Abstract:During the production, storage, and transportation, surimi is susceptible to external factors such as temperature, oxidation, and microorganisms. These factors can lead to the decline of gel properties, thereby affecting surimi quality. Gel properties are affected by various factors, including fish species, rinsing methods, heating methods, and exogenous additives. These properties are critical indicators of surimi quality and major factors influencing consumer purchasing decisions. Polyphenols, compounds with multiple phenolic hydroxyl groups, can interact with proteins and offer advantages such as a wide range of sources and being green and safe. Polyphenols can not only delay protein oxidation and inhibit microbial growth but also enhance the gel properties of surimi, thereby addressing the issues caused by external factors. Therefore, it is essential to understand the mechanism by which polyphenols enhance the gel properties of surimi and review the research progress in this area. The gelation mechanisms of surimi and the interaction mechanisms between polyphenols and proteins were summarized, and the effects of different factors on the gel properties of surimi and the interactions between polyphenols and proteins were discussed. Furthermore, the current applications of polyphenols in improving the gel properties of surimi were reviewed, providing a theoretical basis and reference for expanding the application of polyphenols in the processing of surimi and other aquatic products.

Abstract:[Objective] The aim of the study is to synthesize omeprazole efficiently. [Method] With CHMO_{NCIMB 9871} from Acinetobacter johnsonii （A. johnsonii）and P450 BM3 from Priestia megaterium （P. megaterium） as templates, the monooxygenase with the highest transformation rate was screened out and the optimal conditions for inducing protein expression were determined. Single factor experiments and response surface methodology were employed to optimize the conditions of this recombinant enzyme for producing omeprazole. [Result] The cyclohexanone monooxygenase CHMO_KT2440 from Pseudomonas sp. synthesized omeprazole with the highest transformation rate. The optimal inducing conditions of CHMO_KT2440 expression were as follows: OD₆₀₀ value of 0.6, induction temperature of 17 ℃, and final IPTG concentration of 0.15 mmol/L. The single factor experiment results showed that the reaction with 0.05 mmol/L FAD, 3.0 g/L substrate mass concentration at 33 ℃ and pH 9.0 for 10 h produced omeprazole with a yield of 78.9%. The CHMO_KT2440-catalyzed synthesis of omeprazole was fitted by the regression equation: y=60.78+9.56A-19.74B+9.55C+1.80AB+5.13AC+1.48BC-18.04A²-0.79B²-9.41C². Based on the single factor experimental results, the transformation conditions were finally optimized as follows: the substrate mass concentration of 3.0 g/L, 34 ℃, pH 9.0, 0.05 mmol/L FAD, and reaction time of 10 h. Under these conditions, the yield of omeprazole reached 79.9%, which increased by 90.23% compared with that before optimization. [Conclusion] This study provides an elite cyclohexanone monooxygenase candidate CHMO_KT2440 for omeprazole synthesis.

Abstract:Objective This study aims to enhance the catalytic activity, thermal stability, and pH stability of α-L-rhamnosidase (EC 3.2.1.40).Method Three combined mutants (K423R-R307Y-D508N, K423R-R307Y-N561D, and K389R-R307Y-N561D) were constructed based on improved single-point mutants.Result Compared with the wild type (WT), the combined mutants K423R-R307Y-D508N exhibited an increased half-life of 0.24 min at both 65 ℃ and 70 ℃. Additionally, its half-life improved by 145.64 min and 4.91 min at pH 7.0 and pH 8.0, respectively. However, its relative enzyme activity was only 41.01% of that of the WT. The combined mutant K423R-R307Y-N561D exhibited an increased half-life of 51.42 min and 20.65 min at pH 7.0 and pH 8.0, respectively, and its relative enzyme activity were 63.26% of that of the WT. The combined mutant K389R-R307Y-N561D achieved an relative enzyme activity 167.20% of that of the WT, with an increased half-life of 1.60 min and 1.34 min at 65 ℃ and 70 ℃, respectively. However, its stability in alkaline environments did not show significant improvement. Molecular docking, molecular dynamics simulations, and MM-PBSA analyses revealed that the increased flexibility of the catalytic domain Loop region and partial α helix structure at the bottom of K389R-R307Y-N561D enhanced substrate binding and affinity. However, the R307 mutation induced local perturbations, disrupting the hydrogen bond network and π-π hydrophobic interactions, leading to decreased structural stability.Conclusion The site compensation mechanism triggered by combined mutations is the primary reason for the difficulty in simultaneously enhancing the catalytic activity and structural stability of α-L-rhamnosidase.

Abstract:Objective This study aimed to develop an efficiently secretory expression system for feruloyl esterase (Fae) and xylanase (Xyn) in Escherichia coli (E. coli).Method The optimal signal peptides for the secretory expression of Fae and β-1,4-endoxylanase in E. coli were screened out from 7 signal peptides. The genes fae and xyn were from the rumen metagenomes of China Holstein cow and camel, respectively. E. coli strains expressing Fae and Xyn were constructed, and their fermentation conditions were optimized for efficient expression and secretion of the two enzymes. Furthermore, ferulic acid was prepared by fermentation with de-starched wheat bran (DSWB) as the substrate. The best performed strain and the optimal production process were then determined.Result The yield and release rate of ferulic acid reached 314.1 mg/L and 90.0%, respectively, after shake flask fermentation for 48 h.Conclusion The engineered E. coli strain co-expressing Fae and Xyn was constructed for ferulic acid production, establishing a theoretical foundation to achieve rapid and high-efficiency biosynthesis of this compound.

Abstract:Objective The aim of this study was to prepare a novel aminopeptidase with excellent properties and evaluate its performance in debittering zein peptides.Method Aminopeptidase aminopeptidase YwaD derived from Bacillus licheniformis (B. licheniformis) SQ1 was expressed in Bacillus subtilis (B. subtilis) WB600, and the enzymatic properties and debittering effect on zein peptides were investigated after purification.Result Aminopeptidase YwaD was expressed in a secretory form in B. subtilis WB600, with the relative molecular mass of about 49 000. Aminopeptidase YwaD showed the optimum reaction temperature of 80 ℃, the half-life of 24 h, and the relative activity higher than 90% in the presence of 4.0 mol/L NaCl, which indicated that aminopeptidase YwaD had strong tolerance to high temperatures and salt. Aminopeptidase YwaD had substrate specificity, with the K_m of 0.093 mol/L and the V_max of 18.975 μg/(L·min) in the case of Leu-pNA as the substrate. Compared with zein peptides produced by single-enzyme hydrolysis using alkaline protease alone (ZA), the hydrolysis degree of zein peptides produced by aminopeptidase YwaD combined with alkaline protease in two steps (ZAAP) increased by 12.14%, the mass concentration of bitter amino acids by 4.85 times, the mass concentration of umami amino acids by 4.74 times, the proportion of peptides with molecular mass less than 500 by 21.14%, the DPPH free radical scavenging rate by 34.53%, and the hydroxyl radical scavenging rate increased by 16.59%, while decreasing the bitter value by approximately 80% and increasing the umami value by 27%.Conclusion Aminopeptidase YwaD could release hydrophobic amino acids (mostly bitter amino acids) at the N-terminus of bitter peptides to significantly remove the bitterness and enhance the antioxidant activity of zein peptides, demonstrating a good application prospect in the preparation of functional plant peptides.

Abstract:Objective The aim of the study is to verify the roles of 3-ketosteroid-9α-hydroxylase (KSH) from Mycobacterium neoaurum NRRL B-3805 in hydroxylation at C-9 and dehydrogenation at C-1,2.Method The oxygenase component kshA395 and the reductase component kshB122 from ksh were heterologously expressed in Escherichia coli (E. coli) BL21(DE3) respectively. With 4-androstene-3,17-dione (4-AD) as substrate, the enzyme-catalyzed reactions were carried out with KshA395, KshB122, and mixture of KshA395 and KshB122 in a ratio of 1∶1. Furthermore, the reaction system was optimized, and fusion plasmids such as pET28a-kshB-kshA were constructed for co-expression of the two-component enzyme of KSH. In addition, the enzyme-catalyzed reaction was performed with 4-AD as the substrate.Result Both KshA395 and KshB122 showed dehydrogenation on 4-AD, and the product was 1,4-androstene-3,17-dione (ADD), with no hydroxylation. The two-component enzyme of KSH, which was mixed in a ratio of 1∶1, showed strong hydroxylation at C-9 in phase 1 of the reaction. In phase 2, hydroxylation was shut down, and dehydrogenation at C-1,2 of 4-AD was turned on. The fusion proteins of KSH, such as KshB122-MBP-KshA395, MBP-KshB122-MBP-KshA395, and MBP-KshB122-KshA395, showed hydroxylation at C-9 in phase 1 and dehydrogenation at C-1,2 in phase 2 of the reaction. Nevertheless, KshB122-KshA395 showed only dehydrogenation at C-1,2 and no hydroxylation.Conclusion In the sterol degradation pathway, KSH can play a role in both hydroxylation and dehydrogenation. It first plays a role in hydroxylation at C-9 and then dehydrogenation at C-1,2, which leads to the degradation of the ring-B in steroids.

Abstract:Objective This study prepared the polyclonal antibodies against Chlamydomonas reinhardtii (C. reinhardtii) channelrhodopsin 2 (ChR2) to study the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.Method The constructed expression vector pET-28a(+)-chr2 was transformed into Escherichia coli BL21(DE3) competent cells to induce the expression of the 6×His-ChR2 fusion protein, which was then purified through a nickel column. The fusion protein was used as an antigen to immunize animals for the production of corresponding antibodies. The antiserum was treated with Protien A to enrich the antibodies, and thus high-purity polyclonal antibodies against C. reinhardtii ChR2 were obtained. Western blot was employed to assess the specificity of the prepared antibodies, and immunofluorescence was used to determine the localization of ChR2 in flagella.Result The purification results of the 6×His-ChR2 fusion protein demonstrated a purity exceeding 85% after purification. The indirect ELISA results showed that the titer of the polyclonal antibodies against C. reinhardtii ChR2 reached 1:25 600. The target band (relative molecular mass 93 000) appeared in electrophoresis of the whole protein extract of wild-type C. reinhardtii CC-125 cells but did not appear in that of the whole protein extract of mutant C. reinhardtii CC-5499 (a mutant with knockout of both chr1 and chr2) cells, indicating that the obtained polyclonal antibodies had high specificity. The localization of ChR2 was in the eyespots and flagella of C. reinhardtii cells.Conclusion The findings lay a foundation for subsequent studies on the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.

Abstract:Objective The aim of the study is to investigate the metabolite changes of Lactococcus lactis (L. lactis) IMAU51121 under acid stress.Method L. lactis IMAU51121 was exposed to acid stress in the GM17 liquid medium at pH 4.0 for 0.5 h. The metabolites changes before and after acid stress were detected by liquid chromatography-mass spectrometry (LC-MS). The multivariate statistical analysis was performed for metabolomics data.Result In total, 1 178 metabolites were identified, from which 20 major differential metabolites were screened based on variable importance in projection (VIP)≥1, P<0.05, and fold change (FC)>1.2. After acid stress, 11 metabolites showed increased relative content, including L-histidine, aspartic acid, and isoleucine, while 9 metabolites demonstrated decreased relative content, including citrulline, β-tyrosine, and uridine diphosphate (UDP)-glucose. KEGG pathway enrichment and topological analysis revealed that acid stress affected five key differential metabolic pathways, including arginine biosynthesis and galactose metabolism.Conclusion Acid stress affects the growth and metabolism of L. lactis IMAU51121, which responds to acid stress by adjusting the levels of amino acids, sugars, lipids, and nucleotides and the metabolic pathways.

Abstract:Objective This study aims to screen and identify microbial strains capable of efficiently reducing the mass concentration of organic acids in lemon juice, and evaluate their application effects.Method Target strain was screened through microbial isolation and purification techniques, and the application conditions in the acid-reducing process of lemon juice were optimized.Result After screening, it was found that the yeast strain Saturnispora diversa SD-59 (SD-59) can efficiently reduce the content of organic acids while producing pleasant flavor compounds. Under the optimal culture conditions (inoculation volume fraction of 1.0%, temperature of 31 ℃, rotation speed of 200 r/min, culture time of 60 h), the total acid mass concentration in lemon juice was reduced by 97.9%, with malic acid and citric acid decreasing by 100.0% and 95.9%, respectively.Conclusion SD-59 exhibits remarkable acid-reducing capability, providing a novel microbial resource for biological deacidification and holding great promise for application in highly acidic environments.

Abstract:Objective To provide a theoretical basis for the development and utilization of Actinomycetes resources in Baijiu-making environment,Actinomycetes were isolated and screened from high-temperature Daqu, and their functional characteristics were analyzed.Method Actinomycetes were screened from high-temperature Daqu using the dilution-plate coating method, and identified based on morphological characteristics and molecular biological evidence. Specifically, the metabolites of the strain were detected by high performance liquid chromatography and gas chromatography, and the Illumina platform was used for whole genome sequencing of the strain.Result The starch-degrading strain XJ-DQ-B-0001 was identified as Cellulosimicrobium funkei.The results of metabolite detection showed that strain XJ-DQ-B-0001 produced 14 metabolites, including lactic acid, acetic acid, β-phenyl ethanol, and ethyl acetate. The genome of strain XJ-DQ-B-0001 was 3 043 158 bp in length, with a GC percentage of 74.57% and 2 769 predicted protein-coding genes. The functional annotation of genes showed that the strain had the potential to produce carbohydrate hydrolases, flavor substances, and potential health factors.Conclusion The functional characterization of strain XJ-DQ-B-0001 shows that Actinomycetes can produce hydrolases, flavor substances, and health factors, which are beneficial to Baijiu brewing. The findings provide data support for the development and utilization of Actinomycetes resources in the Baijiu-producing environment.

Abstract:Objective The aim of the study is to explore the effect of adding sodium nitrite (NaNO₂) and tea polyphenols (TP) in fresh pork on the toxicity in vivo of carbon nanoparticles (CNPs) in fried pork.Method CNPs were obtained from the fried pork prepared from fresh pork without additives (control group), added with NaNO₂, and added with TP. BALB/c male mice were gavaged with 5 mg/kg and 15 mg/kg CNPs, respectively.Result CNPs significantly inhibited the weight gain of mice, reduced the red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin (Hb) mass concentration, and platelet (PLT) count (P<0.05), and affected liver and kidney functions in mice. HE staining showed that CNPs induced significant damage of the liver and colon. CNPs weakened the enzyme activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px), and total superoxide dismutase (T-SOD) and decreased the total antioxidant capacity (T-AOC) in the liver and colon, while elevating the levels of malondialdehyde (MDA), the inflammatory factors interleukin-1β (IL-1β) and interferon-γ (IFN-γ) (P<0.05). Compared to mice gavaged with CNPs extracted from the raw pork added with NaNO₂ after frying, those receiving CNPs from the pork added with TP after frying had slow weight losses and mild organ damage. In addition, gut microbiota analysis revealed that CNPs changed the relative abundance and diversity of intestinal flora in mice, especially reducing the relative abundance of Lactobacillus closely associated with the inhibition of colitis and increasing the relative abundance of Bacteroides associated with septicemia and appendicitis. NaNO₂ aggravated the decrease in relative abundance of Lactobacillus and the increase in relative abundance of Bacteroides, while TP effectively mitigated the relative abundance changes of the above intestinal microorganisms.Conclusion The CNPs extracted from the raw pork added with NaNO₂ after frying were more damaging to mice than the control group. Compared to both the control group and CNPs extracted from the raw pork added with NaNO₂ after frying, the addition of TP can reduce the toxicity of CNPs in vivo. The results provide a perspective to prevent the toxicity of CNPs in vivo and a theoretical basis for assessing the health hazards of red meat products.

Abstract:Objective The aim of the study is to evaluate the safety of an endophytic strain Bacillus velezensis (B. velezensis） BR-1 isolated from rice.Method The Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factor Database (VFDB) were employed to annotate the antibiotic resistance genes and virulence genes, respectively, of BR-1 based on the whole genome sequence. The antibiotic sensitivity test and hemolysis test of BR-1 were performed, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to annotate the genes related to probiotic properties of B. velezensis BR-1.Result The total genome length of B. velezensis BR-1 was 1 071 550 432 bp, with an average GC ratio of 46.3%, containing 4 027 protein-coding genes and 14 CRISPR-Cas. It did not contain genes encoding toxins such as hemolysin BL (Hbl), non-hemolytic enterotoxin (Nhe), enterotoxin (cytotoxin K), and deoxynivalenol (cereulide-Ces). The KEGG annotation revealed genes with good gastrointestinal tolerance, antioxidant activity, and excellent probiotic properties such as acid tolerance, high temperature tolerance, and bile salt tolerance. It was predicted that strain BR-1 contained two antibiotic resistance genes vmlR and clbA. This strain was highly sensitive to 9 antibiotics and did not contain drug-resistant plasmids.Conclusion B. velezensis BR-1 does not contain genes encoding toxins or have broad-spectrum antibiotic resistance and hemolytic activity. It carries genes related to probiotic properties, which meets the safety requirements specified in the General Criteria for Quality and Safety Evaluation of Microbial Fertilizer (GB/T 41728—2022), and can be safely applied as microbial fertilizer.

Abstract:Objective The influences of environmental factors on the stability of Lonicera caerulea (L. caerulea) anthocyanins were studied to provide a theoretical basis for the processing and storage of anthocyanin-rich beverages and expand the application of L. caerulea in food processing.Method Anthocyanins of L. caerulea were obtained by ultrasonic-assisted extraction and macroporous resin purification. The retention rates of L. caerulea anthocyanins at different temperatures, pH, light conditions, oxidation-reducing agents, preservatives, sugars, and metal ions were determined by the pH differential method, and the environmental stability of L. caerulea anthocyanins was analyzed. At the same time, the thermodynamic characteristics of L. caerulea anthocyanins were analyzed according to the degradation kinetics at different pH and temperatures.Result Anthocyanins were sensitive to 50~90 ℃ and pH 1.0~7.0. The retention rate of anthocyanins decreased with the increases in pH and temperature, and the degradation conformed to the first-order kinetic equation. The thermal stability of anthocyanins exhibited strong at pH 2.0 and pH 3.0, and the thermal degradation of anthocyanins was a non-spontaneous endothermic reaction. Light, oxidation-reducing agents, and preservatives exerted adverse effects on the stability of anthocyanins. Sucrose outperformed glucose in maintaining the stability of anthocyanins at low concentrations and within a short time period. At high concentrations, glucose was more conducive to the stability of anthocyanins than sucrose. The color of anthocyanins was enhanced in the presence of Zn²⁺, Ca²⁺, and Mg²⁺. In contrast, Fe²⁺, Fe³⁺, Cu²⁺, and Al³⁺ destroyed the color.Conclusion Environmental factors such as temperature, pH, light, oxidation-reducing agents, and preservatives have adverse effects on the stability of anthocyanins, and the concentrations and types of sugars added and the types of metal ions have different effects on the stability of anthocyanins. The findings provide a scientific basis for the application of anthocyanins in food processing.

Abstract:Objective In order to improve the bioactivities of scallop polysaccharides and clarify the structure-activity relationship of polysaccharides derived from marine mollusk.Method Sulfated polysaccharides with different degrees of substitution were prepared from scallop polysaccharides by the chlorosulfonic acid-pyridine method. The basic components, structure, antioxidant activity, and immune activity were compared between the polysaccharides with and without sulfation.Result The mass fractions of sulfate groups in SSP1 and SSP2 were 22% and 32%, respectively. SSP1 and SSP2 were mainly composed of glucose, and they were acidic polysaccharides with α?-pyranose rings and no triple helical structure. SSP1 and SSP2 had strong scavenging abilities against DPPH free radicals but weak scavenging abilities for ABTS and hydroxyl free radicals in vitro. The immune activity of the polysaccharides was weakened after sulfation.Conclusion Sulfation changed the structures of scallop polysaccharides, enhancing the scavenging ability against DPPH free radicals. Moreover, the scavenging ability increased with the increase in sulfate group content. In addition, sulfation weakened the immune activity of the polysaccharides, which, however, had no toxic or side effects on cells. This study provides a reference for improving the bioactivities of scallop polysaccharides through sulfation.

Abstract:Objective The aim of the study is to improve the stability and utilization of Eucommia ulmoides flavonoids.Method Eucommia ulmoides flavonoids were encapsulated with yeast cells as the wall material. The preparation process of microcapsules was optimized by single factor experiments combined with response surface methodology. The effects of high temperature treatment, ultraviolet irradiation, and long-term storage on the stability of microcapsules were studied. Furthermore, the release behaviors of microcapsules in the simulated gastrointestinal tract and food simulants were investigated.Result The optimal preparation process for microcapsules was core-wall mass ratio of 1∶3, encapsulation time of 5.9 h, and encapsulation temperature of 45 ℃, under which the microcapsule encapsulation rate was (78.6±0.8)%. The microcapsules showed a high retention rate after high temperature treatment, ultraviolet irradiation, and long-term storage. The release rate of microcapsules in the simulated intestinal fluid was significantly higher than that in the simulated gastric fluid. Furthermore, the release in 95% ethanol by volume fraction was significantly higher than that in 10% ethanol by volume fraction. The release behaviors in the two food simulants were consistent with Zero-Order and Hixson-Crowell models (R²>0.90), and the K-value of the Ritger and Peppas model was less than 0.45, indicating Fickian diffusion. The microcapsules were ellipsoidal and had smooth surfaces without cracks, while significant dents and cracks appeared after treatment with ethanol.Conclusion The stability and sustained release characteristics of Eucommia ulmoides flavonoids can be significantly improved by encapsulation in yeast cells.

Abstract:?Objective The aim of the study is to explore the feasibility of ion exchange chromatography as a pre-treatment method for rat fecal samples. ?Method Rat fecal samples were collected and pretreated with three different methods: control (Con) group, centrifugation (Cen) group, and ion exchange chromatography (Chr) group. The DNA extraction quality, 16S rRNA amplicon high-throughput sequencing results, and real-time fluorescence quantitative PCR detection results of specific bacterial groups were compared between samples pretreated with different methods. ?Result The mass concentration of DNA extracted from samples of the Chr group was 1.44 times that of the Cen group and 6.49 times that of the Con group. The number of operational taxonomic unites (OTUs) in the Con group was 2 630, while the number increased to 3 220 and 3 223 in the Cen group and Chr group, respectively. α diversity and β diversity analyses indicated that the Chr group had higher microbial diversity, higher similarity and repeatability among the samples within the group. The results of real-time fluorescence quantitative PCR showed that the abundance of Bifidobacterium adolescentis was different among the three groups. Specifically, the abundance of Bifidobacterium adolescentis was the highest in the Chr group, moderate in the Cen group, and the lowest in the Con group. When the volume of bacterial suspension added increased from 100 μL to 200 μL, there was no significant difference in the abundance of Bifidobacterium adolescentis in the Con group (P>0.05), while the abundance increased to 1.32 times in the Cen group and 1.70 times in the Chr group. This indicated that ion exchange chromatography better preserved the microorganisms in rat fecal samples.Conclusion As a pretreatment method of rat fecal samples, ion exchange chromatography can improve the repeatability and sensitivity of the analysis of intestinal flora.

Abstract:Objective The aim of this study is to establish a rapid, efficient, and sensitive method for the detection of Escherichia coli (E. coli) O157:H7.Method The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated method was used to conjugate antibodies with time-resolved fluorescent microspheres (markers), and a fluorescence reader was used for quantitative analysis and detection of E. coli O157:H7. The detection sensitivity was improved by optimizing the pH of buffer for labeling, the amount of time-resolved fluorescent microspheres, the mass concentration of T-line antibodies, and the incubation time of samples and labeled antibodies.Result Under the optimal conditions, the detection sensitivity for E. coli O157:H7 reached 3.2×10³ CFU/mL, which was 10~1 000 times higher than that of colloidal gold immunochromatography, and there was no cross-reaction with other 8 common food-borne pathogens. In addition, the established test strip demonstrated good detection performance for the real samples of artificially contaminated drinking water, milk, and beef.Conclusion The EU-TRFM immunochromatographic test strip is suitable for the detection of E. coli O157:H7.