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Lactic acid bacteria (LAB) are usually freeze-dried into powder for transportation and storage, with the bacterial membrane playing a crucial role in this process. However, different strains exhibit different levels of freeze-drying resistance in their cell membranes. In this study, Lacticaseibacillus paracasei ( L. paracasei ) strains 1F20, K56, and J5, demonstrating survival rates of 59.51, 25.86, and 4.05% after freeze-drying, respectively, were selected. The membrane structure and composition of these strains were subsequently analyzed. Bacterial live/dead staining results indicated that strain 1F20 maintained the highest membrane integrity after drying. Nontargeted lipidomics analysis revealed six differential lipid species that differed in membrane lipid compositions. KEGG functional enrichment analysis revealed 13 significantly different pathways, with glycerophospholipid metabolism being the most critical. This study explored the membrane composition of L. paracasei at the cellular level and identified key lipid species associated with freeze-drying resistance, providing a reference for screening highly resistant strains.

A variety of bioactive peptides with unique and diverse structures could be found in microalgae with various bioactivities including antioxidant, antihypertensive, and antibacterial bioactivities. Food products containing microalgae peptides hold significant health and nutrition potential. Peptide liberation through enzymatic and other processes enhanced protein extraction, and some animal studies were conducted to verify their health-promoting effects. Various studies have focused on developing practical methods for their production, purification, and identification of bioactive peptides. The emerging trends of in silico peptide therapies, computational approaches, artificial intelligence, and the prospects of microalgae peptide research are briefly highlighted. Moreover, this article focused on the potential of microalgae-derived peptides as functional food ingredients their role in promoting health, and their future applications in nutraceutical industries. It also discussed the challenges of bioavailability in functional foods.

Staphylococcus aureus , a common foodborne pathogen, has a close association with agriculture and food. With the rapid emergence and widespread dissemination of antimicrobial resistance, efforts have been directed toward developing and studying new antimicrobial compounds to inhibit the growth of S. aureus and other foodborne pathogens, thereby preventing contamination and ensuring food safety. Herein, we reported eight new aromatic polyketides, naphpyrones A-H ( 1 - 8 ), from the heterologous expression strain Streptomyces coelicolor A3(2)/ spi1 Δ spi H3. Their structures and absolute configurations were elucidated by extensive NMR, MS, theoretical NMR calculations, DP4+ probability analysis, Mosher's method, and ECD analyses. Notably, naphpyrone A ( 1 ) featured an unprecedented 6/6/6/6/5 neocyclic skeleton. Bioactivity evaluation revealed that compounds 1 and 2 exhibited antibacterial activity against S. aureus , with MIC values of 1 μg/mL and 4 μg/mL, respectively. These findings highlight the potential for screening and developing therapeutic agents from actinomycetes -derived aromatic polyketides against food pathogens.

The palatability of protein-based food heavily relies on aminopeptidases with the ability for bitter peptides degradation. However, there is a lack of methods for rational mining of aminopeptidases toward different proteins as well as the evolution direction for substrate specificity remaining unclear. In this study, an in silico simulated enzymolysis-based method for aminopeptidases mining was developed with Crassostrea gigas protein as a model. Results indicated that Ala and Ile were the most frequently exposed hydrophobic amino acids, causing a bitter taste in C. gigas hydrolysates. Furthermore, an aminopeptidase APs1 with putative Ala specificity was heterologously expressed and characterized with high enzyme activity toward Ala (4.92 ± 0.136 U/mg) and Arg (3.50 ± 0.178 U/mg). Site-saturation mutation and molecular docking results revealed that changes in steric hindrance and salt bridge formation within the active site contribute to enhanced catalytic efficiency. Among the mutants, APs1(F316M) showed significantly improved activity toward tested hydrophobic amino acids, especially the activity toward Ala was increased to 14.50 ± 0.137 U/mg. This study presents a directional approach to aminopeptidase mining and evolution, contributing to the rapid selection and combination of protein-degrading enzymes for food quality improvement.

Carrageenan oligosaccharides have shown promising bioavailability and possess a variety of physiological activities, making them highly suitable for use in the food, pharmaceutical, and agricultural industries. The preferred method for producing carrageenan oligosaccharides is using various carrageenolytic enzymes, as it offers mild reaction conditions, high efficiency, and product specificity. However, there is still a lack of specific applications for using these enzymes to prepare odd-numbered carrageenan-oligosaccharides (OCOSs). Our previous research identified a more convenient route for simultaneously preparing OCOSs and 3,6-anhydro-D-galactose (D-AHG) using only two types of carrageenolytic enzymes: κ-carrageenase and exo-α-3,6-anhydro-D-galactosidase (D-ADAGase). In this study, we utilized a CipA-based self-assembly system to cascade κ-carrageenase CaKC16A and D-ADAGase ZuGH129A for one-step preparation of β/κ-carrapentaose, G-(DA-G4S) 2 , and D-AHG from degrading β/κ-carrageenan. This self-assembled enzyme, namely CipA-CaKC16A-ZuGH129A, can be easily obtained through a simple centrifugation process. The final optimized enzymatic process produced 0.74 g/L G-(DA-G4S) 2 and 0.13 g/L D-AHG. This cascade system of different types of carrageenolytic enzymes has the potential to achieve the preparation of various types of carrageenan oligosaccharides.

The application of agarose oligosaccharides has garnered great attention, with their biological activities varying among different structures. However, it still meets a great bottleneck for the targeted production of odd-numbered neoagarooligosaccharides (NAOSs), such as neoagarotriose (NA3), due to the lack of one-step hydrolases. In this work, the α-agarase AgaA33 and β-galactosidase BgaD were synergistically used to prepare NA3 with agarose as a substrate. Additionally, an l-arabinose isomerase CaLAI from Clostridium acetobutylicum was characterized to valorize low-value byproducts (d-galactose) by forming d-tagatose, which exhibited good thermal stability without the need for additional metal ions. Under the optimal reaction conditions, the production of NA3 and d-galactose catalyzed by these three enzymes was 0.40 and 0.15 g/L, respectively. The artificial three-enzyme-based cascade transformation system not only achieved the highest production of NA3 until now but also allowed for the valorization of d-galactose, providing a wiser application route for agarose utilization.

κ-Carrageenases play an important role in achieving the high-value utilization of carrageenan polysaccharides. They can be used in the preparation of even-numbered κ-neocarrageenan oligosaccharides by degrading κ-carrageenan (KC). We previously identified and characterized a κ-carrageenase, CaKC16B, with high specificity for producing a single κ-neocarrabiose. It can produce a single κ-neocarrabiose from degrading KC. However, they also exhibited poor thermal stability and catalytic efficiency. To improve these properties, we introduced noncatalytic domains (nonCDs) from a heat-resistant κ-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct CaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of them exhibited improved enzymatic properties, including optimal reaction temperature, thermal stability, substrate affinity, and catalytic efficiency. Remarkably, the k cat values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and βκ-carrageenan, with mutants exhibiting higher relative activity for βκ-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and βκ-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.K m values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and βκ-carrageenan, with mutants exhibiting higher relative activity for βκ-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and βκ-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.

Laminarin oligosaccharides (LOSs) degraded from laminarin present nutritional functions. Laminarinases with high activity and good stability are significant tools for LOS production. OUC-SaLam66, a novel GH128 laminarinase from Streptomyces albus , was heterologously expressed. OUC-SaLam66 harbored a significant activity advantage up to 2294.80 U/mg at 45 °C and pH 4.0; meanwhile, it could preserve 80% activity at 45 °C for 12 h, indicating good stability. Significantly, its residual activity was over 75% after incubating at 100 °C for 1 h. Differential scanning calorimetry and molecular dynamic modeling revealed that its melting point and RMSD average point were 114.83 °C and 0.125 nm at 100 °C, respectively, which further proved its superior stability. Its apparent K m against laminarin were 6.437 mM and 2.5 μg/min/mg, respectively. Hydrolysis products were mainly composed of laminaritriose and laminaribiose. The high activity and thermostability of OUC-SaLam66 make it a promising candidate for LOS preparation.V max against laminarin were 6.437 mM and 2.5 μg/min/mg, respectively. Hydrolysis products were mainly composed of laminaritriose and laminaribiose. The high activity and thermostability of OUC-SaLam66 make it a promising candidate for LOS preparation.

The efficient, sensitive, and rapid detection of Salmonella typhimurium ( S. typhimurium ) in food and food products is important to ensure food safety and health. This study developed a fluorescence biosensing assay that integrated recombinase-aided amplification (RAA) and CRISPR/Cas12a with a zeolitic imidazolate framework-8@fluorescein sodium (ZIF-8@FLS) nanocomposite for the sensitive detection of S. typhimurium . In this approach, using RAA as a preamplification module, CRISPR/Cas12a-AChE as a target recognition and dual-enzyme cascade amplification module, and the prepared ZIF-8@FLS with high porosity and rapid pH responsiveness as a fluorescence signal explosive amplification module, the RAA-CRISPR/Cas12a-ZIF-8@FLS biosensor was constructed. Under optimal conditions, it exhibited an excellent linear relationship for S. Typhimurium , with a sensitive detection limit as low as 1.3 × 10 2 CFU/mL and could complete sample detection within 2 h relying on the RAA and ZIF-8@FLS explosive fluorescence rapid response, demonstrating its significant advantages in specificity, sensitivity, and reliability in food-borne pathogens detection.

The incidence of pancreatic cancer has been increasing globally in recent years and dietary is a well-defined factor contributing to its carcinogenesis. In this study, we showed that in a cerulein-induced KC (Pdx1-cre; LSL-Kras G12D/+) mouse model, a fasting-mimicking diet (FMD)─comprising fasting for 3 days followed by 4 days of refeeding, repeated over three 1-week cycles─significantly retards the progression of pancreatic carcinogenesis. FMD treatment altered gut microbiota, notably boosting butyrate-producing bacteria and elevating butyric acid levels in pancreatic tissues. Furthermore, lysine pan-crotonylation (pan-Kcr) expression was markedly upregulated in pancreatic intraepithelial neoplasia (PanIN) tissues from FMD-treated mice. Treatment of normal pancreatic duct and pancreatic cancer cells with sodium butyrate also upregulated pan-Kcr expression while reducing cell proliferation. Our findings reveal the pivotal role of dietary factors in the carcinogenesis of the pancreas and support further clinical studies of FMD as an antineoplastic therapeutic measure.

Cascade conversion of chitin into soluble and functional chitooligosaccharides has gained great attention. However, the biotransformation route is still limited to the low catalytic performances of chitin deacetylases (CDAs) and complicated procedures. In this study, a CDA from Arthrobacter sp. Jub115 (ArCDA) was identified and characterized, which showed a higher catalytic stability than the reported CDAs, with residual activity of 80.49%, 71.12%, and 56.09% after incubation at 30, 35, and 40 °C for 24 h, respectively. Additionally, ArCDA was identified to have a broad substrate spectrum toward β-chitin and N -acetyl chitooligosaccharides. Moreover, an engineered chitin-degrading bacteria (CDB) with cell-surface-displayed deacetylase ArCDA and chitinase SaChiB was constructed to simplify catalysis procedures, facilitating the chitobiose production of 294.30 ± 16.43 mg/L in 10 h. This study not only identified a CDA with the desirable catalytic performance but also provided a strategy for constructing CDB, facilitating the high-value utilization of chitin.

Electrostatic adsorption is an enzyme immobilization method that effectively maintains enzyme activity and exhibits considerable binding efficiency. However, enzymes carry different charges at their respective reaction pH levels, which prevents the use of the same carrier to immobilize enzymes with different charges. In this study, we employed a template-mediated polysaccharide-enzyme coupling self-assembly strategy to develop a charge-controllable supramolecular immobilization carrier by regulating the charge properties of carboxymethyl chitosan, enabling the universal immobilization of enzymes with different charge levels across a range of reaction pH values. By using silica nanoparticles of certain sizes as templates, the size of the carrier can be precisely controlled and the hollow network structure formed after removing the template can effectively reduce mass transfer resistance. Trypsin and papain are used as model enzymes, and the experimental results show that the supramolecular self-assembly immobilization strategy does not disrupt the secondary structure of the enzyme molecules. After 2 h of reaction, the enzyme activities of immobilized papain and immobilized trypsin are 13.2% and 7.7% higher than those of the free enzymes, respectively. After 10 consecutive reactions, the enzyme activities of immobilized papain and immobilized trypsin retained 56.3% and 64.3% of their initial values, respectively.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb 3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t . Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.65 °C 1/2 . Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the βκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the βκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing βκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Spotted seabass ( Lateolabrax maculatus ) is the second largest maricultural fish species in China and is the main trigger of food-related allergic reactions. Nevertheless, studies on the allergens of L. maculatus are limited. This study aimed to characterize pan-allergen parvalbumin from L. maculatus . Two proteins of about 11 kDa were purified and confirmed as parvalbumins by mass spectrometry. The IgG- and IgE-binding activities were evaluated through an immunoblotting assay. The molecular characteristics of β-parvalbumin were investigated by combining proteomics, genomics, and immunoinformatics approaches. The results indicated that β-parvalbumin consists of 109 amino acids with a molecular weight of 11.5 kDa and is the major allergen displaying strong IgE-binding capacity. In silico analysis and a dot blotting assay confirmed seven linear B cell epitopes distributed mainly on α-helixes and the calcium-binding loops. In addition, the cross-reactivity among 26 commonly consumed fish species was analyzed. The in-house generated anti- L. maculatus parvalbumin polyclonal antibody recognized 100% of the 26 fish species, demonstrating cross-reactivity and better binding capacity than the anticod parvalbumin antibody. Together, this study provides an efficient protocol to characterize allergens with multiomics methods and supports parvalbumin from L. maculatus as a candidate for fish allergen determination and allergy diagnosis.

This study aimed to rapidly develop novel umami peptides using yeast protein as an alternative protein source. Yeast protein hydrolysates exhibiting pronounced umami intensity were produced using flavorzyme under optimum conditions determined via a sensory-guided response surface methodology. Six out of 2138 peptides predicted to possess umami taste by composite machine learning and assessed as nontoxic, nonallergenic, water-soluble, and stable using integrated bioinformatics were screened as potential umami peptides. Sensory evaluation results revealed these peptides exhibited multiple taste attributes (detection threshold: 0.37 ± 0.10-1.1 ± 0.30 mmol/L), including umami. In light of the molecular docking outcomes, it is inferred that hydrogen bond, hydrophobic, and electrostatic interactions enhanced the theoretically stable binding of peptides to T1R1/T1R3, with their contributions gradually diminishing. Hydrophilic amino acids within T1R1/T1R3, especially Ser, may play a particularly pivotal role in binding with umami peptides. Future research will involve establishing heterologous cell models expressing T1R1 and T1R3 to delve into the cellular physiology of umami peptides. Peptide sequences (FADL, LPDP, and LDIGGDF) also had synergistic saltiness-enhancing effects; to overcome the limitation of not investigating the saltiness enhancement mechanism, comprehensive experiments at the molecular and cellular levels will also be conducted. This study offers a rapid umami peptide development framework and lays the groundwork for exploring yeast protein taste compounds.

Functional oligosaccharides induce specific alterations in gut microbiota, potentially providing physiological benefits. However, the effects of laminaripentaose (LPA) on metabolic syndrome and the mechanism underlying it have not been intensively investigated yet. This study aimed to determine the effects of LPA on obesity and obesity-induced cognition impairment in mice. C57BL/6N mice fed with a high-fat diet received an LPA treatment for 12 weeks. An antibiotic intervention was further applied to evaluate the effects of the gut microbiota on cognitive functions. LPA treatment (500 mg/kg) reduced the weight gain by 32.4%. Furthermore, LPA improved memory functions and reduced hippocampal insulin resistance and neuronal injury. LPA markedly reduced systemic low-grade inflammation and intestinal barrier injury. Moreover, LPA increased gut beneficial bacteria, and Butyricimonas and Bifidobacterium were increased by 94.0 and 422.7%, respectively, accompanied by increased fecal short-chain fatty acids. Interestingly, antibiotic cocktail treatment abrogated the beneficial effects of LPA on cognition, which further suggests that LPA may attenuate obesity-induced cognition impairment via the gut-brain axis. Our findings provide the first evidence for the potential of dietary LPA to prevent obesity and obesity-associated complications.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN) 5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN) 5 . These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN) 5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN) 4 and (GlcN) 5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Microalgae, integral to marine ecosystems for their rich nutrient content, notably lipids and proteins, were investigated by using reversed-phase liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RPLC-Q-TOF-MS/MS). This study focused on lipid composition in three commonly used microalgae species ( Spirulina platensis , Chlorella vulgaris , and Schizochytrium limacinum ) for functional food applications. The analysis unveiled more than 700 lipid molecular species, including glycolipids (GLs), phospholipids (PLs), sphingolipids (SLs), glycerolipids, and betaine lipids (BLs). GLs (19.9-64.8%) and glycerolipids (24.1-70.4%) comprised the primary lipid. Some novel lipid content, such as acylated monogalactosyldiacylglycerols (acMGDG) and acylated digalactosyldiacylglycerols (acDGDG), ranged from 0.62 to 9.68%. The analysis revealed substantial GLs, PLs, and glycerolipid variations across microalgae species. Notably, S. platensis and C. vulgaris displayed a predominance of fatty acid (FA) 18:2 and FA 18:3 in GLs, while S. limacinum exhibited a prevalence of FA 16:0, collectively constituting over 60% of the FAs of GLs. In terms of PLs and glycerolipids, S. platensis and C. vulgaris displayed elevated levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA), whereas S. limacinum exhibited a significant presence of docosahexaenoic acid (DHA). Principal component analysis (PCA) revealed MGDG (16:0/18:1), DG (16:0/22:5), Cer (d18:1/20:0), and LPC (16:1) as promising lipid markers for discriminating between these microalgae samples. This study contributes to a comprehensive understanding of lipid profiles in three microalgae species, emphasizing their distinct biochemical characteristics and potentially informing us of their high-value utilization in the food industry.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. β-Lactoglobulin (β-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in β-Lg. Herein, plasmin inhibitory peptides were explored from β-Lg using experimental and computational techniques. The results revealed that increased denaturation of β-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the β-Lg/plasmin complex. Denatured β-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to β-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from β-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Emerging technologies for cell-cultured fish meat as an environmentally friendly protein source for humans still have many obstacles, including large-scale production of high-quality cells, differentiation and bioassembly of cellular material, and improvement of the quality of meat products. Here, we used edible porous microcarriers as scaffolds to support scalable skeletal muscle cell expansion to prepare centimeter-scale cell-cultured fish (CCM) of Carassius auratus for the first time. The quality of CCM was assessed by analyzing the texture, nutrition, flavor, and safety. The results indicated that CCM demonstrated a softer texture than natural fish due to a high moisture content. CCM contained higher protein and lower fat contents, with no significant difference in energy from natural golden crucian carp meat (NGM). CCM had better digestible properties, and 17 volatile components were identified in CCM, ten cocontained compared to NGM. ELISA quantified penicillin, streptomycin, vitamin D, and insulin residues as risk factors in CCM. In conclusion, we utilized edible porous microcarriers to scale-up the expansion of Carassius auratus skeletal muscle cells and bioassembled high-quality CCM of Carassius auratus for the first time, which represents a state-of-the-art protocol applicable to different fish species and even to other economic animals and provides a theoretical basis for scaling up cell-cultured meat production.

Sea cucumber phospholipids have ameliorative effects on various diseases related to lipid metabolism. However, it is unclear whether it can ameliorate obesity-associated glomerulopathy (ORG) induced by a high-fat diet (HFD). The present study applied UPLC-QqQ-MS/MS and atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry imaging (AP-MALDI MSI) to investigate the effects of sea cucumber phospholipids, including plasmalogen PlsEtn and plasmanylcholine PakCho, on phospholipid profiles in the HFD-induced ORG mouse kidney. Quantitative analysis of 135 phospholipids revealed that PlsEtn and PakCho significantly modulated phospholipid levels. Notably, PlsEtn modulated kidney overall phospholipids better than PakCho. Imaging the "space-content" of 9 phospholipids indicated that HFD significantly increased phospholipid content within the renal cortex. Furthermore, PlsEtn and PakCho significantly decreased the expression of transport-related proteins CD36, while elevating the expression of fatty acid β-oxidation-related protein PPAR-α in the renal cortex. In conclusion, sea cucumber phospholipids reduced renal lipid accumulation, ameliorated renal damage, effectively regulated the content and distribution of renal phospholipids, and improved phospholipid homeostasis, exerting an anti-OGR effect.

The lack of practical platforms for bacterial separation remains a hindrance to the detection of bacteria in complex samples. Herein, a composite cryogel was synthesized by using clickable building blocks and boronic acid for bacterial separation. Macroporous cryogels were synthesized by cryo-gelation polymerization using 2-hydroxyethyl methacrylate and allyl glycidyl ether. The interconnected macroporous architecture enabled high interfering substance tolerance. Nanohybrid nanoparticles were prepared via surface-initiated atom transfer radical polymerization and immobilized onto cryogel by click reaction. Alkyne-tagged boronic acid was conjugated to the composite for specific bacteria binding. The physical and chemical characteristics of the composite cryogel were analyzed systematically. Benefitting from the synergistic, multiple binding sites provided by the silica-assisted polymer, the composite cryogel exhibited excellent affinity toward S. aureus and Salmonella spp. with capacities of 91.6 × 10 7 CFU/g and 241.3 × 10 7 CFU/g in 0.01 M PBS (pH 8.0), respectively. Bacterial binding can be tuned by variations in pH and temperature and the addition of monosaccharides. The composite was employed to separate S. aureus and Salmonella spp. from spiked tap water, 40% cow milk, and sea cucumber enzymatic hydrolysate, which resulted in high bacteria separation and demonstrated remarkable potential in bacteria separation from food samples.

Indole-3-lactic acid (ILA) has exhibited antimicrobial properties. However, its role in inhibiting Helicobacter pylori infection remains elusive. This study investigated the inhibitory effect of ILA produced by Lacticaseibacillus paracasei on H. pylori , which was further confirmed by cell and animal experiments. 5 mg/mL ILA was sufficient to directly inhibit the growth of H. pylori in vitro, with a urease inhibitory activity reaching 60.94 ± 1.03%, and the cell morphology and structure were destroyed. ILA inhibited 56.5% adhesion of H. pylori to GES-1 and significantly reduced the number of apoptotic cells. Furthermore, ILA suppresses H. pylori colonization by approximately 38% to 63%, reduced inflammation and oxidative stress in H. pylori -infected mice, and enhanced the enrichment and variety of gut microbiota, notably fostering the growth of beneficial bacteria such as Lactobacillus and Bifidobacterium strains. The results support that ILA derived from Lactobacillus can be applicated as a novel prebiotic in anti- H. pylori functional foods.

Trimethylamine N-oxide (TMAO), a characteristic nonprotein nitrogen compound, is widely present in seafood, which exhibits osmoregulatory effects for marine organisms in vivo and plays an important role in aquaculture and aquatic product preservation. However, much attention has been focused on the negative effect of TMAO since it has recently emerged as a putative promoter of chronic diseases. To get full knowledge and maximize our ability to balance the positive and negative aspects of TMAO, in this review, we comprehensively discuss the TMAO in aquatic products from the aspects of physiological functions for marine organisms, flavor, quality, the conversion of precursors, the influences on human health, and the seafood ingredients interaction consideration. Though the circulating TMAO level is inevitably enhanced after seafood consumption, dietary seafood still exhibits beneficial health effects and may provide nutraceuticals to balance the possible adverse effects of TMAO.

This study reports a peptide design model for engineering fusion-expressed antimicrobial peptides (AMPs) with the AflR dinuclear zinc finger motif to improve the defense against aflatoxins and Aspergillus flavus . The study identified AflR, a Zn 2 Cys 6 -type sequence-specific DNA-binding protein, as a key player in the regulation of aflatoxin biosynthesis. By integrating the AflR motif into AMPs, we demonstrate that these novel fusion peptides significantly lower the minimum inhibitory concentrations (MICs) and reduce aflatoxin B 1 and B 2 levels, outperforming traditional AMPs. Comprehensive analysis, including bioinformatics and structural determination, elucidates the enhanced structure-function relationship underlying their efficacy. Furthermore, the study reveals the possibility that the fusion peptides have the potential to bind to the DNA binding sites of transcriptional regulators, binding DNA sites of key transcriptional regulators, thereby inhibiting genes critical for aflatoxin production. This research not only deepens our understanding of aflatoxin inhibition mechanisms but also presents a promising avenue for developing advanced antifungal agents, which are essential for global food safety and crop protection.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a β-sandwich fold comprising two antiparallel β-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

Astaxanthin (AST), mainly found in algae and shrimp, is a liposoluble ketone carotenoid with a wide range of biological activities and is commonly used in healthcare interventions and cosmetics. AST has a long chain of conjugated double bonds with hydroxyl and ketone groups at both ends, enabling it to form astaxanthin esters (AST-Es) through esterification with fatty acids. The fatty acid structure of AST plays a key role in the stability, antioxidant activity, and bioavailability of AST-Es. Antarctic krill ( Euphausia superba ) and blood-red algae Haematococcus Pluvialis ( H pluvialis )-derived AST-Es exhibit strong antioxidant activity and numerous biological activities, such as improving insulin resistance, preventing Parkinson's disease, regulating intestinal flora, and alleviating inflammatory bowel disease. This review discusses the significance of AST-Es as functional food ingredients, highlighting their nutritional value, phytochemical structure, biological activities, and potential applications in the food industry.

κ-Carrageenase plays an important role in achieving the high-value utilization of carrageenan. Factors such as the reaction temperature, thermal stability, catalytic efficiency, and product composition are key considerations for its large-scale application. Previous studies have shown that the C-terminal noncatalytic domains (nonCDs) could influence the enzymatic properties, of κ-carrageenases, providing a strategy for exploring κ-carrageenases with different properties, especially catalytic products. Accordingly, two κ-carrageenases (CaKC16A and CaKC16B), from the Catenovulum agarivorans DS2, were selected and further characterized. Bioinformatics analysis suggested that CaKC16A contained a nonCD but CaKC16B did not. CaKC16A exhibited better enzymatic properties than CaKC16B, including thermal stability, substrate affinity, and catalytic efficiency. After truncation of the nonCD of CaKC16A, its thermal stability, substrate affinity, and catalytic efficiency have significantly decreased, indicating the vital role of nonCD in maintaining a good enzymatic property. Moreover, CaKC16A degraded κ-carrageenan to produce a highly single κ-neocarratetrose, while CaKC16B produced a single κ-neocarrabiose. CaKC16A could degrade β/κ-carrageenan to produce a highly single desulfated κ-neocarrahexaose, while CaKC16B produced κ-neocarrabiose and desulfated κ-neocarratetrose. Furthermore, it was proposed that CaKC16A and CaKC16B participate in the B/KC metabolic pathway and serve different roles, providing new insight into obtaining κ-carrageenases with different properties.

To achieve effective separation and enrichment of bacteria, a novel synthetic scheme was developed to synthesize star-style boronate-functionalized copolymers with excellent hydrophilicity and temperature and pH responsiveness. A hydrophilic copolymer brush was synthesized by combining surface-initiated atom-transfer radical polymerization with amide reaction using bovine serum albumin as the core. The copolymer brush was further modified by introducing and immobilizing fluorophenylboronic acids through an amide reaction, resulting in the formation of boronate affinity material BSA@poly(NIPAm- co -AGE)@DFFPBA. The morphology and organic content of BSA@poly(NIPAm- co -AGE)@DFFPBA were systematically characterized. The BSA-derived composites demonstrated a strong binding capacity to both Gram-positive and Gram-negative bacteria. The binding capabilities of the affinity composite to Staphylococcus aureus and Salmonella spp. were 195.8 × 10 10 CFU/g and 79.2 × 10 10 CFU/g, respectively, which indicates that the novel composite exhibits a high binding capability to bacteria and shows a particularly more significant binding capacity toward Gram-positive bacteria. The bacterial binding of BSA@poly(NIPAm- co -AGE)@DFFPBA can be effectively altered by adjusting the pH and temperature. This study demonstrated that the star-shaped affinity composite had the potential to serve as an affinity material for the rapid separation and enrichment of bacteria in complex samples.

The development of food-derived antihyperuricemic substances is important for alleviating hyperuricemia (HUA) and associated inflammation. Here, novel peptides from Thunnus albacares (TAP) with strong antihyperuricemic activity were prepared. TAP was prepared by alkaline protease (molecular weight <1000 Da), with an IC 50 value of xanthine oxidase inhibitory activity of 2.498 mg/mL, and 5 mg/mL TAP could reduce uric acid (UA) by 33.62% in human kidney-2 (HK-2) cells ( P < 0.01). Mice were fed a high-purine diet and injected with potassium oxonate to induce HUA. Oral administration of TAP (600 mg/kg/d) reduced serum UA significantly by 42.22% and increased urine UA by 79.02% ( P < 0.01) via regulating urate transporters GLUT9, organic anion transporter 1, and ATP-binding cassette subfamily G2. Meantime, TAP exhibited hepatoprotective and nephroprotective effects, according to histological analysis. Besides, HUA mice treated with TAP showed anti-inflammatory activity by decreasing the levels of toll-like receptor 4, nuclear factors-κB p65, NLRP3, ASC, and Caspase-1 in the kidneys ( P < 0.01). According to serum non-targeted metabolomics, 91 differential metabolites between the MC and TAP groups were identified, and purine metabolism was considered to be the main pathway for TAP alleviating HUA. In a word, TAP exhibited strong antihyperuricemic activity both in vitro and in vivo.

κ-Carrageenase plays a crucial role in the high-value utilization of carrageenan. Heat resistance is a key factor in the practical application of κ-carrageenase, as carrageenan exhibits gel-like properties. Previous studies have shown that the C-terminal noncatalytic domains (nonCDs) can affect the thermostability of κ-carrageenases. In this study, we expressed and characterized a κ-carrageenase, MtKC16A, which contains three nonCDs, from Microbulbifer thermotolerans . MtKC16A has the highest activity at 80 °C and pH 7.0. Surprisingly, it exhibits excellent heat resistance, with 71.58% relative activity at 100 °C and still retains over 50% residual activity after incubation at 100 °C for 60 min. Additionally, MtKC16A has been shown to have a dual substrate hydrolysis activity. It can degrade κ-carrageenan to produce highly single Nκ4 and degrade β/κ-carrageenan to produce Nκ2 and desulfated Nκ4 DA-G-DA-G4S, suggesting its potential in producing κ- and β/κ-hybrid oligosaccharides. Furthermore, we found that the unknown function domain (UNFD) in MtKC16A plays the most vital role among the three nonCDs. When this UNFD is truncated, the resulting mutants completely lose their catalytic ability at 100 °C. Finally, by introducing this UNFD to the C-terminal of another κ-carrageenase CaKC16B, we were able to improve its heat resistance at 100 °C.

Crustacean shellfish are major allergens in East Asia. In the present study, a major allergic protein in crustaceans, tropomyosin, was detected accurately using multiple reaction monitoring mode-based mass spectrometry, with shared signature peptides identified through proteomic analysis. The peptides were deliberately screened through thermal stability and enzymatic digestion efficiency to improve the suitability and accuracy of the developed method. Finally, the proposed method demonstrated a linear range of 0.15 to 30 mg TM /kg food ( R > 0.99), with a limit of detection of 0.15 mg 2 > 0.99), with a limit of detection of 0.15 mg TM and a limit of quantification of 0.5mg food and a limit of quantification of 0.5mg TM and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.food and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

The use of convertible immobilized enzyme carriers is crucial for biphasic catalytic reactions conducted in Pickering emulsions. However, the intense mechanical forces during the conversion process lead to enzyme leakage, affecting the stability of the immobilized enzymes. In this study, a CO 2 -responsive switchable Janus (CrSJ) nanoparticle (NP) was developed using silica NP, with one side featuring aldehyde groups and the other side adsorbing N , N -dimethyldodecylamine. A switchable Pickering emulsion catalytic system for biphasic interface reactions was prepared by covalently immobilizing lipase onto the CrSJ NPs. The CO 2 -responsive nature of the CrSJ NPs allowed for rapid conversion of the Pickering emulsion, and covalent immobilization substantially reduced lipase leakage while enhancing the stability of the immobilization during the conversion process. Impressively, after repeated transformations, the Pickering emulsion still maintains its original structure. Following 10 consecutive cycles of esterification and hydrolysis reactions, the immobilized enzyme's activity remains at 77.7 and 79.5% of its initial activity, respectively. The K of the CrSJ catalytic system showed no significant change compared to the free enzyme, while its m values were 1.2 and 1.6 times that of the free enzyme in esterification and hydrolysis reactions, respectively.V max values were 1.2 and 1.6 times that of the free enzyme in esterification and hydrolysis reactions, respectively.

The sea cucumber plasmalogen PlsEtn has been shown to be associated with various chronic diseases related to lipid metabolism. However, the mechanism is unclear. Therefore, the present study used the sea cucumber plasmanylcholine PakCho as a structural contrast to PlsEtn and assessed its effect in 8 week high-fat diet (HFD)-fed mice. The lipidomic approach based on high-resolution mass spectrometry combined with molecular biology techniques was used to evaluate the mechanism of PlsEtn. The results showed that both PlsEtn and PakCho significantly inhibited an increase in mouse body weight and liver total triglyceride and total cholesterol levels caused by HFD. In addition, oil red O staining demonstrated that lipid droplets stored in the liver were degraded. Meanwhile, untargeted lipidomic experiments revealed that total lipids (increased by 42.8 mmol/mg prot; p < 0.05), triglycerides (increased by 38.9 mmol/mg prot; p < 0.01), sphingolipids (increased by 1.5 mmol/mg prot; p < 0.0001), and phospholipids (increased by 2.5 mmol/mg prot; p < 0.05) were all significantly elevated under HFD. PlsEtn resolved lipid metabolism disorders by alleviating the abnormal expression of lipid subclasses. In addition, five lipid molecular species, PE (18:1/20:4), PE (18:1/20:3), PE (18:1/18:3), TG (16:0/16:0/17:0), and TG (15:0/16:0/18:1), were identified as the biomarkers of HFD-induced lipid metabolism disorders. Finally, lipophagy-associated protein expression analysis showed that HFD abnormally activated lipophagy via ULK1 phosphorylation and PlsEtn alleviated lipophagy disorder through lysosomal function promotion. In addition, PlsEtn performed better than PakCho. Taken together, the current study results unraveled the mechanism of PlsEtn in alleviating lipid metabolism disorder and offered a new theoretical foundation for the high-value development of sea cucumber.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 μg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti- A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Nanoplastic pollution poses a significant global concern for public health due to the potential toxicity it induces in the human body through food and water intake. Consequently, the urgent task of removing nanoplastics, especially from water resources, is paramount for enhancing food safety, and developing eco-friendly materials capable of efficiently removing nanoplastics is crucial. In this context, we propose the use of biodegradable anionic seaweed cellulose nanofibers (TEMPO-mediated seaweed cellulose nanofibers, TCNFs) and cationic seaweed cellulose nanofibers (quaternized seaweed cellulose nanofibers, QCNFs) for nanoplastic removal in both single- and copollutant systems. In our experiments under simulated practical conditions, we revealed that TCNFs and QCNFs achieved an average removal efficiency of 98.71% against nanoplastic particles. Moreover, TCNFs and QCNFs exhibited higher adsorption capacities compared to those of existing materials, potentially offering a cost-effective advantage. Toxicity assessments conducted with mammalian cells further confirmed the biosafety of TCNFs and QCNFs. This study contributes to the scientific and theoretical understanding of using edible seaweed as well as offers promising solutions for food safety control in an efficient, cost-effective, and eco-friendly manner.

The whole enzymatic conversion of chitin is a green and promising alternative to current strategies, which are based on lytic polysaccharide monooxygenases (LPMOs) and chitinases. However, the lack of LPMOs with high activity toward α-chitin limits the efficient bioconversion of α-chitin. Herein, we characterized a high chitin-active LPMO from Oceanobacillus sp. J11TS1 ( Os LPMO10A), which could promote the decrystallization of the α-chitin surface. Furthermore, when coupled with Os LPMO10A, the conversion rate of α-chitin to N -acetyl chitobiose [(GlcNAc) 2 ] by three chitinases ( Serratia marcescens , ChiA, -B, and -C) reached 30.86%, which was 2.03-folds that without the addition of Os LPMO10A. Moreover, the results of synergistic reactions indicated that Os LPMO10A and chitinases promoted the degradation of α-chitin each other mainly on the surface. To the best of our knowledge, this study achieved the highest yield of N -acetyl chitooligosaccharides ( N -acetyl COSs) among reported LPMOs-driven bioconversion systems, which could be regarded as a promising candidate for α-chitin bioconversion.

Abscisic acid (ABA) is an important plant hormone with a variety of physiological functions such as regulating plant growth and helping plants to resist an adverse growth environment. However, at present, the ABA yield of heterologous biosynthesis by metabolic engineering is still low for industrial production. Therefore, five Botrytis cinerea genes ( bcaba1 , bcaba2 , bcaba3 , bcaba4, and bccpr1 ) related to ABA biosynthesis were expressed in Yarrowia lipolytica PO1h; its ABA production was 24.33 mg/L. By increasing the copy number of IDI and ERG12S , ERG20Y MT , and bcaba3 genes, the yield of ABA was increased to 54.51 mg/L. By locating HMG-CoA reductase and HMG-CoA synthase in mitochondria, acetyl-CoA in mitochondria was converted into mevalonate; this increased the ABA yield to 102.12 mg/L. Finally, in the fed-batch fermentation process with the addition of dodecane, the ABA yield was up to 1212.57 mg/L, which is the highest yield of heterologous production of ABA by metabolic engineering.bcaba1 genes, the yield of ABA was increased to 54.51 mg/L. By locating HMG-CoA reductase and HMG-CoA synthase in mitochondria, acetyl-CoA in mitochondria was converted into mevalonate; this increased the ABA yield to 102.12 mg/L. Finally, in the fed-batch fermentation process with the addition of dodecane, the ABA yield was up to 1212.57 mg/L, which is the highest yield of heterologous production of ABA by metabolic engineering.

Six new 9 H -carbazole derivatives ( 1 - 6 ) and nine previously reported compounds ( 7 - 15 ) were isolated from a fermented solid medium of the Thailand mangrove-derived Streptomyces strain, OUCMDZ-5511, under fluoride stress. Compounds 2 - 5 , 12 , and 15 were exclusively present in the fluoride-supplemented fermentation medium, while compounds 7 - 9 , 13 , and 14 were newly discovered natural products. The molecular structures of the compounds were identified by a spectroscopic analysis. The new compound 2 displayed antiquorum sensing activity against Chromobacterium violaceum ATCC 12472 by reducing the violacein production and inhibiting the biofilm formation in a concentration-dependent manner. The study revealed that compound 2 could be a novel potential inhibitor of quorum sensing.

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.

β- N -Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto- N -triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family β- N -acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p -nitrophenyl N -acetyl-β-d-glucosaminide ( p NP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward p NP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM p NP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from p NP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM p NP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 β- N -acetylhexosaminidases and promoting the high-value utilization of whey powder.

Caseinophosphopeptides have shown great potential to increase zinc bioavailability from phytate-rich diets, but the mechanism of action remains unclear. Here, caseinophosphopeptides from a sodium caseinate hydrolysate dose-dependently retained zinc in solution against calcium phytate coprecipitation under physiologically relevant conditions. The 3 kDa ultrafiltration separation unveiled no added low-molecular-weight chelates of zinc and calcium by caseinophosphopeptides. Tyndall effect, dynamic light scattering measurements, transmission electron microscopy observation, electron diffraction pattern, X-ray diffraction spectrum, and energy-dispersive X-ray analysis demonstrated the caseinophosphopeptides-mediated formation of single-crystal zinc/calcium phytate nanocomplexes (Zn/CaPA-NCs) with a size and ζ-potential of 10-30 nm and -25 mV, respectively. Caseinophosphopeptides-stabilized Zn/CaPA-NCs were found to deliver bioavailable nanoparticulate zinc in mouse jejunal loop ex vivo model and polarized Caco-2 cells, and the treatments with specific inhibitors revealed that intestinal zinc absorption from Zn/CaPA-NCs invoked macropinocytosis, lysosomal release into the cytosol, and transcytosis. Overall, our study proposes a new paradigm for the benefit of caseinophosphopeptides for zinc bioaccessibility and bioavailability in phytate-rich diets.

Milk-derived extracellular vesicles can improve intestinal health and have antiosteoporosis potential. In this paper, we explored the effects of bovine raw milk-derived extracellular vesicles (mEVs) on ovariectomized (OVX) osteoporotic mice from the perspective of the gut-bone axis. mEVs could inhibit osteoclast differentiation and improve microarchitecture. The level of osteoporotic biomarkers in OVX mice was restored after the mEVs intervened. Compared with OVX mice, mEVs could enhance intestinal permeability, reduce endotoxin levels, and improve the expression of TNF-α, IL-17, and IL-10. 16S rDNA sequencing indicated that mEVs altered the composition of gut microbiota, specifically for Bacteroides associated with short-chain fatty acids (SCFAs). In-depth analysis of SCFAs demonstrated that mEVs could restore acetic acid, propionic acid, valeric acid, and isovaleric acid levels in OVX mice. Correlation analysis revealed that changed gut microbiota and SCFAs were significantly associated with gut inflammation and osteoporotic biomarkers. This study demonstrated that mEVs could inhibit osteoclast differentiation and improve osteoporosis by reshaping the gut microbiota, increasing SCFAs, and decreasing the level of pro-inflammatory cytokines and osteoclast differentiation-related factors in OVX mice. These findings provide evidence for the use of mEVs as a food supplement for osteoporosis.

The stimulation of host iron absorption is a promising antianemia strategy adjunctive/alternative to iron intervention. Here, gum arabic (GA) containing 3.14 ± 0.56% hydroxyproline-rich protein with repetitive X-(Pro/Hyp) n motifs was found to increase iron reduction, uptake, and transport to upregulate duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin, and hephaestin to inhibit hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) and to stabilize HIF2α in polarized Caco-2 cell monolayers in a dose-dependent manner, and this was dependent on its protein fraction, rather than the polysaccharide fraction. Three abundant GA-derived hydroxyproline-containing dipeptides of Hyp-Hyp, Pro-Hyp, and Ser-Hyp were detected by liquid chromatography-mass spectrometry in the lysates of polarized Caco-2 cell monolayers at the maximum levels of  0.167 ± 0.021, 0.134 ± 0.017, and 0.089 ± 0.015 μg/mg of protein, respectively, and showed desirable docking affinity energy values of -7.53, - 7.91, and -7.39 kcal/mol, respectively, against human PHD3. GA-derived peptides also acutely increased duodenal HIF2α stability and Dcytb, DMT1, ferroportin, and hephaestin transcription in rats ( P < 0.05). Overall, GA-derived hydroxyproline-rich peptides stimulated intestinal iron absorption via PHD inhibition, HIF2α stabilization, and subsequent upregulation of iron transport proteins.

Alginate lyase degrades alginate by the β-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔ G value calculation with the B -factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

The dysfunction of intestinal microbiota and bile acid metabolism is related to the pathogenesis of atherosclerosis. This study we explored the mechanism of Bifidobacterium animalis subsp. lactis F1-7 ( Bif. animalis F1-7), improving atherosclerosis by regulating the bile acid metabolism and intestinal microbiota in the ApoE -/- mice. The Bif. animalis F1-7 effectively reduced aortic plaque accumulation and improved the serum and liver lipid levels in atherosclerotic mice. The untargeted metabolomics revealed that Bif. animalis F1-7 reduced the glycine-conjugated bile acids and the levels of differential metabolite lithocholic acid (LCA) significantly. Downregulation of LCA decreased the intestinal levels of the farnesoid X-activated receptor (FXR) and regulated the bile acid metabolism through the FXR/FGF15/CYP7A1 pathway. Furthermore, the 16srRNA gene sequencing analysis revealed that structural changes in intestinal microbiota with an increase in the abundance of Bifidobacterium , Lactobacillus , Faecalibaculum , Desulfovibrio , and a decrease in Dubosiella , Clostridium&#95;sensu&#95;stricto&#95;1 , and Turicibacter following the Bif. animalis F1-7 intervention. Correlation analysis showed that the changes in intestinal microbiota mentioned above were significantly correlated with bile acid metabolism in atherosclerotic mice. In conclusion, this study sheds light on the mechanisms by which Bif. animalis F1-7 regulates atherosclerosis.

G-specific alginate lyases are important tools for alginate fragment biodegradation and oligosaccharide production, which have great potential in alginate refining research. In this research, a novel G-specific alginate lyase Aly7Ce was cloned, expressed, and characterized, with the optimal reaction conditions at 30 °C and pH 8.0. By employing the UPSEC-VWD-MS method, Aly7Ce was confirmed as a random endoacting alginate lyase. Its minimum substrate was tetrasaccharide, and the final product majorly consisted of disaccharide to tetrasaccharide. HPAEC-PAD/MS method was employed to investigate the structurally different unsaturated alginate oligosaccharides. The substrate recognition and subsite specificity of Aly7Ce were revealed by detecting the oligosaccharide pattern in the enzymatic products with oligosaccharides or polysaccharides as substrates. Aly7Ce mainly attacked the second glycosidic linkage from the nonreducing end of oligosaccharide substrates. The subsite specificity of Aly7Ce was revealed as -2 (M/G), - 1 (G), + 1 (M/G), and +2 (M/G). The regular oligosaccharide products of Aly7Ce could be applied for the efficient preparation of ΔG, ΔGG, and ΔGGG with high purity. The G-specific alginate lyase Aly7Ce with a well-defined product composition and action pattern provided a novel tool for the modification and structural elucidation of alginate, as well as for the targeted preparation of oligosaccharides.

A sustainable enzymatic system is essential for efficient phosphatidylserine (PS) synthesis in industrial production. Conventional biphasic systems face challenges such as excessive organic solvent usage, enzyme-intensive processes, and increased costs. This study introduces a novel approach using chitin nanofibrils (ChNFs) as an immobilization material for phospholipase D (PLD) in a mixed micellar system stabilized by the food-grade emulsifier sodium deoxycholate (SDC). The immobilized enzyme, ChNF-chiA1, was quickly prepared in a one-step process, eliminating the need for purification. By optimizing the reaction conditions, including l-Ser concentration (1.0 M), SDC concentration (10 mM), reaction time (8 h), and enzyme dosage (1.0 U), a remarkable PS yield of 96.74% was achieved in the solvent-free mixed micellar system. The catalytic efficiency of ChNF-chiA1 surpassed that of the free PLD-chiA1 biphasic system by 6.0-fold. This innovative and green biocatalytic technology offers a reusable solution for the high-value enzymatic synthesis of phospholipids, providing a promising avenue for industrial applications.