Discovery and directed evolution of C-C bond formation enzymes for the biosynthesis of ß-hydroxy-α-amino acids and derivatives.

ß-Hydroxy-α-amino acids (ß-HAAs) have extensive applications in the pharmaceutical, chemical synthesis, and food industries. The development of synthetic methodologies aimed at producing optically pure ß-HAAs has been driven by practical applications. Among the various synthetic methods, biocatalytic asymmetric synthesis is considered a sustainable approach due to its capacity to generate two stereogenic centers from simple prochiral precursors in a single step. Therefore, extensive efforts have been made in recent years to search for effective enzymes which enable such biotransformation. This review provides an overview on the discovery and engineering of C-C bond formation enzymes for the biocatalytic synthesis of ß-HAAs. We highlight examples where the use of threonine aldolases, threonine transaldolases, serine hydroxymethyltransferases, α-methylserine aldolases, α-methylserine hydroxymethyltransferases, and engineered alanine racemases facilitated the synthesis of ß-HAAs. Additionally, we discuss the potential future advancements and persistent obstacles in the enzymatic synthesis of ß-HAAs.

Strategic manipulation of biofilm dispersion for controlling Listeria monocytogenes infections.

Listeria monocytogenes (L. monocytogenes), a gram-positive foodborne pathogen that can easily cause listeriosis. It secretes extracellular polymers and forms biofilms that are highly resistant to disinfection methods, such as UV light and germicides, posing risks to food processing equipment and food quality. Dispersion of biofilm is the cycle of its formation in which the bacteria return to planktonic state and become susceptible to antimicrobials, the strategic manipulation of biofilm dispersion is thus heralded as a novel and promising approach for the effective control of biofilm-related infections. Compared to the traditional methods, it is more effective to start with the composition of biofilms, cut off the production of their constituent substances, and genetically reduce the probability of biofilm formation. Meanwhile, the dispersion of bacteria can be supplemented with exogenous substances, making long-term control possible. This paper provides a brief but comprehensive overview of the mechanisms of L. monocytogenes biofilms or cross-contamination and their resistance properties, and facilitates our understanding and control of the prevention and containment of L. monocytogenes biofilm contamination based on the biofilm's active and passive diffusion strategies. This work provides practical guidelines for the food industry to guard against the enduring threat to food safety due to L. monocytogenes biofilms.

Purification, characterization and inactivation kinetics of polyphenol oxidase extracted from Cistanche deserticola.

MAIN CONCLUSION: PPO was purified from Cistanche deserticola, and its enzymatic characteristics were clarified. It was found that microwave treatment was an efficient way to inactivate PPO. Polyphenol oxidase (PPO) from Cistanche deserticola was obtained and purified through an acetone precipitation and anion exchange column, the enzymatic characteristics and inactivation kinetics of PPO were studied. The specific activity of PPO was 73135.15 ± 6625.7 U/mg after purification, the purification multiple was 48.91 ± 4.43 times, and the recovery was 30.96 ± 0.27%. The molecular weight of the PPO component is about 66 kDa by SDS-PAGE analysis. The optimum substrate of PPO was catechol (Vmax = 0.048 U/mL, Km = 21.70 mM) and the optimum temperature and pH were 30 °C and 7, respectively. When the temperature is above 50 °C, pH < 3 or pH > 10, the enzyme activity can be significantly inhibited. The first-order kinetic fitting shows that microwave inactivation has lesser k values, larger D values and shorter t1/2. It was found that microwave treatment is considered as an efficient and feasible way to inactive PPO by comparing the Z values and Ea values of the two thermal treatments.

Simultaneous detection of enrofloxacin and florfenicol in animal-derived foods based on fluorescence quenching BELISA and a nanozyme catalytic strategy.

Enrofloxacin (ENRO) and florfenicol (FF) are animal-specific drugs, but they present great harm to human health. Therefore, it is essential to rapidly and accurately detect ENRO and FF in animal-derived foods simultaneously. Herein, dual-template molecular imprinted polymers (MIPs) with specific recognition of ENRO and FF were prepared, meanwhile, the molar ratios of templates to monomer and cross-linker were optimized and then applied as a bionic antibody to experiment. Based on the principle that the fluorescence of QDs could be efficiently quenched by the enzymatic fabrication of Prussian blue nanoparticles (PBNPs), a novel and sensitive fluorescence quenching biomimetic enzyme-linked immunosorbent assay (BELISA) was established for simultaneous detection of ENRO and FF by the conversion of the absorption signal into fluorescent signals. Under optimal conditions, the detection limit (IC15) was 4.64 ng L-1 for ENRO and 1.33 ng L-1 for FF. Besides, matrix interference of chicken, eggs, milk and shrimp samples, was investigated in our study, and the result indicates that all of the sample matrices had a profound impact on the fluorescence of QDs, especially for milk samples (with Im of 94.10 %). After performing the matrix-elimination experiments, chicken, eggs, milk and shrimp samples spiked with ENRO and FF were extracted and detected by this proposed method, with recoveries ranging from 82.70 to 113.48 %. The results correlated well with those obtained using HPLC. In conclusion, the developed method could be an alternative and sensitive method for the simultaneous detection of ENRO and FF in animal-derived foods.

Development of two novel ELISAs based on Prussian blue nanoparticles for ultrasensitive detection of norfloxacin in milk.

The development of traditional enzyme-linked immunosorbent assay (ELISA) systems for the detection of small-molecule residues in foods is limited because of the poor stability of biological enzymes and the lower sensitivity of absorption-based signals. Herein, two ELISAs based on Prussian blue nanoparticles (PBNPs) were developed to establish ultrasensitive and stable methods for detecting norfloxacin (NOR) in milk. The results show that the detection limit (IC15) of NOR was 0.77 µg L-1 and the sensitivity (IC50) was 18.28 µg L-1 in the standard solution using the PBNPs-based nano-ELISA. When the fluorescence quenching ELISA based on PBNPs was used, the detection limit was 0.06 µg L-1 and the sensitivity was 4.21 µg L-1 in the standard solution. The recoveries and precision were good, as confirmed by analysis of real milk samples. The results were consistent with those of commercial ELISA kits, indicating the high accuracy of these two methods.

An Operating Stiffness Controller for the Medical Continuum Robot Based on Impedance Control.

Continuum manipulators can conform to curvilinear paths and manipulate objects in complex environments, which makes it emerging to be applied in minimally invasive surgery (MIS). However, different and controllable operating stiffness of the continuum manipulator is required during different stages of surgery to achieve safe access or stable and precise operation. This work proposes an operating stiffness controller (OSC) for the typical tendon-driven continuum manipulator based on the variable impedance control method with Lagrangian dynamic modeling. This controller can adjust the operating stiffness by modifying the driving forces along the driving tendons of the continuum manipulator without changing its material or structure. The proposed OSC converts the damping and stiffness matrices of the impedance control into variable parameters. This merit allows it to dynamically adjust the operating stiffness of the continuum manipulator according to the desired constant or time-varying stiffness. Furthermore, the OSC stability can be proven based on a Lyapunov function, and its stiffness control performances have been analyzed and evaluated in both simulations and experiments. The OSC controller generated average relevant error values of 7.82% and 3.09% for the operating stiffness control experiments with constant and time-varying desired stiffness, respectively. These experimental results indicate that the OSC has high accuracy, stability, and strong robustness in the operating stiffness control tasks.

Application and prospect of microbial food Chlorella.

Modern food is evolving in the direction of green, healthy, and convenient products, and developing natural products with health benefits is an important direction for the food industry. Chlorella is rich in nutrients, such as carotene and fatty acids, which provide it with a variety of health benefits, and therefore widely used in the food industry as a health or functional food. This study reviews the research progress and specific applications of Chlorella in health, functional, and other foods, and expounds on the bottlenecks faced in the use of Chlorella in food industry. This review provides a theoretical basis for the research, utilisation, and production of new food materials involving Chlorella.

Enhancing water retention and mechanisms of citrus and soya bean dietary fibres in pre-fermented frozen dough.

In recent years, the production of prepared and frozen foods has increased with economic development. However, during freezing, moisture migration forms ice crystals that damage food structure and reduce quality. This study investigates moisture migration changes in pre-fermented dough during frozen storage and effectiveness of Citrus fibre (CF) and Soya dietary fibre (SDF) on quality improvement. Pre-fermented frozen dough properties were evaluated at different freezing storage days with CF and SDF. Results showed frozen storage reduced water retention, converting deeply bound water to weakly bound and free water. Freezable water content increased significantly from 53% (fresh) to 56.95% (60d-control), forming disruptive ice crystals in gluten protein structure. SDF had superior water flow restriction compared to CF, preventing large ice crystal accumulation, enhancing water-holding capacity, and maintaining gluten protein structure. These findings lay a theoretical foundation for improving quality and industrial applications of pre-fermented frozen dough.

Preparation of pectin-coated and chitosan-coated phenylethanoside liposomes: Studies on characterization, stability, digestion and release behavior.

In this paper, the effects of extrusion, ultrasound on physicochemical properties of liposomes were studied, and the liposomes were prepared by ethanol injection combined with extrusion-ultrasound. In addition, the quality of PhGs lips, pectin-coated PhGs lips (P-lips) and chitosan-coated PhGs lips (C-lips) was evaluated by the average particle size, encapsulation efficiency (EE) and other indicators, which indicated that the nanoparticles had been successfully prepared. Compared with extrusion or ultrasonic operation alone, the EEs of ethanol injection combined with extrusion-ultrasonic increased by 8 % and 18 % respectively. Subsequently, transmission electron microscopy, Fourier transform infrared spectroscopy and DSC thermal analysis showed that PhGs in PhGs lips may produce hydrogen bonding forces with phospholipids, and pectin and chitosan in P-lips and C-lips were not only coated on the surface of PhGs lips, but also might have some interaction between them. Cell experiments showed that PhGs lips, P-lips and C-lips can effectively improve the bioavailability of PhGs. In addition, the storage stability of P-lips and C-lips was not significantly improved compared to PhGs lips, but their digestive stability was significantly improved, and the final retention rate in simulated intestinal fluid was about 25 % higher than that of PhGs lips.

One-pot enantioselective synthesis of chiral phenyllactic acids by combining stereocomplementary d- and l-lactate dehydrogenases with multi-enzyme expression fine-tuning.

Chiral phenyllactic acid (PLA) is a new type of antiseptic agent and a valuable precursor for active ingredients in pharmaceuticals and agrochemicals. In this study, we designed a multi-enzyme cascade that combined stereocomplementary d- and l-lactate dehydrogenases with threonine aldolase, phenylserine dehydratase, and formate dehydrogenase for the one-pot conversion of achiral glycine and benzaldehyde to synthesize d-PLA and l-PLA. To overcome the imbalance of multi-enzymes in a single cell, two enzyme modules, overexpressing four enzymes, were assembled in Escherichia coli cells to construct whole-cell catalysis systems (WCCSs). Furthermore, by optimizing reaction conditions and components, recombinant E. coli (WCCS 26) was able to produce 100 mM d-PLA with >99 % ee using a fed-batch strategy, while E. coli (WCCS 60) produced 47.2 mM l-PLA with >99 % ee. This study presents a sustainable and efficient method for synthesizing chiral PLAs from food-grade achiral starting materials.

Proposal and Verification of the Theory of Layer-by-Layer Elimination of Biofilm in Listeria monocytogenes.

Biofilms are microbial communities that represent a high abundance of microbial life forms on Earth. Within biofilms, structural changes during clearance processes occur in three spatial and temporal dimensions; therefore, microscopy and quantitative image analysis are essential in elucidating their function. Here, we present confocal laser scanning microscopy (CLSM) in conjunction with ISA-2 software analysis for the automated and high-throughput quantification, analysis, and visualisation of biofilm interiors and overall biofilm properties in three spatial and temporal dimensions. This paper discusses the removal process of Listeria monocytogenes (LM) biofilms using slightly acidic electrolytic water, non-electrolytic hypochlorite water, and alternating the use of strongly acidic and strongly alkaline electrolytic water. The results show that the biofilm gradually thins and gutters from the initial viscous dense and thick morphology under the action of either biocide. This process is consistent with first-level kinetics. After CLSM filming to observe the biofilm structure, analysis software was used to process and quantify the biovolume, average biofilm thickness, biofilm roughness and other indicators; fluorescence enzyme markers were used to verify the remaining amount of extracellular nucleic acid. In this study, we proposed and validated the theory of layer-by-layer elimination of LM biofilm.

[Teaching reform and practice of "food enzymology and enzyme engineering" based on cutting-edge researches].

Food enzymology and enzyme engineering is an important professional course of food science. The course includes the basic theory of enzymology, enzyme engineering technology and the application of enzymes in food industry. Considering the knowledge gap between the teaching contents and the cutting-edge researches, the team constantly adjusted and optimized the course contents to enable students to keep up with state-of-the-art progress by carefully mining the cutting-edge researches. Taking cutting-edge researches as the breakthrough point, we explored the problem-based learning (PBL) teaching model under the guidance of outcome-based education (OBE) concept, and highlighted the importance of the teacher-student and student-student interactions to improve students' enthusiasm and participation. A diversified assessment system was established to evaluate the performance of students in the learning process. The teaching reform consolidated the basic knowledge and expanded the academic frontiers, and fostered students' ability in analyzing problems, designing solutions and achieving team communication. The course may give new insights into the teaching reform of food enzymology and enzyme engineering and other related courses.

Antioxidant Benefits and Potential Mechanisms of Slightly Acidic Electrolyzed Water Germination in Sesame.

Slightly acidic electrolytic water (SAEW) treatment for seed germination is a promising technique for sustainable agriculture. This study investigated the antioxidant activity of germinated sesame seeds treated with SAEW for the first time. Specifically, the impact and correlation of SAEW on the activities of total phenols, total flavonoids, and antioxidant oxidase in sesame seeds were examined. The results showed that SAEW with low ACC inhibited sesame germination, SAEW with high ACC promoted sesame germination, and sesame buds treated with SAEW with 30 mg/L and 50 mg/L ACC showed lower antioxidant activity and total phenolic and flavone content compared to tap water. In contrast, SAEW with 30 mg/L ACC had no significant effect on sesame growth but positively influenced the antioxidant activity of sesame seed germination by promoting phenolic compound synthesis through increased phenylalanine ammonia-lyase (PAL) activity and enhancing antioxidant activity by boosting PAL, polyphenol oxidase (PPO), and peroxidase (POD) activities. Generally, antioxidant ability was the most prominent in SAEW with 30 mg/L ACC, and positive correlations between antioxidation and total phenols and flavonoids content were found in sesame. These findings provide valuable insights into the mechanisms underlying the enhanced antioxidant capacity observed in germinated sesame seeds under SAEW stress.

Application of molecular imprinting technology based on new nanomaterials in adsorption and detection of fluoroquinolones.

Irrational use of fluoroquinolones (FQs) can lead to allergic reactions, adverse reactions to the heart and damage of the liver; thus, it is of great significance to establish rapid, sensitive and accurate detection methods for FQs. Molecularly imprinted polymers (MIPs) with specific structures synthesized by molecular imprinting technology (MIT) are widely used for the detection of FQs due to their high specificity, high sensitivity and stable performance. Recently, new functional nanomaterials with different morphologies and sizes, which can provide rich sites for surface chemical reactions, have attracted more and more attention of the researchers. Thus, the application status and development prospects of MIT based on new nanomaterials in the adsorption and detection of FQs were summarized in this study, providing a theoretical basis and technical guarantee for the development of new and efficient food safety analysis strategies based on MIPs.

Mechanism of acid and alkali electrolyzed water on the elimination of Listeria monocytogenes biofilm based on proteomic analysis.

Acidic electrolyzed water is a relatively mature bactericide, which has a certain inhibitory effect on a variety of microorganisms, and is widely used in the field of food processing for cleaning, sterilization and disinfection. This study investigated the deactivation mechanisms of Listeria monocytogenes by Tandem Mass Tags quantitative proteomics analysis. Samples were treated through A1S4 (Alkaline electrolytic water treatment for 1 min and Acid electrolytic water treatment for 4 min), S3A1S1 (Acid electrolyzed water treatment 3 min, Alkaline electrolyzed water treatment 1 min and Acid electrolyzed water treatment 1 min), S5 (Acid electrolytic water treatment for 5 min). Proteomic analysis showed that the mechanism of acid alkaline electrolyzed water treatment to eliminate the inactivation of the biofilm of L. monocytogenes was related to protein transcription and extension, RNA processing and synthesis, gene regulation, sugar and amino acid transport and metabolism, signal transduction and ATP binding. The study on the influence mechanism and action mechanism of the combination of acidic and alkaline electrolyzed water to remove L. monocytogenes biofilm is helpful to understand the development of the process of removing biofilm by electrolyzed water, and provides theoretical support for the treatment of other microbial contamination problems in food processing by electrolyzed water.

Nutrient Composition of Germinated Foxtail Millet Flour Treated with Mixed Salt Solution and Slightly Acidic Electrolyzed Water.

Germination of millet can improve its consumption quality, optimize its nutritional composition, and promote the accumulation of functional components such as γ-aminobutyric acid (GABA). In the present study, foxtail millet was germinated with tap water, a mixed salt solution of 7.5 mmol/L NaCl and 15 mmol/L CaCl2, and slightly acidic electrolyzed water (SAEW) with three available chlorine concentrations (ACCs; 10.92, 20.25, and 30.35 mg/L). The effects of the salt solution and SAEW on the germination of foxtail millet and the GABA, crude protein, and amino acid composition of the germinated millet flour were analyzed. The results showed that the salt solution and SAEW treatments promoted the growth of millet sprouts, contributed to the accumulation of GABA in germinated millet flour, and optimized the protein and amino acid composition. The GABA content of germinated foxtail millet flour treated with salt solution for 60 h (336.52 mg/100 g) was 29.5 times higher than that of ungerminated millet flour. In conclusion, the highest GABA content and amino acid scores of germinated millet flour obtained by germination treatment with salt solution at 25 °C and 86% humidity for 60 h were more acceptable for human nutritional requirements.

Research Progress and Trends of Phenylethanoid Glycoside Delivery Systems.

BACKGROUND: Phenylethanoid glycosides (PhGs) are obtained from a wide range of sources and show strong biological and pharmacological activities, such as antioxidant, antibacterial and neuroprotective effects. However, intestinal malabsorption and the low bioavailability of PhGs seriously affect their application. Delivery systems are an effective method to improve the bioavailability of active substances. Scope and approach: In this article, the biological activities of and delivery systems for PhGs are introduced. The application statuses of delivery systems for echinacoside, acteoside and salidroside are reviewed. Finally, the problems of the lack of uniform standards for delivery systems and the poor targeted delivery accuracy of PhGs in the current research are proposed and suggestions for future research are put forward based on those problems. KEY FINDINGS AND CONCLUSIONS: Although there are still some problems in the delivery system of phenylethanoside, such as inconsistent standards and inaccurate delivery, phenylethanoside itself has been proven to have a variety of physiological activities. Therefore, the action mechanism and application of phenylethanoside and its delivery system should be studied further.

Bactericidal Effect and Associated Properties of Non-Electrolytic Hypochlorite Water on Foodborne Pathogenic Bacteria.

This study investigated the broad-spectrum bactericidal activity of non-electrolytic hypochlorite water (NEHW) and detected its hydroxyl radical content compared with that of slightly acidic electrolytic water (SAEW). Based on the results of UV scanning and storage stability, higher hypochlorite content and stronger oxidation were found to be responsible for the stronger bactericidal effect of NEHW. NEHW can achieve 99% bacterial disinfection effect by treating with 10 mg/L available chlorine concentration for more than 5 minutes. At the same time, the storage stability of NEHW was higher than that of SAEW. After 20 days of storage under sealed and dark conditions, the pH value only increased by 7.9%, and the effective chlorine concentration remained nearly 80%. The results showed that NEHW had higher germicidal efficacy and storage stability than SAEW.

Effect of high-voltage electrostatic field pretreatment on the antioxidant system in stored green mature tomatoes.

BACKGROUND: High-voltage electrostatic field (HVEF), as a feasible and non-chemical technique, applied to food preservation is a new area of study. Our previous research suggested HVEF could maintain the quality of tomato in storage. The present article intensively investigated the effect of HVEF pretreatment on antioxidant system of green mature tomato in storage. RESULTS: Green mature tomatoes were exposed to negative (or positive) HVEF for 2 h at 20 °C and then stored for 24 days at 13 ± 1 °C, 85-90% relative humidity. The results indicated HVEF significantly reduced contents of O(2)(-) and H(2)O(2) of tomato fruits during storage compared to the control. HVEF treatment also enhanced the activities of antioxidant enzymes including catalase, superoxide dismutase, ascorbate peroxidase and peroxidase. The contents of non-enzyme antioxidant components including reduced glutathione, phenols and ascorbic acid also were increased by HVEF treatment. However, HVEF treatment did not increase the content of lycopene in tomato fruit. CONCLUSION: HVEF treatment could promote the antioxidant capacity of tomato fruits in storage. Further research is be highly recommended to understand the mechanisms improving the antioxidant capacity of tomato fruits by HVEF.

[Catalytic mechanism, molecular engineering and applications of threonine aldolases].

Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish ß-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure ß-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.