Lactic acid‐mediated isolation of alpha‐, beta‐ and kappa‐casein fractions by isoelectric precipitation coupled with cold extraction from defatted cow milk

The present study focused on the isolation of individual caseins (α, β and κ) from defatted cow's milk using lactic acid‐mediated isoelectric precipitation coupled with cold extraction technique, followed by purification using sucrose‐based density gradient centrifugation method. In vitro characterisation of the isolated casein fractions confirmed preservation of micellar morphology throughout the experimental conditions with anionic surface charge and wider particle size range. The RP‐HPLC analysis showed a peak at retention time of 25.13 min and 27.84 min for α‐casein and β‐casein, respectively. Results confirmed isolation of pure casein fractions from cow's milk which could serve as a rich source of bioactive peptides for nutraceutical/pharmaceutical applications.     

猴头菌菌丝多糖的分离纯化及其性质研究

对液体发酵生产的猴头菌丝,利用"水提醇沉"的方法进行多糖的初步提取;粗多糖除去蛋白质杂质之后,进行DEAE-纤维素和Sephadex G-100凝胶分步柱层析纯化,获得的多糖组分经过纯度检验之后,进行多糖性质的研究。结果显示:猴头菌丝多糖主要含有D-木糖和D-果糖,并且,多糖分子容易发生酯化反应而表现出抗病毒作用。     

采用盐析及Q Sepharose FF分离纯化酪蛋白胃蛋白酶水解物中的酪蛋白糖巨肽

本文探讨了利用了（NH4）2SO4分级沉淀、透析脱盐及Q—Sepharose FF层析分离纯化酩蛋白的胃蛋白酶水解物中的CGMP的工艺。结果表明：采用60％饱和度的（NH4）2SO4盐析，可从上清液中直接回收高纯度CGMP，得率为0．71％。低饱和度的（NH4）2SO4可有效脱除杂蛋白。脱盐粗提物CGMP Q—Sepharose FF层析条件为：pH8．5、20mmol／L Tris缓冲液平衡上样，用含0．3mol／L NaCl、20mmol、pH7．1的Tris缓冲液洗脱：浓缩物最大上样量为34．2mg蛋白／ml树脂。利用优化的工艺对水解液层析纯化，纯化产物得率约为2．19％（以酪蛋白汁），总结合态唾液酸回收率为87．4％（以酶解液中的唾液酸汁），糖基化度可提高到0．472以上。整个工艺路线简使、快捷、成本较低，适合工业化生产。     

Improved procedures for the isolation of pure oleic,linoleic, and linolenic acids or their methyl esters from natural sources

The methods normally employed for the isolation of pure methyl oleate, linoleate, and α-linolenate from appropriate natural sources are reviewed and simple procedures for isolating these esters are recommended. Evening Primrose Oil (Oenothera biennis) is a particularly useful source of linoleic acid.     

红景天中高纯度红景天苷制备工艺

研究从红景天粗提物中分离纯化高纯度红景天苷的制备方法。红景天提取物经75% 乙醇提取,乙酸铅沉淀和水饱和正丁醇萃取等三步预处理,可使红景天苷富集,红景天苷含量达43.80%,然后将预处理后得到红景天苷粗品分别用大孔吸附树脂D101 和AB-8 以及硅胶进行柱层析,采用TLC 法和RRLC法分别进行红景天苷的定性和定量分析;结果表明经AB-8 柱层析后,依次用水和5% 乙醇洗脱,得到的红景天苷含量大于90%。该方法工艺安全简单、成本低,非常适合工业化生产。     

梯度淋洗/离子色谱法测定婴儿乳粉中硝酸盐

婴儿乳粉中含有丰富的营养物质,存在干扰硝酸盐分析的未知成分。采用梯度淋洗方式将其与硝酸盐进行分离,达到了准确测定的目的。分析过程中采用乙酸溶液沉淀乳乳粉中蛋白,过滤后用RP柱去除样品中的脂类物质。净化后,直接将试样注入离子色谱仪分析,淋洗液由自动发生器产生,外标法定量。稳定性实验5次进样硝酸盐的峰面积平均值为0.2961μS.min(RSD为0.35%),保留时间平均值为26.721 min(RSD为0.23%)。组分添加回收率分别为99.04%和99.43%,相对偏差(RSD)分别为1.85%和1.02%。     

底物亲和法分离纯化蕲蛇胃蛋白酶

利用酶(胃蛋白酶)与其底物(酪蛋白)的亲和性,从蕲蛇胃中提取得到一种胃蛋白酶.让底物和酶在pH 2.5的乳酸缓冲液中充分结合,然后调pH至4.0(底物的等电点)沉淀底物和酶的结合物,随后让沉淀物再溶解于乳酸缓冲液中,添加低浓度的SDS将底物和酶分离,最后用DEAE离子交换色谱柱进行纯化,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,分离得到的胃蛋白酶为电泳纯,其分子量为33KD.酶的最适pH值小于3.0,最适温度为50℃.该结果证明胃蛋白酶能利用其与酪蛋白的亲合性及SDS沉淀作用有效地分离,这种胃蛋白酶能在食品生产的加热、强酸处理过程中应用.     

A large-scale isolation of native β-lactoglobulin: characterization of physicochemical properties and comparison with other methods

A novel method for β-lactoglobulin (β-lg) isolation from whey was investigated. The method comprised a peptic treatment of whey and membrane filtration under gentle conditions for isolation of native β-lg. The method was applied to 10,000 L batches of processed whey and the product quality obtained was compared with pilot-scale results of three other procedures: 3% trichloroacetic acid (TCA) precipitation, a salting-out procedure and a selective thermal precipitation. The calculated yields of native β-lg were 67.3, 44.9, 46.7 and 49.6% of the β-lg present in whey using the four methods, respectively. The composition of the different β-lg preparations was found to be comparable, however, analysis of purity revealed slight differences as indicated by electrophoresis and fast protein liquid chromatography (FPLC). The isolated β-lg retained a high degree of purity and native properties. The new method for isolation of native β-lg was found to be reproducible, selective and robust as processing of three 10,000 L batches did not indicate any technical problems or variations in the purity of the isolated β-lg.     

反相离子对色谱法测定奶粉中核苷酸质量浓度

为检测奶粉中胞嘧啶核苷酸、尿嘧啶核苷酸、腺嘌呤核苷酸、鸟嘌呤核苷酸、次黄嘌呤核苷酸5种核苷酸的质量浓度,建立起奶粉中5种核苷酸的反相离子对色谱法。样品经质量分数为10%乙酸去除蛋白,在流动相为1.4 mmol/L四丁基硫酸氢胺含0.01 mol/L磷酸二氢钾(pH=4.0)∶甲醇=96∶4的条件下用C18色谱柱分离,紫外检测波长254 nm,流速1 mL/min,标准曲线法定量。5种核苷酸的平均回收率和样品重复测定变异系数分别是95.7%,96.0%,90.8%,98.7%,99.3%和0.583%,0.147%,0.257%,0.387%,0.167%。该方法无需进行固相萃取,快速灵敏,简便准确,适用于奶粉中核苷酸质量浓度的检测。     

牛羊乳酪蛋白制备及电泳分析比较

采采用新鲜和复原的牛羊乳为原料,等电点沉淀,通过洗涤、干燥等步骤分别制得牛乳和羊乳酪蛋白,用凯氏定氮法对其进行定量检测,应用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS—PAGE）分析比较牛羊乳酪蛋白组分差异。结果表明：牛乳酪蛋白得率为86．75％,羊乳酪蛋白得率为90．55％,高含量的酪蛋白主要集中在电泳图谱的中分子量组,可分为ds,CN、as2-CN、β-CN和K—CN,牛羊乳as2-CN分子量羊乳大于牛乳,牛乳a-CN含量比较多,羊乳β—CN含量比较多,鲜乳与复原乳全蛋白主要组分在电泳图谱中除酪蛋白差别外,上端的高分子量组清蛋白区羊乳IgG重链的分子量比牛乳小。应用蛋白质电泳分析技术可以区分羊乳和牛乳的蛋白质组分,等电点沉淀法制备酪蛋白的方法简单,易于操作。     

Electrochemical sensor detecting free sulfhydryl groups: evaluation of milk heat treatment

We describe a new and rapid method for the evaluation of reactive sulfhydryl groups in whey proteins obtained after precipitation of casein by acetic acid at pH 4.6. The procedure is based on the use of a wire tungsten electrode operating at -0.2 V versus saturated calomel electrode in flow injection analysis. The method was applied to raw milks and to commercial pasteurized and UHT milks. Results showed that the tungsten electrode constituted a robust amperometric sensor that could be used to differentiate milks that underwent different heat treatments. The decrease of thiol content in the whey proteins from samples was in agreement with the whey protein content found by HPLC. The procedure is suitable for on-line quality control of heat-treated milks.     

蔷薇藻藻蓝蛋白的分离纯化及其体外抗氧化活性

蔷薇藻(Rhodella reticulata)是属于红藻门的一种海洋单细胞微藻,在生长过程中会产生藻胆蛋白、胞外多糖等生物活性物质。文中研究了蔷薇藻的藻蓝蛋白的分离纯化及抗氧化性能,藻细胞经过破碎,通过硫酸铵沉淀和DEAE Sepharose FF柱层析后,可以分离出较纯的藻蓝蛋白,纯化的藻蓝蛋白在620 nm处有一个最高的吸收峰,纯度为3.92,经SDS-PAGE电泳后,测得藻蓝蛋白含有α和β两个亚基,分子质量分别为18 ku和22 ku。蔷薇藻藻蓝蛋白由17种氨基酸组成。蔷薇藻纯化后的藻蓝蛋白比粗藻蓝蛋白具有更高的抗氧化能力,且呈剂量依赖效应。     

Identification and determination of phosphates in dairy products

This paper outlines the thin-layer chromatographic separation of condensed phosphates on polyamide-starch layers with subsequent digestion of the spots and photometric identification of the phosphates. With cheese and condensed milk the preparation of samples is carried out by isolation of the phosphate solution after protein precipitation by trichloroacetic acid. With pure phosphate solutions a recovery rate of 95.9% +/- 3.1% was established, whereas the recovery rate of added phosphate from processed cheese and condensed milk was 82.7% +/- 5.9%.     

高效液相色谱检测乳制品中的乳铁蛋白含量

目的：借助高效液相色谱法测定乳制品中乳铁蛋白。方法：先通过额外加入的二价铁离子使乳铁蛋白的构型更利于沉淀。然后用体积分数60% 乙醇溶液沉淀蛋白除去糖分,再用醋酸盐缓冲液提取乳铁蛋白并分离掉酪蛋白,最后用高效液相色谱检测,采用LiChrosorb RP-C18 色谱柱,在质量浓度0.1g/100mL 的三氟乙酸溶液协助下,甲醇与水线性梯度洗脱。结果：乳铁蛋白的线性范围是0.4～2mg/mL(R2=0.9980),平均回收率95.4%。结论：此方法可以用于乳制品中乳铁蛋白的测定。     

3种烟用香精的极性偶合柱系统-GC/MS分析

采用DB-5MS和DB-WAXETR毛细管气相色谱柱及由其组成的偶合柱系统对3种烟用香精进行了GC/MS分析。结果表明:①总柱长80m的偶合柱系统比60m的分离效果差;②总柱长60m的极性-非极性偶合柱系统对2种烟用香精的分离效果比单根极性或非极性柱好,但对另一种烟用香精的分离效果比非极性柱好,比极性柱差。总长适宜的极性偶合柱系统适合于某些烟用香精的分离分析;极性-非极性偶合柱系统仅适合于含极性成分较多,且高沸点组分不多、使用单柱中低沸点组分重叠严重的烟用香精的分离;偶合柱系统存在优化比较繁琐、费时,柱连接器的死体积会对组分分离产生不良影响,使用温度偏低,前柱分离的部分组分可能会重新混合等缺点。     

Isolation and characterisation of caseinmacropeptide from bovine,ovine, and caprine cheese whey

Based on the thermostability of caseinmacropeptide (CMP) and on the differences in molecular weight of its polymeric and monomeric forms, we have developed a method of isolating CMP from whey protein concentrate (WPC) and from liquid sweet cheese whey, particularly suited to large-scale industrial production. This procedure includes acidification and heating and ultrafiltration of cheese whey to give a CMP powder with a protein content from 75 to 79%. CMP obtained from WPC and from pure bovine, ovine, and caprine cheese whey were characterized. The CMP recovery was close to 71-76% and the purity determined by RP-HPLC ranged from 75 to 90%.     

蛹虫草胞外多糖的提取纯化及免疫活性研究

目的提取纯化蛹虫草发酵液中的胞外多糖,初步研究其结构及免疫活性。方法乙醇沉淀多糖,用Sevag法除去蛋白质、透析法除去小分子物质,经CM-52纤维素柱层析后得纯品。用紫外光谱测定多糖纯度,用凝胶柱层析法测定相对分子质量,高效毛细管电泳和红外光谱分析其分子结构,检测胞外多糖对小鼠巨噬细胞系RAW264.7产生NO的影响。结果蛹虫草胞外多糖经脱蛋白、CM-52纤维素柱层析后可获得单一对称峰的纯多糖,相对分子质量为3.49×10^4,含羟基、糖醛结构和羰基结构,单糖基之一为甘露糖。多糖可显著刺激巨噬细胞分泌NO,具有活化免疫细胞、增强机体免疫力的作用。结论该方法可获得高纯度蛹虫草胞外多糖,并证明它对巨噬细胞有活化作用。     

Directional isolation of ethanol-tolerant acetic acid bacteria from industrial fermented vinegar

The aim of this study was to isolate indigenous ethanol-tolerant acetic acid bacteria (AAB) from industrial fermented vinegar. As routine phenotypic methods for AAB isolation and identification appear to be very time-consuming and not so accurate, we adopted a two-step isolation strategy in the present study. In the preliminary screening step, GYEC agar plates with 3–10 % (v/v) ethanol as selective stress were utilized to recover potential AAB strains with ethanol tolerance from vinegar samples. In the rescreening process, acetic acid bacterial genus’ specific adhA gene was amplified as an effective DNA target for directional detection of real AAB so that non-AAB isolates could be eliminated rapidly. In this way, an AAB pure isolate named ET-7-3 with 7 % (v/v) ethanol tolerance was successfully isolated and further identified to Acetobacter pasteurianus according to 16S rDNA sequencing and phylogenetic analysis. Summarizing, by the use of ethanol as selective stress combined with molecular identification method, ethanol-tolerant AAB could be isolated from industrial fermented vinegar with both efficiency and accuracy.     

奶粉中三聚氰胺HPLC-IC分析方法的比对

采用反相离子对色谱法、亲水相互作用色谱法及阳离子交换色谱法测定奶粉中三聚氰胺的含量。奶粉样品前处理采用水- 乙酸提取后过RP柱和甲酸提取后乙腈沉淀两种方法,反相离子对色谱法采用C18 柱和10mmol/L庚烷磺酸钠与10mmol/L 柠檬酸的缓冲溶液- 乙腈(85:15,V/V)的流动相;亲水相互作用色谱法采用HILIC 柱和20mmol/L甲酸胺- 乙腈(10:90,V/V)的流动相;阳离子交换色谱法采用IonPac CS17 阳离子交换柱和6mmol/L 硫酸的流动相。流速1.0mL/min,检测波长240nm。结果表明,3 种方法测定三聚氰胺在0.1～25.0mg/L 范围内线性均很好(r=1.0000),样品加标回收率在93.91%～99.96% 范围内。3 种方法中,反相离子对色谱法对三聚氰胺的响应值最高,阳离子交换色谱法则能用最短的时间检测出三聚氰胺,而亲水相互作用色谱法在假阳性的判断上可作为其他两种方法的补充。