大米蛋白抗氧化肽的活性以及组成鉴定研究

采用碱性蛋白酶酶解大米蛋白制备大米蛋白抗氧化肽。研究表明大米蛋白酶解产物具有螯合金属铁离子、清除H2O2、羟自由基、DPPH自由基、抑制亚油酸自然氧化的作用。通过应用高解析离子淌度质谱（HD-MS）分析鉴定米蛋白抗氧化肽的序列主要为Met-Pro-Pro-Ser-Ser-Pro-His（376.68u）,Leu-Ala-Gly-Pro-Lys-Phe-Ala-Leu（408.75u）和Met-Pro-Arg-His-Asp-Pro-Gln（440.70u）。      

Properties of TCA-insoluble peptides in kimoto (traditional seed mash for sake brewing) and conditions for liberation of the peptides from rice protein

It was found that a large amount of TCA (trichloroacetic acid)-insoluble peptides were liberated into the supernatant of kimoto on the 7th-10th day of mashing. These TCA-insoluble peptides had five polypeptide groups (12, 21, 31, 38, and 55 kDa) on SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and a large amount of high molecular weight peptides, higher than 10,000, were observed upon gel filtration chromatography using TSKgel G2000swxl (Tosoh Co.). Four of these peptides (12, 21, 31, and 38 kDa on SDS-PAGE) appeared specifically in kimoto, and were not detected at all either in sokujo-moto or in the main mash for sake brewing. These TCA-insoluble peptides were fractionated from the supernatant of kimoto on the 9th day, and it was revealed that free amino acids were produced abundantly from them in the presence of the enzyme of rice-koji. Therefore, it was assumed that the peptides are related to the abundant production of free amino acids in kimoto. For the liberation of these TCA-insoluble peptides from rice protein, the enzyme of rice-koji was indispensable. The enzyme liberating the TCA-insoluble peptides from rice protein was purified from rice-koji, and was presumed to be identical with acid protease (AP) of rice-koji. The presence of a high concentration of glucose (higher than 20%) was also indispensable for the liberation of the TCA-insoluble peptides. Furthermore, it was revealed that the peptides were liberated from rice protein under a limited pH of around 4.5.     

利用米糠蛋白制备类阿片拮抗肽和降血压肽的研究

采用离体豚鼠回肠(GPI)检定法测定不同米糠蛋白胰蛋白酶水解物的类阿片拮抗活性,结果发现跟新鲜米糠一样,低温脱脂米糠蛋白的酶解物具有较高的类阿片拮抗活性,高温脱脂米糠蛋白的酶解物无类阿片拮抗活性。采用低温脱脂米糠蛋白为原料,研究其制备类阿片拮抗肽后的残留蛋白酶解物的ACE抑制活性,结果发现在6种酶解物中,水解度(DH)为15.4%的碱性蛋白酶水解物的ACE抑制活性最高。     

Purification and characterisation of antioxidative peptides from unfractionated rice bran protein hydrolysates

Crude rice bran protein (CRBP) was prepared by alkaline extraction and then treated with 0.6 m HCl to remove phytic acid. The phytate‐free rice bran protein (PFRBP) was hydrolysed with proteases M, N, S, P and pepsin under optimal conditions. Hydrolysates obtained from various hydrolysis periods were subjected to analysis for their degree of hydrolysis (DH) and functional properties. The hydrolysates were fractionated by reversed‐phase column chromatography on Kaseigel ODS resin (120–140 μm) using a stepwise gradient of aqueous ethanol, and their activities were measured. The 40% ethanol fraction of protease P 4 h‐hydrolysate was separated by successive reversed‐phase high‐performance liquid chromatography and the amino acid sequences of isolated antioxidative peptides were determined by a protein sequencer and matrix‐assisted laser desorption ionisation‐time of flight mass spectrometry. Crude rice bran protein had higher antioxidative activity than PFRBP, due to the presence of phytic acid. Phytate contents of rice bran, CRBP and PFRBP were 2.5%, 1.42% and 0%, respectively. The activity of PFRBP increased upon protease digestion. Protease M hydrolysates showed the highest DH, but the lowest antioxidative activity. Hydrolysates with DH below 10% had higher antioxidative activity than those above 20%. This result indicates that the antioxidative activity of the hydrolysates is inherent to their characteristics amino acid sequences of peptides depending on the protease specificities.     

Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.     

预分离米糠蛋白类阿片拮抗肽的离子交换工艺

研究离子交换层析法对米糠蛋白酶解物超滤透过液中类阿片拮抗肽的初步分离提取。采用强酸性阳离子交换树脂,上样流速:SV0.8,上样pH值:5.0;洗脱液氨水浓度:1mol/mL,洗脱速度:SV1.2。离体豚鼠回肠(GPI)检定法鉴定表明,在此条件下制备的米糠蛋白类阿片拮抗肽活性得到提高。     

Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from Thai jasmine rice bran protein hydrolysates

Rice bran protein hydrolysate (<50 kDa RBPH) from Thai jasmine variety demonstrating a high Angiotensin I converting enzyme (ACE) inhibitory activity was purified and characterised. ACE inhibitory peptides were obtained from a two-step purification process: gel filtration and preparative reverse-phase high-performance chromatography (RP-HPLC) and then identified by mass spectrometer hybrid quadrupole-time-of-flight. A novel peptide GSGYF in the RBPH was firstly identified and found to have a partial sequence homology of Oryza sativa Japonica Group. This sequence was further synthesised to exhibit as good an inhibition potency with IC₅₀ value of 2.11 µg mL⁻¹ as Captopril (1.15 µg mL⁻¹). The cytotoxicity test revealed that this RBPH is non-toxic against Vero cells. In addition, the <50 kDa RBPH was resistant to in vitro digestion by pepsin and trypsin. These findings suggest that the RBPH containing ACE inhibitory peptides is likely to be safer and healthier than synthetic drugs and can be an effective food supplement for lowering blood pressure.     

Functional peptides derived from rice bran proteins

Rice bran has been predominantly used in the feed industry, and only recently it has attracted greater attention in terms of human nutrition with increasing knowledge of its bioactivity. A growing interest is the analysis of physiologically active peptides derived from rice bran proteins. In this paper, the bioactivities of rice bran proteins hydrolysates and peptides are reviewed based on recent studies. These enzymatic hydrolysates and peptides exert various biological activities including antioxidant, antidiabetic, anticancer and inhibitory activity for angiotensin converting enzyme (ACE), which may ultimately prevent certain chronic diseases. Nevertheless, these functionalities can be highly associated with their corresponding structural characteristics, in particular specific sequences and molecular weight distribution. This article may facilitate the expansion of the prospective applications of the bioactive peptides in a number of fields and provide some clues of the relationship between peptides structure and functionality for future research.     

预分离米糠可溶性蛋白类阿片拮抗肽的超滤工艺

采用超滤法从米糠可溶性蛋白酶解物中初步分离提取类阿片拮抗肽,研究了错流方式的板式膜组件时间、压力、温度和料液浓度对膜通量的影响。采用截留分子量为10000的超滤膜,料液浓度为10mg/mL,膜压差为0.14MPa,温度15℃的操作条件。离体豚鼠回肠(GPI)检定法鉴定表明,类阿片拮抗肽活性得到提高。     

大米肽中压离子交换色谱分离及其免疫活性评价

为进一步提高经过DA201-C型大孔吸附树脂脱盐、40%乙醇梯度洗脱的大米免疫活性肽RPHs-A3组分的纯度和免疫活性,采用中压离子交换色谱对其进行进一步分离纯化,并对分离得到组分的免疫活性进行了评价。结果表明,采用WorkBeads 40Q强阴离子交换树脂、2.6cm×40cm色谱柱对RPHs-A3组分进行分离的最佳条件为:上样浓度30mg/mL,上样量5 mL,起始缓冲液A为pH 9.0的0.01mol/L Tris-HCl,洗脱缓冲液B为含1 mol/L NaCl的pH 9.0、0.01 mol/L Tris-HCl,上样、洗脱流速分别为1,10mL/min。共收集到5个组分,其中RPHs-A3-B3组分在MTT试验和小鼠巨噬细胞脂多糖(LPS)炎症模型中皆表现出较高免疫活性,对RAW264.7细胞具有显著增殖作用,其SI值为1.315;且对炎症模型中细胞内NO释放量具有显著抑制作用(P0.05),抑制率为8.28%。采用中压离子交换色谱能有效地对RPHs-A3组分进行分离纯化,使SI值从1.273提高到1.315,且对细胞内NO释放量具有显著抑制作用。     

米糠蛋白肽制备过程中褐变控制及脱色工艺研究

对酶解法制备得到的米糠蛋白肽进行精制,先后抑制制备过程中褐变反应发生、对米糠蛋白肽进行澄清及脱色工艺研究。确定浓度0.15%柠檬酸、0.15%L-半胱氨酸及3.0%抗坏血酸的复配抑制剂为米糠蛋白酶解制备过程中的最佳褐变抑制剂,此时蛋白肽色素抑制率达80.80%、水解度7.42%、氮溶解指数92.46%。灭酶过程的最佳澄清条件为pH4.0、温度100℃、加热10min、3000r/min离心20min,肽液透光度达89.12%。粉末活性炭为最优的脱色吸附剂,其脱色适宜工艺条件:粉末活性炭用量3.0%、pH=4.0、脱色温度60℃、吸附时间50min,此时米糠蛋白肽脱色率达81.45%,肽损失率10.11%。      

Identification of cationic peptides derived from low protein rice by-products and evaluation of their multifunctional activities

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利用米渣为原料制备大米浓缩蛋白

通过对碱蛋白酶两步法、水溶液洗涤法、非淀粉酶法和淀粉酶法四种不同的方法对米渣蛋白提取的比较,发现淀粉酶法在制备大米浓缩蛋白时纯度最高,第二、三种方法的纯度略低于淀粉酶法,相比而言在以米粉为原料制备大米蛋白工艺中广泛采用的第一种方法没有显现出优越性。     

植物源降血压肽研究进展

降血压肽的实质是血管紧张素转化酶抑制肽,它通过抑制血管紧张素转化酶(ACE),阻碍有升高血压作用的血管紧张素Ⅱ的生成,同时抑制具有降血压作用的血管舒缓激肽的分解,从而使血压下降。对血压调节机制,降血压肽的结构和组成对其功能的影响,以及目前植物源降血压肽研究进展进行了综述。     

蛋源降血压肽研究进展

鸡蛋不仅是人们饮食营养的重要来源,而且还含有许多生物活性蛋白。鸡蛋活性蛋白经酶解后会产生许多具有生物活性的多肽,其中最重要的就是具有降血压活性的多肽。本文介绍了鸡蛋来源的降血压多肽的发现、特性、生产和分离及体内外活性,以期为国内相关研究提供参考。     

大米生物活性肽研究进展

对大米生物活性多肽的制备和开发研究进展及关键问题进行综述,以期为大米蛋白的综合利用提供理论支持。     

碎米蛋白ACE抑制肽制备工艺的研究

研究了以碎米为原料,酶法制备血管紧张素转换酶(ACE)抑制肽的工艺。采用不同的蛋白酶对碎米蛋白进行水解,以水解液对ACE抑制率为评价指标,结果得出风味蛋白酶为最佳水解酶;通过单因素和正交试验,确定最佳的酶解工艺条件:pH为7.0,酶解温度为55℃,料液比为3%,酶解时间3h,风味蛋白酶酶添加量4000U/g。在此条件下ACE抑制肽的抑制率为85.4%。     

米糟制备大米蛋白研究

以米糟为原料对四种制备大米蛋白不同方法,即碱蛋白酶两步法、水溶液洗涤法、淀粉酶除杂法和非淀粉酶除杂法进行比较;研究结果发现,后三种方法在制备大米蛋白时得率(>85%)和纯度(>80%)明显高于第一种方法得率(约69.2%)和纯度(约70%)。     

米糠蛋白ACE抑制肽在大鼠体内降压效果的研究

采用灌胃和静脉注射的方法,观察了米糠蛋白ACE抑制肽对应激性高血压(SHR)大鼠的降压效果,并与卡托普利的降压效果进行了比较。分别以50、100、150 mg/kg·bw剂量的米糠蛋白ACE抑制肽一次性给药后,每2 h测量大鼠血压,连续测量8 h;实验持续4周,每天灌胃一次,每周测量血压及体重一次。SHR大鼠给予米糠蛋白ACE抑制肽后,血压均显著下降(p<0.05),并均在灌胃后2 h达到最低值,血压下降最大幅度为(31±6.8)mm Hg;而正常大鼠灌胃米糠蛋白ACE抑制肽后血压无显著变化,灌胃卡托普利则会明显降低其血压;长期灌胃米糠蛋白ACE抑制肽可使SHR大鼠血压下降明显(p<0.05),且降压效果稳定。      

超声辅助酶解制备大米蛋白黄嘌呤氧化酶活性抑制肽的工艺条件优化

摘 要: 本文以大米蛋白为原料,研究了碱性蛋白酶酶解法制备黄嘌呤氧化酶（Xanthine Oxidase,XOD）活性抑制肽,并对其体外功能活性进行评价。研究了蛋白酶种类、底物浓度、酶解温度、加酶量、酶解pH、酶解时间对大米蛋白水解度（degree of hydrolysis,DH）及酶解产物对黄嘌呤氧化酶活性抑制率的影响,探讨了不同超声处理方式对酶解过程的影响,通过响应面法对实验条件进行了优化结果如下：采用碱性蛋白酶,底物浓度5 %,酶解温度为56 ℃,加酶量为8000 U/g,酶解pH为10,超声功率70 W,超声酶解60 min,对所制备的大米蛋白黄嘌呤氧化酶活性抑制肽的功能活性进行了评价。在最优条件下其黄嘌呤氧化酶抑制活性的IC50为1.55 mg/mL, ABTS自由基清除作用、羟基自由基清除作用、DPPH自由基清除作用的IC50值分别为0.49、12.48、3.88 mg/mL。