世界大豆生产和科研的进展(续五)

(接续四)Kunitz胰蛋白酶抑制剂是大豆胰蛋白酶抑制剂的一种,Mandarino和Bolotari分析了13个巴西大豆品种的胰蛋白酶抑制剂含量,结果为10·65~27·23mg/g;Vollmann等利用基因技术培育了一种低胰蛋白酶抑制剂含量的大豆品种。发芽也是调控胰蛋白酶抑制剂含量的有效途径。Bhatia和     

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.     

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。     

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。     

豆制品中胰蛋白酶抑制剂活性测定方法研究

利用紫外分光光度计，对豆制品中胰蛋白酶抑制剂在抑制牛胰蛋白酶活性反应前后的吸光值差异制作差值曲线，从而得出斜率与抑制剂活性之间关系的方法。并通过反复的实验对比，最终可以快速准确地测定大豆制品中胰蛋白酶抑制剂活性。     

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.     

以bar基因为筛选标记转基因大豆的获得及鉴定

在已优化的大豆农杆菌介导转化体系基础上,以发芽1～2 d的成熟大豆子叶节为外植体,以bar基因作为筛选标记基因,采用农杆菌介导法将胰蛋白酶抑制剂基因(Sporamin)和几丁质酶基因(Chitinase KDEL)转入大豆品种YC-2中.T0代得到生根苗115株,其中采用除草剂叶片涂抹法和目的基因PCR检测法鉴定出阳性...     

多年生野生大豆Kunitz型胰蛋白酶抑制剂基因的克隆及分析

利用RT-PCR方法,从对蚜虫高抗的多年生野生大豆短绒野大豆(G.tomentella)(2n=78)未成熟子叶中扩增到了大豆Kunitz型胰蛋白酶抑制剂同源基因单一目的片段,该基因和蛋白分别被命名为KTiPW54和KTiPW54。序列分析表明:该片段为654 bp的完整编码序列,编码218个氨基酸残基,编码的蛋白与栽培大豆(G.max)、野生大豆(G.soja)、短绒野大豆(G.tomentella)的Kunitz型胰蛋白酶抑制剂基因编码蛋白高度保守的区域一致。将KTiPW54编码区克隆到表达载体pTWIN1,在宿主菌BL21(DE3)中经IPTG诱导获得高效表达。     

Effect of cooking on proteinase inhibitors ofDolichos lablab bean (Dolichos lablab perpureus L.)

Proteinase inhibitory activity in ten different varieties ofDolichos lablab perpureus. L. was determined. All the varieties tested exhibited appreciable level of proteinase inhibitory activity (PIA). The trypsin inhibitory activity (TIA) (Mean:20170 TIU/g) was relatively higher than the chymotrypsin inhibitory activity (CIA) (Mean: 15380 CIU/g). Effect of temperature and cooking on PIA was studied. The nature of cooking medium and duration of cooking had profound effect on the PIA. The dry fried seeds lost their PIA very rapidly (91% in 20 min). Seeds cooked in slightly alkaline medium lost their PIA quickly (89% in 30 min) compared to those cooked in acidic (80% in 30 min) and neutral pH (83% in 30 min). The PIA in green pods was also determined and they had only one third of the PIA (8200 TIU/g and 8125 CIU/g) found in the dry seeds.     

Quick-cooking beans (Phaseolus vulgaris L.): II. Phytates,oligosaccharides, and antienzymes

Effects of the quick-cooking processes on phytate and oligosaccharide levels, and on trypsin and chymotrypsin inhibitors, were investigated in three bean varieties (Phaseolus vulgaris L.: Great Northern, red kidney, and pinto). Beans soaked in distilled water had lower levels of phytate-P than those soaked in a mixed salt solution. Leaching losses of oligosaccharides were nearly the same in different soaking treatments for all the beans except kidney beans. Residual trypsin inhibitor activities (TIA) in cooked quick-cooking beans were about 10% compared with about 20% for chymotrypsin inhibitor activities (CTIA) in the same bean products. -Irradiation was more effective in reducing TIA than CTIA and paralleled destruction by moist heat.Utah Agricultural Experiment Station Journal article no. 2480.     

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.     

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。     

黑龙江几个大豆品种中抗营养因子含量的分析

本研究测定了１３个黑龙江大豆品种中的抗营养因子含量,包括胰蛋白酶抑制因子,糜蛋白酶抑制因子,凝集素。发现高蛋白含量的“东农４２”品种抗因子含量最低,ＴＩＡ为３６．７２ｍｇ／ｇ,ＣＩＡ为７．９８ｍｇ／ｇ,凝集素为１．９７ｍｇ／ｇ。脂用型品种“７１４３５”的蛋白质含量低,抗因子成分的含量却是最高,ＴＩＡ６２．２５ｍｇ／ｇ,ＣＩＡ３５．６１ｍｇ／ｇ,凝集素为３．８６ｍｇ／ｇ。     

Assay of some nutritional and antinutritional factors in different cultivars of winged bean (Psophocarpus tetragonolobus (L.) DC) seeds

Seed of 34 cultivars of winged bean of different origin were evaluated for their nutritional composition and the antinutritional factor, the trypsin inhibitor. The values ranged from 14.1–17.6, 6.12–8.73, 21.94–34.89 and 30.86–39.05% for their fat, fibre, carbohydrate and protein contents, respectively. The protein content in defatted seed meal ranged from 33.43–47.25%, whereas the trypsin inhibitor activity (TIA) registered a wide variation from 63–123 mg g^?1 with seed coat and 51–98 mg g^?1 without seed coat. The strain selection-12 and NBRI selection contained the minimum TIA (63 mg g^?1), while the highest value (123 mg g^?1) was in variety V7, a 2-fold higher activity.     

Trypsin inhibitor in moth bean: thermal stability and changes during germination and cooking

Trypsin inhibitor from moth bean was studied for thermal stability and changes during germination and cooking. The application of dry heat did not inactivate the inhibitor. However, autoclaving at 120°C at 15lbs pressure destroyed inhibitor activity completely. The extracted inhibitor lost 70% activity in 60 min when incubated at 100°C. Soaking of moth bean seeds for 8 h decreased trypsin inhibitor activity by 20%. The germination of seeds for 24 h resulted in 70% reduction in inhibitor activity. No activity was detected in 48 h germinated seeds. Germination (for 24 h) followed by cooking of moth bean seeds destroyed the trypsin inhibitor completely.     

30%旱立停(ASA)提高大豆抗旱能力及安全性效果初报

田间试验结果表明,30%ASA是一种高效保水抗旱剂,30%ASA拌种用量为40 g/12 kg,叶喷用量为30～40 g/667m2.在特别干旱的情况下,种植拌种后的种子,并在两片子叶展开后再叶喷一次.此用量及用法对大豆安全,且对大豆生长具有非常明显的促进作用,从而提高了大豆的抗旱、抗逆性,有效时间可达30 d左右,对作物产量有显著的提高作用.     

Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables

Proximate composition, energy, mineral and vitamin contentsand the effect of blanching methods and times on the trypsinand chymotrypsin inhibitor activities were studied usingcabbage, collard, turnip, peanut, and sweet potato leaves.Results of this study indicated that, crude protein, crudefat, carbohydrate and ash contents were in the range of 15.5–25.6%, 1.4–6.5%, 60.4–73.1% and 6.8–7.5%,respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%)while energy content per 100 g of vegetables was highestin sweet potato leaves (402 kcal) and lowest in cabbage(379 kcal). The mineral content per 100 g of vegetableswere in the range of 33.4–249.8 mg, 241.2–471.2 mg,12.1–75.1 mg, 14.9–98.9 mg, 0.5–3.5 mg and 0.9–3.1 mgfor Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbicacid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of45.1–112.7 mg, 0.2–0.3 mg, 0.3–0.8 mg and 2.0–7.3 mg,respectively. The trypsin inhibitory activity per gram ofthe vegetables was highest in collard (60.1 TIU/g) andlowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitoractivity was highest in the peanut (69.6 CIU/g) but lowestin the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p<0.05) reduced by most of the treatments in eitherthe conventional or microwave blanching methods. In the conventional blanching method, trypsin inhibitor activitywas reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves,respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsininhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3and 65.6 percent in cabbage, collard, turnip, sweet potatoand peanut greens, respectively. For the microwave ovenblanching, trypsin inhibitor activity was reduced by 3.8,3.3, 32.7, 5.0 and 9.5 percent in cabbage, collard, turnip,sweet potato and peanut leaves, respectively when thevegetables were blanched for 30 seconds. When blanched for60 seconds, trypsin inhibitor activity was reduced by 16.2,45.8, 46.2, 51.0 and 42.4 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively.Similar trends in the reduction of chymotrypsin inhibitoractivity were observed when the vegetables were conventionally blanched for 2.5, 5 and 10 minutes andwhen blanched by microwave oven for 30, 45 and 60 seconds.Based on the results of this study, the vegetables weregood dietary sources of minerals, vitamins, carbohydrateand proteins. Also, blanching was an effective method forreducing the trypsin and chymotripsin inhibitor activitiesin the leafy vegetables, however, further investigation onthe heating times for both conventional and microwaveblanching methods is suggested.