改良纸层析法测定发酵液中异亮氨酸的含量

传统的氨基酸纸层析法是以特制的滤纸为支持物,用特殊配方的溶剂混合液作展层剂,然后喷刷显色剂茚三酮,烘烤显色,展层与显色分开操作,给实验带来不便.本文对把显色剂直接加到展层剂中进行了研究,用该方法定量测定了发酵液中的异亮氨酸含量.实验表明:改良后的纸层析法简化了操作,节约试剂,测定L-异亮氨酸含量具有较高的准确度和精密度,适合在L-异亮氨酸生产中应用.     

发酵液中L-精氨酸的检测方法

目的建立发酵液中L-精氨酸的定量检测方法。方法用坂口试剂(α-萘酚和2,3-丁二酮)定量测定L-精氨酸的含量。结果最佳测定波长为525nm,显色反应温度为30℃,反应时间为15min,NaOH浓度为15g/L。该方法具有较高稳定性和重复性。结论本方法可对发酵液中L-精氨酸进行定量测定。     

L一鸟氨酸纸层析定量测定法的研究

本文建立了一种纸层析定量测定L-鸟氨酸的新方法,用苯酚氨水水=511为展开剂能将L-鸟氨酸从发酵液中分开;确定了采用茚三酮为显色剂,90℃～100℃显色5min,硫酸铜乙醇洗脱液洗脱10min后比色等测定条件,并发现滤纸上L-鸟氨酸含量为5～40μg比较合适;绘制了L-鸟氨酸标准曲线,并推出发酵液中L-鸟氨酸含量的计算公式为X=38.46×A×n/v.     

薄层色谱-薄层扫描法测定发酵液中 L-亮氨酸的研究

用层析硅胶G板进行薄层色谱分离发酵液中L-亮氨酸与L-缬氨酸,经茚三酮显色得到完整的L-亮氨酸色斑;用CS-9301薄层扫描仪测定L-亮氨酸色斑峰面积;根据公式C=(Y-97.286)×n/(2559.5×V)计算发酵液L-亮氨酸浓度.结果表明该方法平均回收率为97.3%,重现性试验的变异系数为0.045%,说明本方法的准确性和重现性良好,能满足大批量测定发酵液中L-亮氨酸含量的要求.     

发酵液中L-谷氨酰胺的定性定量测定

应用纸层析-分光光度计法对发酵液中L-谷氨酰胺进行了定性分析和定量测定,确立了L-谷氨酰胺定量测定条件.研究结果表明,该法对测定L-谷氨酰胺含量具有较高的准确度和精密度,而且该法操作简单,易于掌握,非常适合在L-谷氨酰胺产生菌筛选中应用.     

分光光度法测定发酵液中L-精氨酸含量

建立了定量测定发酵液中L-精氨酸含量的方法。以百里酚的次溴酸钠溶液为显色剂,用正丁醇萃取显色产物,以分光光度计比色测定有机相的吸光度值。测定发酵液中L-精氨酸的最佳条件为:取待测物稀释液5.0mL,依次加入0.03%的百里酚溶液2.0mL,0.7%的次溴酸钠溶液0.5mL,摇匀,30s内加入正丁醇5.0mL,除去水相;向有机相中加入0.4mL无水乙醇,室温放置1min,用分光光度计测定OD480。方法的检出限为2.5μg/mL,摩尔吸光系数ε为1.2×104L/(mol.cm),发酵液样品相对标准偏差为0.89%～1.10%,回收率为97.3%～102.0%。此方法简便、快速、准确可靠,适合用于发酵液中L-精氨酸含量的定量检测。     

氧化法测定发酵液中L-异亮氨酸含量

L-异亮氨酸通过亚硝酸和高锰酸钾氧化，生成丁酮，再用香草醛显色，测得吸光度。通过与标准曲线相比较，即可求得发酵液中L-异亮氨酸的产量。     

木糖和葡萄糖共发酵生产L-组氨酸

以1株L-组氨酸生产菌Escherichia coli HIS1为研究对象,优化其L-组氨酸合成关键酶His G*的诱导条件。通过摇瓶发酵的方式确定诱导剂木糖的最适质量浓度为10 g/L;通过5 L发酵罐发酵的方式确定最佳诱导时间为发酵8 h时诱导;因发酵过程中木糖会被消耗,故通过多次添加木糖或敲除xyl A基因阻断木糖代谢途径保持发酵液中的木糖浓度,以稳定诱导条件。结果表明,木糖同时作为诱导剂和碳源更有利于L-组氨酸的发酵生产,为此摸索出了木糖和葡萄糖共发酵生产L-组氨酸的发酵工艺。该工艺主要控制要点为发酵8 h之后添加质量浓度为10 g/L的木糖,发酵过程中流加木糖和葡萄糖质量比为1∶5的糖溶液。最终L-组氨酸的产量可达56.5 g/L,是纯葡萄糖发酵时的2倍。     

L-鸟氨酸快速定量检测方法

在参照Chinard的L-鸟氨酸测定方法的基础上,确定了一种有效、高灵敏度的L-鸟氨酸快速定量检测方法。用6 mol/L H3PO4-冰乙酸(1/3,V/V)配制浓度为25 mg/mL茚三酮混合酸溶液作为显色液,在100℃水浴锅中60 min反应后,波长510 nm下比色测定L-鸟氨酸浓度。该反应体系稳定,受其他氨基酸的影响程度很小,或者在发酵液等的实际测定体系中存在量极少而可以忽略不计,线性范围为0~0.20μmol/mL,检测精度为0.01μmol/mL。     

ε-聚赖氨酸产生菌的筛选

改进了筛选ε-聚赖氨酸产生菌的方法。在加有复合抑菌剂的初筛平板上涂布土壤悬液,于28℃培养7 d后喷洒次甲基兰溶液显色,挑出周围形成透明圈的菌落,再次接种培养,7d后挖取菌落周围的琼脂块,利用文中设计的简易转移装置,将琼脂块中的水溶性成分转移到滤纸上,然后分别用茚三酮试剂和Dragendorff试剂检测,挑取对2种试剂都呈阳性的菌落,进一步摇瓶复筛,发现1株放线菌的发酵液中有ε-聚赖氨酸。经生化反应鉴定和分子生物学鉴定,确定该菌株为灰橙链霉菌(Streptomyces griseoaurantiacus)。     

A modified ninhydrin reagent using ascorbic acid instead of potassium cyanide

The use of a modified ninhydrin reagent using ascorbic acid instead of potassium cyanide was investigated for the photometric determination of amino acids. It has been found possible to obtain an almost stoichiometric reaction for glutamic acid with a quantitative yield of "Ruhemann's purple", the end-product of the reaction, using ascorbic acid as a safe and economical reducing agent. The modified ninhydrin reagent was composed of 0.5 g of ninhydrin, and 15 mg of ascorbic acid in 60 ml of methyl cellosolve.     

Amino acid analysis: Reduction of ninhydrin by sodium borohydride

Ninhydrin based monitoring systems remain the widely employed for the quantitative determination of amino acids subsequent to their separation by ion-exchange chromatography. In the reaction of ninhydrin with amino acids the presence of hydrindantin (reduced ninhydrin) is advantageous. The latter can be added to the reaction mixture directly or it can be formed by the addition of a reducing agent. In the present work, the stability of a ninhydrin reagent in which ninhydrin has been reduced with sodium borohydride is compared with that of commercial preparation using stannous chloride or titanium chloride as a reducing agent. Both agents are commonly used in automated amino acid analysis. The advantages of the sodium borohydride-containing preparation are as follows: (a) ninhydrin solution does not form precipitates in the flow lines of the analyser, (b) no waiting period for the maturation of the reagent is required, (c) very stable ninhydrin solution (at room temperature for a month, at 4–5°C even longer), (d) no necessity to keep the solution in refrigerator, (e) amino acid colour yield is about 10% higher for 17 amino acids, (f) this modification based on methylcellosolve as a solvent buffered with 4 M sodium acetate (pH 5.5) overcome the increase of the base line at high pH (pH above 8) connected with ninhydrin reagent using dimethylsulfoxide-lithium acetate buffer.     

Analysis of Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell Ninhydrin Assay

In this study we present a ninhydrin based microwell assay that can be utilized in place of the traditional Kjeldahl method for the determination of the protein content of beer or wine. In addition, the assay is ideal for the determination of free amino acids in beer (FAN), a term understood and used by brewers, and yeast assimilable nitrogen (YAN) used by enologists. The assay only measures alpha amino acids and ammonia so other nitrogen sources are not detected, resulting in a 30% reduction in total protein of a variety of beers compared to the Kjeldahl method, which measures nitrogen from all sources. The results also showed that only 25% of the total “protein” in beer is actually derived from peptides larger than 3,500 Kd. Analysis of beer or wine with the microwell assay for total usable nitrogen was compared to the standard FAN and YAN methods and conditions were determined for maximal efficiency and precision. Superior results were obtained with low reaction volumes and a stable sodium acetate buffered ninhydrin reagent at pH 5.5. As an alternative, for use with cuvettes, a reduced volume FAN assay using the same pH 5.5 sodium acetate buffered ninhydrin reagent gave comparable results. The assay is economical, rapid, accurate and applicable to large numbers of samples.     

Methods for methionine and cyst(e)ine determination. A comparison between two institutes

Methods for determination of sulphur-containing amino acids in protein concentrates were compared between two institutes. Methionine determined by gas-liquid chromatography and colorimetrically by iodoplatinate gave comparable results. For cyst-(e)ine three modifications of liquid chromatography determination after performic acid oxidation, one determination with a cysteine specific ninhydrin reagent and one determination based on transforming cyst(e)ine-S to cadmium sulphide and atomic absorption determination of cadmium, were compared. It is concluded that the best which can be hoped for in a cyst(e)ine method is to obtain results which may be used for comparisons within a laboratory.     

脑白金口服液功效成分的鉴别及低聚异麦芽糖含量的测定

目的研究脑白金口服液中功效成分的鉴别方法与低聚异麦芽糖含量的测定。方法脑白金样品经沉淀、离心、固相萃取(solid-phase extraction,SPE)小柱分离、富集和纯化处理。使用化学反应法、薄层色谱法及高效液相色谱法分别鉴别和测定脑白金口服液中低聚异麦芽糖、山楂、茯苓等原料的相关功效成分。同时采用高效液相色谱双柱法测定了低聚异麦芽糖含量。结果乙醇沉淀法、蒽酮法和苯酚硫酸法有效地鉴别了低聚异麦芽糖;高锰酸钾褪色法、薄层色谱法及高效液相色谱法鉴别了山楂中的水溶性枸橼酸;福林酚试剂法、茚三酮显色法分别鉴别了茯苓中水溶性蛋白质及氨基酸。结论上述鉴别和含量测定的方法专属性强、重现性好、灵敏度高,可管控产品质量。     

基于气相色谱-离子阱法的纺织品及皮革中的富马酸二甲酯检测

为了解决样品基体复杂,干扰测定的难题,建立了一种基于气相色谱-离子阱二级质谱(GC-MS/MS)检侧纺织品及皮革制品中富马酸二甲酯的方法。样品中富马酸二甲酯经乙腈提取,PSA/SAX小柱净化,GC-MS/MS进行定量和定性分析。该法采用化学源电离,甲醇作为反应气,并对前处理过程所用提取溶剂、净化过程,CID电压进行了优化,大大提高灵敏度和选择性。该方法在0.05-10.0μg/mL浓度范围内呈线性关系,相关系数为0.9998,仪器定量检出限LOQ为0.05mg/kg ,外标法定量。在50,500,1000μg/kg 3个水平的相对标准偏差为3.52%～6.12%,回收率为84.0%～99.5%。结果表明,相对于GC、GC-MS方法,GC-MS/MS分析时间短、无杂质峰干扰、灵敏度高,可以满足纺织品及皮革制品中检测大批量筛选要求。     

催化荧光法测定蔬菜中痕量亚硝酸根的研究

该研究建立了催化荧光分析法测定蔬菜中痕量NO2-的方法。NO2-可以催化中性红的荧光褪色反应,且其含量与褪色反应速率成正比。通过单因素试验以及均匀试验优化了实验条件,确定了最佳的试剂因素组合。实验结果表明:在激发波长λex=537 nm和发射波长λem=595 nm,试剂因素组合中性红900μL,溴酸钾1 850μL,硫酸1 050μL时,方法NO2-测定的工作曲线为y=1.58x+0.46,相关系数r=0.999 5,线性范围为0.02~0.11 mmol/L,检出限0.001 2 mmol/L,变异系数为6.5%,平均回收率为100.2%,此法适合于根茎类蔬菜中微量NO2-含量的测定。     

Ninhydrin-Reactive Lysine in Food Proteins

A lithium acetate-dimethyl sulfoxide ninhydrin reagent was shown to be useful for reaction of amino groups in a series of structurally diverse food proteins and flours of variable lysine content. The proteins were bovine serum albumin, casein, hemoglobin, lactalbumin, lysozyme, soybean protein, soybean trypsin inhibitor, trypsin, and wheat gluten. The flours were barley, high lysine corn, high protein rice, lima bean, nonfat dry milk, rice, soy, and wheat. The ninhydrin-reactive lysine content of these proteins varied from 63-109% of the theoretical values. This variation needs to be taken into account in possible applications of the ninhydrin reaction to food proteins.     

茚三酮比色法测定谷氨酸含量的研究

依据α-氨基酸与茚三酮显色反应的原理，研究了比色法定量检测谷氨酸的方法，着重探讨了此显色反应的影响因素、反应条件及注意事项。作为定量检测谷氨酸的方法，它与常规的谷氨酸检测方法相比，具有简便实用的特点。适用于一般实验室的样品分析及谷氨酸生产过程中的产品检测。