亚麻籽降胆固醇活性肽的酶解工艺优化及分级制备

以亚麻籽分离蛋白为原料,利用酶解工艺制备降胆固醇活性肽。对蛋白酶进行了筛选,并通过单因素实验和正交实验确定最优酶解工艺,采用超滤分离技术得到具有较高降胆固醇活性的亚麻籽肽,并对超滤前后亚麻籽肽的氨基酸组成及降胆固醇活性进行了研究。结果表明:最佳酶解工艺条件为采用Protease M进行酶解、加酶量1. 5%、底物质量分数2. 0%、酶解温度50℃、酶解时间3 h,在此条件下亚麻籽肽的降胆固醇活性最强,胆固醇胶束溶解度抑制率达53. 19%;超滤后相对分子质量小于1 kDa的多肽组分降胆固醇活性最强,胆固醇胶束溶解度抑制率达72. 39%,较超滤前提高了19. 20个百分点。氨基酸分析结果表明,超滤后相对分子质量小于1 kDa的多肽组分的总疏水性氨基酸含量明显高于超滤前,提高了15. 97个百分点,而且多肽组分中赖氨酸/精氨酸的比值低于超滤前,这可能是其降胆固醇活性强于超滤前的主要原因。     

酶解乳清蛋白制备降胆固醇活性肽的工艺研究

利用生物酶技术酶解乳清蛋白,制备具有降胆固醇活性的多肽,通过响应面试验、二次旋转回归设计建立回归模型,以胆固醇胶束溶解度抑制率为评价指标,对乳清蛋白的酶解条件进行了优化。结果表明,乳清蛋白的最佳酶解条件为：酶解时间8.5 h、酶解温度55 ℃、酶解pH 8.0、加酶量4.7%、乳清蛋白含量5.8%,在此条件下,酶解乳清蛋白得到的活性肽的胆固醇胶束溶解度抑制率为18.21%。     

酪蛋白源降胆固醇肽的酶解制备工艺优化

以酪蛋白为原料,采用中性蛋白酶、碱性蛋白酶以及胰蛋白酶对酪蛋白进行水解,确定制备降胆固醇肽的最佳蛋白酶;通过单因素实验和响应面试验,研究水解pH、水解温度、酶与底物比、底物浓度和水解时间对酪蛋白水解度和胆固醇胶束溶解度抑制率的影响,确定最佳水解条件;而后通过超滤和凝胶过滤层析确定降胆固醇肽的初步分离工艺。结果表明:制备酪蛋白源降胆固醇肽的最佳水解工具酶是中性蛋白酶,其最佳酶解条件为反应温度51.3 ℃,酶与底物浓度比6.47%,pH6.34,底物浓度5 g/100 mL,反应时间3.5 h,胆固醇抑制率为58.25%±0.59%;Sephadex G-10分离酪蛋白降胆固醇肽条件为上样浓度80 mg/mL,上样体积2.5 mL,洗脱速度3.5 mL/min;经酶解、超滤及层析后制备的酪蛋白源降胆固醇肽峰1和峰2样品在100 μg/mL的胆固醇溶解度抑制率为24.2%±0.24%和4.3%±0.16%。经酶解制备分离后,获得具有抑制降固醇胶束溶解活性的降胆固醇肽,为降胆固醇肽的开发提供理论研究基础。     

降胆固醇亚麻籽蛋白酶解肽的分离纯化及结构鉴定

采用Protease M对亚麻籽蛋白进行酶解,制备具有降胆固醇活性的亚麻籽蛋白酶解肽,对其进行分离纯化及结构鉴定,并合成相应多肽验证其降胆固醇活性。结果表明,经Protease M对亚麻籽蛋白酶解4 h时获得的亚麻籽蛋白酶解肽胆固醇胶束溶解度抑制率最高,为47.57%;继续采用超滤技术将其分离为相对分子质量小于等于3 kDa、3~5 kDa、5~10 kDa、10~30 kDa和大于30 kDa 5个组分,发现相对分子质量小于等于3 kDa的组分胆固醇胶束溶解度抑制率最高,为71.0%;再经大孔树脂对该组分进行吸附后经过不同体积分数乙醇溶液洗脱,发现75%的乙醇洗脱分离得到的组分胆固醇胶束溶解度抑制率最高,为79.8%;采用反相高效液相色谱（RP-HPLC）对其进一步分离纯化,收集到F9组分的胆固醇胶束溶解度抑制率最高,为85.7%;最后采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)从F9组分中鉴定出6种降胆固醇亚麻籽肽,氨基酸序列分别为Ile-Ile-Pro-Ala-Phe(IIPAF)、Leu-Asn-Phe-Phe(LNFF)、Leu-Leu-Gly-Thr-Leu(LLGTL)、Ile-Pro-Pro-Phe(IPPF)、Ile-Ile-Phe(IIF)和Leu-Leu-Gly-Ala(LLGA),其胆固醇胶束溶解度抑制率分别为82.8%、77.8%、88.0%、93.5%、80.3%和87.1%。     

斑点叉尾鮰鱼露生产工艺研究

以斑点叉尾鮰为原料,采用多酶法制取鱼露。通过正交试验优化酶解工艺条件参数。结果表明,最佳生产工艺条件为：酶解时间3.5h、酶解温度50℃、pH8、酶浓度2%。所得鱼露味道柔和,具有鱼露特殊香气。     

罗非鱼蛋白酶解制取鱼露及其抗氧化研究

目的:以罗非鱼为原料,采用生物酶解技术加工传统产品鱼露,提高产品质量,缩短加工时间。方法:采用枯草杆菌碱性蛋白酶(E1)、风味酶(E2)酶解罗非鱼蛋白,制取富含多肽的酶解物;并以酶解物为基本原料制备调味品鱼露。通过对自由基的清除作用,研究其抗氧化作用。结果:采用生物酶解技术生产罗非鱼鱼露,产品色泽好,风味浓,营养高,有良好的抗氧化性。结论:与传统技术生产鱼露比较,生产时间短,工艺条件容易控制,产品质量稳定,该研究为罗非鱼到深加工提供了理论基础。     

鸡肠蛋白酶解多肽功能特性研究

目的:研究鸡肠多肽的提取条件及其功能特性。内容:(1)以鸡肠为原料进行粗蛋白提取并测定粗蛋白中蛋白质含量,粗蛋白提取率为10.03%,蛋白质含量为16.06/100 g。(2)以抑菌圈、水解度和DPPH(1,1-二苯基-2-三硝基苯肼)清除率为指标,选用中性蛋白酶、碱性蛋白酶、复合蛋白酶对鸡肠粗蛋白液进行单酶酶解,筛选出碱性蛋白酶为制备鸡肠多肽的最佳水解酶,运用单因素实验优选出碱性蛋白酶的最终酶解条件:反应温度60℃、加酶量0.4%、p H8.0、酶解时间4h;此条件下,酶水解度可达到24.97%。(3)对鸡肠多肽进行功能性评价,酶解液DPPH清除率为90.10%,羟基自由基清除率为95.19%,总抗氧化能力为1.75 mol/L。随着酶解液稀释倍数增加,DPPH清除率、自由基的清除率和总抗氧化能力都不断下降。酶解液胆固醇胶束溶解度的抑制率为54.18%,酶解液可以降低胆固醇在胶束溶液中的溶解度且抑制效果呈现出浓度依赖性。对酶解液进行抑菌实验表明:鸡肠多肽对大肠杆菌具有一定的抑菌效果。     

降胆固醇活性花生多肽制备工艺的研究

以冷榨花生粕为原料,酶解制备具有降胆固醇活性的花生多肽。以体外结合胆酸盐的能力为指标,考察了酶的种类、温度、p H值、加酶量、酶解时间等因素对花生多肽降胆固醇活性的影响,确定最优蛋白酶为木瓜蛋白酶,通过正交试验的方法,确定了酶解花生粕制备降胆固醇活性花生多肽的最佳工艺条件为:温度50℃、p H 7.0、加酶量4 000 U/g底物、酶解时间4 h。     

鸭肫酶解工艺优化及降胆固醇活性研究

以水解度为指标,选用木瓜蛋白酶对鸭肫蛋白酶解工艺进行优化,以酶解温度、时间、p H值、加酶量、水料比为因素,得到木瓜蛋白酶酶解鸭肫的最佳工艺条件为:酶解温度50℃、酶解时间3 h、p H值6.0、加酶量0.2%、水料比为3:1。鸭肫小肽中含有丰富的氨基酸,其中包含8种人体必需氨基酸,占总氨基酸含量的14.73%,疏水性氨基酸占总含量的25.44%。同时,鸭肫小肽具有一定的降胆固醇功能,经过消化后,鸭肫对胆固醇溶解度抑制率从未消化时的26.41%升高为27.52%,结果表明消化处理可提高小肽的降胆固醇活性。     

鹰嘴豆降胆固醇肽的制备及活性

为了获得降胆固醇肽,本试验以鹰嘴豆为原料,将提取的分离蛋白通过碱性蛋白酶水解,选择加酶量、料液比、酶解时间3个因素,以水解度和胆固醇抑制率为考察指标,进行单因素试验,并在此基础上,以胆固醇抑制率为评价指标,采用Box-Behnken响应面分析法优化酶解鹰嘴豆蛋白,制备降胆固醇活性肽的条件;并将制备的鹰嘴豆肽对Wister大鼠进行试验。结果表明:鹰嘴豆蛋白最佳酶解工艺为:p H 8.0,加酶量2%,料液比2%,酶解温度50℃,酶解时间80 min,在此条件下鹰嘴豆多肽体外胆固醇抑制率可达到71.55%;动物试验显示灌胃100 mg/kg bw剂量的鹰嘴豆肽可使大鼠体内胆固醇含量降低22.39%。     

豆腐蛋白体外消化物抑制胶束转运胆固醇作用分析

大豆蛋白肽具有显著的降血脂功效,但目前研究主要集中在小分子肽段,且对蛋白肽降血脂作用的分子机理探究较少。模拟豆腐蛋白的胃肠道消化,得到豆腐蛋白的体外消化物,按相对分子质量（MR）分为3 个级别：＞10 000、3 000～10 000和＜3 000,利用胆固醇胶束实验（采用两种实验基质及两种检测方法）检测其抑制膳食混合胶粒转运胆固醇的作用。结果表明,在分级的3 种豆腐消化物中,MR＞10 000具有最强的抑制膳食混合胶粒转运胆固醇作用,其胆固醇胶束吸收率为62.40%,小于阳性对照（Val-Ala-Trp-Met-Tyr,其胆固醇胶束吸收率为68.41%）,且MR＞10 000豆腐消化物的抑制胶束转运胆固醇作用随着样品质量浓度增大呈先增强后减弱的趋势。此外,胆固醇胶束实验中采用的两种“膳食混合胶粒”的转运效果及两种检测方法的结果均具有一致性,说明豆腐蛋白消化物具有影响膳食混合胶粒转运和胆固醇吸收的作用。     

Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice

The yield and in vitro hypocholesterolemic activity of low-molecular weight soy protein hydrolysates were studied. The yield and cholesterol micellar solubility inhibitory rate (CMSIR) were higher for soy protein Alcalase hydrolysates (SAPH) than soy protein AS1398 hydrolysates (SNPH). The highest CMSIR of 48.6% was found with SAPH with a degree of hydrolysis (DH) of 18%. Optimal parameters for desalting SAPH with macroporous adsorption resin DA201-C were decided to be pH 4.5, SAPH dispersion to resin ration 75:100, and a loading rate 1 BV/h, at which the adsorption rate of the peptide was 89.7%. Only a 4% decrease in CMSIR was found with SPAH (DH 18%) after gastrointestinal protease digestion. In a mice feeding study for 30 d, the serum level of LDL-C + VLDL-C in the SAPH diet group with 0.5 and 2.5 g/kg BW SAPH decreased by 34% and 45% respectively compared to the mice fed only the hypercholesterolemia diet.     

Fish protein decreases serum cholesterol in rats by inhibition of cholesterol and bile acid absorption

Fish protein has been shown to decrease serum cholesterol content by inhibiting absorption of cholesterol and bile acid in laboratory animals, though the mechanism underlying this effect is not yet fully understood. The purpose of this study was to elucidate the mechanism underlying the inhibition of cholesterol and bile acid absorption following fish protein intake. Male Wistar rats were divided into 2 dietary groups of 7 rats each, 1 group receiving a diet consisting of 20% casein and the other receiving a diet consisting of 10% casein and 10% fish protein. Both experimental diets also contained 0.5% cholesterol and 0.1% sodium cholate. After the rats had been on their respective diets for 4 wk, their serum and liver cholesterol contents and fecal cholesterol, bile acid, and nitrogen excretion contents were measured. Fish protein consumption decreased serum and liver cholesterol content and increased fecal cholesterol and bile acid excretion and simultaneously increased fecal nitrogen excretion. In addition, fish protein hydrolyzate prepared by in vitro digestion had lower micellar solubility of cholesterol and higher binding capacity for bile acids compared with casein hydrolyzate. These results suggest that the hypocholesterolemic effect of fish protein is mediated by increased fecal cholesterol and bile acid excretion, which is due to the digestion products of fish protein having reduced micellar solubility of cholesterol and increased bile acid binding capacity.     

Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates

A novel hypocholesterolemic peptide was fractionated by gradient ethanol elution from a macroporous adsorption resin (MAR DA201-C), and then separated on Sephadex G-15 and RP-HPLC from a soy protein hydrolysate (SAPH DH 18%). Identification of the hypocholesterolemic peptide structure was accomplished with HPLC–MS. The peptide with the highest hypocholesterolemic activity was found in 75% ethanol fraction among the four fractions from gradient ethanol elution with MAR DA201-C. The calculated average hydrophobicity by amino acid composition of each ethanol eluted fraction suggested that the peptides could be separated in terms of hydrophobicity with MAR DA201-C. Four peaks were obtained with further fractionation on Sephadex G-15, the highest cholesterol micellar solubility inhibition rate, 81.3%, was obtained in Peak 2, corresponding to the molecular weight fraction of 300–800 Da. Fifteen main peaks were obtained with RP-HPLC fractionation, the highest cholesterol micellar solubility inhibition rate (94.3%) was in Peak 7. The amino acid sequence of this peptide was identified as WGAPSL with LC–MS and amino acid composition analysis.     

Preventive effect of fermented Maillard reaction products from milk proteins in cardiovascular health

The aim of this study was to determine the dual effect of Maillard reaction and fermentation on the preventive cardiovascular effects of milk proteins. Maillard reaction products (MRP) were prepared from the reaction between milk proteins, such as whey protein concentrates (WPC) and sodium caseinate (SC), and lactose. The hydrolysates of MRP were obtained from fermentation by lactic acid bacteria (LAB; i.e., Lactobacillus gasseri H10, L. gasseri H11, Lactobacillus fermentum H4, and L. fermentum H9, where human-isolated strains were designated H1 to H15), which had excellent proteolytic and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities (>20%). The antioxidant activity of MRP was greater than that of intact proteins in assays of the reaction with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and trivalent ferric ions; moreover, the effect of MRP was synergistically improved by fermentation. The Maillard reaction dramatically increased the level of antithrombotic activity and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitory effect of milk proteins, but did not change the level of activity for micellar cholesterol solubility. Furthermore, specific biological properties were enhanced by fermentation. Lactobacillus gasseri H11 demonstrated the greatest activity for thrombin and HMGR inhibition in Maillard-reacted WPC, by 42 and 33%, respectively, whereas hydrolysates of Maillard-reacted SC fermented by L. fermentum H9 demonstrated the highest reduction rate for micellar cholesterol solubility, at 52%. In addition, the small compounds that were likely released by fermentation of MRP were identified by size-exclusion chromatography. Therefore, MRP and hydrolysates of fermented MRP could be used to reduce cardiovascular risks.     

Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates

BACKGROUND: Rice bran, containing about 100–150 g kg^?1 protein, is a by‐product of rice milling that has only become an available ingredient in recent years owing to the centralisation of rice milling. Rice bran, but not its protein fraction or hydrolysates, has been shown to have a hypocholesterolaemic effect. Peptides from soy, milk and other foods have been proposed to have hypocholesterolaemic effects based on their ability to lower cholesterol solubility in bile acid/phosphotidyl choline micelles. RESULTS: Rice bran protein hydrolysates (RBPHs) were prepared and investigated for their potential to lower cholesterol concentration in micelles. The RBPHs were produced by digestion using four different peptidases, alcalase 2.4L^®, neutrase 0.8L^®, papaya latex papain and porcine pancreas trypsin, and then fractionated by hydrophobicity using styrene/divinylbeneze resins. Alcalase 2.4L^® produced the highest degree of hydrolysis, and the resulting hydrolysates had the highest micellar cholesterol inhibition ability in an in vitro hypocholesterolaemic test. The adsorption dynamics of four different macroporous resins, DA201‐C, Sepabeads SP207 and SP825 and Diaion HP20, were determined using the Langmuir isotherm model. DA201‐C had the highest adsorption capacity with an equilibrium concentration of 220 mg g^?1. The hydrolysates eluted with 25, 50, 75 and 95% (v/v) ethanol lowered the micellar cholesterol concentration by 11.88, 14.76, 19.37 and 7.56% respectively. CONCLUSION: A hydrophobic fraction of RBPH had the highest inhibitory activity on micellar cholesterol, which suggests that it may have hypocholesterolaemic properties. Copyright © 2011 Society of Chemical Industry     

秋刀鱼鱼露速酿加工工艺研究

为了研究鱼露快速发酵生产工艺,以秋刀鱼为原料,采用加曲适量保温的方式生产鱼露,通过单因素实验探讨不同因素(发酵周期、温度、固液比、加盐量、加曲量)对鱼露发酵生产的影响,并在此基础上进行正交实验工艺优化,结果表明,在发酵周期30 d时,鱼露发酵液中氨基酸态氮含量维持稳定,表明鱼肉组织基本分解完全,最佳发酵工艺条件为:温度30℃、固液比1∶0.6、加曲量5%、加盐量12%。在此条件下制得的鱼露氨基酸态氮含量(0.97±0.02)g/100 m L,可溶性固形物含量23%±0.51%,感官评分88分,鱼露颜色棕红至橙红,无异味,富有鱼露特殊香气,达到快速生产鱼露目的。      

Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: Effects of protein hydrolysates prepared from Achatina fulica snail foot muscle

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC₅₀ of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.     

葛花异黄酮精制及体外降脂活性研究

目的:利用HPD-100型大孔树脂优化葛花异黄酮精制工艺,并评价其体外降脂活性。方法:通过大孔树脂柱层析法优化葛花异黄酮精制工艺,通过体外胰脂肪酶活性、胆固醇酯酶活性和胆固醇胶束溶解度法评价葛花异黄酮的降脂活性。结果:花异黄酮精制工艺如下,吸附参数:葛花异黄酮质量浓度100 mg/mL,pH 6.0,上样体积20 mL,解吸参数:洗脱液为pH 5.0体积分数70%的乙醇溶液,洗脱流速2.0 mL/min,精制后葛花异黄酮纯度可达81.47%;精制葛花异黄酮对胰脂肪酶、胆固醇酯酶和胆固醇胶束溶解度的抑制率可达40.73%,52.32%,46.17%,较未精制分别提高了26.65%,47.46%,37.53%,并存在一定的剂量效应关系。结论:葛花异黄酮可通过抑制胰脂肪酶和胆固醇酯酶的活性,降低胆固醇胶束溶解度发挥降脂作用。