work aimed to elucidate the role of phosphorylation on the interfacial associative interaction and then emulsion stabilizing properties of GLP using three structurally relevant phosphates of sodium trimetaphosphate (STMP), sodium tripolyphosphate (STPP) and sodium pyrophosphate (TSPP). A monotonic increment of protein charging treated from STMP, STPP to TSPP caused progressively increased particle de-aggregation, surface hydrophobicity and structural flexibility of GLP. Compared with STMP and TSPP, STPP phosphorylation rendered the most strengthened interfacial equilibrium pressure (11.98 ± 0.24 mN/m) due to sufficient unfolding but moderated charging character conveyed. Desorption curve and interfacial protein microstructure indicated that STPP phosphorylation caused the highest interfacial connectivity between proteins adsorbed onto the same droplet, as was also verified by interfacial elastic modulus (10.3 ± 0.21 mN/m). STPP treated GLP also yielded lowest droplet size (8.16 ± 0.10 µm), flocculation (8.18%) and Turbiscan stability index (8.78 ± 0.36) of emulsion but most improved microrheological properties. Overall, phosphorylation functioned itself in fortifying the intradroplet protein-protein interaction but restraining the interdroplet aggregation, and STPP phosphorylation endowed the protein with most enhanced interfacial stabilization and emulsifying efficiency.

New clues for postbiotics to improve host health: a review from the perspective of function and mechanisms.

Strain activity and stability severely limit the beneficial effects of probiotics in modulating host health. Postbiotics have emerged as a promising alternative as they can provide similar or even enhanced efficacy to probiotics, even under inactivated conditions. This review introduces the ingredients, preparation, and identification techniques of postbiotics, focusing on the comparison of the advantages and limitations between probiotics and postbiotics based on their mechanisms and applications. Inactivation treatment is the most significant difference between postbiotics and probiotics. We highlight the use of emerging technologies to inactivate probiotics, optimize process conditions to maintain the activity of postbiotics, or scale up their production. Postbiotics have high stability which can overcome unfavorable factors, such as easy inactivation and difficult colonization of probiotics after entering the intestine, and are rapidly activated, allowing continuous and rapid optimization of the intestinal microecological environment. They provide unique mechanisms, and multiple targets act on the gut-organ axis, co-providing new clues for the study of the biological functions of postbiotics. We summarize the mechanisms of action of inactivated lactic acid bacteria, highlighting that the NF-κB and MAPK pathways can be used as immune targeting pathways for postbiotic modulation of host health. Generally, we believe that as the classification, composition, and efficacy mechanism of postbiotics become clearer they will be more widely used in food, medicine, and other fields, greatly enriching the dimensions of food innovation. © 2024 Society of Chemical Industry.

Binary probiotic fermentation promotes signal (cyclic AMP) exchange to increases the number of viable probiotics, anthocyanins and polyphenol content, and the odor scores of wolfberry fermented beverages.

The effects and underlying molecular mechanisms of binary probiotics (Lactiplantibacillus plantarum subsp. plantarum CGMCC 1.5953 and Lacticaseibacillus casei CGMCC 1.5956) on the quality of wolfberry fermented beverages (WFB) were investigated. The results indicated that binary probiotics increased the number of probiotics, anthocyanin (89.92 ± 1.64 mg/L), polyphenol content (283.04 ± 3.81 µg/mL), and odor score (24.19) in WFB. Metabolomics found that they could enhance signal exchange (cyclic AMP) between binary probiotics and improve the utilization of citrulline, d-proline, d-glucose, and d-galactose through galactose metabolism and amino acid biosynthesis pathway to promote probiotics growth. Furthermore, HS-SPME-GC-MS and GS-IMS revealed that the improvement in flavor was mainly due to an increase in the content of the aromatic flavor substances 3-heptanol, glutaraldehyde, and 2-heptanone, and a decrease in the content of the off-flavor substances methyl isobutyl ketone-D and 2-undecanone. This is strategically important for the development of WFB with high probiotic content and unique flavor.

Effects of Semen Ziziphi Spinosae extract and binary probiotics co-fermentation on the quality of yogurt and their underlying molecular mechanisms.

The study aimed to investigate the impact of water-soluble extract from Semen Ziziphi Spinosae (SZSE) on yogurt quality and understand the underlying mechanism. The results demonstrated that adding 0.5% (w/v) SZSE had a significant effect on reducing yogurt syneresis and resulted in a more compact and uniform casein gel. Notably, the co-fermented yogurt with binary probiotics (Lacticaseibacillus casei CGMCC1.5956 and Levilactobacillus brevis CGMCC1.5954) along with SZSE led to increased viable probiotics and a higher odor score (23.23). This effect might be attributed to the increased amino acid utilization by binary probiotics through biosynthesis of valine, leucine and isoleucine, metabolic pathways, and amino acid biosynthesis to produce amino acid derivatives such as N5-(l-1-carboxyethyl)-l-ornithine and diaminopyrimidine acid. The yogurt contained 79 volatile flavor compounds, with hexanoic acid, 2-heptanone, and 2-nonanone potentially contributing to the high odor scores. These findings have strategic implications for developing yogurt with high gel characteristics and distinctive flavor.

Recent Advances on Antimicrobial Peptides from Milk: Molecular Properties, Mechanisms, and Applications.

Traditional antibiotics are facing a tremendous challenge due to increased antimicrobial resistance; hence, there is an urgent need to find novel antibiotic alternatives. Milk protein-derived antimicrobial peptides (AMPs) are currently attracting substantial attention considering that they showcase an extensive spectrum of antimicrobial activities, with slower development of antimicrobial resistance and safety of raw materials. This review summarizes the molecular properties, and activity mechanisms and highlights the applications and limitations of AMPs derived from milk proteins comprehensively. Also the analytical technologies, especially bioinformatics methodologies, applied in the process of screening, identification, and mechanism illustration of AMPs were underlined. This review will give some ideas for further research and broadening of the applications of milk protein-derived AMPs in the food field.

Screening of an efficient cholesterol-lowering strain of Lactiplantibacillus plantarum 54-1 and investigation of its degradation molecular mechanism.

In this study, an efficient cholesterol-lowering strain of Lactiplantibacillus plantarum 54-1 was screened and its degradation molecular mechanism was investigated. Furthermore, a novel practical MRS medium for screening cholesterol-lowering lactic acid bacteria (LAB) was developed based on ultrasound treatment. L. plantarum 54-1 was found to have the highest ability to eliminate cholesterol (340.69 ± 5.87 µg/mL). According to SEM and the count of viable LAB results, the morphology of LAB in the cholesterol-containing medium developed in this experiment was close to the normal (full and smooth), and it can grow normally. Metabolomics revealed that L. plantarum 54-1 initially converted a portion of cholesterol to 7α-hydroxy-cholesterol and then to the key metabolite taurine, via the phosphotransferase system. These metabolites were further transformed into L-alanine, L-lysine, N6-Acetyl-L-lysine, (R)-b-aminoisobutyric acid, and 2-oxoarginine, through glycine, serine, and threonine metabolism, citrate cycle, D-arginine and D-ornithine metabolism, lysine degradation, and pyruvate metabolism pathways. Prokaryotic reference transcriptomics found that this may be mainly regulated by the bsh, phnE, ptsP, B0667_RS04545, and B0667_RSRS12300 genes, which was further validated by qPCR. Furthermore, molecular docking results demonstrated that 8 differential metabolites might bind to another portion of cholesterol via PI-PI conjugation and hydrophobic interactions and lower cholesterol via co-sedimentation. This study has strategic implications for developing probiotic powder food that lowers cholesterol.

Astaxanthin nanoparticles ameliorate dextran sulfate sodium-induced colitis by alleviating oxidative stress, regulating intestinal flora, and protecting the intestinal barrier.

This study aimed to develop a novel astaxanthin nanoparticle using gum arabic (GA) and whey protein powder enriched with milk fat globule membranes (MFGM-WPI) as carriers and to investigate its effect and alleviation mechanism on colitis in mice. We demonstrated that MFGM-GA-astaxanthin could improve the bioaccessibility of astaxanthin and cope with oxidative stress more effectively in a Caco-2 cell model. In vivo studies demonstrated that MFGM-GA-astaxanthin alleviated colitis symptoms and repaired intestinal barrier function by increasing the expression of mucin 2, occludin, and zonula occludens-1. This was attributed to the alleviating effect of MFGM-GA-astaxanthin on oxidative stress. Moreover, MFGM-GA-astaxanthin restored the abnormalities of flora caused by dextran sulfate sodium, including Lactobacillus, Bacteroides, Ruminococcus, and Shigella. This study provides a basis for the therapeutic effect of astaxanthin nanoparticles on colon diseases.

Transcriptomic Responses to Nitrite Degradation by Limosilactobacillus fermentum RC4 and Effect of ndh Gene Overexpression on Nitrite Degradation.

The excessive nitrite residue may increase cell damage and cancer risk. Limosilactobacillu fermentum RC4 exhibited excellent nitrite degradation ability. Herein, the molecular mechanism of nitrite degradation by L. fermentum RC4 was studied by integrating scanning electron microscopy analysis, transcriptomics, and gene overexpression. The results demonstrated that the gene profile of RC4 cultured in MRS broth with 0, 100, and 300 mg/L NaNO2 varied considerably; RC4 responded to nitrite degradation by regulating pyruvate metabolism, energy synthesis, nitrite metabolism, redox equilibrium, protein protection, and signaling. High nitrite concentrations affected the morphology of RC4 with a longer phenotype, rough and wrinkle cell and reduced cell surface hydrophobicity. Moreover, an up-regulated expression of gene ndh encoding NADH dehydrogenase, which provides electrons for nitrite reduction by catalyzing NADH, was identified when RC4 was exposed to nitrite. Overexpression of ndh in RC4 increased the nitrite degradation rate by 2-9.5% in MRS broth with 100 mg/L NaNO2. Thus, the findings of this study could be helpful for the application of L. fermentum to reduce nitrite residues and improve food safety in fermented food products.

Protection Mechanism of Metal Ion Pre-Stress on Lactobacillus acidophilus CICC 6074 under Acid Tolerance.

The prerequisite for the probiotic effect of lactic acid bacteria is that they could survive the acid stress environment of production and application. In this experiment, the mechanism for the effect of different metal ion pre-stress on the acid-tolerant survival of Lactobacillus was investigated. Scanning electron microscopy, Fourier infrared spectroscopy, and flow cytometry were used to analyze the condition of bacteria after acid treatment, which revealed that different metal ion pre-stress could improve the survival ability of Lactobacillus acidophilus CICC 6074 under low acid conditions by improving cell morphology, mitigating cell membrane damage, and regulating surface protein expression. Furthermore, Tandem Mass Tags (TMT) proteomic analysis revealed that Mn2+ pre-stress showed relatively more superior protective effects on acid tolerance in L. acidophilus CICC 6074 through activation of DNA replication, RNA synthesis, S-layer protein secretion, H+-ATPase enzyme activity, etc. This study will provide new ideas and a theoretical basis for the development and application of lactic acid bacteria.

Enhancement of gum Arabic/casein microencapsulation on the survival of Lactiplantibacillus plantarum in the stimulated gastrointestinal conditions.

Probiotic products that contain lactobacilli have long histories of safe use as Lactobacillus strains have many physiological functions in the gastrointestinal tract (GIT). However, the viability of probiotics can be affected by food processing and the adverse environment. This study investigated the O/W (Oil-in-water emulsions) emulsions formed by coagulation of casein/GA (Gum Arabic) complexes for Lactiplantibacillus plantarum microencapsulation, and the stability of the strains during gastrointestinal environment were also determined. The results showed that the particle size of the emulsion decreased from 9.72 µm to 5.48 µm when the GA concentration increased from 0 to 2 (w/v), and the emulsion particles were found to be more uniform as observed by CLSM (Confocal Laser Scanning Microscope). The surface of this microencapsulated casein/GA composite forms smooth, dense agglomerates and has high viscoelasticity, which effectively improved casein's emulsifying activity (8.66 ± 0.17 m2/g). After the casein/GA complexes microencapsulation, a higher viable count was detected after gastrointestinal digestion in vitro, and the activity of L. plantarum is more stable (about 7.51 log CFU/mL) during 35 days of storage at 4 °C. The results of study will help to design lactic acid bacteria encapsulation systems based on the GIT environment for the oral delivery strategy.

Relationship of Gene-Structure-Antioxidant Ability of Exopolysaccharides Derived from Lactic Acid Bacteria: A Review.

Polysaccharides derived from lactic acid bacteria (LAB) have widespread industrial applications owing to their excellent safety profile and numerous biological properties. The antioxidant activity of exopolysaccharides (EPS) offers defense against disease conditions caused by oxidative stress. Several genes and gene clusters are involved in the biosynthesis of EPS and the determination of their structures, which play an important role in modulating their antioxidant ability. Under conditions of oxidative stress, EPS are involved in the activation of the nonenzyme (Keap1-Nrf2-ARE) response pathway and enzyme antioxidant system. The antioxidant activity of EPS is further enhanced by the targeted alteration of their structures, as well as by chemical methods. Enzymatic modification is the most commonly used method, though physical and biomolecular methods are also frequently used. A detailed summary of the biosynthetic processes, antioxidant mechanisms, and modifications of LAB-derived EPS is presented in this paper, and their gene-structure-function relationship has also been explored.

Nanocoating of lactic acid bacteria: properties, protection mechanisms, and future trends.

Lactic acid bacteria (LAB) is a type of probiotic that may benefit intestinal health. Recent advances in nanoencapsulation provide an effective strategy to protect them from harsh conditions via surface functionalization coating techniques. Herein, the categories and features of applicable encapsulation methods are compared to highlight the significant role of nanoencapsulation. Commonly used food-grade biopolymers (polysaccharides and protein) and nanomaterials (nanocellulose and starch nanoparticles) are summarized along with their characteristics and advances to demonstrate enhanced combination effects in LAB co-encapsulation. Nanocoating for LAB provides an integrity dense or smooth layer attributed to the cross-linking and assembly of the protectant. The synergism of multiple chemical forces allows for the formation of subtle coatings, including electrostatic attractions, hydrophobic interactions, π-π, and metallic bonds. Multilayer shells have stable physical transition properties that could increase the space between the probiotic cells and the outer environment, thus delaying the microcapsules burst time in the gut. Probiotic delivery stability can be promoted by enhancing the thickness of the encapsulated layer and nanoparticle binding. Maintenance of benefits and minimization of nanotoxicity are desirable, and green synthesized nanoparticles are emerging. Future trends include optimized formulation, especially using biocompatible materials, protein or plant-based materials, and material modification.

Plant-based meat analogs and fat substitutes, structuring technology and protein digestion: A review.

There is currently an increasing trend in the consumption of meat analogs and fat substitutes due to the health hazards by excessive consumption of meat. Simulating the texture and mouthfeel of meat through structured plant-derived polymers has become a popular processing method. In this review, the mechanical structuring technology of plant polymers for completely replacing real meat is mainly introduced in this review, which mainly focuses on the parameters and principles of mechanical equipment for the production of vegan meat. The difference in composition between plant meat and real meat is mainly reflected in the protein, and particular attention should be paid to the digestive characteristics of plant meat protein in the gastrointestinal tract. Therefore, the differences in the protein digestibility properties of meat analogs and real meat is discussed in this review, focusing primarily on protein digestibility and peptide/amino acid composition of mechanically structured vegan meats. In terms of fat substitutes for meat products, the types of plant polymer colloidal systems used for meat fat substitutes is comprehensively introduced, including emulsion, hydrogel and oleogel.

Research Progress of Nitrite Metabolism in Fermented Meat Products.

Nitrite is a common color and flavor enhancer in fermented meat products, but its secondary amines may transfer to the carcinogen N-nitrosamines. This review focuses on the sources, degradation, limitations, and alteration techniques of nitrite. The transition among NO3- and NO2-, NH4+, and N2 constitutes the balance of nitrogen. Exogenous addition is the most common source of nitrite in fermented meat products, but it can also be produced by contamination and endogenous microbial synthesis. While nitrite is degraded by acids, enzymes, and other metabolites produced by lactic acid bacteria (LAB), four nitrite reductase enzymes play a leading role. At a deeper level, nitrite metabolism is primarily regulated by the genes found in these bacteria. By incorporating antioxidants, chromogenic agents, bacteriostats, LAB, or non-thermal plasma sterilization, the amount of nitrite supplied can be decreased, or even eliminated. Finally, the aim of producing low-nitrite fermented meat products is expected to be achieved.

Effect of lentinan on gelling properties and structural changes of goose myofibrillar protein under oxidative stress.

BACKGROUND: The reduction of protein oxidation is important for maintaining the product quality of reconstituted meat. In this study, the dose-dependent effects of lentinan (LNT) on gelling properties and chemical changes in oxidatively stressed goose myofibrillar protein were investigated. RESULTS: Myofibrillar protein (MP) with 200 µmol g-1 protein LNT increased gel strength by 87.90 ± 9.26% in comparison with LNT-free myofibrillar protein after oxidation. Scanning electron microscopy analysis revealed that the gel network containing LNT was compact, with small pores and uniform distribution. The absolute value of the zeta potential reduced significantly following oxidation of LNT with 200 µmol g-1 protein at 4 °C for 12 h compared with the zeta potential without LNT, according to the laser particle size analyzer. The incorporation of LNT increased protein solubility and -SH content, inhibited carbonyl formation, enhanced α-helix content and tryptophan intrinsic fluorescence intensity, and reduced exposure of hydrophobic groups and protein aggregation. CONCLUSION: The results indicated that adding LNT to myofibrillar protein could improve gel. This is related to its protective effect on conformational changes in the oxidation system. Lentinan is therefore recommended for oxidatively stressed goose meat processing to enhance the MP gelling potential. © 2023 Society of Chemical Industry.