超声处理对菜籽蛋白酶解效果的影响

以双低菜籽饼粕蛋白为原料,以常规酶解为对照,以酶解产物的抑制活性为指标,分别考察了超声预处理蛋白酶、超声预处理蛋白原料和酶解过程前期施加超声3种超声处理方式对双低菜籽蛋白酶解制备ACEI 活性肽效果的影响。结果表明,3种超声辅助方式都能有效地促进菜籽饼粕蛋白的酶解,提高产物的ACE抑制活性,其中超声预处理蛋白原料效果最为明显。这种方式的最佳参数为：超声频率20 kHz、超声时间 30 min、超声功率1250 W、作用时间4 s、间歇时间2 s,该条件下酶解产物对ACE的抑制率从对照组的70.83%提高到92.97%,肽得率从29.6%提高到41.4%,半抑制浓度IC50从3.57 mg/mL降到2.48 mg/mL。这表明脉冲超声辅助酶解法是一种高效制备双低菜籽ACEI肽的方法。     

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。     

红娘鱼降血压肽的酶解制备工艺优化及酶解产物的抗氧化活性研究

为有效利用红娘鱼制备降血压肽,以红娘鱼鱼糜为原料提取蛋白,并对其进行酶解制备降血压肽。以血管紧张素转换酶ACE抑制率和水解度为指标,通过响应面分析法对酶解红娘鱼鱼糜蛋白制备降血压肽的工艺条件进行优化,并对最优条件下制备的酶解产物进行分子量和抗氧化活性测定。结果表明,碱性蛋白酶是制备降血压肽的最适蛋白酶,响应面法优化制备降血压肽的最佳酶解条件为pH值9、酶与底物的比值(酶底比)1.4%、温度54℃、时间2 h,此条件下酶解制得的降血压多肽ACE抑制率理论值为88%,实际值为89.3%;经高效液相色谱(HPLC)分析可得酶解产物相对分子量<2 000 Da。通过测定酶解产物样品的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、羟自由基(·OH)清除率及还原力判定其体外抗氧化活性,结果表明酶解产物具有较强抗氧化活性。本研究结果为红娘鱼的高值化利用提供了数据支持和理论基础。     

南瓜籽粕酶法制备血管紧张素转化酶抑制肽工艺优化

用中性蛋白酶水解南瓜籽粕,制备血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制肽,是南瓜籽粕蛋白深度开发的途径之一。为了探寻南瓜籽粕酶法制备ACE抑制肽的最佳工艺,该文以ACE抑制率为响应值,用响应面分析法研究酶浓度、底物质量浓度和水解时间等因素对酶解产物的ACE抑制活性的影响,优化制备工艺。结果表明,各因素对制备ACE抑制肽的活性具有极显著影响（P<0.001）。获得中性蛋白酶水解南瓜籽粕制备ACE抑制肽的最佳工艺条件为：酶体积分数为4.8%、底物质量浓度为4.0g/100 mL、水解时间为320 min。在此条件下,南瓜籽粕蛋白酶解产物对ACE的理论抑制率最高可达80.0％,验证值为80.56％±0.23%,预测模型可靠性高,可应用于南瓜籽粕ACE抑制肽的酶法制备。通过优化,提高了南瓜籽粕ACE抑制肽的活性。     

仿刺参ACE抑制肽的纯化及其分子量的测定

利用仿刺参体壁制备具有抑制血管紧张素转移酶(ACE)活性的多肽,为进一步研究ACE抑制肽提供理论基础。本研究采用动物蛋白水解酶酶解仿刺参体壁,得到具有ACE抑制活性的酶解液;将酶解液粗品通过截留分子量5KDa的超滤系统,将活性部分继续用Sephadex G15分离纯化,采用HPLC法测定各组分的ACE抑制活性,同时利用RP-HPLC测定其相对分子质量分布。结果可知:仿刺参活性较高的ACE抑制肽主要由二肽到五肽组成,且分子量越小,抑制活性越高,得到了具有较高ACE抑制活性且组成比较单一的组分d,其IC50为439μg·mL-1。     

超声辅助竹节虾头酶解及抗氧化肽分离研究

为了实现竹节虾加工副产物的高值化利用,以竹节虾加工废弃物中的虾头副产物为原料,以水解度和DPPH清除率作为评价指标,采用中性蛋白酶酶解,通过响应面法优化超声辅助酶解工艺,并依次通过超滤、凝胶层析色谱和反相高效液相色谱等分离方法,从竹节虾虾头酶解产物中分离制备抗氧化肽,采用超高压液相色谱串联质谱联用技术对肽的结构进行表征。结果表明,在中性蛋白酶添加量为3 000 U·g~(-1)、p H值7.0条件下,最佳超声辅助酶解工艺参数为超声时间41 min,超声温度55℃,超声频率22 k Hz,料液比1∶9(w/v),在此条件下获得的酶解产物DPPH清除率达69.50%。当水解时间为0.5~2.5 h时,超声辅助酶解的酶解产物水解度和DPPH清除率比非超声辅助酶解工艺分别高17.95%和18.83%,该工艺缩短了酶解时间,节约了能耗。酶解产物经超滤初步分离发现,相对分子质量在3k Da(SHP4)的组分具有显著的抗氧化活性。用凝胶层析法进一步分离纯化SHP4组分后得到4个峰,其中SHP4-II的DPPH清除率最高;SHP4-II通过反相高效液相纯化后也得到4个主要肽峰,在多肽含量为1.5 mg·m L~(-1)时,SHP4-II-4的DPPH清除率最高,达到85.69%,并且具有较好的分离度,质谱分析发现,该抗氧化肽的结构为Gly-Asn-Gly-Leu-Pro(455.99 Da)。本研究结果为虾肽抗氧化保健食品的研发提供了一定的科学依据。     

胃蛋白酶水解珠蛋白获得ACE抑制肽的工艺优化(简报)

以猪血中提取的珠蛋白为原料,水解度为指标,对胃蛋白酶水解珠蛋白的工艺条件进行优化,然后测定不同水解阶段产物对血管紧张素I转换酶(ACE)抑制活性的影响,来筛选具有较高抑制ACE活性的生物功能活性肽.结果表明:在酶量为0.3%,初始pH值2.6,蛋白浓度0.7%,温度37℃条件下,时间为12h时,水解度值最大,为11.85%;在其他因素不变条件下,水解时间为4h时,水解产物对ACE抑制率达到最大值,其ACE抑制率为93.2%,水解度为9.64%.     

花椒籽蛋白降血压肽的分离纯化

为了获得高活性、高纯度的花椒籽蛋白降血压肽,本试验采用SephadexG-25凝胶层析对酶解液进行分离纯化,并用Tricine-SDS-PAGE测定其分子量。结果表明,最佳分离条件为:以超纯水为洗脱溶液,洗脱流速为0.6mL·min-1,上样量为2.0mL,样品浓度为30mg·mL-1。在此条件下可收集得到4个组分,其中洗脱峰Ⅱ有较高的ACE抑制效果,IC50为0.021mg·mL-1,该组分的分子量在3000Da以下。本研究为新型降血压药物或者保健食品的研发提供了一定的科学依据。     

海带酶解和菌解工艺优化及其降解产物对菜心抗逆性的影响

  【目的】  海藻 (海带) 降解物对作物具有抗逆促生作用,研究海带菌解和酶解的最佳工艺条件,并比较两个工艺生产的降解产物对菜心的抗逆效果。  【方法】  采用单因素和正交试验,探究不同的底物浓度、接种量、溶液pH、降解温度分别对微泡菌Microbulbifer sp. SH-1和海藻酸裂解酶AlgSH7制备海藻降解产物的影响。同时,借助光学显微镜,研究SH-1菌和AlgSH7酶降解过程中海带细胞形态变化。并在此基础上,利用盆栽试验,研究海带菌解产物、酶解产物对菜心非生物胁迫抗性的影响。  【结果】  海藻酸裂解酶AlgSH7降解海带的最佳工艺条件是底物浓度2%,酶添加量6%,降解体系初始pH 8.5,温度44℃;SH-1菌株降解海带的最佳工艺条件是底物浓度2.5%,接种量1.5%,降解体系初始pH 7.5,温度32℃。与菌解产物相比,酶解产物中海藻酸、总糖、还原糖含量分别提高141.8%、57.6%、150.5%,而褐藻多酚、甘露醇、甜菜碱含量分别下降35.3%、60.6%、62.6%,但菌解工艺的产率比酶解工艺高9.3%。细胞形态观察结果表明,海带原始细胞排列紧密,形状规则饱满,随菌解或酶解时间的延长,海带细胞可视面积变小,细胞之间的间距增大。降解24 h时,酶解海带细胞可视面积仅为菌解的77.2%。在盆栽试验中,中度干旱条件下 (土壤相对含水量50%),酶解液灌根处理的菜心生物量比菌解液处理提高13.7%;酶解液配施水溶肥 (NPK, N:P₂O₅:K₂O=110:50:60) 处理的菜心生物量比菌解液配施NPK处理明显提高10.6%。淹水条件下 (淹水层深度2 cm),酶解液处理的菜心生物量比菌解液处理降低7.1%,但均显著高于对照处理;酶解液配施NPK处理的菜心生物量比菌解液配施NPK处理提高5.6%。盐胁迫条件下 (土壤中NaCl 含量24 g/kg),海带酶解液处理的菜心生物量比菌解液处理有增加趋势,但增加不显著。  【结论】  海带酶解产物中海藻酸、总糖、还原糖含量高于菌解产物,而褐藻多酚、甘露醇、甜菜碱含量低于菌解产物。菌解海带的产率高于酶解海带的产率。逆境胁迫条件下,海带酶解液配施NPK处理的菜心生物量高于菌解液配施NPK处理,海带酶解液对农作物抗逆促生作用优于菌解产物。     

秋刀鱼制备黄嘌呤氧化酶抑制肽的工艺优化

为探讨秋刀鱼制备黄嘌呤氧化酶(XOD,xanthine oxidase)抑制肽的最佳工艺,该文以氮回收率和体外XOD抑制活性为指标,首先通过单因素试验确定中性蛋白酶和胰酶为水解秋刀鱼制备XOD抑制肽的最佳蛋白酶。在此基础上,采用响应面分析法研究加酶量、酶解时间和中性蛋白酶所占比例对酶解氮回收率及酶解产物XOD抑制活性的影响,进一步优化并最终确定了水解秋刀鱼制备XOD抑制肽的最优工艺:料液比1∶2(g/g),总加酶量0.3%(中性蛋白酶∶胰酶质量比=6∶4),在p H值7.0和55℃条件下酶解6 h,其理论氮回收率和酶解产物XOD抑制率分别为72.69%和30.32%,对应实际值分别为72.03%和30.96%,预测模型可靠性高,可用于秋刀鱼XOD抑制肽的酶法制备,为工业化利用秋刀鱼生产降尿酸肽提供一定的理论和技术指导。     

Antihypertensive Effect of Protein Hydrolysate from Azufrado Beans in Spontaneously Hypertensive Rats

The objective of this study was to evaluate the antihypertensive potential of common bean protein hydrolysate. Protein concentrates were obtained, followed by Alcalase enzymatic hydrolysis, and then ultrafiltrated (3,000 molecular weight cutoff); the lyophilized product was named BP3. The angiotensin converting enzyme (ACE) inhibitory activity was determined as IC₅₀ (3.68 ± 0.07 μg/mL). The antihypertensive effect was evaluated in spontaneously hypertensive rats (SHR) by two assays; Captopril ACE inhibitor was used as a reference compound and water as a control. A short‐term assay showed a maximum decrease in mean arterial pressure of –41 ± 5 mmHg in SHR, 3 h after oral administration of 500 mg of BP3/kg of body weight (bw). In a long‐term assay, a significant decrease in systolic blood pressure of –24 ± 5 mmHg was observed in SHR, after 45 days of oral administration of 500 mg of BP3/kg of bw/12 h. In both assays, BP3 treatment showed antihypertensive effect over SHR, similar to Captopril treatment. The sequences of the most abundant peptides present in BP3, determined by mass spectrometry, were identified as KFPWVK, GADFRKK, and PQSPCKRVNRHS. These peptides are reported for the first time in Azufrado Higuera common beans, and they are most likely responsible for the antihypertensive effect of BP3.     

Angiotensin I-converting enzyme inhibitory activity of gelatin hydrolysates and identification of bioactive peptides

In this project we report on the angiotensin I-converting enzyme (ACE)-inhibitory activity of a bovine gelatin hydrolysate (Bh2) that was submitted to further hydrolysis by different enzymes. The thermolysin hydrolysate (Bh2t) showed the highest in vitro ACE inhibitory activity, and interestingly a marked in vivo blood pressure-lowering effect was demonstrated in spontaneously hypertensive rats (SHR). In contrast, Bh2 showed no effect in SHR, confirming the need for the extra thermolysin hydrolysis. Hence, an angiotensin I-evoked contractile response in isolated rat aortic rings was inhibited by Bh2t, but not by Bh2, suggesting ACE inhibition as the underlying antihypertensive mechanism for Bh2t. Using mass spectrometry, seven small peptides, AG, AGP, VGP, PY, QY, DY and IY or LY or HO-PY were identified in Bh2t. As these peptides showed ACE inhibitory activity and were more prominent in Bh2t than in Bh2, the current data provide evidence that these contribute to the antihypertensive effect of Bh2t.     

酸-超声波预处理及糖化水解稻草研究

采用室内实验方法,研究了酸-超声联合预处理稻草对其化学组成以及糖化效果的影响,并与传统酸预处理法的效果进行了对比。结果表明,与未经处理的稻草相比,经酸-超声波处理的稻草其半纤维素、木质素含量最高分别减少了64．46％、62．19％,纤维素含量最高则上升了73．20％,而酸处理的稻草相应数值只能达到56．72％、59．90％及53．41％。同时分别对两种方法的稻草糖化的工艺条件通过正交试验进行了优化,得出两种方法的稻草最佳糖化条件均为：pH值为4．8,温度为45℃,酶浓度为20mg·g-1。在该条件下,对于酸一超声波预处理稻草,在糖化108h以后还原糖浓度稳定并达到最大值26．4g·L-1,而对于酸预处理稻草,在糖化120h以后还原糖浓度才稳定并达到最大值26．2g·L-1,且前者能比后者产生更多的葡萄糖以及更少的木糖,更有利于提高后续酒精发酵的效率。     

Porcine skeletal muscle troponin is a good source of peptides with Angiotensin-I converting enzyme inhibitory activity and antihypertensive effects in spontaneously hypertensive rats

In the search for novel peptides that inhibit the angiotensin I-converting enzyme (ACE), porcine skeletal troponin was hydrolyzed with pepsin, and the products were subjected to various types of chromatography to isolate active peptides. Glu-Lys-Glu-Arg-Glu-Arg-Gln (EKERERQ) and Lys-Arg-Gln-Lys-Tyr-Asp-Ile (KRQKYDI) were identified as active peptides, and their 50% inhibitory concentrations were found to be 552.5 and 26.2 microM, respectively. These are novel ACE inhibitory peptides, and the activity of KRQKYDI was the strongest among previously reported troponin-originated peptides. KRQKYDI was slowly hydrolyzed by treatment with ACE, and kinetic studies indicated that this peptide was a competitive inhibitor of the enzyme. When KRQKYDI was administered orally to spontaneously hypertensive rats (SHR) at a dose of 10 mg/kg, a temporary antihypertensive activity was observed at 3 and 6 h after administration.     

超声辅助酶解促进草鱼鳞胶原肽水解进程的内在机制解析

为探究超声辅助酶解促进草鱼磷胶原肽水解进程的内在原因，分别研究了超声对底物蛋白（草鱼鳞）的分子结构、表面疏水性、粒径等理化特性和蛋白酶酶解能力的影响机制，在此基础上，对超声酶解进程进行了动力学拟合，从酶解动力学角度进一步评估了促进草鱼鳞胶原肽水解进程的"超声-酶"耦合效应。结果表明，适当的超声强度（300 W、20 min）可以使底物蛋白的结构展开，此时其表面疏水性最大、粒径最小，使之更适合后续酶解，但当超声功率大于300 W时，底物蛋白会重新聚集，其中的部分疏水基团被掩埋，不利于酶解的进行。同时，当超声功率为300 W、时间为20 min时，单酶酶解组和分步酶解组的酶解能力从2.35×105 U/g、3.41×105 U/g分别提高至3.44×105 U/g、3.86×105 U/g，且表现出显著性差异（P<0.05）。通过动力学模型对超声辅助单酶酶解（r2=0.988 8）和分步酶解进程（r2=0.960 7）进行了动力学拟合，依据建立的反应动力学方程，证实了超声辅助分步酶解进程更快，研究结果为超声辅助酶解工艺制备胶原肽提供了一定理论基础。     

Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max)

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, catalyzes the conversion of Angiotensin I to the potent vasoconstrictor Angiotensin II and plays an important physiological role in regulating blood pressure. Inhibitors of angiotensin 1-converting enzyme derived from food proteins are utilized for pharmaceuticals and physiologically functional foods. ACE inhibitory properties of different enzymatic hydrolysates of glycinin, the major storage protein of soybean, have been demonstrated. The IC50 value for the different enzyme digests ranges from 4.5 to 35 microg of N2. The Protease P hydrolysate contained the most potent suite of ACE inhibitory peptides. The ACE inhibitory activity of the Protease P hydrolysate after fractionation by RP-HPLC and ion-pair chromatography was ascribed to a single peptide. The peptide was homogeneous as evidenced by MALDI-TOF and identified to be a pentapeptide. The sequence was Val-Leu-Ile-Val-Pro. This peptide was synthesized using solid-phase FMOC chemistry. The IC50 for ACE inhibition was 1.69 +/- 0.17 microM. The synthetic peptide was a potent competitive inhibitor of ACE with a Ki of 4.5 +/- 0.25 x 10(-6) M. This peptide was resistant to digestion by proteases of the gastrointestinal tract. The antihypertensive property of this peptide derived from glycinin might find importance in the development of therapeutic functional foods.     

His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo.

It has been reported that soybean peptide fractions isolated from Korean fermented soybean paste exert angiotensin I converting enzyme (ACE) inhibitory activity in vitro. In this study, further purification and identification of the most active fraction inhibiting ACE activity were performed, and its antihypertensive activity in vivo was confirmed. Subsequently, a novel ACE inhibitory peptide was isolated by preparative HPLC. The amino acid sequence of the isolated peptide was identified as His-His-Leu (HHL) by Edman degradation. The IC(50) value of the HHL for ACE activity was 2.2 microg/mL in vitro. Moreover, the synthetic tripeptide HHL (spHHL) resulted in a significant decrease of ACE activity in the aorta and led to lowered systolic blood pressure (SBP) in spontaneously hypertensive (SH) rats compared to control. Triple injections of spHHL, 5 mg/kg of body weight/injection resulted in a significant decrease of SBP by 61 mmHg (p < 0.01) after the third injection. These results demonstrated that the ACE inhibitory peptide HHL derived from Korean fermented soybean paste exerted antihypertensive activity in vivo.