Effects of amino acids and peptides on rumen microbial growth yields

Experiments were conducted using mixed rumen bacterial cultures to determine which amino acids limited growth. Complete amino acid mixtures stimulated microbial growth alone and when added to casein. Amino acid subgroups did not stimulate growth alone or when added to casein or casein hydrolysates. Results were similar whether growth was limited by periodic addition of low amounts of carbohydrate or when higher amounts were added to batch cultures. Little growth occurred with ammonia as sole N source. Addition of 100 mg/L of amino acids and peptides quadrupled growth, peptides at 10 mg/L resulted in higher growth than the corresponding amount of free amino acids. Apparent saturation of growth occurred when 10 mg/L of a complete amino acid mixture or trypticase was added to cultures. The Michaelis constant values for amino acids and trypticase were determined to be .5 and 1.0 mg/L, respectively. Growth was a linear function of amount of carbohydrate fermented with the coefficient of slope increasing with increasing amino acid concentrations. These experiments demonstrate that growth stimulation from amino acids and proteins is due to the number of amino acids provided in a given mixture rather than specific growth limiting amino acids. Rumen bacterial growth is greatly stimulated by amino acids and peptides, with low affinity constant values, allowing good growth in the concentrations of amino acids and peptides found in vivo     

GROWTH OF NORMAL AND ARGININE-REQUIRING STRAINS OF SACCHAROMYCES CEREVISIAE ON SELECTED NITROGEN COMPOUNDS

Two arginine-requiring mutant yeast strains grew on arginosuccinic acid or argininic acid in presence of ammonium sulphate but failed to do so on ornithine, citrulline or glycocyamine. The metabolic lesion in these mutants appears to lie therefore in the enzyme system involved in completing the guanidino group of arginine. Arginine could not be replaced by various peptides containing it. Histidine depressed growth on arginine of both the deficient yeast and its parent but growth was somewhat stimulated by certain other amino acids. To some extent the need for all amino acids was met by the use of hexamethylenediamine.     

Chemometric analysis of proteolysis during ripening of Ragusano cheese

Chemometric modeling of peptide and free amino acid data was used to study proteolysis in Protected Denomination of Origin Ragusano cheese. Twelve cheeses ripened 3 to 7 mo were selected from local farmers and were analyzed in 4 layers: rind, external, middle, and internal. Proteolysis was significantly affected by cheese layer and age. Significant increases in nitrogen soluble in pH 4.6 acetate buffer and 12% trichloroacetic acid were found from rind to core and throughout ripening. Patterns of proteolysis by urea-PAGE showed that rind-to-core and age-related gradients of moisture and salt contents influenced coagulant and plasmin activities, as reflected in varying rates of hydrolysis of the caseins. Analysis of significant intercorrelations among chemical parameters revealed that moisture, more than salt content, had the largest single influence on rates of proteolysis. Lower levels of 70% ethanol-insoluble peptides coupled to higher levels of 70% ethanol-soluble peptides were found by reversed phase-HPLC in the innermost cheese layers and as the cheeses aged. Non-significant increases of individual free amino acids were found with cheese age and layer. Total free amino acids ranged from 14.3 mg/g (6.2% of total protein) at 3 mo to 22.0 mg/g (8.4% of total protein) after 7 mo. Glutamic acid had the largest concentration in all samples at each time and, jointly with lysine and leucine, accounted for 48% of total free amino acids. Principal components analysis and hierarchical cluster analysis of the data from reversed phase-HPLC chromatograms and free amino acids analysis showed that the peptide profiles were more useful in differentiating Ragusano cheese by age and farm origin than the amino acid data. Combining free amino acid and peptide data resulted in the best partial least squares regression model (R(2) = 0.976; Q(2) = 0.952) predicting cheese age, even though the peptide data alone led to a similarly precise prediction (R(2) = 0.961; Q(2) = 0.923). The most important predictors of age were soluble and insoluble peptides with medium hydrophobicity. The combined peptide data set also resulted in a 100% correct classification by partial least squares discriminant analysis of cheeses according to age and farm origin. Hydrophobic peptides were again discriminatory for distinguishing among sample classes in both cases.     

发酵酸化技术对乳饼蛋白质降解及蛋白肽生物活性的影响

通过发酵酸化技术研制发酵型乳饼,以传统的直接酸化技术为对照,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）、液相色谱-质谱测定乳饼蛋白质降解情况和游离氨基酸含量,通过用液相色谱-串联质谱鉴定乳饼蛋白肽。并通过测定体外抗氧化活性、α-葡萄糖苷酶抑制率和血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制率表征乳饼蛋白肽的生物活性。SDS-PAGE结果表明,两种酸化技术均能促进乳饼蛋白质的降解,发酵酸化技术对乳清蛋白的降解程度更高,能促进游离氨基酸的释放。从发酵酸化乳饼中鉴定出潜在的ACE抑制肽23?条、抗氧化肽6?条和降糖肽5?条,而传统的直接酸化乳饼中分别为13、4?条和1?条。发酵酸化技术能显著提升乳饼蛋白肽的α-葡萄糖苷酶抑制活性和ACE抑制活性（P＜0.05）,其中分子质量＜3?kDa超滤组分对二者抑制率的IC50分别为1.250?mg/mL和0.416?mg/mL。发酵酸化技术能促进乳饼蛋白质降解,释放生物活性多肽,同时产生游离氨基酸,提升乳饼的生物学价值。     

清酱肉多肽的抗氧化活性和氨基酸分析

以清酱肉为研究对象,提取并通过超滤分离得到清酱肉多肽（分子质量＜10 kDa）,测定不同质量浓度多肽液对1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、羟自由基（·OH）的清除效果、Fe2＋螯合能力和还原能力,分析清酱肉多肽的氨基酸组成。结果表明：当清酱肉多肽液的质量浓度为5 mg/mL时,其对·OH和DPPH自由基的清除率分别可达51.49%和57.50%,与相同质量浓度的谷胱甘肽（glutathione,GSH）相比差异显著（P＜0.05）;清酱肉多肽的Fe2＋螯合能力随其质量浓度的增加而显著提高,当质量浓度为5 mg/mL时,其对Fe2＋的螯合率可达50.97%;清酱肉多肽还具有一定的还原性。此外,清酱肉多肽液的氨基酸组成较为丰富,其中必需氨基酸含量为29.14%;与抗氧化活性相关的碱性、酸性及疏水性氨基酸的总量达到87.75%。综上所述,清酱肉多肽液具有一定的抗氧化能力及营养价值。     

陶瓷膜微滤耦合大孔树脂处理对罗非鱼肉酶解多肽理化性质的影响

本文研究无机陶瓷膜微滤和大孔树脂吸附联合处理对罗非鱼肉酶解多肽组分的影响,采用50 nm陶瓷膜处理及AB-8大孔树脂动态吸附罗非鱼肉酶解多肽,应用国标分析基本成分、高效体积排阻色谱(HPSEC)分析分子量分布、氨基酸分析仪测定氨基酸含量、反相液相分析氨基酸疏水性。结果表明,陶瓷膜微滤处理对酶解多肽的氨基酸组成影响不大,且陶瓷膜微滤处理后酶解多肽中1~2、2~3、3~5、5~8 ku组分的比例分别减少了7.29%、17.13%、33.93%和50.0%,说明陶瓷膜微滤对多肽有截留作用,且分子量越大截留比例越大;AB-8大孔树脂吸附使氨基酸总量降低1.99%,必需氨基酸含量降低5.15%,苦味氨基酸与疏水性氨基酸含量分别降低12.35%和8.44%,对2~8 ku多肽的吸附较敏感,对游离氨基酸吸附较少;反相液相分析结果显示,AB-8大孔树脂吸附的洗脱组分含有更多的疏水性肽段。综上所述,AB-8大孔树脂动态吸附可以有效改善罗非鱼肉酶解多肽的苦味。     

不同分子质量大豆蛋白和鸡肉酶解物的美拉德反应产物风味特征分析

为了研究不同分子质量的肽在美拉德反应后的风味特征,以大豆蛋白和鸡肉为原料酶解得到蛋白酶解物。对酶解物超滤分离得到小于1000Da、1000~5000Da和大于5000Da的不同分子质量肽组分,并对大豆蛋白和鸡肉酶解物完全酸水解得到复合氨基酸。在得到的肽段和氨基酸中添加一定量的木糖,构建美拉德反应模型。对美拉德反应产物感官特性进行评价,再采用气相色谱-质谱联用、高效液相色谱对挥发性和不挥发性化合物进行分析。结果表明:在2种不同蛋白来源的酶解物的美拉德反应产物中,共鉴定出挥发性化合物69种,其中吡嗪和呋喃硫醇等肉香味化合物的含量最高。统计分析表明:不同分子质量的肽和游离氨基酸的挥发性化合物存在明显差异。小于1000Da的肽在美拉德反应过程促进了吡嗪类化合物的生成,其中包括了吡嗪、5-甲基-2-乙基吡嗪、2,5-二甲基-3-乙基吡嗪、三甲基吡嗪、2-乙酰基吡咯等挥发性化合物。与肽相比,游离氨基酸有利于含硫化合物如呋喃硫醇类和噻吩类物质的生成。有29种挥发性化合物的相对气味活性值大于1。偏最小二乘回归研究了气味化合物与感官评价之间的相关性,发现小于1000Da的肽与基本的肉味和浓厚味密切相关,而大于5000Da的肽与苦味和焦煳味密切相关。在美拉德反应后,分子质量大于5000Da的肽数量显著减少；分子质量1000~5000Da的肽降解可以补充消耗的游离氨基酸；分子质量小于1000Da的肽容易发生交联和聚合现象而含量增加,其产物对风味的影响明显增强,能更好地体现基本的肉味、鲜味和浓厚味。希望研究可以为利用蛋白质酶解物生产美拉德反应产物的应用提供理论支撑和指导。     

云南产6种蜂蜜中35种游离氨基酸的测定

目的采用全自动氨基酸分析仪测定蜂蜜中35种游离氨基酸的含量。方法蜂蜜样品经水溶解,采用全自动氨基酸分析仪进行定量检测。检测器为荧光检测器,脯氨酸在波长440 nm处测定,其余34种氨基酸在570 nm波长下测定。结果蜂蜜中含有丰富的游离氨基酸成分,且氨基酸组成及含量存在较大区别。脯氨酸是蜂蜜中的主要氨基酸,其平均含量为0.042978 g/100g,占氨基酸组分总含量的71.21%。35种游离氨基酸的线性相关系数均达到0.99以上,回收率在92.4%~109.4%范围内。结论该方法快速、准确,可用于蜂蜜中游离氨基酸含量的测定。     

Elucidation of a major IgE epitope of Pacific mackerel parvalbumin

Ten overlapping peptides (20 amino acids in length with an offset of 10 amino acids) spanning the amino acid sequence of Pacific mackerel, Scomber japonicus parvalbumin (Sco j 1), were synthesized and evaluated for IgE-binding ability by fluorescence ELISA using fish-allergic patient sera. As a result, the region 21–40 was judged to contain a major IgE epitope of Sco j 1. However, this region was not necessarily assumed to be a major IgE epitope for the parvalbumins from seven other species of fish (sardine, Japanese eel, cod, horse mackerel, crimson sea bream, skipjack and flounder). Ala-scanning experiments revealed that eight amino acid residues (Ser-23, His-26, Lys-27, Lys-28, Lys-31, Cys-33, Leu-35 and Lys-38) are important for the IgE-binding of Sco j 1. Some of these residues are replaced by different residues in the parvalbumins of the other seven species of fish.     

肉杂鸡鸡骨架酶解工艺优化及其分析评价

以肉杂鸡鸡骨架为原料,测定其基本成分和氨基酸组成,对蛋白质进行营养评价,发现鸡骨架是进行酶解的良好原料.从碱性蛋白酶、风味蛋白酶、胰酶和复合蛋白酶中筛选出鸡骨架酶解的最佳用酶,利用单因素实验和响应面法优化了鸡骨架酶解的工艺条件.结果表明:在温度50℃、pH7.0、酶添加量7000 U/g的条件下酶解4 h,酶解效率最高...     

蜂蜜中17种水解氨基酸的测定

目的采用酸水解法和全自动氨基酸分析仪同时测定蜂蜜中17种氨基酸的含量。方法 8种不同蜜源的蜂蜜经酸水解后,采用全自动氨基酸分析仪进行定量检测;检测器为荧光检测器,脯氨酸在波长440 nm处测定,其余16种氨基酸在570 nm波长下测定。结果 17种氨基酸的线性回归系数均达到0.99以上;方法检出限为5~10 nmoL/L,样品平均回收率为92.1%~99.3%,相对标准偏差RSD小于8.5%。经检测实际样品发现,蜂蜜中不含胱氨酸,氨基酸总量在32.214~78.439 mg/100 g范围。结论该方法快速、准确,可适用于蜂蜜中水解氨基酸含量的测定。     

锌螯合肽的两端排序法定量构效关系

为了研究锌螯合肽结构与生物活性之间的关系,构建定量构效关系（quantitative structure-activity relationship,QSAR）模型。采取两端排序法将56 条不同长度的合成肽规格化,采用18 种氨基酸描述符,利用偏最小二乘法进行分析。发现5 种氨基酸描述符对应的QSAR模型的相关系数达到建模要求,分别为描述符FASGAI、Z、HESH、C和ST,其中描述符FASGAI最优（R2＝0.827 3、Q2＝0.602 2、估计均方根误差＝0.168 6、Q2ext＝0.717 2、预测方根误差＝0.255 8）。对描述符FASGAI所构建的模型进一步分析发现,多肽序列中的氨基酸位置对多肽锌螯合活性的影响力依次为C3＞N3＞C1＞N1＞N2＞C2,同时,多肽各位置上氨基酸残基的立体属性会影响其螯合活性。该模型的成功建立为锌螯合肽定量构效关系的研究提供了探索性思路。     

不同处理方法对草菇呈味物质释放的影响

以草菇子实体为研究对象,以可溶性糖及糖醇、有机酸、5’-核苷酸、游离氨基酸组成和含量、分子质量分布为指标,研究常压蒸煮、高压蒸煮和酶解3?种处理方法对草菇呈味物质释放的影响,结果表明：常压蒸煮制备液可溶性糖及糖醇的总量显著降低,高压蒸煮制备液可溶性糖及糖醇的总量和原液基本相同,而酶解作用显著地提高了样品中可溶性糖的含量;和原液相比,常压蒸煮和高压蒸煮提取后有机酸总量显著增加,而复合酶解制备液有机酸总量显著降低;3?种处理方法作用后的游离氨基酸总量都有所增加,以高压蒸煮增加最显著,同时高压蒸煮制备液的鲜味氨基酸、甜味氨基酸以及苦味氨基酸的含量均显著增加;常压蒸煮和高压蒸煮制备后,大于3?000?Da组分的含量有所减少,高压蒸煮制备液中小于3?000?Da的组分含量最多,是制备呈味肽的重要来源。因此,不同处理方法对草菇呈味物质的释放有着明显差异,可以根据所需要的呈味物质采用不同的处理方法。     

西式发酵火腿粗肽的制备及抗氧化活性和氨基酸组成分析

采用磷酸盐缓冲液和盐酸溶液提取西式发酵火腿的粗肽,测定并比较2种溶液提取的粗肽1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、羟自由基清除能力、Fe2+螯合能力及总抗氧化能力,同时对氨基酸组成进行分析.结果表明:磷酸盐法提取得到的粗肽粉A的质量和肽含量显著高...     

基于模拟胃肠道消化的云南民族乳制品蛋白肽研究

乳扇、乳饼和奶渣是云南民族传统乳制品,具有历史悠久、特色鲜明、蛋白质含量高等特点.本研究基于模拟胃肠道消化,采用液相色谱-串联质谱、氨基酸分析仪研究乳扇、乳饼和奶渣的肽谱及游离氨基酸含量,并借助蛋白肽数据库分析潜在的生物活性肽.结果表明,乳扇蛋白肽221条,分子质量集中在500～750Da,潜在的功能活性肽包括血管紧张...     

利用牡蛎制备ACE抑制肽的工艺优化

运用正交试验和响应面法优化牡蛎血管紧张素转换酶(angiotensin-Ⅰconverting enzyme,ACE)抑制肽的分步酶解制备工艺。采用复合蛋白酶和碱性蛋白酶对牡蛎进行分步酶解,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析和ACE抑制率为考核指标优化工艺参数。结果表明,第1步复合蛋白酶最佳工艺参数为:料液比1∶4、加酶量1.2%、pH 7.0、反应温度50℃、酶解时间1 h;第2步碱性蛋白酶最佳工艺参数为:加酶量0.42%、pH 8.3、反应温度53℃、酶解时间73 min。凝胶过滤色谱法测得牡蛎酶解液分子质量在3 000 D以下小肽所占比例为99.72%,其ACE抑制活性IC50为0.8 mg/mL。同时,氨基酸组成分析表明,牡蛎肽中Glu含量最高,Cys含量最低;必需氨基酸和疏水性氨基酸含量分别占总氨基酸的36.6%和37.2%。本研究制备的低分子质量、高ACE抑制活性牡蛎肽,可为牡蛎资源的开发利用提供理论参考。     

Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

酶膜反应器制备的蛋清水解物的营养评价

对酶膜反应器(EMR)制备的蛋清水解物进行营养评价。用配备截留分子质量10kD 超滤膜的EMR 制备并经大孔吸附树脂层析得到精制的蛋清水解物。以天然蛋清为对照物,分析水解物的游离氨基酸组成、相对分子质量分布及其氨基酸组成,并根据氨基酸分析结果进行氨基酸评分。结果发现：水解物的游离氨基酸质量分数为0.386%;水解物中2～4 个氨基酸残基组成的肽的质量分数约占71%;EMR 蛋清水解物所含的必需氨基酸占氨基酸总量的44.8%,氨基酸评分值为113.3,比天然蛋清的氨基酸评分值提高105.6%。该水解物易被人体吸收、营养均衡而价值较高,可广泛应用于营养保健食品中。     

纳豆菌液态发酵荞麦产物中抗氧化活性物质的分离鉴定

本实验通过超高效液相色谱-串联质谱（ultra performance liquid chromatography-tandem mass spectrum,UPLC-MS/MS）技术鉴定纳豆菌液态发酵荞麦的发酵产物多酚提取物中多酚组成与结构,且通过超滤、乙醇分级沉淀、大孔树脂柱层析定向分离抗氧化肽,并采用UPLC-MS/MS鉴定多肽组成与结构。从发酵产物中共分离鉴定出8种多酚类化合物：咖啡酸、原儿茶酸、丁香酸、绿原酸、儿茶素、芦丁、牡荆素、金丝桃苷。采用超滤、乙醇分级沉淀及XAD-16大孔树脂对发酵产物进行分离纯化,获得富含抗氧化肽的体积分数40%乙醇溶液洗脱组分（蛋白质量分数为66.51%、氧化自由基吸收能力为1 731.52 μmol/g）。从发酵产物中共分离鉴定出27 条多肽,搜索Mascot数据库得到23 条均为七肽以上多肽,在BIOPEP数据库中搜索均未经报道,且所有肽段都包含疏水性氨基酸,其中有11 条肽段含有至少一个强抗氧化氨基酸。GDKIYNVF中的IY片段、CRNLLLYPAGA中的LY片段、PWFFTST中的PW片段以及QGSGGPPIV中的GPP片段等在BIOPEP数据库中均被报道具有抗氧化活性。所鉴定的多酚、多肽是发酵产物发挥抗氧化活性重要的物质基础。     

Identification of Small Peptides Generated in Spanish Dry-cured Ham

ABSTRACT: A water‐soluble extract of dry‐cured ham was fractionated by gel filtration chromatography. Collected fractions (below 1200 Da) were sensory‐analyzed and subjected to amino acid analysis with and without previous acid hydrolysis, in order to determine the amino acid and peptide distribution. Those fractions with the highest peptide amount were further separated by reverse‐phase and cation‐exchange high‐performance liquid chromatography in order to isolate the peptides and sequence them. The results demonstrated the presence of small peptides, mainly dipeptides. The combination of these peptides and free amino acids may contribute to the characteristic taste in the savory fractions of dry‐cured ham.