酱油二次沉淀蛋白质的分离、鉴定及氨基酸分析

以酱油上清液中可溶性蛋白质为对照,以静置-真空冷冻干燥法制备酱油二次沉淀,采用N-三(羟甲基)甘氨酸-十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)、基质辅助激光解析电离飞行时间串联质谱(MALDI-TOF/TOF MS)和高效液相色谱(HPLC)法对比研究了酱油二次沉淀蛋白质的分子量分布、氨基酸组成特征和蛋白质的疏水性,并对酱油二次沉淀蛋白质进行了分子鉴定。结果表明:酱油二次沉淀蛋白质由分子量约为22.7ku(80.20%)和34.1ku(19.80%)的蛋白质组成,质谱鉴定结果证实前者为大豆球蛋白(Glycinin)G4亚基碱性端B3,后者为大豆球蛋白(Glycinin)G1酸性端A1a。氨基酸组成分析表明酱油二次沉淀蛋白质与酱油上清蛋白质氨基酸组成存在一定差异,酱油二次沉淀蛋白质的疏水性明显高于酱油上清蛋白质。本研究进一步深化了对酱油二次沉淀蛋白质的认识,为酱油二次沉淀问题的解决提供了新的依据。     

食醋二次沉淀及其蛋白质组成特征

通过离心和真空冷冻干燥法制备了食醋二次沉淀样品。以其上清液为对照,通过SDS-PAGE、高效液相色谱等方法,对比研究了食醋二次沉淀的常规理化指标和其蛋白质组成特征。实验结果表明,食醋二次沉淀主要由蛋白质和少量NaCl、游离氨基酸组成,二次沉淀蛋白质主要以高分子量蛋白质或蛋白质聚合物(>116kDa)的形式存在,其氨基酸组成与其对应上清液中的蛋白质氨基酸组成存在明显差异,其蛋白质平均疏水值(HΦavg)明显高于对应上清液蛋白质的平均疏水值。以上结果说明,食醋二次沉淀蛋白质的氨基酸组成和蛋白质平均疏水值偏高是导致食醋二次沉淀形成的重要原因。     

大豆高压短时蒸煮显著提高酱油二次沉淀蛋白降解率

酱油二次沉淀严重影响其外观质量和商品价值。本研究以大豆低压长时(0.24 MPa/18 min)蒸煮工艺为对照,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和基质辅助激光解吸附电离串联飞行时间质谱(MALDI-TOF/TOF MS)等方法研究了大豆高压短时(0.54MPa/14min)蒸煮工艺对酱油二次沉淀蛋白和二次沉淀生成量的影响。实验结果表明大豆球蛋白G4中的B3亚基(20 ku)和G1中的A1a亚基(35 ku)是酱油二次沉淀蛋白的主要成分,采用大豆高压短时蒸煮工艺制备的酱油(样品)比其对照酱油中B3亚基减少65.38%,样品中A1a亚基被完全降解;样品中二次沉淀含量比其对照减少72.69%。同时,采用大豆高压短时蒸煮工艺可提高酱油原料蛋白质利用率9.37%、氨基酸态氮14.14%、总氮10.74%、总糖22.36%、还原糖27.87%和无盐固形物12.38%(p0.05)。因此,采用大豆高压短时蒸煮工艺可显著提高酱油二次沉淀蛋白降解率、减少酱油二次沉淀生成量和提高酱油滋味物质含量,同时改善酱油外观质量和滋味。     

纸质食品包装材料中松香酸含量测定抽提方案的优化

目的对纸质食品包装材料中松香酸抽提方案进行优化。方法分别采用超声萃取法、微波萃取法、索氏萃取法、Na OH/尿素/硫脲混合碱溶液溶解-超声提取法对纸质食品包装材料中的松香酸进行提取,并对提取产物进行了高效液相色谱法分析,比较4种提取方法的提取效果,并对经4种提取方法提取后的样品进行了电子显微镜分析,观察提取后的样品中松香的残留情况。结果经混合碱溶液-超声提取法处理的样品,松香酸含量测定值明显高于其他三种方法。经超声萃取法、微波萃取法和索氏萃取法处理的样品中均残留有松香,而经混合碱溶液-超声提取法处理的样品中未观察到松香残留,与高效液相色谱分析的结果一致。结论 Na OH/尿素/硫脲混合碱溶液溶解-超声提取法可以完全提取样品中的松香酸。     

大豆球蛋白G4蛋白B3亚基导致中式高盐稀态酱油二次沉淀的形成

该文以日式酱油为对照,系统分析了pH、Fe3+/Fe2+、多酚、大豆多糖、大豆蛋白酶解物、NaCl、乙醇和温度对中式高盐稀态酱油二次沉淀生成量的影响,并对比分析了它们的游离氨基酸组成和敏感蛋白。结果显示除乙醇（<1.8%）外,上述其他因素均对中式高盐稀态酱油二次沉淀生成量具有显著影响（p<0.05）,仅pH（6.5）、大豆蛋白酶解物、NaCl和温度（60 ℃）对日式酱油二次沉淀生成量有显著影响（p<0.05）;中式高盐稀态酱油15种游离氨基酸含量及其平均疏水值（HΦavg）显著低于日式酱油相应值;中式高盐稀态酱油中大豆球蛋白中的B3亚基（敏感蛋白）含量显著高于日式酱油（未检出）。综合上述结果推测大豆球蛋白G4蛋白B3亚基是中式高盐稀态酱油二次沉淀形成的关键物质,中式高盐稀态酱油二次沉淀可能是通过B3亚基-Fe3+/Fe2+-多酚复合物和B3亚基-大豆多糖-Na+-Cl-复合物途径形成。上述研究结果将为解决中式高盐稀态酱油二次沉淀问题提供理论参考。     

酱油沉淀成分研究

本文研究了国产酱油原沉淀、一次沉淀及二次沉淀的成分组成和氨基酸组成情况，提出高蛋白质和多糖是造成酱油沉淀的主要原因，高盐份不会促进沉淀产生。文章中对糖类和蛋白质的来源作出了分析。     

纸质食品包装材料中松香酸含量测定 抽提方案的优化

分别采用超声萃取法、微波萃取法、索氏萃取法、NaOH/尿素/硫脲混合碱溶液溶解-超声提取法对纸质食品包装材料中的松香酸进行提取,比较了4种提取方法的提取效果,并对提取产物进行了高效液相色谱法分析,结果表明,经混合碱溶液-超声提取法处理的样品,松香酸含量测定值明显高于其它三种方法。对经4种提取方法提取后的样品进行了电子显微镜分析,结果表明,经超声萃取法、微波萃取法和索氏萃取法处理的样品中均残留有松香酸,而经混合碱溶液-超声提取法处理的样品中未观察到松香酸残留,与高效液相色谱分析的结果一致。     

饮料中提取酪蛋白的方法选择

目的探索4种沉淀蛋白质的方法在提取饮料酪蛋白过程中对其复溶率的影响,为建立超高效液相色谱-三重四极杆串联质谱法鉴别饮料中酪蛋白亚型及相应含量的测定寻找最佳前处理方法。方法选取乳粉、以乳粉或牛乳为原料的饮料、添加酪蛋白或酪蛋白酸钠的饮料作为测试样品,用pH 8.5的Tris-HCl溶液溶解,采用等电点沉淀法、乙腈沉淀法、丙酮沉淀法和乙醇沉淀法提取样品中的蛋白质,低温离心后对沉淀的蛋白质进行复溶,用考马斯亮蓝法分别测定沉淀前和复溶后样品溶液中可溶性蛋白质的含量,计算类酪蛋白的复溶率。结果乳粉和3类饮料中的酪蛋白经等电点沉淀法提取后,乳粉的类酪蛋白复溶率在64.95%~66.20%之间,3种饮料中的类酪蛋白复溶率在69.73%~84.95%之间,等电点沉淀法优于其他3种沉淀方法。αs-酪蛋白标准物质和β-酪蛋白标准物质的复溶率分别为70.16%和87.17%,且经高分辨质谱确证其复溶后的主成分为酪蛋白。另外对45种含酪蛋白饮料中的类酪蛋白含量进行了测定,其含量在0.10~42.36 mg/g(mg/mL)之间。结论等电点沉淀法提取类酪蛋白的复溶率较其他3种沉淀方法相对稳定,为饮料中酪蛋白的亚型鉴别及相应含量测定提供了回收率较高的前处理方法,为今后测定酪蛋白含量的液相质谱法的研究奠定了较好基础。     

基于统计学方法的酱油二次沉淀形成的初步研究

本文以市售25种酱油为研究对象,以pearson相关分析法研究了酱油pH、总糖、还原糖、NaCl、总氮、氨基酸态氮、无盐固形物含量与酱油二次沉淀形成的相关性 . 同时,以T检验法研究了酱油不同发酵方式、类型、主要原料、质量等级和产地对酱油二次沉淀形成的影响.研究结果表明:酱油二次沉淀的形成与酱油中总糖、总氮和无盐固形物的含量呈显著正相关,与酱油PH、还原糖、NaCl、氨基酸态氮含量无统计学意义上的相关性.此外,分析结果显示酱油的发酵方式、主要原料、质量等级和产地对酱油二次沉淀的形成存在较大影响,而酱油类型对酱油二次沉淀的形成不存在明显影响.     

国产酱油二次沉淀研究进展和展望

国产酱油普遍存在二次沉淀问题,严重影响产品外观和市场竞争力。该文为开发国产酱油二次沉淀去除技术提供依据和参考。参考国外研究结果,系统对比分析了国产酱油二次沉淀的组成、形成机制和去除技术。国产酱油二次沉淀主要由蛋白质(G4蛋白中的碱性B3多肽和G1蛋白中的酸性A1a多肽)、多糖和Na Cl组成,蛋白质的强疏水性是导致其形成二次沉淀的关键因素之一。要彻底解决国产酱油二次沉淀问题,必须对其二次沉淀形成机制进行深入研究。另外,在酱油灌装之前添加促进絮凝共沉淀因子或借助发酵结合超声技术去除"二次沉淀物质"是解决其二次沉淀现象的可行方法。     

发酵枸杞酒沉淀物成分鉴定及稳定性提升研究

发酵枸杞酒营养丰富,风味独特,市场前景广阔,但在货架期内易出现沉淀,影响消费者的偏爱。本研究以发酵枸杞酒及其沉淀物作为研究对象,采用物理化学方法对枸杞酒沉淀物进行定性、定量研究,探究了添加不同澄清剂（皂土、明胶、蛋清粉和蛋白酶等）对枸杞酒化学组成及稳定性的影响。结果表明,发酵枸杞酒沉淀物主要包括蛋白质、有机酸、多糖、多酚和金属元素等,其中,蛋白质占24%～34%,有机酸占22%～28%,多糖占15%～23%。添加不同种类的澄清剂对枸杞酒沉淀组分的影响不同,皂土类能够降低蛋白含量,其中皂土NC处理后的枸杞酒与对照组相比蛋白含量降低了28.13%。而明胶类能降低有机酸和多糖含量,其中明胶W处理后的枸杞酒多糖含量下降为初始对照的53.84%。添加明胶类澄清剂效果较好,可以减少枸杞酒的沉淀量,提高枸杞酒的稳定性。     

Measurement of protein in tannin-protein precipitates using ninhydrin

A method was tested for quantifying protein in precipitated tannin-protein complexes in which protein was hydrolysed with acid and the amino acids released were measured with ninhydrin. Unlike previously published methods, this technique requires no prior separation of tannin and protein and can be used to compare the binding of different soluble proteins to tannins under identical conditions. The method was used to compare the precipitation of bovine serum albumin, porcine pancreatic protease, β-glucosidase and γ-globulin by tannic acid. The amount of tannic acid required to precipitate maximal amounts of protease and β-glucosidase was approximately 7–8 times that required to cause maximal precipitation of albumin and γ-globulin when tested with 2 mg ml^?1 protein. All protein preparations contained a fraction (10–40% of total protein) which was not precipitated by tannic acid. Protein in tannin-protein precipitates produced in a standard haemanalysis assay were also measured with ninhydrin. Per cent precipitation at each tannic acid concentration as measured with ninhydrin was identical to that determined by haemanalysis within the linear portions of the binding curves produced by the two methods. These results confirm that the ninhydrin method accurately measures precipitation of protein by tannin.     

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。     

Characterization and formation mechanism of proteins in the secondary precipitate of soy sauce

To reveal the characteristics and formation mechanism of proteins in the secondary precipitate of soy sauce (SPSS), proteins in the supernatant of soy sauce were used as control, and SPSS was prepared by centrifugation in combination with lyophilization. The proteins in SPSS were isolated and identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. Acidic polypeptide A_1a of soy glycinin G1 (32–35 kDa) and basic polypeptide B₃ of soy glycinin G4 (23 kDa) were verified as the predominant proteins (95.2 %) in SPSS. The amino acid composition, average hydrophobicity (HΦ _avg), and the secondary structure of the proteins in SPSS were investigated by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Results revealed that SPSS contained not only proteins but also free amino acids; the significantly higher average hydrophobicity and much lower contents of β-sheet and random coil were the main characteristics and reasons for the formation of proteins in SPSS.     

Reaction of Tetrazolium Salts with Sulfated Polysaccharides and Other Hydrocolloids

SUMMARY— Triphenyltetrazolium chloride, (TTC), selectively precipitates carrageenan and other sulfated hydrocolloids in the presence of neutral, carboxylic and phosphorylated polysaccharides. Agar is not precipitated. Tetrazolium blue (TB) precipitates both the sulfated and carboxylic polysaccharides but does not precipitate phosphorylated and neutral polysaccharides.
Except for "weak" reactions with mucin, gum karaya, sodium alginate, and polysaccharides B-1459 (NRRL); tetrazolium red, neotetrazolium chloride and tetrazolium violet precipitate only sulfated polysaccharides. Although in aqueous dispersion the proteins were not precipitated by any of the tetrazolium salts, when these salts are added to milk (containing no added carrageenan), copious precipitation occurred.
Carrageenan was isolated from milk, milk products and other complex milieu by precipitation with 2, 3, 5, -triphenyl-tetrazolium chloride. The proteins were precipitated simultaneously. The mixed carrageenan-protein precipitate was then analyzed for carrageenan by hydrolyzing it with hydrochloric acid and assaying for sulfate by the barium chloranilate method.
Mixtures of sulfated, carboxylic and neutral polysaccharides were resolved by precipitating the sulfated polysaccharide with 2, 3, Atriphenyltetrazolium chloride. After centrifugation and/or filtration to remove the precipitate, the filtrate was treated with tetrazolium blue to precipitate the carboxylic polysaccharide. The neutral polysaccharide remained in the supernatant. Solubility studies have shown that the carrageenan-TT precipitate is highly insoluble in sodium chloride, sodium sulfate, magnesium chloride and other salt solutions.
The method developed allows the use of tetrazolium salts to differentiate between and to separate the main groups (sulfated, carboxylic, and neutral) polysaccharides. After separation, the individual polysaccharides may be determined quantitatively by suitable analytical methods.     

Comparison of physicochemical properties of beef potentiators prepared by synergistic fermentation and traditional method

Beef potentiator was prepared by beef qu, which was previously prepared by a novel synergistic fermentation method (BPSF). Three kinds of other beef potentiators were prepared by commercial enzymes (protamex, flavourzyme and papain, BPCEs), which were used as controls to investigate the physicochemical properties of BPSF. Results showed that BPSF possessed a higher protein recovery (70.99%) and degree of hydrolysis (DH, 51.39%) than BPCEs. The tests of molecular weight distribution and free amino acids (FAAs) indicated that the moderate molecular weight peptides (1–5 kDa) were dominant (70.07–74.43%) in all beef potentiators. The low molecular weight peptides (<1 kDa), umami amino acids and total amino acids in BPSF, were 1.03‐ to 5.56‐, 5.25‐ to 26.50‐ and 1.57‐ to 4.79‐fold higher than those in BPCEs, respectively. Meanwhile, hierarchical cluster analysis indicated that BPSF was significantly different to BPCEs, and sensory evaluation showed that BPSF had apparently stronger umami taste than BPCEs.     

Amino acid profile of raw and as-eaten products of spinach (Spinacia oleracea L.)

The aim of the work was to evaluate the amino acid composition of fresh spinach and spinach products prepared for consumption. The investigation included fresh and cooked spinach and two kinds of frozen product: one obtained using the traditional method (blanching-freezing-refrigerated storage) and then cooked; and the other, of the ready-to-eat type, obtained using the modified method (cooking-freezing-refrigerated storage) and then prepared for consumption in a microwave oven. In 100 g of edible parts of the as-eaten products, the content of amino acids exceeded that in the raw material from which they were obtained, except for sulphur amino acids and tyrosine. In the as-eaten products, the content of amino acids in protein calculated in 16 g N was similar to that in the raw material, except for lower tyrosine and higher arginine content in the traditional frozen product prepared for consumption. Cystine with methionine was the amino acid limiting the quality of protein in the investigated samples, except for the traditional frozen product prepared for consumption, where the Chemical Score (CS) index was 100.     

蚕蛹蛋白及其水解产物中氨基酸组成分析

采用FMOC-OPA柱前衍生化-高效液相色谱法测定蚕蛹蛋白及水解液氨基酸含量,同时测定血管紧张素转化酶(ACE)抑制活性,研究蚕蛹蛋白水解后氨基酸组成的改变及对ACE抑制活性的影响。结果表明:水解液10kD以下组分(SN2)中疏水性氨基酸含量为49.15%,显著高于10kD以上组分(SN1)中的40.55%及蚕蛹蛋白中的45.21%,其中对ACE抑制活性影响较大的脯氨酸、芳香族氨基酸含量为7.75%和19.20%,高于SN1中的5.62%和13.59%及蚕蛹蛋白中的7.42%和15.81%,并且SN2中ACE抑制率为47.6%显著高于SN1和蚕蛹蛋白中的抑制率,表明水解后疏水性氨基酸组成的改变与ACE抑制活性的变化趋势相同,且ACE抑制活性主要集中于SN2。     

An investigation into the possible occurrence of peptide-bound lanthionine in locust muscle

The amino acid composition of the protein of the wing muscle of the African migratory locust (Locusta migratoria migratorioides) was examined by qualitative and quantitative methods. the possible presence of lanthionine was investigated. Lanthionine could not be detected by two-dimensional paper chromatography nor by ion-exchange chromatography operated under three different conditions of temperature and pH. the analysis of hydrolysates prepared after a preliminary oxidation revealed no changes in composition that would arise had lanthionine been present together with other acids.