Expression and Purification of Recombinant Hepatitis Delta Virus(HDV) Antigen for Use in a Diagnostic ELISA for HDV Infection Using the High-Density Fermentation Strategy in Escherichia coli

Objective Hepatitis Delt a Virus（HDV） antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose. Methods Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method. Results The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified（98%） by immobilized metal ion affinity chromatography（IMAC） using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity（97% for IgM and 100% for IgG） and specificity（100% for IgG and IgM） for the detection of anti-HDV antibodies. Conclusion Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells

AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.

Glycation of high-density lipoprotein triggers oxidative stress and promotes the proliferation and migration of vascular smooth muscle cells

Background In type 2 diabetes mellitus （T2DM）, high-density lipoprotein （HDL） impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated HDL （G-HDL） was modified with D-glucose （25 mmol/L） in vitro. Diabetic HDL （D-HDL） was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofiuorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species （ROS） was detected based on ROS-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration. Conclusions HDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.

OXIDIZED HIGH-DENSITY LIPOPROTEIN PROMOTES MATURATION AND MIGRATION OF BONE MARROW DERIVED DENDRITIC CELLS FROM C57BL/6J MICE

Objective To explore the influence of oxidized high-density lipoprotein （oxHDL） on the maturation and migration of bone marrow-derived dendritic cells （BMDCs） from C57BL/6J mice. Methods The C57BL/6J mice bone marrow cell suspension was prepared and purified. Recombinant granulocyte-macrophage colony-stimulating factor （rmGM-CSF） and recombinant interleukin-4 （rmlL-4） were used to promote monocytes to differentiate and suppress lymphocytes. Then 50μg/mL oxHDL was added to stimulate BMDCs, using 50μg/mL high-density lipoprotein （HDL） as homologous protein control, PBS as negative control, and 1 μg/mL lipopolysaccharide （LPS） as positive control. The CD86 and MHCII expression rates were detected with fluorescence-activated cell sorting （FACS）. Liquid scintillation counting （LSC） was used in mixed lymphocyte reactions （MLRs） to reflect the ability of BMDCs in stimulating the proliferation of homologous T cells, Levels of cytokines IL-12 and IL-10 were detected by ELISA. The cell migration was evaluated with the transwell system. Results Compared with PBS group, the expressions of CD86 and MHCII, counts per minute of MLRs, secretion of IL-12 and IL-10, and number of migrated cells in oxHDL group and LPS group significantly increased （all P 〈 0.05）, while the increment was less in oxHDL group than LPS group. The number of migrated cells in oxHDL group was about twice of that in HDL group. Conclusion OxHDL may promote the maturation and migration of BMDCs in vitro.

Current status and challenges for cell-cultured milk technology: a systematic review

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture.While most cellular agriculture is predominantly centered on the production of cultured meat,there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture.This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production.Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture.Currently,various companies synthesize milk components through precision fermentation technology.Nevertheless,several startup companies are pursuing animal cell-based technology,driven by public concerns regarding genetically modified organisms in precision fermentation technology.Hence,this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components,specifically emphasizing the structural,functional,and productive aspects of mammary epithelial cells,providing new information for industry and academia.

Fermentation Conditions on the Production of Single Cell Protein Feed by Water Chestnut Peel

In order to improve the comprehensive utilization value of water chestnut peel and the income of farmers. Contents of crude protein （ CP）, total sugar and reducing sugar were taken as indicators. Effects of initial pH, ratio of yeast to fungi species, incubation time and liquid volume on production of single cell pro- tein （SCP） feed was studied, and technological conditions on production of SCP feed by water chestnut peel were optimized by orthogonal test. Results showed that the production of SCP feed by water chestnut peel was optimal when pH was 5.0, ratio of yeast to fungi species was 2： 1, fermentation time was 2 d and the liquid volume was 70 mL / 250 mL. Under the optimum conditions, content of fermentation CP was 64.25%, content of total sugar was 19.8%, content of reducing sugar was 5.0%, content of coarse fibre was 0.0% and content of ash was 8.04%.

Enhanced straw fermentation process based on microbial electrolysis cell coupled anaerobic digestion

The low quality and yield of methane severely hinder the industrial application of straw biogas fermentation, and no effective solution has been found so far. In this study, a novel method was developed when a microbial electrolysis cell(MEC) was coupled with normal anaerobic fermentation to enhance methane yield and purity. The fermentation process achieved a methane purity of more than 85%, which is considerably higher than that of previously published reports. With microbial stimulation and an electric current, the degradation of fibers has been greatly enhanced. The MEC system substantially improved the yield and purity of biogas, bringing a new path to the synthesis of methane by carbon dioxide and hydrogen ions in solution under electron irradiation. Electrochemical index analysis showed extra methane synthesis, due to the external circuit electron transfer. The results of the gas chromatography and solid degradation rate showed that the carbon source of extra methane was CO_(2) produced during normal fermentation and additional volatile solid degradation. These results show that the MEC considerably enhanced the quality and yield of methane in the straw fermentation process, providing insights into normal anaerobic fermentation.

Cathode catalyst prepared from bacterial cellulose for ethanol fermentation stillage treatment in microbial fuel cell

Bacterial cellulose doped with P and Cu was used as a catalyst for a microbial fuel cell(MFC) cathode,which was then used to treat ethanol fermentation stillage from food waste.Corresponding output power,coulombic efficiency(CE),and biological toxicity were detected.Through a series of characterization experiments,the addition of the cathode catalyst was found to improve catalytic activity and accelerate the consumption of the substrate.The resulting maximum output power was 572.16 mW·m^(-2).CE and the removal rate of chemical oxygen demand(COD) in the fermentation stillage by P-Cu-BC reached 26% and 64.5%,respectively.The rate of biotoxicity removal by MFC treatment reached 84.7%.The aim of this study was apply a novel catalyst for MFC and optimize the treatment efficiency of fermentation stillage.

Membrane Microfiltration Fermentation of Glucose Oxidase with Cell Recycling

Membrane microfiltration fermentation (MMF) with cell recycling was successfully applied to the production of glucose oxidase (GOD). A plate microfiltration module was found suitable for such purpose. By feeding whole medium in MMF, the productivity of GOD was much higher than that by feeding glucose alone. With increasing dilution rate the enzyme productivity increased and average enzyme activity decreased. The enzyme productivity of MMF under D = 0.12h-1 and 0.20h-1 were 3871 and 3945U·h-1 respectively, which was about 3 times as that of batch fermentation (BF) and the average enzyme activity was still as high as STU·mL-1 under D = 0.12h-1. The relative efficiency of MMF applied to low yield strain was higher than that applied to high yield strain.

High-density lipoprotein endocytosis in endothelial cells

AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

Immobilization techniques for beverage production using yeast cell systems: challenges, types, and future perspectives - a mini review

Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventional methods.Research highlights that immobilized yeast cell systems have several benefits as compared to free yeast cells.The immobilized yeast cell systems improve fermentation rates,especially when paired with continuous fermentation and appropriate immobilization techniques.Understanding various immobilization techniques,continuous fermentation processes,yeast metabolic activity related to beverage flavor production,and bioreactor designs is vital for optimizing the use of immobilized yeast cells systems on industrial scale.This review provides an overview of recent basic research on immobilized yeast cell systems,with a focus on continuous beverage fermentation.In this study,different reactor configurations and immobilization techniques are explored.The study focus on the impacts of immobilization on the yeast cells,and discuss the recent advancements in these techniques.The review concludes with a discussion on the practical applications of immobilized yeast cells and continuous fermentation in beverage production.

Theoretical Analysis of the Application of Immobilized Cells to the Continuous Fermentation Process

This paper theoretically studies the continuous immobilized cell (IMC) fer mentation system. A kinetic model of IMC is established, and the relational expressions between production rate, substrate concentration, biomass concentration and dilution rate in the IMC continuous stirred tank reactor (CSTR) are derived. These equations and some numerical calculations show that as compared with the free cell system the IMC system has many advantages: high production rate, steady operation, and being independent of the dilution rote. They also indicate that the diffusion of substrate is a constraint to the production of metablite.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 （LFWGE）. Methods The HT-29 cells were transplanted via subcutaneous injection of 1×10^7cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE （high-dose 2 g/kg/d; low-dose 1 g/kg/d） and for 7 d with 5-fluorouracil （5-FU, 25 mg/kg/d） by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. Results Tumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group （2 g/kg/d, 60.2%＋4.4%; 1 g/kg/d, 58.6%＋6.9%） was significantly higher than that of the control group （11.5%＋1.6%） and 5-FU group （32.1%＋3.5%） as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. Conclusion The results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Barley Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells was established in nude mice to study the anticarcinogenic activities and apoptosis-regulatory mechanistic effect of aqueous extract of fermented barley with Lactobacillus plantarum dy-1 （LFBE）. Methods HT-29 cells were transplanted via subcutaneous injection of 1 × 107cells into the right flank of each nude mouse. Then, nude mice were treated for 30 days with LFBE （high-dose 2 g·kg-1·d-1; low-dose 1 g·kg-1·d-1） and for 7 days with 5-fluorouracil （5-FU, 25 g·kg-1·d-1） by gavage and intraperitoneal injection, respectively. Results Tumor volume and weight decreased significantly in both groups of nude mice treated with LFBE. In addition, the cell apoptosis rate of the LFBE group was significantly higher than that of the control group and 5-FU groups as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot methods further confirmed these apoptosis-enhancing and growth-inhibiting effects. The involvement of LFBE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and cyclin D1. Conclusion The results showed that LFBE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be used as a natural nutrient supplement or chemopreventive agent in the treatment of human colon cancer.

Research of Submerge Fermentation Conditions on <i>Agaricus blazei</i>Murill

Rotational speed, temperature, inoculation amount and pH are the four factors that affect the submerge fermentation of Agaricus blazei Murill. The orthogonal experiment was on the basis of single factor experiments. And the optimal submerge fermentation condition of Agaricus blazei Murill was determined through orthogonal test. The results were as follows: temperature (25℃), rotational speed (150 r/min), inoculation amount (6%) and pH (7), in this condition the cell dry weight reached 1.32 g/100mL;temperature (25℃), rotational speed (150 r/min), inoculation amount (8%) and pH (6.5) in this condition the extracellular polysaccharide reached 6.95 mg/mL.

Fermented Herbal Decoction Selectively Targeting Human Cancer Cell Line and Human Pathogenic Microorganism

Introduction: Prolonged immuno-suppressed status promised to induce internal growth of malignant cell and infectious agent, yet, only a small part of affected individuals seek medical attention or berried by commercially over-flowed fake information. Several studies have described complementary and alternative medicine as effective strategies for improving anti-infectious agent including malignant cell. The purpose of this study was to investigate the effect of a fermented herbal decoction (FHD) both in vitro and in vivo to malignant cells and microorganism by regulating leukocyte subset proportioning FHD as dietary material. Methods: In this approach of alternative study, selective anti-cancer effect by fermented decoction was tried to show first in vitro system both, cancer cell and virus strain. The fermented herbal decoction consisting of 80 sorts of herbs and fruits. The selective toxicity was set up and then for immunological factors in animal and human. The most important factor is to reduce side effect for a normal cell. Results: First, FHD was proved as safe by animal test. FHD regulated also the proportion of granulocyte and lymphocyte ratio both animal and human. In vitro culture showed selective toxicity by FHD against human melanoma and leukemia cell line but reduced toxicity was showed by normal cell line. As for the anti-virus activity, anti-virus effect was tested on the feeder layer of human fibroblast cell, after 9 days of culture. Second, FHD inhibits colon cancer growth in 3-methylholanthrene induced cancer in rat. Conclusion: The present results suggest that our fermented herbal decoction showed selective anti-cancer activities and anti-virus activities, together with the regulative effect on the immune system.

Biosynthesis of Ethanol and Hydrogen by Glycerol Fermentation Using <i>Escherichia coli</i>

Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.

Optimization of Fermentation Condition of Yeast Culture

Culture condition of every phase for fermentation of yeast culture was studied, and its solid and liquid conditions of elaboration were optimized to improve the total counts of living cells. Results showed that microzyme grew best at 30℃ when solid fermented, and the count of the living cells reached the tiptop with pH 5.5. The count of Candida tropicalis could reach 137.96× 10^9 cfu·g^-1, the count of Saccharomyces cerevisia could reach 134.62× 10^9 cfu·g^-1 the best liquid fermentation condition for cell-wall broken was 50℃ for 28 h, the rate of cell-wall broken could reach 80% at least; the rate of vitamin loss in yeast could be the minimun, the loss rate of vitamin B6 in Candida tropicalis and Saccharomyces cerevisiae was 8.71% and 19.54% respectively, the loss rate of vitamin B2 was 19.39% and 13.18%, respectively, and the loss rate of vitamin B6 was 6.3% and 3.04%, respectively.

乳酸菌的乙醇耐受机制及其在食醋生产中的应用

传统固态发酵食醋因其丰富的菌群在风味、品质等方面有着不可比拟的优势,乳酸菌作为固态食醋发酵中重要的功能微生物,广泛应用于食品、生物等领域。然而,食醋在发酵过程中乳酸菌会受到高乙醇环境的胁迫,因此探究乳酸菌如何在高乙醇环境下生存具有积极意义。该研究选择耐乙醇副干酪乳杆菌(PC-5)和不耐乙醇植物乳杆菌(PR-7)作为实验对比菌株,通过检测生物指标发现:在体积分数为8%的乙醇环境下,PC-5的多糖含量占比和细胞膜通透性分别提高至0.56%和75%,且显著高于PR-7。此外,代谢途径中的己糖激酶(Hexokinase,HK)、6-磷酸果糖激酶(6-Phosphofructokinase,PFK)、丙酮酸激酶(Pyruvate Kinase,PK)的活性均高于PR-7,并分别提高至99.82、2.78、3.43 U/mg。最后,在食醋不同发酵阶段中加入PC-5使其与酵母菌实现共同发酵,结果发现:共发酵食醋体系中多酚的生成和总抗氧化能力比单菌发酵分别提升了32.14%和55.56%。因此乳酸菌对食醋发酵有着良好的促进作用,为乳酸菌参与食醋共发酵提供了良好的理论依据。