Improving the level of the cytidine biosynthesis in E. coli through atmospheric room temperature plasma mutagenesis and metabolic engineering.

AIMS: Cytidine, as an important commercial precursor in the chemical synthesis of antiviral and antitumor drugs, is in great demand in the market. Therefore, the purpose of this study is to build a microbial cell factory with high cytidine production. METHODS AND RESULTS: A mutant E. coli NXBG-11-F34 with high tolerance to uridine monophosphate structural analogs and good genetic stability was obtained by atmospheric room temperature plasma (ARTP) mutagenesis combined with high-throughput screening. Then, the udk and rihA genes involved in cytidine catabolism were knocked out by CRISPR/Cas9 gene editing technology, and the recombinant strain E. coli NXBG-13 was constructed. The titer, yield, and productivity of cytidine fermented in a 5 l bioreactor were 15.7 g l-1, 0.164 g g-1, and 0.327 g l-1 h-1, respectively. Transcriptome analysis of the original strain and the recombinant strain E. coli NXBG-13 showed that the gene expression profiles of the two strains changed significantly, and the cytidine de novo pathway gene of the recombinant strain was up-regulated significantly. CONCLUSIONS: ARTP mutagenesis combined with metabolic engineering is an effective method to construct cytidine-producing strains.

De novo transcriptome profiling reveals the patterns of gene expression in plum fruits with bud mutations.

Bud mutation is a common technique for plant breeding and can provide a large number of breeding materials. Through traditional breeding methods, we obtained a plum plant with bud mutations (named "By") from an original plum variety (named "B"). The ripening period of "By" fruit was longer than that of "B" fruit, and its taste was better. In order to understand the characteristics of these plum varieties, we used transcriptome analysis and compared the gene expression patterns in fruits from the two cultivars. Subsequently, we identified the biological processes regulated by the differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed that these DEGs were highly enriched for "single-organism cellular process" and "transferase activity". KEGG analysis demonstrated that the main pathways affected by the bud mutations were plant hormone signal transduction, starch and sucrose metabolism. The IAA, CKX, ARF, and SnRK2 genes were identified as the key regulators of plant hormone signal transduction. Meanwhile, TPP, the beta-glucosidase (EC3.2.1.21) gene, and UGT72E were identified as candidate DEGs affecting secondary metabolite synthesis. The transcriptome sequencing (RNA-seq) data were also validated using RT-qPCR experiments. The transcriptome analysis demonstrated that plant hormones play a significant role in extending the maturity period of plum fruit, with IAA, CKX, ARF, and SnRK2 serving as the key regulators of this process. Further, TPP, beta-glucosidase (EC3.2.1.21), and UGT72E appeared to mediate the synthesis of various soluble secondary metabolites, contributing to the aroma of plum fruits. The expression of BAG6 was upregulated in "B" as the fruit matured, but it was downregulated in "By". This indicated that "B" may have stronger resistance, especially fungal resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01472-3.

A pH-Stable Tb-MOF as Luminescence Sensor for Highly Sensitive Detection of Amino Acids through Diverse Sensing Mechanism.

A luminescent Tb-MOF with excellent stability and dual-emitting properties was constructed with an amide-functionalized tetracarboxylate ligand. Tb-MOFs were initially assembled on one-dimensional Tb3+ chains, then formed a two-dimensional double-decker layer through the synergistic linking of organic ligands and bridging formic acid anions, and further fabricated the final three-dimensional structure through the connection of the organic ligands. Powder X-ray diffraction experiments revealed that Tb-MOFs not only exhibited excellent stability in water but also maintained structural integrity in the pH range of 2-12. Importantly, this Tb-MOF provided the first example of a metal-organic framework (MOF)-based luminescence sensor that can simultaneously detect two acid amino acids (aspartic and glutamic acids) through a turn-off sensing mechanism and two basic amino acids (lysine and arginine acids) through unusual turn-on and turn-off-on sensing mechanisms. Moreover, high sensitivity, low detection limit, and excellent recyclability of this sensor endow Tb-MOFs with great potential as a highly efficient amino acid fluorescence sensor in chemical detection and biological environments.

Transcriptome analysis reveals the mechanism of different fruit appearance between apricot (Armeniaca vulgaris Lam.) and its seedling.

BACKGROUND: Apricot fruit has great economic value. In the process of apricot breeding using traditional breeding methods, we obtained a larger seedling (named Us) from the original variety (named U). And Us fruit is larger than U, taste better. Therefore, revealing its mechanism is very important for Apricot breeding. METHODS: In this study, de novo assembly and transcriptome sequencing (RNA-Seq) was used to screen the differently expressed genes (DEGs) between U and Us at three development stages, including young fruits stage, mid-ripening stage and mature fruit stage. RESULTS: The results showed that there were 6,753 DEGs at different sampling time. "Cellulose synthase (UDP-forming) activity" and "cellulose synthase activity" were the key GO terms enriched in GO, of which CESA and CSL family played a key role. "Photosynthesis-antenna proteins" and "Plant hormone signal transduction" were the candidate pathways and lhca, lhcb, Aux/IAA and SAUR were the main regulators. CONCLUSION: The auxin signaling pathway was active in Us, of which Aux/IAAs and SAUR were the key fruit size regulators. The low level of lhca and lhcb in Us could reveal the low demand for exogenous carbon, but they increased at mature stage, which might be due to the role of aux, who was keeping the fruit growing. Aux and photosynthesis maight be the main causes of appearance formation of Us fruits. Interestingly, the higher expression of CESA and CSL proved that Us entered the hardening process earlier than U. The advanced developmental progress might also be due to the role of Aux.

Overexpression of uracil permease and nucleoside transporter from Bacillus amyloliquefaciens improves cytidine production in Escherichia coli.

Cytidine is an important raw material for nucleic acid health food and genetic engineering research. In recent years, it has shown irreplaceable effects in anti-virus, anti-tumor, and AIDS drugs. Its biosynthetic pathway is complex and highly regulated. In this study, overexpression of uracil permease and a nucleoside transporter from Bacillus amyloliquefaciens related to cell membrane transport in Escherichia coli strain BG-08 was found to increase cytidine production in shake flask cultivation by 1.3-fold (0.91 ± 0.03 g/L) and 1.8-fold (1.26 ± 0.03 g/L) relative to that of the original strain (0.70 ± 0.03 g/L), respectively. Co-overexpression of uracil permease and a nucleoside transporter further increased cytidine yield by 2.7-fold (1.59 ± 0.05 g/L) compared with that of the original strain. These results indicate that the overexpressed uracil permease and nucleoside transporter can promote the accumulation of cytidine, and the two proteins play a synergistic role in the secretion of cytidine in Escherichia coli.

Insight into the Construction of (3,6)-Connected rtl, ant, and Chiral anh Nets Based on Structural Investigation of Several MOFs via Steric Tuning of Linkers.

Six cobalt-organic frameworks (1-6) were solvothermally constructed with a tritopic pyridine-carboxylate linker (L-H) and its methyl- and methoxy-functionalized derivatives (L-OCH3 and L-CH3). Due to incorporated multiple substituents with various steric hindrances, the tritopic linkers adopt different molecular configurations, Y-shaped and T-shaped, which further combine octahedral or trigonal-prismatic inorganic nodes to afford diverse (3,6)-connected nets. Consequently, 1 and 2 are rtl nets and 3 and 4 are ant nets. Notably, 5 and 6 present rarely observed chiral anh (flu-3) networks with left-handed double helical chains. The structural investigation indicates that the steric tuning of linkers may essentially dictate the resulting diverse MOF structures. Furthermore, the MOFs presented here can be regarded as an ideal structural platform for a better understanding of the assembly of (3,6)-connected rtl, ant, and chiral anh nets, which are closely related to the shape and geometric configuration/conformation of tridentate organic nodes as well as inorganic building nodes.

Trends in urban/rural inequalities in physical growth among Chinese children over three decades of urbanization in Guangzhou: 1985-2015.

BACKGROUND: Great growth inequalities between urban and rural areas have been reported in China over the past years. By examining urban/rural inequalities in physical growth among children < 7 years old over the past three decades from 1985 to 2015 in Guangzhou, we analyzed altering trends of anthropometric data in children and their association with economic development during the period of rapid urbanization in Guangzhou. METHODS: The height, body weight and nutrition status of children under 7 years old were obtained from two successive cross-sectional surveys and one health surveillance system. Student's t-test, Spearman's rank-order correlation and polynomial regression were used to assess the difference in physical growth between children in urban and rural areas and the association between socioeconomic index and secular growth changes. RESULTS: A height and weight difference was found between urban and rural children aged 0-6 years during the first two decades of our research (1985-2005), which gradually narrowed in both sex groups over time. By the end of 2015, elder boys (age group ≥5 year) and girls (age group ≥4 year) in rural areas were taller than their counterparts in urban areas (p < 0.05).The same trend could be witnessed in the weight of children aged 6 years, with a - 1.30 kg difference (P = 0.03) for boys, and a - 0.05 difference (P = 0.82) for girls. When GDP increased, the gap in boys' weight-for-age z-score (WAZ from 0.25 to 0.01) and height-for-age z-score (HAZ from 0.55 to 0.03) between urban and rural areas diminished, and disappeared when the GDP per capita (USD) approached 25,000. In either urban or rural areas, the urbanization rate and GDP were positively associated with the prevalence of obesity (all R > 0.90 with P < 0.05) and negatively correlated with the prevalence of stunted growth (all R < -0.87 with P < 0.05). CONCLUSION: Growth inequalities gradually decreased with economic development and urbanization, while new challenges such as obesity emerged. To eliminate health problems due to catch-up growth among rural children, comprehensive intervention programs for early child growth should be promoted in rural areas.

Double-Gap Magnetic Flux Concentrator Design for High-Sensitivity Magnetic Tunnel Junction Sensors.

To improve the sensitivity of the magnetic tunnel junction(MTJ)sensor, a novel architecture for a double-gap magnetic flux concentrator (MFC) was studied theoretically and experimentally in this paper. The three-dimensional finite element model of magnetic flux was established to optimize the magnetic field amplification factor, with different gaps. The simulation results indicate that the sensitivity of an MTJ sensor with a double-gap MFC can be significantly better than that of a sensor with a traditional single-gap MFC, due to the fact that the magnetic magnification sharply increases with the decrease in effective gap width. Besides this, the half-bridge MTJ sensors with the double-gap MFC were fabricated using photolithography, ion milling, evaporation, and electroplating processes. Experimental results show that the sensitivity of the MTJ sensor increased by ten times compared to the sensor without the double-gap MFC, which underlines the theoretical predictions. Furthermore, there is no significant increase in the sensor noise. The work in this paper contributes to the development of high-performance MTJ sensors.

Fermentation by Multiple Bacterial Strains Improves the Production of Bioactive Compounds and Antioxidant Activity of Goji Juice.

Microorganisms can be used for enhancing flavors or metabolizing functional compounds. The fermented-food-derived bacterial strains comprising Bacillus velezensis, Bacillus licheniformis, and Lactobacillus reuteri mixed with Lactobacillus rhamnosus and Lactobacillus plantarum were used to ferment goji berry (Lycium barbarum L.) juice in this study. The fermentation abilities and antioxidant capacities of different mixtures of multiple strains in goji juice were compared. The results showed that the lactic acid contents increased 9.24-16.69 times from 25.30 ± 0.71 mg/100 mL in goji juice fermented using the SLV (Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus velezensis), SZP (Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus licheniformis), and SZVP (Lactobacillus rhamnosus, Lactobacillus plantarum, Bacillus velezensis, and Bacillus licheniformis) mixtures, and the protein contents increased 1.31-2.11 times from 39.23 ± 0.67 mg/100 mL. In addition, their contents of volatile compounds increased with positive effects on aroma in the fermented juices. Conversion of the free and bound forms of phenolic acids and flavonoids in juice was influenced by fermentation, and the antioxidant capacity improved significantly. Fermentation enhanced the contents of lactic acid, proteins, volatile compounds, and phenols. The antioxidant capacity was strongly correlated with the phenolic composition.

Assembly of Two Metal-Organic Frameworks Based on Distinct Cobalt Dimeric Building Blocks Induced by Ligand Modification: Gas Adsorption and Magnetic Properties.

Solvothermal reaction of 3,5-di(pyridin-4-yl) benzoic acid (HDPB) with Co(II) leads to a novel metal-organic framework, [Co2O(DPB)2(DMF)2]· xS (1), which represents a rare reo-type net with trigonal prismatic cobalt dimer, [Co2O(CO2)2N4], as building blocks to construct a 3D framework containing three different types of nanoscale M12L12 and M24L12 polyhedron cages. More interestingly, under the same condition, the assembly of 4-methyl-3,5-di(pyridin-4-yl) benzoic acid (HMDPB) with Co(II) facilitates the formation of a cationic framework, [Co2(MDPB)3(DMF)](NO3)· xS (2), with cobalt dimer, [Co2(CO2)3N4], as building blocks. Complex 2 represents the first example of a zeolite-like network with 48-nuclear SOD cage. The significant effect of subtle modification of ligand on the overall MOFs is discussed. Moreover, the gas adsorption studies reveal that 1 exhibits permanent porosity and selective CO2 uptake. Variable-temperature magnetic susceptibility measurements show that both 1 and 2 exhibit antiferromagnetic behavior.

Construction of luminescent dye@MOF platforms for sensing antibiotics with enhanced selectivity and sensitivity.

The fabrication of luminescent dye@MOF composites has received extensive attentions in the development of realistic sensing applications. Herein, based on two anionic In-MOFs with different pore structure (1 and 2), the charge and size dependent ion-exchange of cationic dyes was investigated, and consequently four luminescent dye@MOF composites (DMASM@1/2 and RhB@1/2) were successfully fabricated and importantly can be regarded as ideal platforms for better understanding of the factors affecting the construction of dye@MOF composites, which may closely related to a well match between the intrinsic properties and size/charge of the fluorescent molecules and the porosity, structure character of the MOF hosts. Furthermore, these four dye@MOF composites were utilized for sensing of different kinds of antibiotics, demonstrating enhanced selectivity and sensitivity. DMASM@1/2 demonstrated excellent selectivity and sensitivity for NFT and NFZ antibiotics, while RhB@1/2 exhibited excellent selectivity and sensitivity for MDZ and DTZ antibiotics. Systematic analysis of the detection mechanism revealed that different energy transfer efficiency and interaction between MOF frameworks and different types of guest dyes led to different selectivity and detection mechanisms for antibiotics. Moreover, high selectivity and sensitivity, low LOD and extraordinary recycling capacity of four dye@MOF composites in the detection of antibiotics promote their excellent prospect in the further practical application.

Polyphenol Extracts from Ziziphus jujuba Mill. "Junzao" Attenuates Ulcerative Colitis by Inhibiting the NLRP3 and MAPKs Signaling Pathways and Regulating Gut Microbiota Homeostasis in Mice.

SCOPE: Polyphenols are the major active substances in red jujube fruit, and their anti-inflammatory and antioxidant activities suggest their potential utility in the prevention of ulcerative colitis (UC). METHODS AND RESULTS: In this study, the effect of polyphenol extracts from red jujube (Ziziphus jujuba Mill. "Junzao") (PERJ) on the dextron sulfate sodium (DSS)-induced UC mice is investigated. The result shows that PERJ effectively improves clinical symptoms, including food and water intake, the disease activity insex (DAI) and spleen index, and routine blood levels, and alleviates the shortening of the colon, in mice with DSS-induced UC. Meanwhile, PERJ remarkably decreases the expression of proinflammatory factors. Moreover, PERJ repairs intestinal barrier damage by increasing the expression level of mucin 2 and mucin 3, and the result is also confirmed in the histological assessment. Besides, the expression levels of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and mitogen-activated protein kinase cascade (MAPKs) signaling pathway-related proteins are inhibited by the PERJ administration. Finally, 16S rRNA sequencing analyses reveal that PERJ reverses intestinal microbiota dysbiosis by enhancing the abundance of Firmicutes and decreasing that of Proteobacteria and Bacteroidetes. CONCLUSION: PERJ probably inhibits the development of UC by suppressing the NLRP3 and MAPKs signaling pathways and regulating gut microbiota homeostasis, and can be considered as a potential resource for preventing UC.

Genome-wide identification of the MADS-box gene family in Avena sativa and its role in photoperiod-insensitive oat.

Background: Traditional spring-summer sown oat is a typical long-day crop that cannot head under short-day conditions. The creation of photoperiod-insensitive oats overcomes this limitation. MADS-box genes are a class of transcription factors involved in plant flowering signal transduction regulation. Previous transcriptome studies have shown that MADS-box genes may be related to the oat photoperiod. Methods: Putative MADS-box genes were identified in the whole genome of oat. Bioinformatics methods were used to analyze their classification, conserved motifs, gene structure, evolution, chromosome localization, collinearity and cis-elements. Ten representative genes were further screened via qRT‒PCR analysis under short days. Results: In total, sixteen AsMADS genes were identified and grouped into nine subfamilies. The domains, conserved motifs and gene structures of all AsMADS genes were conserved. All members contained light-responsive elements. Using the photoperiod-insensitive oat MENGSIYAN4HAO (MSY4) and spring-summer sown oat HongQi2hao (HQ2) as materials, qRT‒PCR analysis was used to analyze the AsMADS gene at different panicle differentiation stages under short-day conditions. Compared with HQ2, AsMADS3, AsMADS8, AsMADS11, AsMADS13, and AsMADS16 were upregulated from the initial stage to the branch differentiation stage in MSY4, while AsMADS12 was downregulated. qRT‒PCR analysis was also performed on the whole panicle differentiation stages in MSY4 under short-day conditions, the result showed that the expression levels of AsMADS9 and AsMADS11 gradually decreased. Based on the subfamily to which these genes belong, the above results indicated that AsMADS genes, especially SVP, SQUA and Mα subfamily members, regulated panicle development in MSY4 by responding to short-days. This work provides a foundation for revealing the function of the AsMADS gene family in the oat photoperiod pathway.

Strategies to Enhance l-Isoleucine Synthesis by Modifying the Threonine Metabolism Pathway in Escherichia coli.

l-threonine as an important precursor substance of l-isoleucine and improving its accumulation in Escherichia coli became an important idea to construct a chassis strain with high l-isoleucine production. Meanwhile, the effect of l-threonine metabolic pathway disruption in E. coli for the improved production of l-isoleucine remains unrevealed. In the present study, a mutant strain of E. coli was engineered by inactivating specific metabolic pathways (e.g., Δtdh, ΔltaE, and ΔyiaY) that were associated with l-threonine metabolism but unrelated to l-isoleucine synthesis. This was done with the aim to reduce the breakdown of l-threonine and, thereby, increase the production of l-isoleucine. The results obtained demonstrated a 72.3% increment in l-isoleucine production from 4.34 to 7.48 g·L-1 in the mutant strain compared with the original strain, with an unexpected 10.3% increment in bacterial growth as measured at OD600. Transcriptome analysis was also conducted on both the mutant strain NXU102 and the original strain NXU101 in the present study to gain a comprehensive understanding of their physiological attributes. The findings revealed a notable disparity in 1294 genes between the two strains, with 658 genes exhibiting up-regulation and 636 genes displaying down-regulation. The activity of tricarboxylic acid (TCA) cycle-related genes was found to decrease, but oxidative phosphorylation-related genes were highly up-regulated, which explained the increased activity of the mutant strain. For instance, l-lysine catabolism-related genes were found to be up-regulated, which reconfigured the carbon flow into the TCA cycle. The augmentation of acetic acid degradation pathway-related genes assisted in the reduction in acetic acid accumulation that could retard cell growth. Notably, substantial up-regulation of the majority of genes within the aspartate pathway could potentially account for the increased production of l-isoleucine in the present study. In this paper, a chassis strain with an l-isoleucine yield of 7.48 g·L-1 was successfully constructed by cutting off the threonine metabolic pathway. Meanwhile, transcriptomic analysis revealed that the cutting off of the threonine metabolic pathway induced perturbation of genes related to the pathways associated with the synthesis of l-isoleucine, such as the tricarboxylic acid cycle, glycolysis, and aspartic acid pathway.

The efficacy and safety of Bazi Bushen Capsule in treating premature aging: A randomized, double blind, multicenter, placebo-controlled clinical trial.

PURPOSE: It is unclear whether traditional Chinese patent medicines can resist premature aging. This prospective study investigated the effects of Bazi Bushen Capsule (BZBS) which is a traditional Chinese patent medicine for tonifying the kidney essence on premature senility symptoms and quality of life, telomerase activity and telomere length. STUDY DESIGN AND METHODS: It was a parallel, multicenter, double-blind, randomized, and placebo-controlled trial. Subjects (n = 530) aged 30-78 years were randomized to receive BZBS or placebo capsules 12 weeks. The primary outcome was the clinical feature of change in kidney deficiency for aging evaluation scale (CFCKD-AES) and tilburg frailty indicator (TFI). The secondary outcomes were SF-36, serum sex hormone level, five times sit-to-stand time (FTSST), 6MWT, motor function test-grip strength, balance test, walking speed, muscle mass measurement, telomerase and telomere length. RESULTS: After 12 weeks of treatment, the CFCKD-AES and TFI scores in the BZBS group decreased by 13.79 and 1.50 respectively (6.42 and 0.58 in the placebo group, respectively); The SF-36 in the BZBS group increased by 98.38 (23.79 in the placebo group). The FTSST, motor function test grip strength, balance test, walking speed, and muscle mass in the elderly subgroup were all improved in the BZBS group. The telomerase content in the BZBS group increased by 150.04 ng/ml compared to the placebo group. The fever led one patient in the placebo group to discontinue the trial. One patient in the placebo group withdrew from the trial due to pregnancy. None of the serious AEs led to treatment discontinuation, and 3 AEs (1.14%) were assessed as related to BZBS by the primary investigator. CONCLUSIONS: BZBS can improve premature aging symptoms, frailty scores, and quality of life, as well as improve FTSST, motor function: grip strength, balance test, walking speed, and muscle mass in elderly subgroups of patients, and enhance telomerase activity, but it is not significantly associated with increasing telomere length which is important for healthy aging. TRIAL REGISTRY: https://www.chictr.org.cn/showproj.html?proj=166181.

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens.

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

Advances in Drug Discovery Targeting Lysosomal Membrane Proteins.

Lysosomes are essential organelles of eukaryotic cells and are responsible for various cellular functions, including endocytic degradation, extracellular secretion, and signal transduction. There are dozens of proteins localized to the lysosomal membrane that control the transport of ions and substances across the membrane and are integral to lysosomal function. Mutations or aberrant expression of these proteins trigger a variety of disorders, making them attractive targets for drug development for lysosomal disorder-related diseases. However, breakthroughs in R&D still await a deeper understanding of the underlying mechanisms and processes of how abnormalities in these membrane proteins induce related diseases. In this article, we summarize the current progress, challenges, and prospects for developing therapeutics targeting lysosomal membrane proteins for the treatment of lysosomal-associated diseases.

Immobilization of Enzyme Electrochemical Biosensors and Their Application to Food Bioprocess Monitoring.

Electrochemical biosensors based on immobilized enzymes are among the most popular and commercially successful biosensors. The literature in this field suggests that modification of electrodes with nanomaterials is an excellent method for enzyme immobilization, which can greatly improve the stability and sensitivity of the sensor. However, the poor stability, weak reproducibility, and limited lifetime of the enzyme itself still limit the requirements for the development of enzyme electrochemical biosensors for food production process monitoring. Therefore, constructing sensing technologies based on enzyme electrochemical biosensors remains a great challenge. This article outlines the construction principles of four generations of enzyme electrochemical biosensors and discusses the applications of single-enzyme systems, multi-enzyme systems, and nano-enzyme systems developed based on these principles. The article further describes methods to improve enzyme immobilization by combining different types of nanomaterials such as metals and their oxides, graphene-related materials, metal-organic frameworks, carbon nanotubes, and conducting polymers. In addition, the article highlights the challenges and future trends of enzyme electrochemical biosensors, providing theoretical support and future perspectives for further research and development of high-performance enzyme chemical biosensors.

Physicochemical properties and in vitro digestibility of proso millet starch modified by heat-moisture treatment and annealing processing.

Heat-moisture treatment (HMT) and annealing (ANN) were applied to modify the proso millet starch, and then the physicochemical properties as well as the in vitro digestion of the modified starch were investigated systematically. Results indicated that HMT and ANN did not change the typical A-type crystallinity. However, both processes cause cracks and dents on the surface of the granule. The gelatinization temperature increased while peak viscosity value, relative crystallinity and gelatinization enthalpy of proso millet starch decreased significantly after HTM and ANN. Meanwhile, a remarkable increase of the slowly digestible starch(SDS) and resistant starch(RS) content was noticed after HTM and ANN modification (the highest content of SDS and RS after HTM and ANN were 9.52 ± 0.82 %, 12.03 ± 1.36 % and 12.15 ± 0.89 %, 8.75 ± 1.63 %, respectively). Those results indicated that the ANN and HMT processes could modify the physicochemical properties and in vitro digestion of proso millet starch efficiently and showed potential application to produce healthy starch food with lower digestion.

Construction of Probiotic Double-Layered Multinucleated Microcapsules Based on Sulfhydryl-Modified Carboxymethyl Cellulose Sodium for Increased Intestinal Adhesion of Probiotics and Therapy for Intestinal Inflammation Induced by Escherichia coli O157:H7.

The decreased number of viable bacteria and the ability of Bifidobacterium to adhere to and colonize the gut in the gastrointestinal environment greatly limit their efficacy. To solve this problem, thiolated carboxymethyl cellulose sodium (CMC) probiotic double-layered multinucleated microcapsules with Bifidobacterium adolescentis FS2-3 in the inner layer and Bacillus subtilis SN15-2 embedded in the outer layers were designed. First, the viable counts and release rates of microcapsules were examined by in vitro simulated digestion assays, and it was found that microcapsules were better protected from gastrointestinal digestion than the controls. Compared with free Bifidobacterium strains, double-layered multinucleated microcapsules have higher viable bacterial survival rates and storage stability. Second, through in vitro rheology, tensile tests, isotherm titration calorimetry, and adhesion tests, it was observed that thiolated CMC could enhance the strong interaction of Bifidobacterium with intestinal mucus and significantly promote the proliferation and growth of probiotics. Finally, double-layered multinucleated microcapsules containing B. adolescentis FS2-3 and B. subtilis SN15-2 modified with sulfhydryl-modified CMC were studied in the intestine. Alleviation of Escherichia coli O157:H7 induced intestinal inflammation. The results showed that microencapsulation could significantly increase the colon content of Bifidobacterium, relieve intestinal inflammation symptoms in mice with bacterial enteritis, and repair the intestinal microbiota disorder caused by inflammation. The probiotic double-layered multinucleated microcapsules prepared in this study can improve the survival rate of probiotics and promote proliferation, adhesion, and colonization of probiotics.