琥珀酸脱酰胺对小麦面筋蛋白酶解产物抗氧化性的影响

研究琥珀酸不同脱酰胺程度对小麦面筋蛋白胰酶的酶解产物抗氧化性的影响。在相同水解度下,以DPPH.清除率、.OH清除率、ORAC值为抗氧化指标,结果表明低脱酰胺程度(22.4%)的小麦面筋蛋白酶解产物的抗氧化性有较大幅度的提高,高脱酰胺程度(60.4%)的小麦面筋蛋白酶解产物的抗氧化性降低。水解度15%的酶解产物的肽分子量分布表明,低脱酰胺的小麦面筋蛋白酶解产物中分子量小于3000u的肽段增加,高脱酰胺的小麦面筋蛋白酶解产物中分子量小于3000u的肽段减少;在相同水解度下的氨基酸分析结果表明,脱酰胺后的小麦面筋蛋白酶解生成的肽的疏水性和抗氧化性的氨基酸含量提高。因此,低脱酰胺的小麦面筋蛋白酶解产物抗氧化性提高的主要原因是产物中含疏水性和抗氧化性氨基酸的小分子肽含量提高。     

不同蛋白酶对小麦蛋白酶解物抗氧化活性的影响

小麦蛋白是小麦淀粉加工的副产物,酶解是提高小麦蛋白溶解性和功能性的有效方式,而酶解用酶种类可能对酶解产物的功能性如抗氧化活性有一定影响。采用碱性蛋白酶、中性蛋白酶、胃蛋白酶、风味蛋白酶、胰蛋白酶、木瓜蛋白酶6种常用的蛋白酶分别对小麦蛋白进行酶解,并对酶解4 h后酶解物的多肽得率、分子质量分布、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率、超氧阴离子自由基(O_2~-·)清除率、羟自由基(·OH)清除率等反映水解程度和抗氧化能力的主要指标进行评价。结果表明,风味蛋白酶酶解物中多肽得率最高,达91.44%,且分子质量小于3 000 D的多肽含量达76.9%;酶解物质量浓度为3 mg/m L时,木瓜蛋白酶酶解物对DPPH自由基清除作用最好,清除率为65.12%(P0.01),其次是风味蛋白酶(58.43%)和碱性蛋白酶(55.29%);碱性蛋白酶酶解物对O_2~-·清除率效果最好,清除率为58.68%(P0.01),其次是风味蛋白酶(49.25%);碱性蛋白酶和木瓜蛋白酶酶解物对·OH清除效果最佳,清除率分别为59.23%和58.16%。结果说明,蛋白酶种类对小麦蛋白酶解物抗氧化活性影响显著,风味蛋白酶对提高蛋白水解程度和生成小分子质量多肽的作用明显,而碱性蛋白酶、木瓜蛋白酶和风味蛋白酶对提高酶解产物抗氧化活性效果较好。     

Investigation of the susceptibility of acid-deamidated wheat gluten to in vitro enzymatic hydrolysis using Raman spectra and free amino acid analysis

BACKGROUND: The number and surface nature of amino acids (AAs) in substrate proteins available to hydrolytic enzymes are critical. Among them, the micro‐environmental properties of specific AAs in substrates before hydrolysis would probably dominate the susceptibility of substrates to enzymatic hydrolysis. Fundamental knowledge concerning this regard is lacking. The objective of this work was to investigate the relationship between the exposure level of AAs in acid‐deamidated wheat gluten and their susceptibilities to in vitro enzymatic hydrolysis by pancreatin through both high‐performance liquid chromatography and Raman spectra. Wheat gluten deamidated with HCl (HDWG), citric acid (CDWG), succinic acid (SDWG) and acetic acid (ADWG) at the same degree of deamidation under the same heat treatment were chosen as the substrates. Substrate characterisations including degree of hydrolysis, surface hydrophobicity and structural characteristics before hydrolysis, together with analysis of free AAs of the corresponding hydrolysates during hydrolysis, were investigated. RESULTS: Hydrolysates from SDWG had the highest value for the degree of hydrolysis. The susceptibility of CDWG to pancreatin hydrolysis was the lowest, lower than native wheat gluten (CK) after the initial 36 h. Compared with free AAs, the mole increase profiles of CK, Arg production levelled off in HDWG after 12 h whereas it was inhibited in ADWG. For SDWG, Arg release was dramatically inhibited after 12 h and was replaced by Trp. Investigations using Raman spectra of the micro‐environment of Cys, Trp, Tyr and His and the mole increase trend of them indicated that the exposure level of these amino acids in substrates was positively related to their susceptibilities to pancreatin hydrolysis especially after 24 h of hydrolysis. CONCLUSION: Deamidation by four acids has a distinct influence on the structural characteristics of wheat gluten substrates. Although the substrates were selected at the same level of deamidation by the same heat treatment, their resultant conformational differences significantly influenced the exposure level of amino acids for binding to enzymes and the susceptibility of substrates to in vitro enzymatic hydrolysis. Therefore, it had an influence on changing enzyme cutting sites of pancreatin. This information will provide a better understanding of specific behaviour of AAs in wheat gluten during enzymatic hydrolysis from a new perspective. Copyright © 2012 Society of Chemical Industry     

Antioxidant activities of enzymatic‐hydrolysed proteins of dromedary (Camelus dromedarius) colostrum

This work investigated the antioxidant activities of dromedary colostrum proteins before and after hydrolysis by pepsin, trypsin, α‐chymotrypsin, pancreatin and papain. The enzymatic hydrolysis affected the degrees of hydrolysis, electrophoretic profiles, molecular weight distribution and hydrophobic/hydrophilic properties of the generated peptides. The antioxidant activities were evaluated using four antioxidant assays, including 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) radical‐scavenging activities, ferric reducing power and ferrous ion chelating activity. Interestingly, the antioxidant activities of dromedary colostrum proteins were enhanced after enzymatic hydrolysis. The highest antioxidant potential was obtained by pancreatic hydrolysates (P ≤ 0.05). These results suggest that dromedary colostrum protein hydrolysates are an important source of natural antioxidant peptides.     

Preparation and antioxidant activity of wheat gluten hydrolysates (WGHs) using ultrafiltration membranes

BACKGROUD: Many hydrolysates from animal and plant proteins have been found to possess physiological activities. Wheat gluten, an important by‐product of the wheat starch industry, is produced worldwide in enormous quantities. In this study, wheat gluten hydrolysates (WGHs) were obtained by enzymatic hydrolysis and fractionated using ultrafiltration membranes. The antioxidant activities of the hydrolysates were investigated by various antioxidant assays, including the ability to inhibit the autoxidation of linoleic acid and the scavenging effect on free radicals. Amino acid composition and molecular weight distribution were also evaluated to determine their relationship with antioxidant activity. RESULTS: The pepsin hydrolysate (PeWGH) had the highest activity and was ultrafiltrated into three major types, PeWGH I (5–10 kDa), PeWGH II (3–5 kDa) and PeWGH III (<3 kDa). PeWGH III showed stronger inhibition of the autoxidation of linoleic acid and higher scavenging activity against 2,2‐diphenyl‐1‐picrylhydrazyl, superoxide and hydroxyl free radicals. Furthermore, PeWGH III had the highest total hydrophobic amino acid content (45.11 g per 100 g protein), and its molecular weight distribution ranged from 1700 to 100 Da. CONCLUSION: The low molecular weight and amino acid composition of PeWGHs were found to be strongly correlated with their antioxidant activity. PeWGH could be used as a natural antioxidant in the pharmaceutical and food industries in the future. Copyright © 2008 Society of Chemical Industry     

谷朊粉酶解条件的优化及其抗氧化活性的研究

以溶解性为指标,采用单酶(Alcalase 2.4 L)和双酶(Alcalase 2.4 L+Flavourzyme 500 MG)2种方法水解谷朊粉,在单因素试验的基础上通过正交试验,优化了单酶和双酶水解的最佳条件,并分析了最优酶解条件下得到的2种产物的抗氧化活性、相对分子质量分布及其氨基酸组成。结果表明,单酶和双酶水解物的最大溶解性分别达到了61.38%和82.21%。水解物质量浓度为3.0 mg/m L时,单酶水解物的还原力、DPPH自由基、超氧阴离子、羟自由基的清除率分别达到0.81、71.82%、56.83%和70.82%,而双酶水解物相应的指标分别达到1.01、85.33%、74.84%和84.33%。结果表明,双酶水解不仅能提高谷朊粉的溶解性,而且也能明显提高其抗氧化活性。双酶水解物中小分子肽(分子质量<1 500 u)和疏水性氨基酸的质量分数分别达到61.63%和33.74%,均高于单酶水解物,表明谷朊粉酶解物的抗氧化活性与小分子肽含量、疏水性氨基酸呈现正相关。     

Evaluation of free radical‐scavenging activities of sweet potato protein and its hydrolysates as affected by single and combination of enzyme systems

To evaluate the free radical‐scavenging activities of sweet potato protein (SPP) and its hydrolysates, single enzymes alone (alcalase, neutrase, protamex) or in combination with flavourzyme were employed. Compared with SPP, free radical‐scavenging activities of the resulting hydrolysates were all significantly increased (P < 0.05). Alcalase (ALC) hydrolysates exhibited the highest superoxide, hydroxyl and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activities (P < 0.05), which was 18.71 ± 0.22, 27.13 ± 0.24 and 90.10 ± 0.15% respectively. Compared with SPP hydrolysates by single enzymes, the hydrolysates obtained by combination of enzyme systems exhibited higher degree of hydrolysis, but lower free radicals scavenging activities. In addition, the content of several antioxidant amino acid residues, such as His, Met, Tyr and Phe, in ALC hydrolysates was much higher compared with SPP and other hydrolysates using amino acids composition assay. The results suggested that peptides with free radical‐scavenging activity could be released from entire SPP chain via moderate enzymatic hydrolysis.     

小麦面筋蛋白盐酸脱酰胺工艺优化及其酶解敏感性

文以小麦面筋蛋白为原料,优化了盐酸对小麦面筋蛋白的脱酰胺工艺,比较了最佳脱酰胺工艺下的小麦面筋蛋白在胰酶、风味蛋白酶和碱性蛋白酶酶解过程中蛋白回收率和水解度的变化,并对其酶解36 h酶解液的自由基吸收能力(ORAC)抗氧化特性进行分析评价。研究结果表明,盐酸脱酰胺的工艺条件是:小麦面筋蛋白浓度为24%,0.30 mol/L的HCl,65℃,24 h脱酰胺;在该工艺条件下,胰酶酶解液蛋白回收率和水解度最高,酶解效果最好;高脱酰胺程度小麦面筋蛋白在胰酶酶解36h后酶解液的蛋白回收率和水解度高于低脱酰胺程度的酶解液;ORAC抗氧化特性分析表明高脱酰胺程度小麦面筋蛋白酶解液的ORAC值高于低脱酰胺程度的酶解液,其ORAC值最高为(689.67±10.22)μmol Trolox/g。     

Evaluation of Antioxidant Activities of Zein Protein Fractions

Zein protein was extracted from the by‐product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC‐ESI‐Q‐TOF MS/MS as Pro‐Phe (263.13 Da) and Leu‐Pro‐Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry.     

Improvement of the Functionality of Minced Mackerel by Hydrolysis and Subsequent Lactic Acid Bacterial Fermentation

ABSTRACT: To improve functionality and add nutritional value to mackerel, its muscle protein was hydrolyzed by the proteases from Aspergillus oryzae , and further fermented by lactic acid bacteria (LAB). Compared with minced mackerel, the antioxidative ability against linoleic acid (from 10% to 42%), α,α-diphenyl-β-picrylhydrazyl scavenging capability (from 38% to 85%), reducing power (from 0.58 to 0.83), Fe²⁺ chelating capability (from 42% to 72%), and trolox equivalent antioxidant capacity (from 2.5 to 3.5 m M ) of the hydrolysate significantly increased ( P < 0.05) after 1 h of hydrolysis and 12 h of LAB fermentation. Both hydrolysate and LAB fermented samples could stimulate the proliferation of both human hybridoma HB4C5 and mouse macrophage J774.1 cells. These data suggest that the enhancement of cell viability and antioxidative ability could be substantially improved after hydrolysis and 12 h of LAB fermentation.     

发酵法制备鱼鳔多肽及其抗氧化活性研究

将米曲霉接种于鱼鳔匀浆液中对其进行直接发酵,利用菌产蛋白酶的作用水解鱼鳔蛋白。随着发酵时间的延长,发酵液水解度不断上升,发酵液中的多肽含量不断增加。对发酵所得鱼鳔多肽进行超滤,采用产O2·和·OH 化学模型研究其清除自由基的活性,采用亚油酸自氧化体系研究其抗氧化活性。结果显示,经发酵40h,水解度(22 ± 0.8)% 所得鱼鳔多肽体外抗氧化能力较强;超滤片段中,平均肽链长度为12 个氨基酸残基的多肽超滤液体外抗氧化能力较强;分子量分布在1000D 左右。发酵法所得鱼鳔多肽不仅对自由基具有强烈的清除作用,还能明显抑制脂质的氧化。     

发酵肉制品中高抗氧化肉品发酵剂的筛选鉴定

为获得高抗氧化活性的天然肉品发酵剂,通过清除自由基、还原能力和抗脂质过氧化实验对来源于5种发酵肉制品中30株乳酸菌的发酵上清液、菌体细胞和胞内提取物的抗氧化活性进行研究,并按照肉品发酵剂的筛选标准对高抗氧化性的菌株进行筛选,鉴定出符合要求的菌株。结果表明:这30株乳酸菌具有不同的抗氧化能力且不同组分的抗氧化活性之间存在很大差异,发现菌株L23、L26和L28具有较高的抗氧化活性,其中L23符合肉品发酵剂的筛选标准,可作为天然高抗氧化性肉品发酵剂用于生产。经鉴定L23为希腊魏斯氏菌。     

小麦蛋白酶解物中抗氧化肽的纯化与鉴定

以自由基清除能力为指标,分别采用4种蛋白酶酶解小麦蛋白,其中3 h复合蛋白酶酶解物显示出最高抗氧化活性。用超滤、凝胶过滤色谱和RP-HPLC的方法纯化抗氧化活性肽,并对抗氧化活性高的多肽进行质谱分析。结果显示,分子质量Mr1 ku组分的抗氧化活性较高(5 mg/m L时达到76.08%),对该组分进行纯化后,经ESI-TOF-MS质谱分析可知其荷质比m/z为730.83,氨基酸序列为Gln-Gln-Gln-ProArg(QQQPR)。     

固态发酵对麸皮抗氧化特性及全麦面包感官品质的提升作用

用植物乳杆菌LB-1和酿酒酵母对小麦麸皮进行固态发酵,利用发酵麸皮重组全麦粉制备全麦面包,并对固态发酵麸皮的抗氧化特性及全麦面包的色泽、组织结构、风味、口感等感官品质进行分析.结果表明:经过乳酸菌LB-1与酵母菌协同发酵36?h后,麸皮的总游离氨基酸、可溶性蛋白、可溶性多酚和可溶性阿拉伯木聚糖分别增加了0.44、0.8...     

超滤法分离富集谷朊粉抗氧化活性肽

为了研究谷朊粉酶解产物中发挥抗氧化活性的肽片段,采用截留不同相对分子质量的超滤膜对谷朊粉酶解产物进行初步分离富集,并通过测定各级多肽的亚铁离子螯合能力、对亚油酸的自氧化抑制能力以及对ABTS自由基的清除能力来评价其体外抗氧化能力.结果表明:抗氧化能力由弱到强依次为:大于10 000＜10 000～5 000＜小于5 000.对于相对分子质量在5000以下的组分,当质量浓度为0.2、0.4 mg/mL时,亚铁离子螯合能力是超滤前样品的2倍左右;对亚油酸自氧化的抑制能力在36 h后比其它相对分子质量片段的组分效果明显;而当质量浓度为4 mg/mL时,TEAC值达到1.45 mmol/L Trolox;并且由高效液相色谱法对其相对分子质量进行分析发现,小分子肽占了绝大部分,说明抗氧化肽主要集中在相对分子质量2 000以下.     

Comparison of ACE inhibitory and DPPH radical scavenging activities of fish muscle hydrolysates

To utilise Atlantic salmon, Coho salmon, Alaska pollack, and southern blue whiting as components of neutraceutical food and to clarify the potential physiological function of those fishes, their muscles were hydrolysed with pepsin, pancreatin or thermolysin. Methanolic extracts of fish muscle and their hydrolysates were prepared for analysis of angiotensin converting enzyme (ACE) inhibitory and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. Pepsin hydrolysates of all the fish samples did not increase degree of hydrolysis, extractive nitrogen content and bioactivities. Pancreatin and thermolysin improved the ACE inhibitory activity, and the activity was expressed following the peak of size exclusion chromatography (SEC). The DPPH radical scavenging activity was increased following pancreatin and thermolysin hydrolysis, but this activity was not related to the SEC result. Except for the lower DPPH radical scavenging activity of Alaska pollack pancreatin hydrolysate than those of the others, there was no significant difference in the ACE inhibitory and DPPH radical scavenging activities by fish species.     

保加利亚乳杆菌发酵乳清的抗氧化性研究

通过控制初始发酵条件:pH6.0、发酵温度39℃,可以提高发酵乳清清除羟基自由基和DPPH.的能力。在发酵20h内,随着乳清蛋白水解度的增加,发酵乳清清除羟自由基和DPPH.的能力也随之增大,在发酵20h时达到最大,分别为48.06%和73.52%,发酵乳清清除羟自由基和DPPH.的能力与未发酵乳清相比分别提高了22.73%和46.09%,与保加利亚乳杆菌菌体相比分别提高了23.75%和40.63%。探讨了蛋白水解度和保加利亚乳杆菌活菌数和抗氧化活性之间的关系。20h后,发酵液自由基清除能力与水解度之间没有正相关性,因此不能采用蛋白水解度作为评价发酵乳清抗氧化性的指标。本研究对可能影响保加利亚乳杆菌发酵乳清抗氧化性的因素进行探讨,为开发功能性乳清产品奠定了理论和实践基础。     

酶水解鲫鱼蛋白质及产物抗氧化活性初探

本实验主要研究了碱性蛋白酶对鲫鱼的水解作用及其水解液的抗氧化活性,探讨了酶浓度、底物浓度、水解温度、时间、pH对鲫鱼蛋白质水解度的影响,同时用DPPH(二苯基苦基苯肼)自由基法和改进的邻苯三酚法测定分析水解液的抗氧化活性。结果表明:酶解条件为E为4%,S为10%,pH8.0,时间为4h,温度为50℃时可以获得较高水解度,同时水解液对DPPH自由基和超氧阴离子自由基(O2·)具有较高清除率。     

Effect of citric acid deamidation on in vitro digestibility and antioxidant properties of wheat gluten

The effects of citric acid deamidation on the physiochemical properties of wheat gluten were investigated. In vitro digestion was carried out to determine changes of molecular weight distribution, amino acids composition and antioxidant efficacy of wheat gluten hydrolysates. Results indicated that citric acid deamidation significantly increased gluten solubility and surface hydrophobicity, at a neutral pH. Deamidation induced molecular weight distribution change of gluten with little proteolysis. Results from FTIR indicated that the α-helix and β-turn of deamidated gluten increased accompanied by a decrease of the β-sheet structure. After deamidation, in vitro pepsin digestibility of wheat gluten decreased, while in vitro pancreatin digestibility increased. The oxygen radical absorbance capacity (ORAC) activity of the in vitro digests decreased with increase of deamidation time. The high Lys and total essential AAs amounts in the final digests suggested that the nutritional values of wheat gluten after deamidation might be enhanced.     

Proteolysis characteristics of sarcoplasmic, myofibrillar, and stromal proteins separated from grass carp and antioxidant properties of their hydrolysates

Proteolysis of grass carp sarcoplasmic, myofibrillar, and stromal proteins by 5 commercial proteases were studied. Sarcoplasmic and myofibrillar protein could be well hydrolyzed by Alcalase 2.4 L to reach high protein recoveries (PR) (71.86±2.46 and 80.77±3.05%, respectively), while the maximum PR for stromal protein was only 42.83±2.84%. However, stromal hydrolysates, containing mostly 6–10 kDa fraction, exhibited higher ·OH scavenging activities due to its high content of antioxidant-assisting amino acids. Alcalase 2.4 L and pancreatin 6.0, which produced hydrolysates with relative high degree of hydrolysis (DH), were used for further hydrolysis of whole grass carp protein with the assistance of response surface methodology (RSM). The results showed that serine proteases (Alcalase 2.4 L and pancreatin 6.0) could produce sarcoplasmic, myofibrillar, or stromal hydrolysates with relatively high PR, DH, and strong ·OH scavenging activity, which may be used to prepare antioxidant hydrolysates from grass carp.