Abstract:Fatty acid desaturase, namely Δ12/Δ15 (FADS12/15) are widespread in plants, animals, and microorganisms. They are involved in the biosynthesis of polyunsaturated fatty acids (PUFAs) and play an important role in the production of PUFAs. PUFAs provide various biological and physiological benefits, however, due to the lack of FADS12/15 in the human body, linoleic acid and α?-linolenic acid cannot be synthesized. Therefore, their intake must be derived from the diet. Furthermore, other PUFAs are essential for improving diet and human health. Thus, it is urgent to develop high-level PUFA producers. Microorganisms are potential fatty acid producers, and FADS12/15 genetic engineering has been performed to increase the yield of ω-3/ω-6 PUFAs, which provide humans with high-quality lipid resources. This article summarizes the fatty acid biosynthetic pathways and the catalytic mechanism of FADS12/15. It also reviews FADS12/15 overexpression system and genetically engineered microorganisms for PUFA production as well as the homologous or heterologous expression of FADS12/15 gene to produce PUFAs. The objective of this review is to provide the fundamental theory and efficient expression strategy for FADS12/15 genetic engineering to produce PUFAs.

Abstract:Biofilms are sessile microbial communities growing on surfaces, which are encased in some self-produced polysaccharides, proteins, lipids, and extracellular DNA. Biofilms were often supposed to be potentially destructive to the environment and detrimental to medical, food, and marine industries due to their tough structures and difficult to remove from surface. However, the biofilms formed by bacteria such as Bacillus can be widely used in agriculture, food, medicine, environment and other fields, and can promote biofilm formation by interfering with quorum sensing and improving the environmental conditions of culture. The author summarizes the process of biofilm formation, focuses on the application of biofilms in the pre-harvest and post-harvest biological control of agricultural products, and explains some strategies currently used to promote the formation of biofilms, which aims to provide reference for further development and application of biofilms in the fields of agriculture and food.

Abstract:Constipation is a common gastrointestinal disorder. Some Bifidobacterium animalis subsp. lactis has been shown to have an ameliorative effect on constipation, but there is variation in their effectiveness. In order to investigate the effect of different B. lactis strains on constipation, an animal model of constipation was established, and the constipation-related indexes, gut microbiota and their metabolite levels were determined through the intervention experiments of three B. lactis strains, to investigate the effects of constipation alleviation of the strains and their mechanisms. The results showed that CCFM1278 and BB12 significantly improved the weight of defecation, number of fecal pellets, fecal water content, time to first black stool, and small intestinal propulsion rate in constipated mice, while the effect of FJSSZ6M5 was not significant. B. lactis CCFM1278 modulated the relative abundance of Akkermansia, Ruminiclostridium 6, Ruminococcus 1 and Alloprevotella in the gut microbiota of constipated mice, improved the abnormal levels of short-chain fatty acids and deoxycholic acid, significantly improved the secretion of gastrin and somatostatin and inhibited the expression of aquaporin 3 and aquaporin 8. In conclusion, B. lactis CCFM1278 has the potential to relieve constipation. This study provides a new direction for the treatment of patients with constipation.

Abstract:In order to investigate the quality differences of bud-type Congou black tea produced from different tea cultivars, high-performance liquid chromatography (HPLC), solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS), quantitative descriptive analysis (QDA), and chemometrics were employed to analyze the characteristic taste substances and aroma components of bud-type Congou black tea made from ten tea cultivars. The results of QDA showed that the overall aroma of the bud-type Congou black tea from the 10 cultivars was predominantly sweet and floral, while the taste was mainly sweet and mellow. Sixteen differentiated internal components (non-volatile compounds) were detected across the different samples, with an average coefficient of variation of 21.63%, and the greatest variation observed in theaflavin. A total of 49 volatile compounds were identified in the black tea from 10 tea cultivars, with alcohols being the most abundant class, followed by esters, and ketones being the least abundant. Geraniol was found to have the highest mass fraction among the volatile components. Orthogonal partial least squares-discriminant analysis (OPLS-DA) effectively distinguish ‘Golden Peony’ and ‘Fuyun No. 7’ from other cultivars, and 12 markers closely related to bud-type Congou black tea cultivars were identified. Correlation analysis revealed a significant positive correlation between caffeine and tea polyphenols with aftertaste sweetness, while caffeine and theobromine were significantly positively correlated with body. Geranyl alcohol, cis-?α,α?-5-trimethyl-5-vinyltetrahydrofuran-2-methanol, methyl salicylate, and linalool were positively correlated with floral and fruity aromas. This study provides a theoretical basis for the production, processing, and quality improvement of bud-type Congou black tea.

Abstract:Melittin is one kind of small molecule active peptide with anti-inflammatory and antitumor effects. The soluble expression and purification of melittin to obtain active peptide without fusion label has always been a problem. The authors conducted a recombinant expression strain Escherichia coli/pET21a-ELP-Intein-MET based on the phase transition property of elastin-like polypeptide (ELP) and the self-cracking property of intein. Soluble expression of fusion protein was achieved in E. coli BL21 (DE3) cells. The supernatant of the recombinant cells was purified by a temperature-induced reversible phase transformation cycle, and optimization of pH, temperature and time of the fusion protein self-lysis. The melittin without fusion label with a purity of over 95% was obtained after lysis at pH 7.0 and 30 ℃ for 32 h, and the following gel chromatography. The inhibition experiments of purified melittin against Staphylococcus aureus, Bacillus subtilis 168 and Escherichia coli JM109 were performed. Melittin showed a stronger inhibitory effect for gram-positive bacteria than gram-negative bacteria. The minimum inhibitory concentrations of melittin for 3 strains of bacteria were 12.5 μg/mL, 50 μg/mL and 100 μg/mL, respectively. The authors realized the high expression of melittin by the introduction of elastin and endopeptide, and non-fusion labelled melittin was purified by a simple isolation. The bacteriostatic functions of purified active melittin were very similar to that of commercial melittin, which is expected to provide new insights for the industrial preparation of melittin.

Abstract:T-2 toxin is a highly toxic trichothecene compound, which exhibits multi-organ toxicity. To investigate the toxic effects of T-2 toxin on different cell lines and the related molecular mechanisms, we analyzed the effects of T-2 toxin exposure on oxidative stress, Ca²⁺ levels, and apoptosis in four cell lines. The key genes and potential signaling pathways of T-2 toxin on sensitive cells were analyzed using transcriptomics. Results revealed that BV2 microglial cells and HepG2 cells exhibited sensitivity to T-2 toxin, displaying a significant increase in intracellular reactive oxygen species (ROS) and Ca²⁺ levels, leading to elevated cell apoptosis. Transcriptomic data and RT-PCR analysis indicated that T-2 toxin triggered apoptosis in BV2 and HepG2 cells through distinct signaling pathways. Additionally, BV2 cells showed an inflammatory response via the MAPK/AP-1 and TNF-NF-κB pathways, upregulating factors like AP-1 and IL-6. Conversely, HepG2 cells underwent apoptosis through the PI3K/AKT and PERK/P53/Casp3 pathways, downregulating Akt and SIRT1, while upregulating superoxide dismutase. Overall, T-2 toxin induced apoptosis in BV2 and HepG2 cells, respectively through inflammatory and oxidative stress-related pathways, highlighting its potential to affect different organs via diverse molecular mechanisms.

Abstract:Immunoassay technology plays an important role in the field of rapid detection of food safety, with antibodies being the core component of immunoassay. However, the current detection of antibodies used in foods is mainly based on polyclonal antibodies or monoclonal antibodies obtained in large quantities through the in vivo cultivation of hybridoma cells, which not only face animal welfare and viral contamination, but also face problems, such as non-targeted antibody contamination and loss of cellular antibody genes. This study focused on the common fungal toxin zearalenone (ZEN) in foods. Based on the self-made anti-ZEN monoclonal hybridoma cell line (Z14) in our laboratory, sub-type specific primers were used to amplify the full-length gene sequences of the heavy and light chains of the anti-ZEN antibody. The expression vector of the ZEN recombinant full-length antibody was constructed and the PEI mediated transient transfection expression of the ZEN recombinant full-length antibody in Expi 293F cells was achieved. The results of the indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) showed that the half inhibitory mass concentration (IC₅₀) of the ZEN inhibition standard curve established was based on recombinant full-length antibodies, which was 2.43 ng/mL. Moreover, the mass concentration in the linear range (IC₂₀~IC₈₀) was 0.66~8.88 ng/mL, while the affinity constant (K_a) was 3.87×10¹⁰ L/mol. The performance of the ZEN monoclonal antibodies prepared from ascites was superior to that of traditional ascites, providing a basis for a large-scale production of recombinant full-length antibodies against ZEN based on in vitro cell culture in the later stage.

Abstract:Alginate lyases could effectively degrade alginates into alginate oligosaccharides (AOS). AOS have good physical characteristics and excellent physiological functions, and are widely applied in the fields of food, cosmetics, and pharmaceuticals. To efficiently prepare AOS, a novel alginate lyase belonging to the PL-7 family and derived from Reinekea thalattae was screened and identified, designated as Reth-aly. The encoding gene of Reth-aly was cloned and overexpressed in Escherichia coli BL21. Under conditions of pH 7.5 (Tris-HCl buffer), 35 ℃ and 600 mmol/L NaCl, Reth-aly demonstrated its highest catalytic activity. With sodium alginate as the substrate, its catalytic activity reached 765.7 U/mg. Degradation products included AOS with a degree of polymerization (DP) of 2~4. Compared with other PL-7 family alginate lyases, it showed good stability with a half-life of 5.4 h at 35 ℃. Reth-aly showed robust catalytic activity in high-salt environments, indicating promising application prospects in AOS bioproduction.

Abstract:Cordyceps militaris strain ZA10-C4, a newly developed strain through hybrid breeding, exhibits high cordycepin production. In order to obtain a suitable medium formulation for its growth and further improve cordycepin yield, the fermentation medium need to be optimized and the response surface method was applied. Cordycepin was extracted and purified from the fermentation broth using the macroporous resin NKA-II. The optimal fermentation medium formulation was determined to contain maltose17.99 g/L, yeast extract powder 41.13 g/L, L-alanine 11.83 g/L, KH₂PO₄ 2.00 g/L, MgSO₄ 1.50 g/L, and vitamin B₁ 12 mg/L, respectively. Under these optimized conditions, the yield of cordycepin reached (4 503.14±15.54) mg/L, a 1.66-fold increase compared to before optimization levels. Meanwhile, the optimal extraction and purification process was identified as follows: natural pH, a sample loading volume of 600 mL, a sample loading and elution flow rate of 240 mL/h, and ethanol elution with 70% volume fraction at 800 mL. The resulting cordycepin achieved a purity of 94.89% with a yield of 89.57%. This study provides technical reference for the large-scale production of novel Cordyceps militaris strain ZA10-C4 and the efficient preparation of cordycepin.

Abstract:Partially hydrolyzed guar gum (PHGG) with different relative molecular mass was prepared using controlled enzymatic hydrolysis technology, and the mass fraction of galactomannan in PHGG was calculated by ion chromatography. The molecular structure of PHGG was characterized by infrared spectroscopy, nuclear magnetic resonance and X-ray diffraction. Thermodynamic properties of PHGG were investigated by differential scanning calorimetry and thermogravimetric analysis. The results showed that the critical factors influencing the enzymatic hydrolysis of PHGG were substrate mass concentration, enzyme addition amount, and enzymatic hydrolysis time. By manipulating these factors, medium and high relative molecular mass PHGG (molar mass 8.92×10⁵ g/mol), medium relative molecular mass PHGG (molar mass 5.07×10⁵ g/mol) and low relative molecular mass PHGG (molar mass 0.62×10⁵ g/mol) were prepared. Among them, the mass fraction of galactomannan in medium relative molecular mass alcohol-precipitated PHGG reached 84.09%. In PHGG with different relative molecular mass, the mass fraction ratio of mannose to galactose decreased, and the main chemical structure remained unchanged, but there were minor disruptions to the crystalline region structure, indicating that PHGG exhibited good thermal stability even at elevated temperatures. Overall, PHGG with different relative molecular mass, prepared by controlled enzymatic hydrolysis, has good stability in thermally processed foods, which provides a theoretical basis for the application of guar gum.

Abstract:To comprehensively understand the influence of column polarity on the analysis of volatile compounds of Fu brick tea, the separation and identification performance of three chromatographic columns with varying polarities (polar column HP-INNOWAX, medium-polarity column DB-35MS, and non-polar column HP-5MS) on the volatile compounds of Fu brick tea were compared. The results indicated that a total of 169 volatile compounds were identified by three chromatographic columns with different polarities, among which 29 volatile compounds were common. HP-INNOWAX, DB-35MS, and HP-5MS identified 77, 66, and 116 volatile compounds, respectively, with unique volatile compounds being 32, 12, and 64 for each column type. In terms of the mass fraction of the identified compounds, HP-INNOWAX provided the highest mass fraction, accounting for 43% of the total compound mass, followed by DB-35MS with 31%, and HP-5MS with the lowest at 26%. Three chromatographic columns with different polarities exhibited specific advantages in the analysis of the volatile compounds in Fu brick tea. The polar column HP-INNOWAX performed best in separating alcohols, the medium-polarity column DB-35MS was the most effective for ketones, and the non-polar column HP-5MS was excelled in separating hydrocarbons, ketones, and aldehydes. The volatile compounds of Fu brick tea are analyzed using chromatographic columns of varying polarities, providing a more comprehensive understanding of its aroma profile and revealing its unique flavor characteristics.

Abstract:The secondary structure of protein has an extremely important impact on their spatial structure and function. Using machine learning methods to predict the secondary structure of grain proteins is one of the important topics in the fields of biology, food, etc.In this experiment, three grain proteins of corn, wheat, and soybeans were selected from the existing protein data bank,using multi-feature fusion method to extract features of protein sequence, and propose to combine multi-core learning with least square support vector machine classifier, the linear weighted combination of multiple kernel functions is used to replace the traditional single kernel function, and the kernel weight is used to adjust the fusion effect to construct a multi-core LSSVM prediction model to predict the secondary structure of grain proteins.The particle swarm optimization algorithm is used to optimize the model hyper parameters and find the best hyper parameter combination to improve the prediction performance of the model. The experimental results prove that the multi-kernel LSSVM algorithm proposed in this paper can make up for the limitations of single kernel function high-dimensional mapping, integrate the advantages of each kernel function, and find the best hyperparameter combination through the PSO algorithm, combine multiple feature extraction methods to significantly improve the Q₃ accuracy of grain protein secondary structure prediction.

Abstract:Bovine casein is one of the main protein components of infant formula milk powder. Its phosphorylation level is higher than that of human casein, which limits its direct application in infant foods. In order to make bovine casein closer to human casein at the molecular level, the author considered milk protein concentrate (MPC) as the research object. A series of MPCs with different dephosphorylation degrees were prepared by using potato acid phosphatase (PAP) as the dephosphorylation modification treatment. Urea-PAGE, microwave digestion-UV/visible absorption spectrometry and ultra high-performance liquid tandem mass spectrometry (UPLC-MS) were applied to investigate the changes in dephosphorylation degree. Moreover, the digestibility of dephosphorylated MPC was also investigated using an in vitro digestion model. The results showed that a series of MPC with dephosphorylation rate of 0~85.8% was obtained after the modification of MPC by PAP dephosphorylation. With the increase in dephosphorylation degree, the looser clots were observed, and the gastrointestinal digestibility of MPC increased under in vitro conditions.

Abstract:Heparin sodium production wastewater (HSW) is rich in nutrients, making it a potential culture medium for microalgae. To explore its feasibility for microalgal cultivation, three strains of Chlorella sorokiniana (GT1, XY3-3, and CMBB276) were cultured in HSW at varying volume fractions (10%~40%), and Chlorella sorokiniana CMBB276 with high tolerance to HSW was screened. To determine the optimal volume fraction of wastewater, the author investigated the removal processes of nutrients, along with the variations in biochemical composition and calorific value, during the growth of the strain CMBB276 in HSW at different volume fractions. Results demonstrated that as the wastewater volume fraction increased, the maximum cell density, total nitrogen (TN) removal efficiency, mass fractions of carbohydrates and fatty acids, and protein productivity of CMBB276 gradually decreased, while the total organic carbon (TOC) and TN removal rates and protein mass fraction gradually increased. The TOC removal efficiency and calorific value remained relatively unchanged. The optimal wastewater volume fraction was determined to be 20%. At the end of cultivation, the cell density of CMBB276 was 6.4×10⁷ cells/mL, with TOC and TN removal efficiencies of 83.3% and 63.0%, respectively. The mass fraction and productivity of protein was 52.5% and 22.22 mg/(L·d), respectively, with a calorific value of 20.37 MJ/kg. Therefore, cultivating C. sorokiniana in HSW at an appropriate volume fraction can efficiently remove TOC and TN from the wastewater, while the cultured CMBB276 strain, characterized by its high protein content and calorific value, holds significant potential for resource utilization.

Abstract:Insufficient aroma and unremarkable typicality of wolfberry wine limit the development of the wolfberry wine industry. Twenty-six strains of non-Saccharomyces cerevisiae (non-S.cerevisiae) were isolated from Lycium barbarum 'Ningqi No. 5' fruit and orchard soil. Six non-S. cerevisiae strains with excellent β-glucosidase (β-G) production ability were screened. Their physiological characteristics were determined using growth curve analysis and tolerance tests to ethanol and osmotic pressure. The co-fermentation of Wickerhamomyces anomalus (W. anomalus) Y11 with S. cerevisiae M-23-7-14 was optimized for wolfberry wine production, with quality assessed based on conventional physicochemical indices, flavor compounds, and sensory evaluation. Results indicated that the optimal fermentation performance was achieved when S. cerevisiae M-23-7-14 and W. anomalus Y11 were inoculated at an inoculum amount of 10∶1, with W. anomalus Y11 inoculated first, followed by S. cerevisiae M-23-7-14 after a 12 h interval. In comparison to single-strain fermentation, the total mass concentration of flavor compounds increased by 21.7% during the mixed-strain fermentation, with a 85.8% increase in the concentration of esters. This study could provide a superior yeast for the wolfberry wine industry, optimize the fermentation process, and enhance the aroma quality of wolfberry wine, offering theoretical support for industrial production.

Abstract:To optimize the method for determining ester compounds in wine using dispersive liquid-liquid microextraction (DLLME), a simulated wine solution was used as the test material. The effects of seven factors, including type and volume of dispersant, type and volume of extractant, sample volume, salt addition amount, and extraction time, on the extraction efficiency of 16 ester compounds in simulated wine were investigated. Single-factor experiments and Box-Behnken response surface methodology were combined to optimize the extraction conditions, and qualitative and quantitative analyses were performed using gas chromatography-mass spectrometry (GC-MS). The results showed that the optimal extraction conditions for ester compounds in the simulated wine were 1 200 μL of methanol as the dispersant, 600 μL of dichloromethane as the extraction solvent, a sample volume of 7 mL, no adjustment of ionic strength, and an extraction time of 1.0 min. Under these conditions, all 16 esters compounds under investigation exhibited good linearity with R² exceeding 0.995, while the limits of detection (LOD) and quantitation (LOQ) ranged from 0.004 mg/L to 0.078 mg/L and 0.012 mg/L to 0.261 mg/L, respectively. The spiked recoveries at three mass concentration levels ranged from 83.3% to 111.3%, with intra-day and inter-day relative standard deviations (RSDs) between 0.02% to 7.69%. When applied to ‘Pinot Noir’ dry red wine and Italian ‘Riesling’ ice wine samples from 2018 to 2020, the method showed good applicability, confirming that the types and concentrations of ester compounds were closely related to the wine type and aging year. This study could provide a basis for the in-depth study of the metabolism of ester compounds in wine.

Abstract:In order to investigate the role of lactic acid bacteria in controlling spoilage during the opening and consumption of sauce products, the salt tolerance and antimicrobial capabilities of lactic acid bacteria were examined, along with their impacts on the number of viable lactic acid bacteria, total bacterial colony count, bacterial community composition and the main volatile compounds responsible for unpleasant odors in a simulated household consumption scenario. The results indicated that Lactobacillus plantarum can tolerate a NaCl mass fraction of 7% and possesses strong antimicrobial properties; during the simulated household consumption of sauce, Lactobacillus plantarum effectively suppressed the total number of bacterial colonies and maintained the stability of viable lactic acid bacteria counts; the relative abundance of Lactobacillus in the sauce, with the addition of Lactobacillus plantarum, was 83.6%, ensuring Lactobacillus as the dominant genus in the sauce; the relative mass fraction of the main volatile compounds causing unpleasant odors was reduced to only 4.71% of that in the blank control, effectively inhibiting the production of unpleasant odors in the sauce. Thus, Lactobacillus plantarum can extend the actual shelf life of sauce products during household use and holds potential for application in green food preservation.