分步酶解制备花生短肽的研究

为了高效制备花生短肽,在花生蛋白Alcalase酶水解的研究基础上,进一步采用N120P酶水解花生蛋白并对各种影响因素进行了系统地研究;建立了短肽得率与各种影响因素的回归模型;确定出了N120P继续酶解花生蛋白Alcalase酶解物制备花生短肽的最佳工艺参数为pH值6.0,水解时间65 min,水解温度57℃,酶与底物比2061 U/g,。在此条件作用下,体系中短肽得率为89.01%,比Alcalase 单独酶解提高10.86个百分点;水解度为23.76%,平均肽链长度为4.21。经高效液相色谱测定,大部分水解产物的相对分子质量小于1000。     

中性蛋白酶水解鸡骨泥制备短肽工艺优化

为了探究酶法制备鸡骨泥短肽的最佳工艺,该文研究了以中性蛋白酶酶解鸡骨泥时各因素对短肽得率和羟自由基清除率的影响,以及鸡骨泥肽清除超氧阴离子自由基和1,1-二苯基-2-三硝基苯肼自由基(2,2-diphenyl-1-picrylhydrazyl)的能力。试验探究了酶解过程中不同底物浓度、酶浓度、反应温度和反应时间对短肽得率和羟自由基清除率的影响,采用响应曲面优化得到了以短肽得率为目标的中性蛋白酶酶解鸡骨泥的最优工艺,当反应液p H值为7.2,反应温度42℃,反应时间2 h,底物质量分数5%,酶添加量200 mg/g,该条件下制备鸡骨泥肽,测得其短肽得率为56.16%,羟自由基清除率为54.12%。采用该酶解工艺制备鸡骨泥肽,测得其对超氧阴离子自由基最高清除率为56.01%,对DPPH自由基最高清除率为81.57%,说明鸡骨泥肽具有一定的抗氧化活性,研究结果为酶法制备鸡骨泥肽提供参考。     

酶解鹰嘴豆蛋白制备抗氧化肽工艺优化研究

为优化 Alcalase 蛋白酶酶解鹰嘴豆蛋白制备抗氧化肽的工艺条件,采用响应面分析法,以还原能力、超氧阴离子捕获率为响应值,研究了酶与底物的比值([E]/[S])、酶解温度和酶解时间对制备抗氧化肽工艺的影响.综合考虑成本和工艺要求等问题,最终确定酶解鹰嘴豆蛋白制备抗氧化肽的工艺条件为:底物浓度2%,pH值8.0,[E]/[S]2.72%,温度52℃,时间31 min.该条件下制备的鹰嘴豆酶解产物还原能力和超氧阴离子捕获率分别为0.667和61.55%,与理论预测值的相对误差在±1%以内,说明利用该文建立的模型在实践中进行预测是可行的.     

高压均质处理对甘薯热变性蛋白Alcalase酶解肽乳化特性影响研究

采用高压均质处理甘薯热变性蛋白Alcalase酶解肽(SPHP-Al)的乳化液,以乳化活性指数(EAI)和乳化稳定性指数(ESI)为指标,探讨了不同高压均质条件下(均质压力0.1～80MPa,均质时间0～2min),多肽浓度为0.1％ (w/V)与油相体积分数为25％(V/V)形成的SPHP-Al乳化液乳化特性的变化.在所得到最佳的均质条件下,以EAI、ESI、乳化颗粒平均粒径(d4,3)和表观粘度为指标0考察了SPHP-Al浓度(0.1、0.25、0.5、1.0、1.5和2.0％,w/V)和油相体积分数(5、15、25、35和45％,V/V)对SPHP-Al的乳化特性的影响.结果显示,均质压力为50MPa和均质时间为1min时,SPHP-Al乳化效果最佳,SPHP-Al乳化液的EAI和ESI分别比未高压均质的提高了4倍(158.14m2·g-1)和8倍(318.06min);且随着SPHP-Al浓度的增加,乳化液的EAI、d4,3和表观黏度均显著减小,ESI显著增大(p＜0.05);与此相反,随着油相体积分数的增大,乳化液的EAI、d4,3和表观黏度均显著增大,ESI显著减小(p＜0.05).因此,高压均质可明显改善SPHP-Al的乳化性,且SPHP-Al浓度与油相体积分数对SPHP-Al乳化性影响显著.上述结果为SPHP-Al在食品行业中的潜在应用提供了数据支撑.     

酶解法制备豆粕水解肽工艺优化

为制备苦味低且生物活性高的豆粕水解肽,本试验在综合考虑水解酶酶切位点后选取7种酶组合对低温豆粕进行水解,以蛋白质转化率、水解度、分子量分布及智能感官评价等作为水解肽的评价指标,比较不同酶组合的作用效果。结果表明,优化得到豆粕水解肽的最佳酶法制备工艺条件:复合酶的最佳组合为碱性蛋白酶+中性蛋白酶+脱苦蛋白酶,酶解pH值依次为8.5、7.5、7.5,反应温度保持55℃,反应时间分别为3、2、2 h,经最佳酶组合制得的水解肽水解度为16%,分子量≤1 000 Da的水解肽可达89.8%,制备的水解肽理化性质及口感更优。本研究结果为豆粕资源开发及功能性肽产品的应用奠定了理论基础。     

超声预处理大米蛋白制备抗氧化肽

为优化中性蛋白酶酶解大米蛋白制备抗氧化肽的工艺条件,采用超声波预处理大米蛋白.以1,1-二苯基-2-苦基苯肼(DPPH)清除率为响应值,用响应面分析法研究超声功率、超声处理时间和超声处理初始温度对制备抗氧化肽工艺的影响.同时研究了其抗氧化肽清除超氧自由基、DPPH、羟自由基能力及螯合Fe2 和还原能力.结果表明:最佳超声预处理大米蛋白的工艺为:超声功率1500 W、超声处理时间20 min、超声处理初始温度40℃,该条件下的抗氧化肽(2.2057 g/L)清除DPPH可达到74.8%,抗氧化肽得率为33.2%.与未经超声预处理比较,抗氧化肽得率提高了43.7%,DPPH的半抑制率(IC50)降低了17.7%.抗氧化试验表明,大米蛋白抗氧化肽清除超氧自由基、DPPH和羟自由基的IC50值分别为0.0678、0.9315和0.1173 g/L,分别是维生素C的IC50值的1.8、175.8和1.4倍;其螯合Fe2 的IC50值是乙二胺四乙酸的42.2倍,还原能力是维生素C的0.7倍.     

大米蛋白的酶水解动力学研究

以米渣为材料,研究碱性蛋白酶、1398中性蛋白酶和木瓜蛋白酶在不同起始pH值、加酶量和温度下水解大米蛋白的进程,建立大米蛋白酶水解动力学方程,为大米蛋白酶水解特性的研究提供依据.在一级化学反应动力学方程基础上,建立了考虑温度、起始pH值和加酶量影响的大米蛋白酶水解动力学模型,该模型可较好的描述不同蛋白酶对大米蛋白的水解进程.木瓜蛋白酶适于在较低的起始pH值下水解,加酶量、起始pH值对其水解进程的影响较大,温度变化对其水解进程的影响较小.1398中性蛋白酶适于在中性条件下水解,加酶量和水解温度对其水解进程的影响程度处于木瓜蛋白酶和碱性蛋白酶之间,起始pH值变化对其水解进程的影响不大.碱性蛋白酶适于在较高pH值下水解,加酶量对其水解进程影响不大,温度的变化对水解进程影响较大.     

酶解制备鱼鳞蛋白降血压肽的工艺优化

为有效利用鱼鳞制备降血压肽,以罗非鱼鱼鳞为原料,在121℃条件下进行热预处理15 min后,运用响应面分析法优化酶解制备鱼鳞蛋白ACE抑制肽的工艺条件。结果表明,以水解度和ACE抑制率为评价指标,筛选出碱性蛋白酶为最优酶。在单因素试验的基础上,根据Box-Behnken中心组合试验设计原理,最终确立最优的酶解工艺参数为:酶解时间2 h、酶解温度56.3℃、pH值8.0,酶底比1.1%,此条件下ACE抑制率理论值为87.95%,实际值为88.26%。最优条件下制得的酶解产物相对分子质量集中在300～3 000 Da之间,水解效果较好。本研究结果对酶解法制备鱼鳞蛋白降血压活性肽具有一定的实践参考价值。     

高压均质-酶解改性提高酸性条件下花生蛋白的溶解性及其稳定性研究

花生蛋白在酸性条件下会絮凝沉淀,溶解性降低,限制了其在酸性饮料加工领域的应用.本研究通过利用高压均质-中性蛋白酶酶解复合改性提高花生蛋白在pH值4.0条件下的溶解度,并根据Turbiscan多重光散射稳定性分析仪的背散射光强值和体系不稳定指数(TSI)分析不同浓度改性蛋白添加量对酸性果汁稳定性的影响.响应面分析结果表明...     

丝胶蛋白抗氧化肽的酶法制备及功能评价

为进一步开发利用废蚕丝,充分挖掘丝胶蛋白的可利用程度,该文研究了6种蛋白酶水解制备丝胶肽及产品的抗氧化活性。筛选出Alcalase碱性蛋白酶为制备丝胶抗氧化肽的理想蛋白酶,并以ABTS体系评价了丝胶肽的抗氧化活性。通过单因素试验研究了反应时间、底物浓度、反应温度、pH值、酶底物比对酶水解制备丝胶肽的影响;并通过回归正交旋转试验设计对水解条件进行优化,建立各因素与水解度和抗氧化活性的数学模型,确定Alcalase酶水解丝胶蛋白的最佳水解条件为:反应时间240min,底物浓度2%,反应温度55℃,pH8.5,酶底物比2.375%。最佳水解条件下水解度为33.35%,ABTS清除活性为43.19%。该研究为开发丝胶抗氧化肽功能产品提供了技术依据。     

Germination of peanut kernels to enhance resveratrol biosynthesis and prepare sprouts as a functional vegetable

Bioactive benefits of resveratrol in the diets have attracted extensive interests of the public. Peanut is one of the potent natural sources of resveratrol. In this study, germination of peanut kernels to enhance resveratrol biosynthesis and preparation of sprouts as a functional vegetable was conducted. When the rehydrated kernels of three peanut cultivars were germinated at 25 degrees C and relative humidity 95% in dark for 9 days, resveratrol contents increased significantly from the range of 2.3 to 4.5 microg/g up to the range of 11.7 to 25.7 mug/g depending upon peanut cultivar. In comparison with the sprout components, resveratrol contents were highest in the cotyledons, slightly lower in the roots, and not detected in the stems. When the sprouts were heated in boiling water for 2 min, resveratrol contents varied in a limited range. Methanol extracts of the freeze-dried sprouts exhibited potent 1,1-diphenyl-2-picryl-hydrazyl scavenging activity and antioxidative potency against linoleic acid oxidation. These activities increased with an increase of germination time. After 9 days of germination, total free amino acid, sucrose, and glucose contents increased significantly while crude protein contents decreased and the large sodium dodecyl sulfate polyacrylamide gel electrophoresis protein molecules of the kernels were extensively degraded. From a practical viewpoint, it is of potency to prepare peanut sprouts as a functional vegetable.     

超声复合酸处理促进花生分离蛋白的亚基解离

为了改善蛋白质的功能特性,该研究分析了超声复合酸处理对花生分离蛋白的溶解性、紫外光谱、荧光光谱、二级结构及纳米结构等的影响,并探讨不同处理条件下蛋白聚集体结构变化的机理。结果表明,超声作用可以明显促进花生分离蛋白的不溶性聚集体向可溶性聚集体转变,单独超声及超声复合酸处理使其溶解度相对于对照分别增加了12.9%和15.3%（P<0.05）;电泳、紫外和荧光光谱表明,超声和酸作用均有助于亚基解离及蛋白质结构展开,从而促进更多的酪氨酸、色氨酸和苯丙氨酸等疏水性基团暴露;圆二色谱分析显示,与对照相比,在超声复合酸处理使花生分离蛋白的a-螺旋增加21.9%,β-折叠减少3.6%,无规则卷曲增加1.8%（P<0.05）;纳米结构表明,超声复合酸处理最大程度地降低了花生分离蛋白的颗粒大小。该研究证实,在酸性条件下进行超声处理,能显著促进花生分离蛋白的亚基解离和结构展开。该研究为后期蛋白亚基重新相互作用形成不同功能的改性蛋白提供参考。     

Release of soluble protein from peanut (Arachis hypogaea, Leguminosae) and its adsorption by activated charcoal

Peanut (Arachis hypogaea, Leguminosae) allergy is a major cause of food-induced anaphylaxis. The potential use of activated charcoal (AC) to adsorb and reduce the bioavailability of peanut protein allergens for use in the moderation of hypersensitivity reactions was investigated. The rate and extent of protein release from peanut and the adsorption of the solubilized protein by AC was determined under physiological pH values and confirmed in vivo using a porcine animal model system. Peanut proteins were adsorbed with equal efficiency at pH 2 and 7 and are completely removed from solution by an AC/protein ratio of approximately 80:1. This suggests that AC can bind protein under gastric (pH 2) or intestinal (pH 7) conditions. The rapid adsorption of soluble peanut allergens and the continuous binding of allergens released from peanut particulate material suggest the potential efficacy of using AC for gastric decontamination and possible elimination of a biphasic allergic reaction.     

Localized production of phytoalexins by peanut (Arachis hypogaea) kernels in response to invasion by Aspergillus species

Peanuts respond to fungal infection by synthesizing phytoalexins, most of which are antibiotic stilbenes. The mechanism and dynamics of phytoalexin formation in the peanut have not been studied. One of the most popular peanut cultivars in the southeastern United States, Georgia Green, was investigated for its ability to produce phytoalexins in response to infection by soil fungal strains. The experimental design allowed for study of phytoalexin production in peanut kernels layer-by-layer. The layers were dissected from different depths of the kernel starting from the infected area down to healthier tissues. Six peanut phytoalexins, trans-resveratrol, trans-arachidin-1, trans-arachidin-2, trans-arachidin-3, trans-3-isopentadienyl-4,3',5'-trihydroxystilbene, and SB-1, were detected in the kernel slices and quantitated. All of the fungal strains triggered phytoalexin production; however, the composition of phytoalexins varied significantly by layer. After incubation for 24 h, tissues remote from the infected area produced almost exclusively trans-resveratrol, whereas closer to the infected area tissues synthesized all six phytoalexins. In all of the experiments, after 48 h of fungal growth, deeper layers produced all tested phytoalexins. There was a significant difference in phytoalexin production elicited by some fungal isolates. No association was observed between phytoalexin production and toxigenic potential of fungal strains that elicited the production in mature peanut kernels.     

Soil sickness of peanuts is attributable to modifications in soil microbes induced by peanut root exudates rather than to direct allelopathy

The quantity and quality of peanut yields are seriously compromised by consecutive monoculture in the subtropical regions of China. Root exudates, which represent a growth regulator in peanut–soil feedback processes, play a principal role in soil sickness. The growth inhibition of a species in an in vitro bioassay enriched with root exudates and allelochemicals is commonly viewed as evidence of an allelopathic interaction. However, for some of these putative examples of allelopathy, the results have not been verified in more natural settings with plants continuously growing in soil. In this study, the phenolic acids in peanut root exudates, their retention characteristics in an Udic Ferrosol, and their effects on rhizosphere soil microbial communities and peanut seedling growth were studied. Phenolic acids from peanut root exudates were quickly metabolized by soil microorganisms and did not accumulate to high levels. The peanut root exudates selectively inhibited or stimulated certain communal bacterial and fungal species, with decreases in the relative abundance of the bacterial taxa Gelria glutamica, Mitsuaria chitosanitabida, and Burkholderia soli and the fungal taxa Mortierella sp. and Geminibasidium hirsutum and increases in the relative abundance of the bacterial taxon Desulfotomaculum ruminis and the fungal taxa Fusarium oxysporum, Bionectria ochroleuca and Phoma macrostoma. The experimental application of phenolic acids to non-sterile and sterile soil revealed that the poor performance of the peanut plants was attributed to changes in the soil microbial communities promoted by phenolic acids. These results suggest that pathogenic fungal accumulation at the expense of such beneficial microorganisms as plant growth promoting rhizobacteria, mycorrhizal fungi induced by root exudates, rather than direct autotoxicity induced by root exudates, might represent the principal cause underlying the soil sickness associated with peanut plants. We hope that our study will motivate researchers to integrate the role of soil microbial communities in allelopathic research, such that their observed significance in soil sickness during continuous monocropping of fields can be further explored.     

反向重复RNA介导转基因烟草对花生条纹病毒抗性

构建携带花生条纹病毒（Peanut stripe virus, PStV）外壳蛋白基因（cp）反向重复序列植物表达载体pKcp，转化农杆菌菌株GV3101，叶盘法转化本生烟，卡那霉素筛选得到的转化植株5~6叶期摩擦接种PStV。通过症状观察和ELISA检测，T0代转基因烟草植株87%表现免疫，T1代不同系植株的免疫比例为60~100%，T2代多数系植株免疫比例达到100%。利用PStVcp特异探针对转基因植株T1代进行特异siRNA的Northern blot检测，转基因植株都含有不同量的特异siRNA，而非转基因植株不含特异siRNA；接种前，免疫植株比感病植株体内siRNA含量高；不同转化系免疫植株在接种前、接种15天和50天后的叶片中都检测到特异siRNA，同一植株不同时期，siRNA含量相对稳定；不同转化系植株体内siRNA含量存在差异。本试验成功利用dsRNA获得对PStV具有较高比例抗性的转基因烟草植株。     

Signaling by glutamate dehydrogenase in response to pesticide treatment and nitrogen fertilization of peanut (Arachis hypogaea L.).

The responses of glutamate dehydrogenase (GDH) to NH(4)(+) and herbicides offer a new approach for probing the effects of NH(4)(+)-pesticide interactions at the whole-plant level. Although pesticides and fertilizers have greatly enhanced food production, their combined biochemical effects are not known in detail. Peanut plants were treated with different rates of Basagran (3-(1-methylethyl)-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide), Bravo 720 (tetrachloroiso-phthalonitrile), and Sevin XLR Plus (1-naphthyl N-methylcarbamate), with and without 25 mM NH(4)Cl fertilization. Isoelectric focusing, followed by native 7.5% polyacrylamide gel electrophoresis (PAGE) fractionated the peanut seed GDH fully to its isoenzyme population patterns. The pesticide treatments induced positive skewing of the GDH isoenzymes, but NH(4)Cl-pesticide cotreatments induced a negatively skewed distribution. Basagran, Sevin, and Bravo increased the amination activities of GDH from 30.0 +/- 2.8 units in the control assay to 479.0 +/- 20.7, 63.0 +/- 5.8, and 35.2 +/- 2.2 units, respectively, therefore indicating a direct GDH-pesticide interaction. Neither the NH(4)(+) nor the pesticides increased the peanut seed protein yields above the threshold of 3.8 +/- 0.7 g per pot. But in the GDH combination of the signals from a pesticide and NH(4)(+), at least 70% of the pesticide signal was overridden by NH(4)(+) with concomitant increases in peanut seed protein yields to 7.0 +/- 1.8 g per pot. Basagran, Sevin, and Bravo possess different pesticidal properties, but their effects on GDH activity were related in the decreasing order of their nucleophilicity, viz. Basagran > Sevin > Bravo.     

Study on the binding of propiconazole to protein by molecular modeling and a multispectroscopic method

Propiconazole (PCZ) is an N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. Although the triazole fungicides have shorter half-lives and lower bioaccumulation than the organochlorine pesticides, possible detrimental effects on the aquatic ecosystem and human health also exist. To evaluate the toxicity of PCZ at the protein level, its effects on human serum albumin (HSA) were characterized by molecular modeling and multispectroscopic method. On the basis of the fluorescence spectra, PCZ exhibited remarkable fluorescence quenching, which was attributed to the formation of a complex. The thermodynamic parameters ΔH and ΔS were calculated to be -14.980 KJ/mol and 26.966 J/(mol K), respectively, according to the van't Hoff equation, which suggests hydrophobic and electrostatic interactions are the predominant intermolecular forces in stabilizing the PCZ-protein complex. Furthermore, HSA conformation was slightly altered in the presence of PCZ. These results indicated that PCZ indeed affected the conformation of HSA.     

用大豆蛋白膜改善预油炸春卷微波加热后的脆性

针对预油炸春卷经冷冻、微波加热后因水分外迁造成产品品质下降的问题,研究了涂布大豆蛋白膜对保持春卷皮脆性的影响。通过对大豆蛋白膜的成型条件及机理的研究,由单因素与正交实验得出大豆蛋白膜的最佳配方为大豆分离蛋白40 g/L、甘油15 g/L、谷氨酰胺转胺酶1.5 g/L;应用此涂膜后,电镜观察显示春卷皮层较未涂膜前致密而平整,经微波加热后能保持良好的脆性。进一步对涂膜春卷的微波升温特性进行考察,发现大豆蛋白膜的应用提高了春卷皮对微波的感受能力,缩小了春卷表皮和馅心之间的温度梯度,进而可缩短微波复热时间,对保持微波复热后的脆性有帮助。