β-伴大豆球蛋白酶解物抑制致病菌粘附Caco-2细胞

以黄大豆和黑大豆为原料调制了β-伴大豆球蛋白,用SDS-PAGE和PAS染色分析了黄大豆及黑大豆来源的β-伴大豆球蛋白各个亚基构成及糖含量,并通过体外模拟人体消化方法制备了胃蛋白酶及胰蛋白酶酶解物,利用动物细胞培养的方法分析了两种β-伴大豆球蛋白酶解物对肠道致病菌大肠杆菌(EScherichia coli stralns serotype 026)和沙门氏菌(Salmonella typhimuryum LT2)粘附Caco-2细胞的抑制作用.结果表明:黄大豆及黑大豆来源的β-伴大豆球蛋白均含有相同的亚基,糖含量上基本一致,为3.5%-3.7%;二者加热处理后再体外消化得到的酶解产物与未加热处理相比可以显著抑制大肠杆菌对Caco-2细胞的粘附,而对鼠伤寒沙门氏菌的抑制作用不明显.这一结果说明,摄取加热后的β-伴大豆球蛋白,在经胃蛋白酶、胰蛋白酶等消化作用后,其消化产物有可能具有保护肠道上皮细胞不被大肠杆菌致病菌粘附的作用.     

固相酶法分离大豆胰蛋白酶抑制剂研究

以硅胶-壳聚糖为载体,通过戊二醛偶联胰蛋白酶,制备固定化酶,分离纯化大豆中胰蛋白酶抑制剂.对戊二醛浓度、反应温度、酶浓度等影响固定化酶制备的因素进行了研究.戊二醛的浓度为2.0%,反应温度为20℃,胰蛋白酶与载体比为15∶ 1 000时,分离纯化的大豆胰蛋白酶抑制剂活性最高,达1872.51 U·mg-1,比粗品高46.22倍.结果表明通过该法能够获得较高活性大豆胰蛋白酶抑制剂,为大豆胰蛋白酶抑制剂的开发利用提供了依据.     

大豆Kti基因ihpRNA种子特异性表达载体的构建及转化

大豆Kunitz型胰蛋白酶抑制剂(trypsin inhibitor,Kti)是大豆品质的主要营养抑制因子之一,培育低Kti含量的大豆品种是提升大豆品质的关键。本研究采用RT-PCR技术克隆得到大豆Kti基因的核心保守序列,并构建成种子特异性表达的、具有Kti基因反向重复结构的RNAi载体,利用农杆菌介导法侵染大豆品种吉林30子叶节,通过潮霉素筛选和PCR检测,获得4株转基因阳性植株。RT-PCR检测表明,构建的种子特异性RNAi载体可以在转基因植株的种子中特异表达,且Kti基因表达量均有降低;通过胰蛋白酶抑制剂活性检测发现,转基因大豆籽粒中胰蛋白酶抑制剂活性平均降低了77.5%。为进一步评价RNAi技术对大豆Kti基因的表达调控效果奠定良好基础。     

不同大豆加工制品中主要抗营养因子免疫及抑制活性的比较

大豆经适当加工可减少或去除其中的抗营养因子,因而大豆制品所含的抗营养因子成分不同.通过竞争ELISA法和胰蛋白酶抑制法检测豆粕、膨化大豆、脱壳豆粕、大豆分离蛋白、大豆浓缩蛋白、全脂豆粉、脱脂豆粉和热处理大豆粉大豆加工制品中胰蛋白酶抑制因子、大豆凝集素、大豆球蛋白及β-伴大豆球蛋白的活性,并进行SDS-PAGE分析,进而比较其主要抗营养因子灭活的程度.结果表明,在现取样的10种大豆加工制品中,膨化处理除去了绝大多数的主要抗营养因子,是一种较理想的加工工艺;去皮豆粕中胰蛋白酶抑制因子和大豆凝集素几乎被灭活,但仍存在一定量的大豆球蛋白及β-伴大豆球蛋白.此项工作为评价大豆加工工艺提供理论基础.     

大豆胰蛋白酶抑制剂与大豆抗大豆胞囊线虫的关系

对大豆胞囊线虫3号小种的抗病品种Hartwig和小粒黑豆,感病品种辽豆15和中黄28分别接种线虫,检测大豆根系中大豆胰蛋白酶抑制剂的表达量对大豆抗胞囊线虫的抗性.经过胰蛋白酶抑制剂活性测定结果表明,接种大豆胞囊线虫的抗感品种的大豆胰蛋白酶抑制剂(STI)表达量明显高于未接种的品种,各个品种的STI表达量也不同.说明大豆胰蛋白酶抑制剂参与抗病过程.但是各个品种的变化趋势并不相同,表明抵御线虫的抗病作用机制存在品种间差异.     

糖基化大豆蛋白酶解物的分离纯化及耐消化稳定性的研究

以糖基化大豆蛋白为原料,经碱性蛋白酶酶解制得具有抑制致病菌粘附功能的糖肽,并分析功能性糖肽的消化稳定性。酶解物用Sephadex G-25层析可获得表观分子量分别为20 000 Da、6 500 Da和小于3 000 Da组分。对酶解物胃蛋白酶和胰蛋白酶体外模拟依次消化2 h后,20 000~6 500 Da的大豆糖肽分子量变化不大,较稳定,而分子量小于3 000 Da的大豆糖肽中有5%被消化,稳定性略有变化。说明糖基化大豆蛋白酶解物其功能性成分可以较稳定存在于消化系统。     

大豆糖蜜上清液中胰蛋白酶抑制剂的去除

胰蛋白酶抑制剂是广泛存在于大豆及大豆制品中的抗营养剂,主要有Kuntiz型和Bowman-Birk型两种,为了得到高品质的产品,需要在加工过程中将其灭活或去除。通过研究大豆糖蜜上清液中胰蛋白酶抑制剂的活性、鉴定抑制剂的种类,采用超滤法将其去除。经测定,固形物含量约10%的大豆糖蜜上清液中胰蛋白酶抑制剂活性约为5.05×105TIA·g-1蛋白,约为鲜豆奶的5倍。用截留分子量3 k Da的超滤膜将大豆糖蜜上清液中的大分子蛋白富集,通过还原-Tricine-SDS-PAGE分析,截留部分在图谱上显示为分子量约10 k Da的模糊的条带。通过用2%巯基乙醇还原再用NEM封闭游离巯基的方式处理样品,使得截留部分在电泳图上显示为分子量约7~8 k Da的两条清晰的条带。综合判断,大豆糖蜜上清液中主要含有Bowman-Birk型胰蛋白酶抑制剂。通过对比超滤效果,选择用截留分子量10k Da的超滤膜截留胰蛋白酶抑制剂,能够得到无抑制活性的透过液。     

大豆疫霉菌诱导的β-1,3-葡聚糖酶与寄主抗病性的关系

为明确-1,3-葡聚糖酶与大豆抗大豆疫霉根腐病的关系,分别测定了具有不同抗性的3个大豆品种的真叶和第1片复叶被大豆疫霉菌侵染后β-1,3-匍聚精酶的活性,并分析其与抗病性的关系.侵染大豆真叶和第1片复叶后备品种的β-1,3-葡聚糖酶活性均明显升高.接种大豆真叶后抗病品种48 h出现酶活性高峰,酶活性增加105.9%;中感晶种和感病品种72 h出现酶活性高峰,酶活性增加量分别为66.3%和65.7%.接种大豆复叶后酶活性的变化与真叶反应一致.说明β-1,3-葡聚糖酶的活性与大豆品种的抗病性呈正相关,抗病品种较感病品种酶活性峰值出现早,酶活性增加量高.对酶的部分性质的研究结果表明:酶的最适反应温度为55℃,最适反应pH为5.0,在55℃以下,pH 4.5～7.5范匍内酶活性较稳定.Ca2+、Mn2+、K+、Al3+对酶均有激活作用,Mg2+、Cu2+、Zn2+、Fe3+、Fe2+、Hg2+、Ba2+对酶有抑制作用.抑菌试验表明,β-1,3-葡聚糖酶粗酶液对大豆疫霉菌的菌丝生长和游动孢子萌发有明显的抑制作用.     

酶解法制备大豆小肽的工艺研究

以豆粕为发酵原料,利用复合酶酶解方法制备大豆小肽,筛选碱性、中性蛋白酶和胰蛋白酶进行复配酶解豆粕。结果表明:复合酶的最佳配比为碱性蛋白酶∶中性蛋白酶∶胰蛋白酶为3∶2∶1;酶解条件:p H8.5,反应温度50℃,反应时间4.5 h,水解度为94.55%,苦味值为3;小肽显示分子量分布范围:≤1 000 Da可达74.67%以上,其中≤500 Da占55.61%以上。综上试验结果可知,对比单酶、双酶及3种酶酶解豆粕的水解度和苦味值两项指标,3种酶组合使用更适合于制备水解度高、苦味低的大豆小肽。     

测定豆类胰蛋白酶抑制剂活性和种类的电泳方法改良

以赤豆胰蛋白酶抑制剂(ABTI)及绿豆的胰蛋白酶抑制剂(MBTI)为材料,采用明胶-PAGE技术对其进行活性和种类检测。同时对影响明胶-PAGE技术体系的明胶浓度、酶解时间、脱酶液及电泳的灵敏度等因素进行探讨。结果表明:改良后的明胶-PAGE技术,鉴定胰蛋白酶抑制剂活性和种类的时间大大缩短,成本低,技术操作可行性强,重复性好,且检测ABTI的种类达7种,灵敏度达到每孔1.0 BAEE、蛋白点样量为每孔0.154μg。     

Trypsin inhibitors and nutritive value in cereals

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet heat treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.     

Effects of heat treatments on trypsin inhibitor and hemagglutinating activities in winged bean

The effect of different heat treatments on inactivation of trypsin inhibitor and hemagglutinin of winged bean was investigated. Trypsin inhibitor extracted from winged bean meal was stable at 60 °C for 60 min. At 80 °C, the activity of the extracted inhibitor decreased by 25% within 5 min, and continued to decline gradually to a loss of 45% of activity after 30 min. When the extracted inhibitor was incubated at 100 °C, it exhibited a triphasic pattern of inactivation. The winged bean extract incubated at 60 °C lost 60% of its hemaggluinating activity within 30 min. At 80 °C, there was a complete loss of activity within 5 min. The microwave treatment to winged bean meal had no effect on trypsin inhibitor or hemagglutinating activities in the meal. However, infrared treatment to winged bean seeds for 60 seconds was effective in destroying most of the trypsin inhibitor and hemagglutinating activities. Autoclave treatment (120 °C at 15 lb pressure) for 10 min inactivated trypsin inhibitor and hemagglutinin in winged bean meal almost completely. Cooking of presoaked beans in boiling water for 30 min was effective in destroying most of the trypsin inhibitor and hemagglutinating activities.     

福建主要乌龙茶品种PPO酶活特性的研究

对四种乌龙茶茶树鲜叶的多酚氧化酶(PPO)进行粗提,测定并比较不同品种酶的特性。结果表明:在邻苯二酚与被测酶液的反应体系中,当底物邻苯二酚(C6H6O2)量为0.3~0.9ml时,毛蟹、铁观音、黄旦、肉桂酶活力随底物浓度的增加呈曲线增长,含量太高会产生抑制作用,当底物含量∶酶含量=1.5∶1时,酶活力表现最高;毛蟹、铁观音、黄旦、肉桂酶活力在45~65℃范围内均呈ω型变化趋势,依次在45℃、55℃、65℃时出现高峰。在50~90℃下水浴处理1h,毛蟹、铁观音、黄旦、肉桂酶活力分别丧失34.66%~55.91%,22.68%~50.89%,23.14%~53.57%,31.58%~57.89%;酶活力的适宜pH相对偏碱,在pH8.6时表现为一个活力高峰。相同浓度的五种抑制剂对酶均有一定的抑制效果,其中NaHSO3对酶活力的抑制作用最强,L-半胱氨酸其次,NaCl的抑制效果最差。     

基于感官组学分析不同足火方式对金牡丹工夫红茶香气的影响

为探究不同足火方式对红茶香气的影响,以金牡丹茶树秋季鲜叶为原料,使用高温热风（80℃）、低温热风（40℃）、脱水热风（热泵）、真空冷冻4种干燥方式加工工夫红茶,采用顶空固相微萃取（Headspace solid-phasemicroextraction, HS-SPME）结合气相色谱-质谱联用（Gaschromatography-massspectrometry,GC-MS）、气相色谱-嗅辨联用（Gas chromatography-olfactometry,GC-O）、香气活度值（Odor activity value,OAV）、香气特征影响值（Aroma character impact value,ACI）以及香气审评方法,比较不同干燥方式工夫红茶关键香气化合物种类、含量及其对香气的贡献程度。结果表明,4种干燥方式红茶在香型、香气化合物的种类与含量等方面均存在显著差异。香气审评表明,高温干燥红茶呈现不愉悦的闷味,低温干燥红茶花香不显且青草气较重,热泵干燥红茶呈现清爽的花香,冷冻干燥红茶香气协调,花果香保留好且青气不显。GC-MS结果表明,4-戊烯醛、1-辛烯-3-酮、(E)-3-...     

Effects of extrusion cooking on functional properties of mixtures of full-fat soy and sweet potato

Full-fat soy and sweet potato were extruded at 0:100, 25:75, 50:50, 75:25 and 100:0 ratios in an Insta-Pro 600 single screw extruder operated at 180, 200, 220 rpm respectively. Functional properties: bulk density, expansion ratio, water absorption and solubility indices and trypsin inhibitor were assayed. Results showed that addition of soybeans to sweet potatoes considerably increased the protein, fat, ash and trypsin inhibitor levels of the raw material mixtures. It also led to decreases in viscosities of raw mixtures, with corresponding increases in pasting temperatures. Addition of soybean significantly (p<0.05) affected expansion ratio, water absorption and solubility indices and trypsin inhibitor but not bulk density. Variation in screw rotation speed (rpm) significantly (p<0.05) affected only water solubility index at increasing speed. Extrudates generally received severe heat treatment, rendering them applicable only in soup bases, flour mixes and breakfast foods.     

Digestibility of Quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis> Willd.) Protein Concentrate and Its Potential to Inhibit Lipid Peroxidation in the Zebrafish Larvae Model

Quinoa protein concentrate (QPC) was extracted and digested under in vitro gastrointestinal conditions. The protein content of QPC was in the range between 52.40 and 65.01% depending on the assay used. Quinoa proteins were almost completely hydrolyzed by pepsin at pH of 1.2, 2.0, and 3.2. At high pH, only partial hydrolysis was observed. During the duodenal phase, no intact proteins were visible, indicating their susceptibility to the in vitro simulated digestive conditions. Zebrafish larvae model was used to evaluate the in vivo ability of gastrointestinal digests to inhibit lipid peroxidation. Gastric digestion at pH 1.2 showed the highest lipid peroxidation inhibition percentage (75.15%). The lipid peroxidation activity increased after the duodenal phase. The digest obtained at the end of the digestive process showed an inhibition percentage of 82.10%, comparable to that showed when using BHT as positive control (87.13%).     

The use of enzymic hydrolysis in vitro to study the digestibility of some phaseolus seed proteins

In vitro proteolysis was used to study the digestibility of the major seed protein phaseolin, and the trypsin inhibitor, purified fromPhaseolus vulgaris seeds. Whereas denatured phaseolin was fully digested by the enzymic method used [4], its native form was only partially digested. Trypsin inhibitor, both native and after heat treatment, was poorly digested. It was also found that native trypsin had some resistance to digestion conferred upon it by the presence of the inhibitor.     

浸泡介质及浸泡条件对豆浆中抗营养因子及品质的影响

为了最大程度降低豆浆中抗营养因子含量的同时保证品质优良,以干豆制浆为对照,利用不同浸泡介质(水、柠檬酸溶液、NaHCO_3溶液)及不同浸泡条件,探究前处理工艺条件以及介质的改变对豆浆中抗营养因子及营养品质的影响。结果表明:浸泡处理能有效降低豆浆中胰蛋白酶抑制因子活性和植酸含量,但不同介质对不同抗营养因子的效果不同。经浸泡处理后的胰蛋白抑制因子的最低活性为30.88±1.35 TIU·mg^-1,相对于未处理组降低了54%;植酸最低含量为3.46±0.15 mg·mL^-1,相对于未处理组降低了77.4%;而经浸泡处理后的单宁含量却显著上升(P<0.05)。从降低胰蛋白酶抑制因子活性的效果来看,NaHCO_3>水>柠檬酸;从降低单宁含量的效果来看,柠檬酸>NaHCO_3>水;从降低植酸含量的效果来看,柠檬酸>水>NaHCO_3。除抗营养因子外,豆浆的品质指标也发生了显著变化,NaHCO_3浸泡组的总体品质最优,除了个别品质指标(蛋白质含量)与水浸泡组相比差异不显著(P>0.05),其它品质成分含量均显著高于水浸泡组,而柠檬酸浸泡组的品质为3组中最差。选取NaHCO_3溶液作为浸泡介质进行正交工艺优化,当浸泡温度为25℃,浸泡时间为16 h,浸泡豆水比为1∶4,NaHCO_3浓度为0.45%时,豆浆的抗营养因子的去除效果最好,品质最优。     

工夫红茶可视化富氧发酵机设计及试验研究

针对目前红茶发酵设备与发酵工艺结合不紧密而造成的发酵温湿度不稳定、难翻拌、缺氧、品质不稳定等问题,设计了一种工夫红茶可视化富氧发酵机。文章描述了机器的总体设计方案,并对发酵筒、翻拌装置和隧道加热等装置进行了设计,确定了其关键参数值。该研究进行了发酵性能试验,最佳工艺参数为发酵温度30℃、湿度大于90%、发酵时间3.5βh。与传统发酵相比,香气、滋味等有明显提高,感官品质总分高于传统发酵2.1分。本研制机型结构简单、操作方便,能很好地满足优质红茶的发酵工艺技术要求,具有很好的推广应用前景。     

Effects of processing and in vitro proteolytic digestion on soybean and yambean hemagglutinins

Some conventional processing methods were applied on yambean and soybean seeds and flour samples. They include soaking, fermentation, cooking whole seeds in the presence and absence of trona, autoclaving and dry heat treatment of flour samples. Hemagglutinating activity was assayed for after processing treatments. The hemagglutinating proteins from these seeds were classified based on their solubility properties. Effects of the presence of 0.01% concentration of trypsin, pepsin and proteases on agglutination of human red blood cells were also evaluated. Most processing methods, particularly cooking whole seeds for 1–2h, soaking and fermentation, reduced hemagglutinating activity on cow red blood cells. Size reduction accompanied by heat treatment was effective in eliminating hemagglutination. Both the albumin and globulin fractions of the soybean showed hemagglutinating activity but only the albumin fraction of the yambean had agglutinating properties. Proteolytic action of proteases was more effective in reduction of hemagglutinating activity than that of trypsin and pepsin.