EFFECTS OF HEAT TREATMENT AND PEPSIN DIGESTION ON TRYPSIN INHIBITORY ACTIVITY AND OVOMUCOID ANTIGENICITY OF JAPANESE QUAIL EGG WHITE

Changes in trypsin inhibitory activity and ovomucoid antigenicity in the egg white of Japanese quail egg were observed after subjecting the quail egg to boiling, saúteing and microwave irradiation. Boiling for 10 min caused about 50% loss of the initial trypsin inhibitory activity. With respect to antigenicity, the boiling caused an initial increase followed by a drop to the original level. Saúteing for 3 min resulted in about 90% loss of the inhibitory activity and about 75% loss of the initial antigenicity. Microwave irradiation for 90 s resulted in about 85% loss of the inhibitory activity and about 80% loss of the initial antigenicity. After pepsin digestion of the egg white boiled for 10 min, 100% of the starting trypsin inhibitory activity was retained even after 24 h digestion, while the antigenicity decreased to about 10% of the initial activity. These results suggest that about 50% of the original trypsin inhibitory and 10% of the original antigenic activity remain after a 24 h pepsin digestion of the egg white boiled for 10 min. Thus, we concluded that intact ovomucoid or ovomucoid degradation products with some trypsin inhibitory activity and antigenicity were introduced into the small intestine after ingestion of the cooked egg white of Japanese quail.     

A comparison study of the impact of boiling and high pressure steaming on the stability of soybean trypsin inhibitor

The aim of the study is to compare the effect of boiling and high pressure steaming (HPS) on the degradation, inhibitory activity reduction and gastric digestibility of soybean trypsin inhibitor (STI). Thermal stability analysis showed that HPS treatment was effective in eliminating the inhibitory activity of STI than boiling. SDS‐PAGE and Western blot analysis indicated that boiling has less impact on the gastric digestibility of STI than HPS. More importantly, boiling‐pretreated STI revealed high inhibitory activity against trypsin even after digestion by pepsin in simulated gastric fluid (SGF), while HPS treatment was more effective. SDS‐PAGE analysis further verified that after boiling, STI still revealed strong binding ability to trypsin, while STI could be completely degraded by trypsin after HPS treatment for 30 min.     

Effect of digestive enzymes on the bioactive properties of goat milk protein hydrolysates

The aim of this research was to study the influence of the gastrointestinal digestion on the bioactivity of goat milk protein hydrolysates prepared with subtilisin, trypsin and a combination of these two enzymes. All hydrolysates had excellent angiotensin converting enzyme (ACE) inhibitory activity, antioxidant activity and bile acid-binding capacity. Peptide profiles and bioactivities were mainly altered during the intestinal digestion, whereas the effect of the gastric digestion was negligible. The influence of the intestinal digestion varied depending on the hydrolysate and the bioactivity studied. In the case of ACE inhibitory activity, it exclusively decreased when peptides were produced with trypsin. In contrast, antioxidant activity and bile acid-binding capacity improved after the gastrointestinal digestion, regardless the enzymatic treatment conducted. Hydrolysis employing mixtures of subtilisin and trypsin is considered a good approach to produce peptides that maintain, or even enhance, their bioactivity after digestion.     

The allergenicity of ovomucoid and the effect of its elimination from hen's egg white

In order to remove the ovomucoid from hen's egg white, chitin and hydrazide polystyrene beads were used as affinity ligands with 8.9 and 7.1 mg trypsin g^?1 ligand respectively. Ovomucoid was successfully depleted using the trypsin affinity column without hydrolysation of the other egg white constituents. The components of the egg white were purified by high‐performance liquid chromatography, and then the allergenicity of each of these components was compared with that of pooled human serum derived from patients who are allergic to hen's eggs. The importance of using pure protein for studies of the allergenicity of egg white is highlighted, and it was determined (using an enzyme‐immunosorbent assay) that ovomucoid and ovalbumin are major allergenic proteins in egg white. The ovomucoid‐eliminated egg white preparation exhibited significantly less IgE‐binding activity than normal egg white. The ovomucoid‐specific IgE antibodies may have important implications with regard to the egg‐allergic reaction in humans. © 2001 Society of Chemical Industry     

体外消化对三文鱼皮胶原低聚肽抗氧化活性的影响

本研究以三文鱼皮为原料通过酶解法制备三文鱼皮胶原低聚肽,并进行体外模拟消化实验,通过分子量分布分析、DPPH自由基清除能力、羟自由基清除能力、总抗氧化能力（ABTS法）以及活性氧ROS实验,探究三文鱼皮胶原低聚肽经模拟胃、肠道消化后抗氧化活性的变化。结果表明:经模拟消化实验后,三文鱼皮胶原低聚肽的重均分子量轻微减少,变化不超过4%;胃蛋白酶消化前后,DPPH自由基清除能力IC₅₀值分别为11.1、12.3 mg/mL,胰蛋白酶消化前后,IC₅₀值分别为12.5、14.6 mg/mL;胃蛋白酶消化前后,羟自由基清除能力IC₅₀值分别为12.2、13.2 mg/mL,胰蛋白酶消化前后,IC₅₀值分别为10.7、11.5 mg/mL;胃蛋白酶消化后,总抗氧化能力下降不超过13%,胰蛋白酶消化后,总抗氧化能力下降不超过19%;胃蛋白酶消化后,ROS清除能力提高不超过3%,胰蛋白酶消化后,ROS清除能力提高不超过5%。这表明三文鱼皮胶原低聚肽具有良好的消化稳定性和抗氧化活性,为其在抗氧化功能性食品的开发提供了理论基础。     

Studies on the processing and properties of soymilk. II. Effect of processing conditions on the trypsin inhibitor activity and the digestibility in vitro of proteins in various soymilk preparations

Soymilks prepared from beans which had been soaked either in water or 0.4 M sodium carbonate solution for 24 h as a pretreatment were subjected to heat treatment under different conditions for varying lengths of time, to destroy the trypsin inhibitor activity. The rate of inactivation oftrypsia inhibitor in soy milks prepared from carbonate presoaked beans was faster than that of the water presoaked preparation when processed at 98°C and this effect was primarily associated with the change that occurred in the pH of the former system; The effect of alkaline pH's at 98°C on the inactivation of trypsin inhibitor was examined and it was found that the rate of inactivation was changed from zero order at pff 6.8 to first-order kinetics at pH 9.9. Regression equation relating pH of the system and time of heating at 98°C for 100% destruction of the inhibitor activity is presented. This effect of pretreatment was eliminated when both milks were processed at 115°C in cans because of the constancy of pH under these conditions. The influence of heat processing conditions on the enzymic digestibility of proteins in both soymilks was also studied. The pepsin digestion showed no significant differences between milks prepared from the water and the carbonate presoaked soybeans, and was highest in milks which had not been heat treated. With trypsin the digestibility increased with the degree of heal treatment up to the point where the trypsin inhibitor was destroyed, after which further heating resulted in lower digestibilities. A 19% increase in digestibility by trypsin was observed in the milk prepared from carbonate presoaked beans when compared with that from water presoaked beans and after both milks had been heated at 98°C for just sufficient time to destroy the trypsin inhibitor. When the milk prepared from water presoaked beans was autoclaved at 115°C to the same end point, its digestibility increased but it was still about 6% lower than that of the milk prepared by alkali presoaking method and processed at 98°C. The digestibility of casein using pepsin under the conditions used was lower than that of both soymilks. When trypsin was used the digestibility of casein was approximately the same as that of the adequately processed soymilk from carbonate presoaked soybeans or the autoclavtd soymilk from water presoaked beans. Other effects of using a carbonate presoaking treatment far the production of soymilk have also been discussed.     

In vitro study of the inhibitory effect of Fe(II), Fe(III) and Zn(II) ions on the activity of trypsin

The effect of Fe(II), Fe(III) and Zn(II) addition on the in vitro digestibility of casein was examined using trypsin as the proteolytic enzyme. The in vitro digestibility was determined by change in trichloroacetic acid (TCA) solubility of casein and by potentiometric titration. The effect of all three metal ions was also examined on the digestion of casein heated at 80°C for 15 min prior to the enzymic hydrolysis. Heat treatments did not cause significant changes in the proteolysis of casein. The addition of Fe(II) and Fe(III) clearly slowed the proteolysis in both heated and unheated casein. The decrease in rate of proteolysis was in a direct linear relationship with the amount of Fe(II) and Fe(III) addition. The inhibitory effect of Fe(III) was more pronounced as compared to Fe(II). The addition of Zn(II) did not affect the proteolytic activity in both heated and unheated casein.     

Antigenicity of Ovomucoid Remaining in Boiled Shell Eggs

Antigenicity of egg white proteins which remained in heated shell eggs was analyzed quantitatively by using rabbit antibodies specific to egg white proteins and human antibody from patients allergic to egg. The major antigenic component in heated (100°C for 20 min) and coagulated egg white was ovomucoid. A considerable amount of ovomucoid remained immunoreactive even after heating at 100°C for 15 min, though ovomucoid in stored eggs was a little less stable to heating than that of fresh eggs. Even long-time heating (100°C for 45 min) could not completely eliminate the ovomucoid immunoreactivity to human IgE antibody.     

Assessment of the in vitro digestibility of dietary proteins and of the degree of the breakdown of their antigenic structures by using polyenzyme systems

A comparative assessment was made of the digestion of bovine serum albumin (BSA), chicken ovalbumin (OVA), and casein by means of the gastric juice--duodenal contents floccular gel structures (FGS) system and a four-enzymic system including trypsin, chymotrypsin, peptidase, and bacterial protease preparations. Decomposition of the BSA and OVA antigenic structures with the use of the two systems was also studied. Significant differences in BSA and OVA digestion by the gastric juice--FGS system were detected both with respect to amino nitrogen content and to the degree of their antigenic structure decomposition, whereas no such differences were observed when the four-enzymic system was used. The systems most accurately simulating the 'proteolytic conveyer' conditions of the gastrointestinal tract are preferable for the studies. The developed method is recommended for use in comparative assessment of the nutrient protein sensitizing properties.     

Factors affecting the in vitro protein digestibility of chickpea albumins

In vitro protein digestibility (IVPD) of chickpea albumins and its possible relationship to their structure and the presence of trypsin inhibitor activity (TIA) have been studied. Trypsin digestion of the albumin fraction under non‐reducing conditions was incomplete, while the reduction of inter‐ and intramolecular disulphide bonds caused an improvement in the accessibility of sites susceptible to trypsin digestion. Trypsin inhibitor activity in the chickpea albumin fraction was dependent upon both temperature and heating time. Although heating the albumin fraction at 100 °C for 30 min reduced the TIA by more than 50% with respect to the initial activity, an important TIA rate was attributable to heat‐resistant trypsin inhibitor. The TIA decrease was not related to an increase in the rate of IVPD. However, we observed a significant (P ≤ 0.05) increment in IVPD in the presence of β‐ME, confirming the essential role of disulphide bonds in stabilising the protein structure of the albumin fraction. © 2000 Society of Chemical Industry     

苦瓜皂苷与乳清蛋白水解物协同对二肽基肽酶-IV的抑制活性

为评价动植物源天然产物的二肽基肽酶-IV（dipeptidyl peptidase-IV,DPP-IV）抑制活性,以苦瓜皂苷（momordica saponins,MS）、乳清蛋白胃蛋白酶水解物（whey protein pepsin hydrolysates,WPPHs）、乳清蛋白胰蛋白酶水解物（whey protein trypsin hydrolysates,WPTHs）为原料,采用单因素试验和响应面分析优化水解条件,再将MS与WPPHs、WPTH进行复配,结合模拟体外消化,探究DPP-IV抑制活性。结果表明,MS、WPPHs及WPTHs具有DPP-IV抑制活性,最佳水解条件为：当酶添加量4%、pH 2.7酶解2 h时,WPPHs的DPP-IV抑制率最高为15.43%;当酶添加量4%、pH 7.6酶解4 h时,WPTHs的DPP-IV抑制率最高为14.62%。苦瓜皂苷与乳清蛋白水解物具有协同对二肽基肽酶-IV抑制活性作用,当MS与WPPHs、WPTHs的复配体积比均为1:1时,显著高于两者单独累加（p<0.05）。经胃消化后,WPPHs的DPP-IV抑制率降低2.02%,MS和WPTHs的DPP-IV抑制率升高0.99%、7.01%;经肠道消化后,WPPHs的DPP-IV抑制率升高2.23%,而MS和WPTHs的DPP-IV抑制率降低4.12%、2.70%。研究结果可为天然动植物源产物协同对二肽基肽酶-IV抑制活性提供了基础性数据,为降血糖功能性产品的开发提供新的方向。     

In vitro study on digestion of peptides in Emmental cheese: analytical evaluation and influence on angiotensin I converting enzyme inhibitory peptides

A simple in vitro protocol simulating gastrointestinal digestion of proteins and peptides to investigate the effect of digestive enzymes on the biological activity of peptides present in dairy products was developed. This protocol consisted in a 30 min incubation with pepsin followed by a 4 h incubation with trypsin or pancreatin. It was applied to an Emmental cheese water-soluble extract (WSE) and to a casein solution (as a control). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) allowed to monitor the digestion of proteins. Reversed-phase high-performance liquid chromatography (RP-HPLC) allowed to monitor the conversion of proteins and peptides into peptides and amino acids: it is proposed to use the mean retention time corresponding to the overall retention time distribution of molecules to assess the effect of digestive enzymes. The biological activity focused in this study was the angiotensin I converting enzyme (ACE) inhibitory activity. Digestion of Emmental WSE induced an increase of the ACE inhibition as compared to undigested WSE while a 10 kDa ultrafiltered WSE lost a part of its ACE inhibitory activity after digestion process. These results strongly suggest that digestive enzymes diminished the ACE inhibition by the peptides present in Emmental cheese WSE, while the digestion of peptides of high molecular weight would generate new ACE inhibitory peptides.     

Proteinase inhibitors of redgram (Cajanus cajan)

Proteinase inhibitory activity of 35 varieties of redgram (Cajanus cujun L) was determined. Chymotrypsin inhibitory activity was more pronounced than trypsin inhibitory activity in all redgram varieties tested. Both trypsin and chymotrypsin inhibitory activities were found to be markedly reduced on germination.     

糖基化大豆蛋白体外消化产物的生物活性研究

为了分析体外消化作用及超滤处理对糖基化大豆蛋白抗氧化活性及抑菌活性的影响,分别利用胃蛋白酶及胰蛋白酶酶解糖基化大豆蛋白,在一定的条件下水解度分别达到了4.9%和6.4%,此时可溶性蛋白质含量分别为13.6、15.0 mg/m L;再经过超滤处理得到相对分子质量低于5 000的组分,两种超滤组分的可溶性蛋白质含量分别为9.65、10.65 mg/m L。糖基化大豆蛋白体外消化产物的抑菌活性没有发生显著变化,但是抗氧化活性下降;进一步的超滤处理能够获得具有较高生物活性的组分:胃蛋白酶消化产物超滤组分的亚铁离子螯合率提高了约1倍;胰蛋白酶消化产物超滤组分的还原力及羟基自由基清除率显著提高;同时,胃蛋白酶消化产物超滤组分对大肠杆菌具有抑制活性,而胰蛋白酶消化产物超滤组分则具有较强的抑制作用。结果表明,结合体外消化作用及超滤处理能够显著改善糖基化大豆蛋白的生物活性。     

Multiple molecular forms of finger millet subtilisin and amylase inhibitors

Nine varieties of finger millet (Eleusine coracana Gaertn) including a wild form were screened for proteinase and α-amylase inhibitory activities. Subtilisin inhibitory activity was present in all the varieties examined and was highest in the wild form, while the cultivated varieties had more trypsin inhibitory activity. Isoelectric focusing of a 60% ammonium sulphate fraction of the wild form, before and after complexing with subtilisin, showed multiple forms of subtilisin inhibitors one of which was also active against trypsin. Isoforms of finger millet subtilisin inhibitors were isolated from the ammonium sulphate fraction by ion exchange chromatography on DEAE-Sephadex followed by SP-Sephadex chromatography. Copurification of trypsin and α-amylase inhibitory activities with subtilisin inhibitory activity was observed during cation exchange chromatography. PAGE analysis revealed them to be charge isomers with pI in the range 5·5–6·0. SDS–PAGE gave an estimate of 12 000 mol wt for each of them. Finger millet subtilisin inhibitors were more active on bacterial proteinases than on bovine trypsin and chymotrypsin.     

动植物源蛋白体外消化产物结构性质及ACE抑制活性

为揭示动植物源蛋白对于血压调节功能上的差异是否由蛋白肽的血管紧张素转化酶（angiotensin conversion enzyme,ACE）抑制活性所引起,动植物蛋白经体外消化后,对蛋白肽的ACE活性抑制进行研究。采用胃蛋白酶-胰蛋白酶两步消化法,水解植物蛋白（大米、燕麦、大豆、豌豆）、红肉蛋白（猪肉、牛肉）和白肉蛋白（鸡肉）。测定所获蛋白肽的水解度、分子质量分布、氨基酸组成及其ACE抑制率。结果表明,随着蛋白质水解度的增加和分子质量的减小,3类蛋白消化产物的ACE活性抑制率均相应增大。其中,植物蛋白消化产物ACE抑制率最高（60.41%）,红肉蛋白最低（40.13%）。对蛋白质消化产物的氨基酸组成进行分析,结果表明,植物蛋白和白肉蛋白消化产物的疏水性氨基酸含量均显著高于红肉蛋白。3类蛋白中消化产物的疏水性氨基酸含量越高,则ACE抑制活性越高,说明疏水性氨基酸含量高可能是植物蛋白肽具有高的ACE抑制活性的主要原因之一。     

体外模拟消化芝麻蛋白产生多肽的抗氧化性分析

为证实芝麻蛋白在人体内消化可产生抗氧化肽,采用体外模拟消化芝麻蛋白,分析芝麻蛋白水解产生多肽的组成与抗氧化活性。结果显示:在体外模拟消化模型中,芝麻蛋白在1 h内丧失原有亚基结构,最终水解为相对分子质量在15 kDa以下的多肽;水解产生的多肽具有抗氧化性,其中胃蛋白酶水解产生的多肽具有较强的DPPH自由基清除能力,而胰蛋白酶与胰凝乳蛋白酶的水解有助于提升多肽的ABTS自由基清除能力。上述结果表明,芝麻蛋白在人体内消化会产生抗氧化肽,不同消化阶段的芝麻多肽组成有差异,从而造成抗氧化活性的差别。     

Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates

This work evaluated the angiotensin-converting enzyme (ACE)-inhibitory activities of bovine, ovine, and caprine kappa-casein macropeptides (CMPs) and their tryptic hydrolysates. The results obtained indicate that bovine, ovine, and caprine CMPs exhibited moderate in vitro ACE-inhibitory activities that increased considerably after digestion under simulated gastrointestinal conditions. Active peptides could also be produced from CMPs via proteolysis with trypsin, with tryptic hydrolysates exhibiting a more extensive ACE-inhibitory activity than intact CMPs during simulated gastrointestinal digestion. Two active fractions were chromatographically separated from the tryptic hydrolysate of the bovine CMP, but their complexity hampered the assignment of the ACE-inhibitory activity to specific peptide sequences. Evidence for the release of the strong ACE-inhibitory tripeptide IPP was found upon simulation of the gastrointestinal digestion of peptides released by trypsin from the CMP sequence. These findings might help to promote further exploitation of cheese whey in the preparation of nutraceuticals for inclusion in the composition of functional food products with high added values.     

大豆胰蛋白酶抑制剂的异源表达与生化特性解析

本文对一疑似大豆胰蛋白酶抑制剂(STI;sti)的开放阅读框在毕赤酵母中进行了克隆表达与生化特性分析。结果表明,在摇瓶水平上,重组菌GS115(pPIC-STI)分泌表达了30 mg/L STI;重组STI在(40~80)℃或pH 2.0~11.0孵育1 h后,仍能保持85%以上的抑制活性;K+、Zn2+和Mg2+对其胰酶抑制活性有明显的激活作用,而Cu2+、Mn2+、Ca2+、Fe2+和Fe3+则有明显的抑制作用;重组STI对胰蛋白酶具有较强的、专一性的和非竞争性的抑制作用,是一种典型的多肽类胰酶抑制剂。良好的酶学性质使STI在食品、医药等行业具有潜在的应用价值。     

富硒螺旋藻蛋白水解多肽的制备及其对ACE活性的抑制作用

从富硒螺旋藻(SeSP)中提取含硒总蛋白(SeSP-TP),建立蛋白酶水解制备含硒多肽(SeSP-PP)的最佳条件,体外检测SeSP-PP对血管紧张素转化酶(ACE)活性的抑制作用,采用ICP法快速测定藻粉及总蛋白中的硒含量。结果发现,5种常见蛋白酶对SeSP-TP的水解度趋势为:胃蛋白酶-胰蛋白酶-胰凝乳蛋白酶联合酶>碱性蛋白酶>木瓜蛋白酶>胰凝乳蛋白酶>胰蛋白酶>胃蛋白酶。利用胃蛋白酶-胰蛋白酶-胰凝乳蛋白酶联合酶水解,可显著提高蛋白水解度,达到87.94%,且联合水解的多肽对ACE抑制效率最高(89.47%)。碱性蛋白酶水解得到的多肽对ACE的抑制率也较高。当荧光检测出的含硒多肽高于50μg/mL时,对NO的合成有明显的促进效应,而含硒蛋白效果不明显。水溶性和非水溶性组分中测定的硒含量之和占总硒的95.47%,可能在提取制备SeSP-TP过程中,部分含硒蛋白质和含硒小分子会在沉淀、复溶和透析等步骤中丢失。