In vitro digestibility of the cancer-preventive soy peptides lunasin and BBI

Lunasin and BBI (Bowman Birk protease inhibitor) are bioactive soy peptides that have been shown to be effective suppressors of carcinogenesis in in vitro and in vivo model systems. Since they are subject to digestion in the gastrointestinal tract, we investigated here the stabilities of lunasin and BBI to digestion in vitro by simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). Samples containing lunasin and BBI of varying purities were subjected to in vitro digestion by SIF and SGF at different times and analyzed by Western blot. While the pure BBI reaction is stable after SIF and SGF digestions, the purified lunasin from soybean and synthetic lunasin are easily digested after 2 min in both in vitro digestions. In contrast, lunasin from soy protein containing BBI is comparatively stable after SIF and SGF digestions. Both lunasin and BBI are able to internalize into the cell and localize in the nucleus even after digestion, suggesting that some of the peptides are intact and bioactive. These data suggest that BBI plays a role in protecting lunasin from digestion when soy protein is consumed orally. The role of other soy protease inhibitors such as Kunitz Trypsin Inhibitor (KTI) cannot be excluded from these experiments.     

Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate

Cold gelation of whey proteins is a two-step process. First, protein aggregates are prepared by a heat treatment of a solution of native proteins in the absence of salt. Second, after cooling of the solution, gelation is induced by lowering the pH at ambient temperature. To demonstrate the additional formation of disulfide bonds during this second step, gelation of whey protein aggregates with and without a thiol-blocking treatment was studied. Modification of reactive thiols on the surface of the aggregates was carried out after the heat-treatment step. To exclude specific effects of the agent itself, different thiol-blocking agents were used. Dynamic light scattering and SDS-agarose gel electrophoresis were used to show that the size of the aggregates was not changed by this modification. The kinetics of gelation as determined by the development of pH and turbidity within the first 8 h of acidification were not affected by blocking thiol groups. During gelation, formation of large, covalently linked, aggregates occurred only in the case of unblocked WPI aggregates, which demonstrates that additional disulfide bonds were formed. Results of permeability and confocal scanning laser microscope measurements did not reveal any differences in the microstructure of networks prepared from treated or untreated whey protein aggregates. However, gel hardness was decreased 10-fold in gels prepared from blocked aggregates. Mixing different amounts of blocked and unblocked aggregates allowed gel hardness to be controlled. It is proposed that the initial microstructure of the gels is primarily determined by the acid-induced noncovalent interactions. The additional covalent disulfide bonds formed during gelation are involved in stabilizing the network and increase gel strength.     

Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation

Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.     

Role of disulfide bonds upon the structural stability of an amaranth globulin.

Analysis of globulin-P, the polymerized amaranth globulin, gave a low amount of free sulfhydryls (10.2 +/- 0.5 micromol/g) from which 7 +/- 1 micromol/g was buried inside the molecule. In addition, its disulfide content was high (51 +/- 1 micromol/g) and similar to soybean 11S globulin content. The more exposed disulfide bridges were found to be stabilizing polymers, whereas the less reactive bridges were either linking P(20) and P(30) polypeptides or forming intrachain linkages. It was found that the buried bonds participate in the stabilization of folded polypeptides and the quaternary structure of the globulin. In turn, the dissociation of polymers and disruption of the quaternary structure by the action of 2-mercaptoethanol reverted upon removal of the reducing agent. This demonstrates that the polymerized state and the quaternary structure of the molecules are most favorable from the thermodynamic point of view. The similar content of SH and SS in globulin-P and globulin-S found in this laboratory suggests that the differences between these proteins may be ascribed to other compositional differences.     

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

高温分解与乳酸菌分步发酵提高秸秆饲料消化率及适口性

以提高秸秆饲料的消化率和适口性为目的,该研究1)以干玉米秸秆为材料,接种木质纤维素分解菌复合系WDC2,进行高温分解发酵;2)以乳酸菌复合系SFC-2为接种物,进行乳酸菌液体发酵;3)将乳酸菌发酵液以质量体积比1∶1比例均匀喷洒到发酵秸秆中,制备发酵秸秆饲料。该研究从营养学和分子生态学角度探讨了高温分解与乳酸菌分步发酵提高秸秆饲料消化率和及适口性的可行性。结果表明,经高温分解发酵,秸秆中微生物多样性丰富,不含致病菌。以南阳黄牛为研究对象,秸秆的干物质、中性洗涤纤维和酸性洗涤纤维体外消化率分别提高了13.94%、22.56%和21.12%,采食量提高21.71%;部分分解的秸秆经乳酸菌发酵液吸附后,粗蛋白增加36.17%。秸秆的高温分解发酵与乳酸菌的乳酸发酵相结合的饲料制作工艺,即提高了秸秆的消化率和营养价值,也改善了秸秆的适口性。     

油脂预乳化提高大豆拉丝蛋白素食香肠品质

为了提高大豆拉丝蛋白(Textured Fibril Soy Protein,TFSP)素食香肠的品质,该研究将油脂预乳化工艺应用于TFSP素食香肠的加工,利用响应面试验设计优化了预乳化工艺条件,采用质构分析仪测定分析了产品的质构特性,并进行了感官评价.结果表明优化后的最佳预乳化工艺条件为菜籽油含量445 g/L、大豆...     

还原剂对大豆蛋白/聚乙烯醇复合薄膜性能的影响

为了改善大豆蛋白/聚乙烯醇复合薄膜的综合性能,该研究以薄膜拉伸强度、断裂伸长率、透光率、吸水率为评价指标,通过隶属度函数综合评分方法,研究了几种还原剂对大豆蛋白/聚乙烯醇复合薄膜性能的影响。试验结果表明：与对照组相比,加入抗坏血酸或半胱氨酸,薄膜拉伸强度显著增大,吸水率和断裂伸长率显著降低;加入半胱氨酸,薄膜透光率显著提高;添加亚硫酸钠,薄膜拉伸强度和透光率显著提高,吸水率降低但不显著,断裂伸长率无明显变化。当亚硫酸钠质量分数为0.15%时,薄膜综合性能较佳,拉伸强度为6.904?MPa,断裂伸长率为66.076%,透光率为32.310%,吸水率为45.695%。该研究为大豆蛋白/聚乙烯醇复合薄膜性能改善及生产应用提供理论基础。     

In vitro protein digestibility and physicochemical properties of dry red bean (Phaseolus vulgaris) flour: effect of processing and incorporation of soybean and cowpea flour

A study was carried out to determine the effect of germination and drying temperature on the in vitro protein digestibility and physicochemical properties of dry red bean flours. A 2 x 3 factorial experiment with two treatments (germination and nongermination) and three drying temperatures was used for this purpose. The effect of particle size on water absorption capacity of bean flour was investigated. In addition, the effect of incorporating soybean and cowpea into the red bean flour on functional properties was equally investigated. Results reveal that protein digestibility increased with germination and also with drying temperature. Drying at 60 degrees C produced flours of optimum functional characteristics, although the hydrophilic/lipophilic index was high and the solubility index reduced. Germination and particle size as well as drying temperature all affected the water uptake properties of bean flours. Incorporation of soybean and cowpea flour into germinated bean flour at levels of 10 and 30%, respectively, produced a composite with higher functional properties.     

Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation

The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.     

Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour: dependence on wheat variety and intermolecular disulfide bridges

The antigenic and allergenic activities of ovomucoid (OM) remaining in soluble fractions of pasta-like model samples of wheat flour mixed with egg white were investigated by ELISA competitive inhibition and immunoblotting analyses using a rabbit anti-OM IgG and the serum IgE specific for OM in patients allergic to egg white. The mixture of egg white and wheat flour of soft, hard, and durum varieties was kneaded for 10-50 min and benched for 1 h at RT, and then small pieces of the dough were heated in boiling 1% NaCl solution for 15 min. Even before heating, only after the kneading for 30 min or more, but not after kneading for only 20 min, followed by the benching, the antigenic activity of OM which remained in the phosphate-buffered saline extract from the dough markedly decreased. Almost no antigenic activity of OM was detected in the extracts of heated samples. Furthermore, in the extracts of heated durum samples, only a trace of or almost no IgE-reactive OM was detected against the five patients' sera. These reductive effects of wheat on the OM antigenicity and allergenicity were more remarkable in the durum variety than in the others. No detectable proteins were extracted with 1% SDS from the heated samples, whereas OM was extracted with 1% SDS containing 10% 2-mercaptoethanol, suggesting heat-induced polymerization through intermolecular disulfide bonds among OM and wheat.     

Histologic detection of cardiac musculature, soy flour, and partially defatted tissue in ground beef: interlaboratory study

A collaborative study was designed and conducted to evaluate the accuracy of a procedure for the histologic detection of cardiac muscle, soy flour, and partially defatted tissue that may occur as adulterants in ground beef. Ground beef samples were prepared containing 0, 3, 5, 10, and 15% of each of the 3 adulterants. Five samples of each composition at each of the 5 dilutions, for a total of 75 unknown samples, were analyzed at each of 5 participating laboratories. The study revealed that this technique is reliable for the detection of these adulterants in ground beef.     

Effect of storage temperature and water activity on the content and profile of isoflavones, antioxidant activity, and in vitro protein digestibility of soy protein isolates and defatted soy flours

The aim of this work was to study the effect of 1 year of storage at different temperatures and 1 month of storage at different water activities on the content and profile of isoflavones, antioxidant activity, and in vitro protein digestibility of defatted soy flours (DSF) and soy protein isolates (SPI). The storage for up to 1 year, at temperatures from -18 to 42 degrees C, had no effect on the total content of isoflavones, but the profile changed drastically at 42 degrees C, with a significant decrease of the percentage of malonylglucosides with a proportional increase of beta-glucosides. A similar effect was observed for SPI stored at a(w) = 0.87 for 1 month. For DSF, however, there was observed a great increase in aglycons (from 10 to 79%), probably due to the action of endogenous beta-glucosidases. The antioxidant activity decreased after storage at 42 degrees C, but the in vitro protein digestibility did not change.     

磷肥减量结合硫酸铵配施提高西北地区旱地春玉米磷素利用效率

  【目的】  西北旱地春玉米种植区磷肥过量施用,磷肥利用效率偏低。本研究旨在探究春玉米磷肥减施的可行性以及实现磷素高效利用的优化施肥方式,以期为该地区春玉米磷肥高效利用提供参考。  【方法】  在西北典型雨养农业区设置3年的定位田间试验,共设5个处理:对照 (CK,不施磷肥)、农户模式 (FP,磷用量P₂O₅ 120 kg/hm2,撒施)、减磷撒施 (RP,磷用量P₂O₅ 70 kg/hm2,撒施)、减磷条施 (BF,磷用量P₂O₅ 70 kg/hm2,条施)、硫酸铵模式 (SA,采用硫酸铵氮肥替代尿素氮肥,其他同减磷撒施处理)。在玉米四叶期、五叶期以及成熟期采集植物和土壤样品 (根际土和非根际土),测定玉米根系、地上部生物量及其磷含量,土壤碱性磷酸酶活性、pH、有效磷、丛枝菌根侵染率,并采用WinRHIZO根系扫描系统测定根长、根表面积、根体积等指标。  【结果】  三年试验结果表明,RP、BF、SA处理玉米籽粒产量、生物量与FP处理无显著差异 (P > 0.05),但磷肥偏生产力显著提高,平均增幅68.0%。RP、BF、SA处理成熟期玉米籽粒磷含量较FP处理降低7.1%～12.9%,磷累积量降低了8.8%～17.0%,其中RP和BF处理降低幅度达到显著水平,但SA处理籽粒磷含量和磷累积量与FP处理相当。与FP处理相比,RP和SA的磷肥回收利用率均有提高,其中SA处理显著提高7.2个百分点;RP、BF、SA处理均有促进春玉米苗期根系生长的趋势,其中SA处理根长、根表面积以及细根长 (直径小于0.50 mm) 分别提高13.9%～37.9%、8.6%～46.1%、12.2%～43.0%。此外,与FP处理相比,RP和SA处理提高了玉米苗期根系丛枝菌根侵染率,增幅在16.2%～21.7%;SA处理非根际土碱性酶活性有增加趋势,五叶期差异达显著水平,BF处理非根际土有效磷含量提高了18.8%～56.3%。  【结论】  在西北旱地春玉米种植区,磷肥施用量由现在的P₂O₅ 120 kg/hm2减少至70～75 kg/hm2仍可保证玉米稳产。在此基础上,用生理酸性肥料硫酸铵代替尿素可促进玉米根系生长以及丛枝菌根侵染,促进玉米对磷素的吸收利用。     

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.     

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.     

Determination of soyasaponins in soy with LC-MS following structural unification by partial alkaline degradation

High-performance liquid chromatography coupled with electrospray ionization mass spectrometer was used to study the soyasaponins in soy. It was found that each soyasaponin belonging to group A existed mainly in their genuine acetylated forms. The partially to fully deacetylated structures coexisted in various proportions. Likewise, the soyasaponins belonging to group B in soy were detected as both 2,3-dihydro-2,5-dihydroxy-6-methyl-4-pyrone (DDMP) conjugated forms and non-DDMP forms. The structural diversity of soyasaponins hinders the separation and determination of the individual compounds in soy. In the present studies, the soyasaponins extracted from soy were treated with sodium hydroxide under mild conditions to cleave the acetyl groups from soyasaponins in group A as well as the DDMP from soyasaponins in group B, while the glycoside structures remained unaffected. By doing so, all soyasaponins originating from the same initial structures were unified into well-defined structures and then quantified individually using the selective ion recording of their [M-H](-) ions. The pure deacetyl and non-DDMP soyasaponins were used as the external standards. The quantification limits of soyasaponins in group A and group B were 1.74 and 1.89 ng injected on column with recovery rates of 94.1% +/- 4.2% and 96.9% +/- 2.9%, respectively.