Extraction of Pumpkin Polysaccharide by Complex Enzyme Method and Its Antioxidant Research

[Objective] This paper aims to study a new method of extracting pumpkin polysaccharide from pumpkin. Single factor experiments were conducted to examine the effects of extracting time,temperature,the solid-liquid ratio and pH value on the extraction yield of polysaccharide from pumpkin. [Method] The best enzyme ratio and extraction conditions for complex enzymes extraction were determined through orthogonal tests. Scavenging ·OH and O-2 activities of pumpkin polysaccharides were also investigated by salicylic acid and improved self-oxidation of o-pheno methods respectively. [Results] The results showed that the biggest extraction yield of polysaccharide from pumpkin can be got when adding 1% cellulose enzyme,1.5% pectinase,1.0% papain and Na2HPO4-citric acid buffer solution (pH was 4.6),and oscillating for 30 min under water at 40 ℃ with the solid-liquid ratio of 1:30. In addition,pumpkin polysaccharides had a strong activity of eliminating ·OH,but very weak activity to scavenge O-2. [Conclusion] This study provided basic data for research and application of Pumpkin polysaccharide.

Role of renin-angiotensin system/angiotensin converting enzyme-2 mechanism and enhanced COVID-19 susceptibility in type 2 diabetes mellitus

Coronavirus disease 2019(COVID-19)is a disease that caused a global pandemic and is caused by infection of severe acute respiratory syndrome coronavirus 2 virus.It has affected over 768 million people worldwide,resulting in approx-imately 6900000 deaths.High-risk groups,identified by the Centers for Disease Control and Prevention,include individuals with conditions like type 2 diabetes mellitus(T2DM),obesity,chronic lung disease,serious heart conditions,and chronic kidney disease.Research indicates that those with T2DM face a hei-ghtened susceptibility to COVID-19 and increased mortality compared to non-diabetic individuals.Examining the renin-angiotensin system(RAS),a vital regulator of blood pressure and pulmonary stability,reveals the significance of the angiotensin-converting enzyme(ACE)and ACE2 enzymes.ACE converts angiotensin-I to the vasoconstrictor angiotensin-II,while ACE2 counters this by converting angiotensin-II to angiotensin 1-7,a vasodilator.Reduced ACE2 exp-ression,common in diabetes,intensifies RAS activity,contributing to conditions like inflammation and fibrosis.Although ACE inhibitors and angiotensin receptor blockers can be therapeutically beneficial by increasing ACE2 levels,concerns arise regarding the potential elevation of ACE2 receptors on cell membranes,potentially facilitating COVID-19 entry.This review explored the role of the RAS/ACE2 mechanism in amplifying severe acute respiratory syndrome cor-onavirus 2 infection and associated complications in T2DM.Potential treatment strategies,including recombinant human ACE2 therapy,broad-spectrum antiviral drugs,and epigenetic signature detection,are discussed as promising avenues in the battle against this pandemic.

Metal 2-Hydroxy-1-Naphthaldehyde Thiosemicarbazone (Me-HNT) Complexes-A New Kind of Biomimic Enzyme Catalyst

Metal (Me=Fe(III), Mo(VI), Mn(II), Co(II), Ni(II), Zn(II) and Cu(II)) 2-hydroxy-1-naphthaldehyde thiosemicarbazone complexes (MeHNT) were synthesized and used as mimic-enzyme catalysts to mimic the active group of horseradish peroxidase (HRP). The results showed that Fe-HNT, Mo-HNT are effective catalysts, which have similar catalytic activity as HRP. The sequence of catalytic activities of tested biomimic peroxidas is Mo-HNT > Fe-HNT > Zn-HNT > Ni-HNT > Mn-HNT. Among them, Fe-HNT is used as a mimic-enzyme catalyst in determination of ascorbic acid and glucose by coupling the catalytic reaction of glucose oxidase.

Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

Effects of Non-starch Polysaccharide Complex Enzymes on Meat Quality in Broilers

[Objective] The study aimed to research the effect of several commercial NSP complex enzymes products on broiler meat quality, and provide scientific basis for feed enterprise and breeding farmers choosing NSP complex enzymes. [ Method] Two hundred ROSS broilers at age of 21 days were designed to five treatment groups, including the positive and negative control treatments, and Group 1 to 3 with feed additive of the commercial non-starch polysaccharide complex enzymes each on base of the negative group diet. At 56 days of age, broilers were killed and meat quality was analyzed. [ Result] The thigh meat color CIELAB a value for the negative control broilers was lower （ P 〈 0.05） than those of the positive control and Group 1. The drip loss of breast meat for Group 1 was the lowest, and the drip loss of thigh meat for the negative control was the highest among all treatments. The shear force for the negative control and Group 3 were higher than those of other three groups. There were no differences （P 〉 0.05） on the contents of chemical components, crude moisture, crude protein, crude fat, crude ash, and inosine acid for breast and thigh meat among all treatments. [ Conclusion] NSP complex enzymes with complete enzyme categories and high enzyme activity can improve meat quality in broilers.

Nickel-Carnosine complex:A new carrier for enzymes immobilization by affinity adsorption

Immobilization is an effective method to promote the application of enzyme industry for improving the stability and realizing recovery of enzyme.To some extent,the performance of immobilized enzyme depends on the choice of carrier material.Therefore,the development of new carrier materials has been one of the key issues concerned by enzyme immobilization researchers.In this work,a novel organic–inorganic hybrid material,nickel-carnosine complex(NiCar),was synthesized for the first time by solvothermal method.The obtained NiCar exhibits spherical morphology,hierarchical porosity and abundant unsaturated coordination nickel ions,which provide excellent anchoring sites for the immobilization of proteins.His-tagged organophosphate-degrading enzyme(Opd A)and x-transaminase(ω-TA)were used as model enzymes to evaluate the performance of NiCar as a carrier.By a simple adsorption process,the enzyme molecules can be fixed on the particles of NiCar,and the stability and reusability are significantly improved.The analysis of protein adsorption on NiCar verified that the affinity adsorption between the imidazole functional group on the protein and the unsaturated coordination nickel ions on NiCar was the main force in the immobilization process,which provided an idea way for the development of new enzyme immobilization carriers.

A Fiber Optic Sensor for the Simultaneous Measurement of Dual-parameter Based on Hydrogel-immobilized Enzyme Complex

A novel fiber optic sensor based on hydrogel-immobilized enzyme complex was developed for the simultaneous measurement of dual-parameter,the leap from a single parameter detecting fiber optic sensor to a fiber optic sensor that can continuously detect two kinds of parameters was achieved.By controlling the temperature from high to low,the function of fiber sulfide sensor and fiber DCP sensor can be realized,so as to realize the continuous detection of dual-parameter.The different variables affecting the sensor performance were evaluated and optimized.Under the optimal conditions,the response curves,linear detection ranges,detection limits and response times of the dual-parameter sensor for testing sulfide and DCP were obtained,respectively.The sensor displays high selectivity,good repeatability and stability,which have good potentials in analyzing sulfide and DCP concentration of practical water samples.

Study on Process Conditions of Preparation of Microporous Potatoes Starch by Complex Enzyme Method

[Objective] The technology of using c-amylase and glucoamylase to prepare microporeus potato starch was studied.[ Method ] Taking potato starch as raw materials, starch hydrolysis rate and the oil absorption as a measure of index, the influences of the reaction temperature, two enzymes proportion, the quantity of enzyme, the chroma of substrate, buffer solution pH and reaction time on microporous potato starch were inves- tigated. [ Result] The experimental results showed that the best technological conditions were reaction temperature 45 ℃, enzyme ratio （ glucoamy- lase/α-amylase）6, the quantity of enzyme （amount of enzyme and starch quality than） is 1.0%, the substrate quantity chroma of 0.14 g/ml, buffer solution pH 4, the reaction time 8 h. In such process condition, the oil adsorption rate of hydrolyzed potato starch was as high as 70.2%, starch hydrolytic ratio was 34.16%. [ Condmion] The study provided a basis for the development and utilization of microporous starch. Key words Microporous starch; Hydrolysis rate; Oil absorption rate; Preparation; Complex enzyme method; China

Effects of A Multi-Enzyme Complex or Probiotic Supplementation on Performance and Nutrient Digestibility of Broiler Chicks

This experiment was conducted to determine whether the performance of broilers fed diets based on corn and soybean meal could be enhanced with enzymes or probiotics. A total of 120 male broilers, three days of age, were assigned to one of four treatments in a completely randomized design, and housed in groups of five with six cages per treatment. The control diet was based on corn and soybean meal while the three experimental diets consisted of the basal diet supplemented with 0.1% of enzyme I, enzyme II, or probiotic. Enzyme I provided α-galactosidase and fl-mannanase, while enzyme II provided protease, amylase, α-galactosidase, xylanase, and cellulase. The probiotic was composed of Bacillus coagulance, Bacillus lichenformis , Bacillus subtilis , and Clostridium butyricum. Over the 28 day experiment, the weight gain of birds fed the probiotic treatment was superior （P = 0.03 ） to the control, while gains for the enzyme treatments were intermediate to those of the control and probiotic. Feed intake and feed conversion did not differ among treatments （P 〉 0.05 ）. Ammonia production was significantly （ P 〈 0.01 ） higher in the control compared with either of the enzyme or probiotic treatments. Compared with the control, supplementation with enzyme H significantly reduced the digestibility of arginine, isoleucine, and lysine （P 〈 0.05 ）. In contrast, the digestibility of energy was higher （P 〈 0.01 ） for birds supplemented with enzyme II than the control. Digestibility coeffi- cients did not differ for any other parameter with the exception of energy which was significantly higher for birds fed the probiotic treatment than the control （P 〈 0.01 ）. In summary, the performance of broilers was significantly enhanced by the addition of a probiotic to the diet. However, under the conditions of this experi- ment, supplementation with a multi-enzyme complex containing either α-galactosidase and fl-mannanase or the combination of protease, amylase, galactosidase, xylanase, and cellulase failed to improve broiler performance.

Electrophoretic Purification and Characterization of Human NADH-Glutamate Dehydrogenase Redox Cycle Isoenzymes Synthesizing Nongenetic Code-Based RNA Enzyme

NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the GDH isoenzymes that synthesize nongenetic code-based RNA enzymes degrading superfluous mRNAs thereby aligning the cellular reactions with the environment of the organism. The aim was to electrophoretically purify human hexameric GDH isoenzymes and to characterize their RNA enzyme synthetic activity as in plants. The outcome could be innovative in chemical dependency diagnosis and management. Multi metrix electrophoresis including free solution isoelectric focusing, and through polyacrylamide and agarose gels were deployed to purify the redox cycle isoenzymes of laryngeal GDH, and to assay their RNA enzyme synthetic activities. The laryngeal GDH displayed the 28 binomial isoenzymes typical of higher organisms. Isoelectric focusing purification produced pure GDH. Redox cycle assays of the GDH isoenzymes produced RNA enzymes that degraded human stomach total RNA. In the reaction mechanism, the Schiff-base intermediate complex between α-ketoglutarate and GDH is the target of nucleophiles, resulting to the disruption of synthesis of glutamate, and RNA enzyme. The strongest nucleophiles are the psychoactive alkaloids of tobacco, cocaine, opium poppy, cannabis smoke because they are capable of reacting with GDH Schiff base intermediate to stimulate synthesis of aberrant RNA enzymes that degrade cohorts of mRNAs thereby changing the biochemical pathways and exacerbating drug overdose and chemical dependency. Electrophoretic purification, and characterization of the RNA enzyme synthetic activity set the forecourt for innovative application of GDH redox cycles in the diagnostic management of chemical dependency.

复合酶法提取诺丽多糖的工艺优化及其体外抗氧化活性

采用单因素试验以及响应面法优化复合酶法提取诺丽多糖的工艺,探讨提取时间、料液比、提取温度、pH值对多糖提取率的影响。通过紫外光谱、红外光谱扫描技术对诺丽多糖的结构进行分析,并检测其体外抗氧化作用。结果表明:诺丽多糖复合酶法最佳提取工艺为提取时间1.9 h、料液比1∶39(g/mL)、提取温度62℃、pH5.5。经光谱扫描鉴定,该工艺下的诺丽多糖含有少量蛋白质,具有典型的多糖结构,是一种酸性多糖。体外抗氧化试验证明,诺丽多糖对DPPH自由基、ABTS+自由基、羟自由基、超氧阴离子自由基均有较强的清除能力,在抗氧化方面有良好的效果。

玫瑰花渣多糖提取工艺优化及体外抗氧化活性分析

玫瑰花渣是玫瑰精油提取过程中产生的主要废弃物之一。以玫瑰花渣为原料,采用单因素实验结合BoxBehnken响应面法优化了超声辅助纤维素酶与果胶酶复合提取玫瑰花渣多糖的提取工艺,并对多糖体外抗氧化活性进行了研究。结果表明,在料液比为1:15,复合酶(纤维素酶:果胶酶=1:1)添加量为1.9%,酶解温度47℃,酶解时长84.5 min的条件下,玫瑰花渣多糖得率为4.308%±0.03%。体外抗氧化实验表明,玫瑰花渣多糖总抗氧化能力较强,当多糖溶液浓度为5 mg/mL时,其FRAP值为0.30,对DPPH·、ABTS^(+)·的清除能力较强,清除率分别为98.93%和92.19%,对O_(2)^(-)·的清除能力较弱,清除率为24.93%。同时,玫瑰花渣多糖对DPPH·、ABTS^(+)·、O_(2)^(-)·清除能力的IC_(50)值大小分别为0.357、0.608、64.206 mg/mL。该研究为玫瑰花渣的回收再利用提供了理论依据。

体外产气法评价饲粮添加复合菌酶制剂对肉牛瘤胃发酵特性、纤维素降解酶和菌群的影响

本试验旨在研究添加不同水平复合菌酶制剂对肉牛体外瘤胃发酵特性、纤维素降解酶活性和菌群的影响。试验在基础全混合日粮(TMR)中分别添加0[对照组(CON组)]、0.3(D1组)、0.6(D2组)、1.2(D3组)和2.4 g/kg(D4组)复合菌酶制剂作为发酵底物,选取3头健康西门塔尔牛作为瘤胃液供体动物,体外发酵48 h。结果表明:1)D3组6、12、24和48 h产气量、产气速率以及干物质(6 h除外)、中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)降解率显著高于CON组(P<0.05)。D3组和D4组12和48 h甲烷(CH 4)产量显著低于CON组(P<0.05)。2)与CON组相比,体外发酵48 h时,D3组丁酸比例显著提高(P<0.05),各复合菌酶制剂添加组氨态氮(NH 3-N)浓度和乙酸比例显著提高(P<0.05)。D3组总挥发性脂肪酸(TVFA)浓度显著高于其他各组(P<0.05)。3)D3组纤维素酶、β-葡聚糖酶、木聚糖酶和果胶酶活性显著高于CON组(P<0.05)。4)2bRAD-M瘤胃菌群测定结果表明,D3组Simpson指数显著高于CON组(P<0.05)。通过线性判别分析效应大小(LEfSe)对组间差异物种进行分析,筛选出2组在不同分类水平上的显著差异物种共11种,其中普通拟杆菌属(Cryptobacteroides)、解琥珀酸菌属(Succiniclasticum)等在CON组中富集,琥珀酸弧菌属(Succinivibrio)、毛螺菌科(Lachnospiraceae)等在D3组中富集。体外瘤胃发酵参数与优势菌属相对丰度相关性分析结果表明,琥珀酸弧菌属相对丰度与丁酸比例以及TVFA和NH 3-N浓度显著正相关(P<0.05),而解琥珀酸菌属相对丰度与乙酸比例以及TVFA和NH 3-N浓度呈显著负相关(P<0.05)。综上所述,在体外发酵条件下,添加1.2 g/kg TMR的复合菌酶制剂可以提高产气量和营养物质降解率,降低CH 4产量,提高纤维素降解酶活性,提高琥珀酸弧菌属相对丰度,降低解琥珀酸菌属相对丰度。

卵黄高磷蛋白α-淀粉酶抑制肽的制备、鉴定及酶抑制动力学分析

为探索卵黄高磷蛋白肽(phosvitin phosphopeptide,PPP)对α-淀粉酶的抑制作用,使其能够调控血糖水平从而缓解Ⅱ型糖尿病,本研究以α-淀粉酶抑制率为指标酶解卵黄高磷蛋白制备PPP,利用酶抑制动力学实验分析PPP对α-淀粉酶的抑制类型,采用液质联用鉴定后用分子对接筛选出高活性的α-淀粉酶抑制肽,最后验证其活性。结果表明,最优酶解条件为先经胰蛋白酶(加酶量7000 U/g)再经胃蛋白酶(加酶量60000 U/g)分别酶解6 h,制得的PPP在浓度7.81×10-3 mg/mL时具有最高α-淀粉酶抑制率70.69%±1.71%。PPP对α-淀粉酶的抑制类型为混合型抑制。鉴定筛选出了两种新型的高活性α-淀粉酶抑制肽FGTEPDAK和IWGR,经验证其IC50值分别为(0.80±0.14)×10-3 mg/mL和(1.80±0.31)×10-3 mg/mL,极显著低于阳性对照阿卡波糖(3.17±0.47)×10-3 mg/mL(P<0.01)。本研究揭示PPP可作为新型降糖物质,应用于缓解Ⅱ型糖尿病的功能性食品开发中。

Effects of Rare Earths and Its Complex with Extract from Seaweed (Laminaria japonica) on Growth and Immunization of Penaeid Shrimp (Penaeus vannamei)

The effects of rare earths (RE) and its complex with extract from seaweed (Lamiaria japonica) on the growth and immunization of penaeid shrimp (Penaeus vannamei) were investigated. The results show that the survival rate, body length and weight of the shrimp treated by RE and its complex with extract from seaweed are higher than those of the control, and that the activities of the phenoloxidase (PO), lysozyme (LSZ), superoxide dismutase (SOD), peroxidase (POD), acid phosphatase (ACP) and alkaline phosphatase (ALP) in the shrimp treated by RE and the complex are also much higher than those from the control.

Antibody Therapies Targeting Complex Membrane Proteins

In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.

IMMOBILIZATION OF GLUCOSE OXIDASE AND CELLULASE BY CHITOSAN— POLYACRYLIC ACID COMPLEX

This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD)and cellulase. The optimum emperature of the immobilized GOD (IG) was determined to be 60℃ which is higher than that of the native GOD about 40℃. The optimum temperature of the immobilized cellulase (IC) was determined to be about 30℃ higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.

酶提醇沉提取荞麦蜂花粉粗多糖工艺优化及其抗氧化活性分析

以荞麦蜂花粉为研究对象,采用Box-behnken响应面试验优化酶提醇沉法提取荞麦蜂花粉粗多糖,并对提取得到的粗多糖进行结构鉴定和抗氧化活性分析。结果表明,当果胶酶、纤维素酶、中性蛋白酶的添加量分别为0.02%,0.03%,1.0%时,以75%乙醇为提取剂,在39℃提取时提取率最高可达(30.74±0.23)%。经红外光谱和紫外光谱分析,粗多糖具有明显的多糖特征吸收峰,且不含有核酸和蛋白质,经HPLC分析,荞麦蜂花粉粗多糖主要是由Glc组成,其含量高达54.74%,并含有少量的Man、鼠李糖、GlcN、GlcUA、Gal、Fuc;其单糖含量比为Man∶鼠李糖∶GlcN∶GlcUA∶Glc∶Gal∶Fuc为1.472∶1.723∶0.119∶0.129∶54.740∶11.458∶0.490。通过对荞麦蜂花粉粗多糖进行抗氧化活力测定结果表明,O_(2)^(-)·清除率IC_(50)值为(0.636±0.005)mg/mL,ABTS+自由基清除率IC_(50)值为(1.678±0.006)mg/mL,DPPH自由基清除率IC_(50)值为(0.734±0.003)mg/mL,羟基自由基(·OH)清除率IC_(50)值为(0.527±0.004)mg/mL,均显著优于阳性对照物荞麦粉多糖、香菇多糖的抗氧化能力(p<0.01)。

昆布多糖的复合酶法提取工艺优化及其对α-葡萄糖苷酶的抑制活性

优化复合酶提取昆布多糖的工艺参数,并考察其抑制α-葡萄糖苷酶的能力。以昆布多糖得率为评价指标,通过正交试验确定复合酶配比,采用响应面法评价酶解时间、pH、液料比和温度对昆布多糖得率的影响。采用体外酶抑制实验测定昆布多糖对α-葡萄糖苷酶的抑制活性。结果表明,复合酶最佳添加量为纤维素酶100 mg、果胶酶90 mg、木瓜蛋白酶55 mg,最佳酶法提取工艺为酶解时间1.8 h、酶解温度49.4℃、pH6.1、液料比59:1 mL/g,最佳工艺条件下昆布多糖预测得率18.183%,实测多糖得率18.19%±1.04%,其中性糖、酸性糖、蛋白质及硫酸根含量分别52.72%、11.76%、2.66%、19.49%;在1~5 mg/mL范围内其对α-葡萄糖苷酶的抑制作用随浓度增加而升高,最大抑制率为79.04%±3.17%,IC50为1.443 mg/mL。复合酶法提取的昆布多糖得率高,其对α-葡萄糖苷酶具有明显的抑制作用。

雨生红球藻中复合酶提取虾青素的工艺优化

以虾青素提取率为评价指标,利用复合酶法提取虾青素,选取复合酶比例、酶浓度、pH值、反应温度、反应时间,通过单因素实验和正交实验优化其关键工艺参数。实验结果表明,当酶配比为纤维素∶果胶酶=1∶1时,最佳工艺参数为:酶浓度7000U·mL^(-1),酶解p H值为4.0,酶解温度50℃,酶解时间1h,其平均提取率约为68.89%。本文采用复合酶法提取虾青素的工艺具有可行性,且可使其提取率显著提高,可实际应用于雨生红球藻中虾青素的提取。