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.     

Implications of nitrogen nutrition for grapes, fermentation and wine

This review discusses the impacts of nitrogen addition in the vineyard and winery, and establishes the effects that nitrogen has on grape berry and wine composition and the sensory attributes of wine. Nitrogen is the most abundant soil‐derived macronutrient in a grapevine, and plays a major role in many of the biological functions and processes of both grapevine and fermentative microorganisms. Manipulation of grapevine nitrogen nutrition has the potential to influence quality components in the grape and, ultimately, the wine. In addition, fermentation kinetics and formation of flavour‐active metabolites are also affected by the nitrogen status of the must, which can be further manipulated by addition of nitrogen in the winery. The only consistent effect of nitrogen application in the vineyard on grape berry quality components is an increase in the concentration of the major nitrogenous compounds, such as total nitrogen, total amino acids, arginine, proline and ammonium, and consequently yeast‐assimilable nitrogen (YAN). Both the form and amount of YAN have significant implications for wine quality. Low must YAN leads to low yeast populations and poor fermentation vigour, increased risk of sluggish/stuck/slow fermentations, increased production of undesirable thiols (e.g. hydrogen sulfide) and higher alcohols, and low production of esters and long chain volatile fatty acids. High must YAN leads to increased biomass and higher maximum heat output due to greater fermentation vigour, and increased formation of ethyl acetate, acetic acid and volatile acidity. Increased concentrations of haze‐causing proteins, urea and ethyl carbamate and biogenic amines are also associated with high YAN musts. The risk of microbial instability, potential taint from Botrytis‐infected fruit and possibly atypical ageing character is also increased. Intermediate must YAN favours the best balance between desirable and undesirable chemical and sensory wine attributes. ‘Macro tuning’, of berry nitrogen status can be achieved in the vineyard, given genetic constraints, but the final ‘micro tuning’ can be more readily achieved in the winery by the use of nitrogen supplements, such as diammonium phosphate (DAP) and the choice of fermentation conditions. This point highlights the need to monitor nitrogen not only in the vineyard but also in the must immediately before fermentation, so that appropriate additions can be made when required. Overall, optimisation of vineyard and fermentation nitrogen can contribute to quality factors in wine and hence affect its value. However, a better understanding of the effect of nitrogen on grape secondary metabolites and different types of nitrogen sources on yeast flavour metabolism and wine sensory properties is still required.     

THE INFLUENCE OF FREE ALPHA-AMINO NITROGEN IN SORGHUM BEER FERMENTATIONS

The interactions of wort free α-amino nitrogen (FAN) and sugar in sorghum beer fermentations were quantified and a simple equation derived. This equation describes the wort FAN demand as a function of the sugar concentration necessary to produce a fully fermented beer within 48 hours. The influence of wort FAN on sorghum beer fermentations had not been quantified so research was undertaken to define sorghum beer yeasts' requirement for wort FAN and the interactions that occur between wort FAN and sugar. Laboratory, sorghum malt and adjunct, worts mashed to cover a wide range of FAN and wort sugar concentrations, were fermented and analysed. The initial wort FAN affects the ethanol production rate, FAN uptake and sugar utilisation rates.     

羊乳酒在发酵过程中主要参数变化的研究

以新鲜羊奶为原料,采用乳酸菌和酵母菌共发酵方法,在29 ℃条件下振荡发酵羊乳,以pH值、酒精度、总氮及非蛋白氮含量为评价指标,通过凯氏定氮法、十二烷基硫酸钠-聚丙烯酰氨凝胶电泳法（SDS-PAGE）,对羊乳酒在发酵过程的不同时期蛋白质降解的动态变化进行了研究。研究结果表明,羊乳酒在发酵的过程中的pH值呈现先降低后升高再降至稳定的趋势;酒精度呈现缓慢增长的趋势;总氮含量变化不大;非蛋白氮含量随着乳酒发酵的进行缓慢升高;SDS-PAGE结果显示,羊乳酒中蛋白质发生了不同程度的降解,小分子蛋白增多,蛋白分子质量主要分布在17～35 kDa。     

氮源对低温发酵脐橙果酒挥发性成分的影响

为解决低温发酵周期长的问题,并进一步分析其挥发性成分。通过考察不同氮源质量浓度对低温发酵脐橙果酒酒精度和发酵时间的影响,确定氮源添加量,并采用顶空-固相微萃取-气质联用法(HS-SPME-GC-MS)分析其挥发性成分。结果表明,与未添加氮源的空白组相比,添加DAP 0.4 g/L、硫酸铵1.0 g/L、碳酸氢铵1.5 g/L的脐橙果酒酒精度可提高4.6%~5.3%,缩短发酵时间22%~56%。四种低温发酵脐橙果酒共检测出挥发性成分32种,其中高级醇5种、萜烯类化合物10种、中链脂肪酸2种、高级醇乙酸酯3种、中链脂肪酸乙酯7种、醛酮类5种。添加三种氮源的果酒中,萜烯类和乙酸酯类挥发性成分总含量均大于空白组,中链脂肪酸和乙基酯类挥发性成分总含量均小于空白组。其中,添加磷酸氢二铵的果酒中乙酸酯类挥发性成分总含量最高(1.170 mg/L),添加硫酸铵的果酒中萜烯类挥发性成分总含量最高(11.378 mg/L);与另外两种氮源相比,添加磷酸氢二铵的果酒中乙酸-2-苯乙酯、庚酸乙酯等挥发性成分含量相对更高,添加硫酸铵的果酒中D-柠檬烯、乙酸乙酯、癸酸乙酯等挥发性成分含量相对更高,添加碳酸氢铵的果酒中β-水芹烯、4-萜烯醇、辛酸乙酯等挥发性成分含量相对更高。实验证明了氮源对促进脐橙果酒低温发酵具有明显效果,并对挥发性香气成分含量具有一定的促进作用,为脐橙果酒低温发酵工艺的应用提供了理论依据。     

Über das Protein in Kartoffel-, Mais- und ReisstärkeHerrn Prof. Dr. Erwin Welte,Direktor des Instituts für Agrikulturchemie der Universität Göttingen,zum 60. Geburtstag gewidmet.

On the Protein in Potato, Maize and Rice Starch The protein content of potato, maize and rice starch was determined, and 21 amino acids could be identified. With the usual hydrochloric acid hydrolysis to set free the amino acids a portion (residual protein) remains in the dark residue (melanine), and can be isolated only after destruction of the latter, e.g. with sodium chlorite, and analyzed for amino acids. As shown by the nitrogen determination after Kjeldahl and amino acid analysis potato starch has the lowest, maize starch a mean and rice starch a high content of amino acids. The total amount of nitrogen in the amino acids compared with the Kjeldahl nitrogen is only 83% for potato protein and 80% for maize and rice protein. The residue from hydrolysis can vary substantially.     

Free α-amino nitrogen production in sorghum beer mashing

Free α-amino nitrogen (FAN) is an essential nutrient for yeast growth during fermentation. Under normal conditions of sorghum beer mashing, 60°C at pH 4.0, production of FAN by proteolysis accounts for approximately 30% of wort FAN, the remaining 70% being preformed in the malt and adjunct. The quality of the FAN in sorghum beer worts is good as it does not contain a high percentage of proline. Optimum conditions for FAN production during mashing are 51°C and pH 4.6. Wort FAN was increased proportionally by raising the ratio of sorghum malt to adjunct and conversely decreased by raising the ratio of adjunct to malt. FAN was also increased by the addition to the mash of a microbial proteolytic enzyme. Wort FAN is directly proportional to malt FAN.     

可同化氮含量对赤霞珠葡萄酒发酵和香气成分的影响

选取山西戎子酒庄有限公司不同位置的三个葡萄园(驮腰坡,东廒北,东廒南),对各葡萄园进行土壤含氮量、赤霞珠葡萄及葡萄酒发酵过程中可同化氮(YAN)含量、葡萄酒理化指标与香气成分的检测分析。结果表明,葡萄果实可同化氮含量可能主要与土壤铵态氮含量有关。不同葡萄园的赤霞珠葡萄汁可同化氮的含量不同,均超过了150 mg/L,能保证发酵顺利进行。如果需要添加可同化氮时,应以此时测定的可同化氮含量为依据,避免添加过量。原酒中酯类和醇类相对含量由高到低依次为：驮腰坡＞东廒南助剂＞东廒北＞东廒南,这与葡萄汁中可同化氮含量呈正相关。     

THE EBC-NINHYDRIN METHOD FOR DETERMINATION OF FREE ALPHA AMINO NITROGEN

A ninhydrin method is recommended as the EBC Standard Method for the determination of free alpha-amino nitrogen (FAN) in malts, worts and beers. The method, which is described in detail, has been tested in several trials and found to yield highly reproducible results, within and between the participating laboratories. The correlation is reported between the recommended ninhydrin method and a method using tri-nitro-benzene-sulphonic acid.     

葡萄及葡萄酒中有机酸测定的衍生化气相色谱法

利用衍生化气相色谱法分离测定葡萄、葡萄酒中多元有机酸;通过不同酯化方法的比较,选择在N,N-二甲基甲酰胺非质子溶剂中,利用碘乙烷与多元有机酸的四甲基铵盐反应制备各酸相应的乙酯,在SE-30色谱柱上利用程序升温进行分离,同时采用内标法鉴定分析了葡萄、葡萄酒中的多种多元有机酸,并比较测定了葡萄和葡萄酒中多元有机酸的含量。揭示了葡萄酒制备过程中主要有机酸的变化。该方法定量测定的回收率都〉95%,相对标准偏差〈5.0%,适合于各种葡萄酒及啤酒等酒类饮品中有机酸的测定。     

Effect of Malting on Protein Metabolism in Two Varieties of Sorghum

The metabolism of albumin, globulin, glutelin and prolamin in two varieties of sorghum recommended as alternatives to barley malt for brewing in Nigeria has been studied. There was a continuous degradation of prolamin and glutelin (storage proteins) with a concomitant rise in albumin and globulin (enzyme proteins) resulting in a synchronous rise in free amino nitrogen (FAN) during malting of SK 5912. On the other hand, all the major proteins in farafara increased at the peak of malting without a synchronous increase in FAN. When compared to the unmalted sorghum digested with external enzymes, only a quarter to half of the groups of amino acids required for yeast nutrition were obtained with malted sorghum. The production of FAN in SK 5912 malt is higher than farafara malt. FAN produced in SK 5912 malt is high enough for lager beer production therefore its recommendation as a local substitute is supported, in part, by this study. © 1997 SCI.     

不同酵母和氮源对笃斯越橘果酒香气的影响

采用顶空固相微萃取与气相色谱-质谱联用技术,对不同酵母和氮源发酵的笃斯越橘果酒中的香气成分进行分析。结果表明,4?种果酒中共检测出86?种香气成分,包括酯类32?种、醇类18?种、萜烯类13?种、酸类10?种、醛酮类7?种,以及其他类6?种,有78?种香气成分是4?种酒共有的。添加不同酵母和氮源种类导致笃斯越橘果酒在香气化合物的种类和含量呈现出一定的差异性;采用酵母Red?Fruit和补充氮源磷酸氢二铵会促进笃斯越橘发酵进程,且果酒中异戊醇、异丁醇、乙酸乙酯以及乙偶姻等香气化合物含量较多,因此选择适宜的酵母和氮源会提升笃斯越橘果酒的香气。     

Amino acid and total protein content of the edible mushroom Cantharellus cibarius (Fries)

It is difficult to determine total protein content in fungi, mainly because chitin and other nitrogenous compounds interfere with total nitrogen analysis (eg Kjeldahl analysis) which is commonly used. Therefore, a few different methods have been compared, and total amino acid concentration and composition in the edible mushroom Cantharellus cibarius (Fries) have been analysed using one sophisticated and one rough method based on acid hydrolysis and ninhydrin. For fungal material, it is better to analyse total amino acids. Only 99 g kg^?1 dry weight of C cibarius was protein which implies that former values based on total N analysis given in literature are 70–200% too high. The nutritional value of mushrooms should therefore not be over emphasised, even though the protein content is higher than that of most vegetables.     

Proteolysis in reduced sodium Feta cheese made by partial substitution of NaCl by KCl

Feta cheeses (five trials) of different sodium content were made, using ewes’ milk, from split lots of curd by varying the salting procedure, i.e. dry salting with NaCl (control) or mixtures of NaCl/KCl (3:1 or 1:1, w/w basis) and filling the cans with brine made with NaCl or the above NaCl/KCl mixtures, respectively, in order to study the influence of the partial substitution of NaCl by KCl on the proteolysis during cheese ripening. The extent and characteristics of proteolysis in the cheeses were monitored during aging by using Kjeldahl determination of soluble nitrogen fractions (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, phosphotungstic acid-soluble nitrogen), the cadmium–ninhydrin method for the determination of total free amino acids (FAA), urea–polyacrylamide gel electrophoresis of cheese proteins followed by densitometric analysis of the α_s1- and β-casein fractions, reverse-phase HPLC analysis of the water-soluble extracts of cheeses, and ion-exchange HPLC analysis of FAA. The results showed that proteolysis was similar in control and experimental cheeses at all sampling ages, indicating that the partial substitution of NaCl by KCl in the manufacture of Feta cheese had no significant effect on the extent and characteristics of proteolysis during cheese aging.     

Wine lees valorization: Biorefinery development including production of a generic fermentation feedstock employed for poly(3-hydroxybutyrate) synthesis

This study demonstrates the development of a novel wine lees (WL) based integrated biorefinery for the production of several added-value products. WL were initially fractionated for the production of antioxidants, tartrate and ethanol and the remaining stream was converted into a fermentation nutrient supplement for poly(3-hydroxybutyrate) (PHB) production using the strain Cupriavidus necator DSM 7237. Hydrolysis of pretreated WL was carried out using crude enzyme consortia produced via solid state fermentation of Aspergillus oryzae. Optimization of hydrolysis was based on the enhancement of total Kjeldahl nitrogen to free amino nitrogen (FAN) conversion yield by evaluating the effect of the initial pH value, temperature, initial proteolytic activity and initial WL concentration. WL hydrolysates and crude glycerol were used as nutrient and carbon sources, respectively, in batch and fed-batch fermentations for the production of PHB. Bacterial growth and PHB production were influenced significantly by the FAN content of the WL derived hydrolysates and by the addition of trace elements. Using an initial FAN concentration of 700 mg L^− 1 and supplementation with trace elements led to the production of 30.1 g L^− 1 of PHB concentration with an intracellular content of 71.3% (w w^-1) and a productivity of 0.56 g L^− 1 h^− 1 during fed-batch fermentation.     

Occurrence and stability of inorganic and organic arsenic species in wines, rice wines and beers from Central European market

We investigated in total 80 wine samples of different types and seven grape juice and 23 beer samples purchased from markets in Central Europe in order to understand the arsenic (As) speciation and help assess the potential As toxicity via intake of alcoholic beverages. Generally, total As concentrations in most samples investigated were below the drinking water limit 10 μg l(-1) published by the World Health Organization (WHO); ranging from 0.46 to 21.0 μg l(-1) As in red and white wines and from 0.75 to 13.4 μg l(-1) As in beers. In addition, concentrations of total As in rice wine and in rice beer were 0.63-6.07 and 3.69-8.23 μg l(-1) As, respectively. The total As concentrations in ice wine ranged from 7.94 to 18.8 μg l(-1) As, significantly higher than in white and red wine. Arsenite predominated as the As species in most of the wine samples, whereas arsenate was the dominant species in rice wine, beer and rice beer. Methyl As components were usually minor components in all wine and beer samples. Monomethylarsonic acid, dimethylarsinic acid and two additional unknown As species were frequently found in grape juice, late harvest and ice wine with higher sweetness. After air exposure, arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid were stable at 4°C for months, probably due to the acidic conditions of wine and beer samples. The presence of sulfite had little influence on As speciation in wine. Despite the predominance of more toxic arsenite and arsenate in wine and beer, the estimated weekly exposure to As (via consumption of beer, wine and rice wine) is low. The As intake per capita is 6.81 μg from beer, <1.93 μg from wine and 0.88 μg from rice wine, estimated using the median of total As concentration multiplied by the average consumption per capita of the corresponding beverage.     

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.     

GAS CHROMATOGRAPHIC DETERMINATION OF TRIHYDROXYOCTADECENOIC ACIDS IN BEER

A rapid and quantitative method for measuring the content of trihydroxyoctadecenoic acids in beer is described. The acids are extracted from degassed beer with ethyl acetate and methylated with diazomethane. After washing, the methylated compounds are silylated and analysed by gas chromatography. The coefficient of variation of the method is 3·2%. The amounts of total trihydroxyoctadecenoic acids in commercial beer samples varied from 4 to 12 mg/litre. The effects of these acids on beer flavour and head retention are discussed.     

Occurrence and stability of inorganic and organic arsenic species in wines,rice wines and beers from Central European market

We investigated in total 80 wine samples of different types and seven grape juice and 23 beer samples purchased from markets in Central Europe in order to understand the arsenic (As) speciation and help assess the potential As toxicity via intake of alcoholic beverages. Generally, total As concentrations in most samples investigated were below the drinking water limit 10?µg?l^?1 published by the World Health Organization (WHO); ranging from 0.46 to 21.0?µg?l^?1 As in red and white wines and from 0.75 to 13.4?µg?l^?1 As in beers. In addition, concentrations of total As in rice wine and in rice beer were 0.63–6.07 and 3.69–8.23?µg?l^?1 As, respectively. The total As concentrations in ice wine ranged from 7.94 to 18.8?µg?l^?1 As, significantly higher than in white and red wine. Arsenite predominated as the As species in most of the wine samples, whereas arsenate was the dominant species in rice wine, beer and rice beer. Methyl As components were usually minor components in all wine and beer samples. Monomethylarsonic acid, dimethylarsinic acid and two additional unknown As species were frequently found in grape juice, late harvest and ice wine with higher sweetness. After air exposure, arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid were stable at 4°C for months, probably due to the acidic conditions of wine and beer samples. The presence of sulfite had little influence on As speciation in wine. Despite the predominance of more toxic arsenite and arsenate in wine and beer, the estimated weekly exposure to As (via consumption of beer, wine and rice wine) is low. The As intake per capita is 6.81?µg from beer, <1.93?µg from wine and 0.88?µg from rice wine, estimated using the median of total As concentration multiplied by the average consumption per capita of the corresponding beverage.     

ESTIMATION OF NITROGEN WITH AN AMMONIA PROBE

An ammonia probe has been used to replace the distillation-titration procedure for estimating the ammonium content of Kjeldahl digest solutions. A procedure is described for estimating the total nitrogen content of barley, malt, wort and beer. The semi-micro digestion stage is performed as a batch procedure and the ammonium content of the acid digest estimated automatically in a continuous flow system at a rate of 60 samples per hour. The method is more rapid than the classical distillation-titration procedure and gives results of comparable accuracy and precision.