A High-Performance Liquid Chromatographic Method for Simultaneous Determination of 21 Free Amino Acids in Tea

Free amino acids are closely related to the savory taste and beneficial effects of tea, and high-performance liquid chromatography (HPLC) is the most widespread analytical approach for simultaneous determination of free amino acids in tea. However, the reported HPLC methods have drawbacks such as long run times, low resolution, or poor efficiency. In this study, a special amino acid analysis column was used to separate and verify 21 amino acids including l-theanine, the predominant amino acid in tea. The mobile phases, including the sodium acetate and acetic acid concentration in buffer B, and the pH and concentration of sodium acetate in buffer A were optimized. The elution gradients were optimized using DryLab 2000 Plus software. In this way, an online o-phthaldialdehyde precolumn derivatization HPLC-fluorescence detection method was developed for simultaneous determination of 21 amino acids. Comparison to other HPLC methods for simultaneous determination of free amino acids in tea showed that our method is easy (automated derivatization), quick (30 min), inexpensive, and green (using a minimum of solution). It has good resolution (≥1.8) and high selectivity (interpark time?≥?0.5 min). Free amino acids in six tea samples were analyzed. This work provides an HPLC method to simultaneously measure 21 amino acids in tea and potential in other food products.     

全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量

建立一种测定贝类肌肉中谷胱甘肽和游离氨基酸的全自动氨基酸分析仪法,以4种常见贝类为研究对象,采用0.02 mol/L盐酸溶液作为提取剂,体积分数5%磺基水杨酸溶液为蛋白沉淀剂,同时优化了缓冲溶液的组成及洗脱程序,并对该方法的线性范围、检出限、精密度以及回收率进行测定。结果表明:贝类肌肉中的谷胱甘肽和游离氨基酸有着较好的分离效果,线性关系良好,相关系数为0.999 1~0.999 9,检出限(RSN=3)为0.07~0.27μmol/L,加标平均回收率为86.40%~102.42%,日内和日间相对标准偏差分别为0.31%~0.73%和1.14%~2.60%。4种贝类肌肉组织中的游离氨基酸含量比较丰富,总游离氨基酸含量分别为1 396.39、1 016.04、911.15、287.01 mg/100 g,文蛤青蛤缢蛏牡蛎,主要的游离氨基酸为牛磺酸(Tau)、谷氨酸(Glu)、丙氨酸(Ala)、精氨酸(Arg);谷胱甘肽含量分别为103.20、82.53、61.77、33.37 mg/100 g,青蛤文蛤缢蛏牡蛎。该方法适用于贝类肌肉中谷胱甘肽和游离氨基酸含量的测定与分析。     

传统发酵豆酱的微生物群落结构和游离氨基酸组成及其相关性分析

研究传统发酵豆酱产品质量的差异,通过定量描述分析法对吉林省长春、黑龙江省桦南、黑龙江省宝泉岭和黑龙江省黑河4 个地区发酵豆酱样品（分别为ChangC、HuaN、BaoQL、HeiH）进行感官评价,运用高通量测序技术和氨基酸分析技术测定其微生物群落结构和游离氨基酸组成,并探讨两者的相互关系。结果表明：BaoQL感官评价最好。不同豆酱样品的菌群组成差异较大,菌群中优势细菌和优势真菌的种类也各不相同,其中样品HuaN和HeiH的优势细菌为四联球菌（Tetragenococcus）,样品ChangC的优势细菌为乳杆菌（Lactobacillus）,样品BaoQL的优势细菌为魏斯氏菌（Weissella）;样品HuaN和HeiH的优势真菌为青霉菌（Penicillium）,样品ChangC的优势真菌为unclassified-k-Fungi,样品BaoQL的优势真菌为曲霉菌（Aspergillus）。通过对其游离氨基酸进行主成分分析和综合性评价,可得东北不同地区传统发酵豆酱游离氨基酸含量存在差异,样品BaoQL的游离氨基酸综合评分最高,样品ChangC次之,HeiH评分最低。采用偏最小二乘回归模型分析豆酱的微生物多样性和游离氨基酸组成的相关性可知,菌群的种类和丰度对游离氨基酸的组成和含量影响较大,从而影响豆酱的质量,其中对游离氨基酸含量影响较大的细菌为Weissella,真菌为毕赤酵母菌（Millerozyma）和Aspergillus。本研究揭示了豆酱中游离氨基酸与微生物群落组成之间的相关性,为豆酱产品的工业化生产提供理论依据。     

Nitrogen-to-protein conversion factors for some cereal products in Japan

ABSTRACT:  To evaluate a practical method of determining more accurately conversion factors for calculating the protein contents of foods from the total nitrogen content, 19 cereal products found in Japan were analyzed for total nitrogen, amino acid nitrogen, and amide nitrogen, and then the nitrogen-to-protein conversion factors were calculated. The average conversion factors were 5.75 for rice, 5.81 for wheat, and 5.95 for others. These values, corresponding to the proportion of the amino acid residue to amino acid nitrogen recovered from 20 amino acids, were lower than the currently applied factors to these foods, except for wheat flour and amaranth. The use of this factor for estimating the protein content results in a considerable difference from the estimate based on amino acid residue concentrations, due to the wide variations in amino acid composition and to the presence of a significant level of nonprotein nitrogen. The distribution of the protein nitrogen recovered from the amino acids to total nitrogen averaged 93%. Adjusted conversion factors corresponding to the proportion of the amino acid residue to total nitrogen averaged 5.26 for rice, 5.47 for wheat, and 5.54 for other cereal products. Protein contents estimated using these factors are in good agreement with the contents defined as amino acid residues.     

Multi‐Cereal Beverage Fermented by Lactobacillus Helveticus and Saccharomyces Cerevisiae

A novel multi‐cereal‐based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and Saccharomyces cerevisiae was developed. Twenty‐seven lactobacilli strains were screened for acid production (pH and titratable acidity) in a mixture of malt, rice, and maize substrates. It was found that Lactobacillus helveticus KLDS1.9204 had the greatest acid production among 27 lactobacilli tested. The fermentation performance of L. helveticus KLDS1.9204 was also assayed and the fermentation parameters were optimized using Plackett–Burman design and steepest ascent method. L. helveticus KLDS1.9204 showed good proteolytic capability, however, the strain could not utilize starch. The optimum substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL), and 25% maize (30 g/100 mL). The inoculum was 5% with a ratio of S. cerevisiae to L. helveticus KLDS1.9204 of 2.5:1. The optimum temperature was 37 °C and the time was 22 h. Lastly, the quality of the multi‐cereal‐based fermented beverage was evaluated. This beverage was light yellow, transparent, and it tasted well with a pleasant acid and a unique flavor of cereals. The beverage was rich in free amino acids and organic acids. The pH and titratable acidity of the beverage were 3.5 and 29.86 °T, respectively. The soluble solids content of the beverage was 6.5 °Brix, and the alcohol content was 0.67%.     

Amino Acid Profiles of Common Cultivated Mushrooms Including the Identification of N-(N-γ-L-Glutamyl-3-Sulfo-L-Alanyl)Glycine in Flammulina velutipes

Amino acid profiles of nonprotein nitrogen (NPN) and protein nitrogen (PN) fractions of common cultivated mushrooms were determined. Free amino acids in the NPN fractions were generally characterized by the predominant occurrence of alanine and amino acids metabolically related to the glutamic acid family and also by the common presence of unique nonprotein amino acids, such as saccharopine, cystathionine and ethanolamine. A novel glutathione analog, N-(N-γ-L-glutamyl-3-sulfo-L-alanyl)glycine, was found in Flammulina velutipes. The proportion of the NPN to the total nitrogen was relatively high and large amounts of the NPN were made up of free amino acids and related compounds. Protein amino acids in the NPN and PN fractions accounted for about 65% of the total nitrogen, suggesting that a practical nitrogen-protein conversion factor for the mushrooms may be considered to be about 4 on the average.     

复配酶法制备南极磷虾鲜味酶解液的工艺优化

以脱脂南极磷虾粉为原料,采用复配酶法制备鲜味酶解液。以感官评价、多肽得率、水解度等评价手段,进行外源酶的筛选及单因素实验。为进一步优化酶解液滋味,以呈鲜味的氨基酸总量为指标进行酶解工艺优化。结果表明:最佳酶解条件为:胰蛋白酶和胃蛋白酶复配比1:100,总加酶量为2800 U/g,料液比1:4 g·mL?1,酶解pH7.0,酶解温度40 ℃,酶解时间3 h,该条件下获得的酶解产物中鲜味游离氨基酸含量为331.79 mg/100 mL。除色氨酸外,其余八种必需氨基酸含量占总氨基酸的63.66%,游离苦味氨基酸占总氨基酸的27.83%,游离鲜甜味氨基酸占总氨基酸26.22%,与鲜味形成具有关键作用的游离谷氨酸和游离天冬氨酸共占总游离氨基酸的10.26%。本研究可为南极磷虾鲜味调味料等产品的开发提供理论基础和技术指导。     

Changes in the amino acid compounds of the wholecrop wheat during ensiling and after fermentation

Changes in amino acid fractions of chopped low-to-medium protein whole-crop wheat were studied at four stages of maturation and at two stages after a 24 h wilting period. Tests were carried out after an ensiling period of 90 days, and after a further 7-day post-fermentation aerobic exposure. Total amino acids contents in the dry matter during the fermentation period remained stable. Quantities of essential amino acids decreased during maturation and fermentation and remained stable during post fermentation aerobic exposure. The highest amino acid concentrations recorded in the fresh material were those of arginine, lysine, glutamic acid, alanine, leucine, glycine and aspartic acid. Free amino acids varied from a low level in the fresh material < 10% of the total amino acids to ca 65% after the aerobic process. Ammonia-N content increased in the silage and remained relatively stable on post fermentation aerobic exposure. The concentration of all free amino acids increased except arginine and glutamine during the ensiling period and post fermentation aerobic exposure. After wilting, an increase in free amino acids and a decrease in insoluble amino acids was recorded. The changes in concentration of 18 amino acids during ensilage and following post fermentation aerobic exposure are given.     

Protein and free amino acid patterns in maize ensiled with or without urea

Free and protein amino acid contents of whole-plant maize ensiled with or without urea were determined at intervals during the fermentation process. The free amino acid content of both untreated and urea-treated silage increased with time in storage, but the increases were generally larger in the treated silage. The true protein of untreated silage decreased with time in storage. In urea-treated silage many individual amino acids increased considerably. Among those were the essential amino acids isoleucine, lysine, threonine and valine. The result was a net gain of several amino acids in the treated silage while there was a general decline of most amino acids in the untreated silage. Urea-treated silage at day 20 contained 38% more true protein and 150% more free amino acids than did untreated silage. The implications of these changes for dairy cattle nutrition are discussed.     

高效液相色谱法检测食品中17种游离氨基酸

目的使用异硫氰酸苯酯衍生-高效液相色谱法对食品中17种游离氨基酸进行检测。方法对17种游离氨基酸及其苯胺硫甲酰基衍生物稳定性的分析,研究游离氨基酸的最佳衍生条件。同时,对盐沉淀法和C_(18)固相萃取(solid phase extraction, SPE)小柱法2种样品除杂方法的加标回收率进行测定。结果胱氨酸加热干燥5h后,回收率为68.46%±4.01%,热稳定性差;其它16种氨基酸加热1～5h后回收率均在90%～110%之间,热稳定性较好。胱氨酸的苯胺硫甲酰基衍生物,6h后回收率仅为11.04%±1.65%,稳定性差;其它氨基酸的苯胺硫甲酰基衍生物24 h后回收率均大于90%,比较稳定。另外,前处理中使用C_(18) SPE除杂时,样品中游离氨基酸的加标回收率均大于70%,而使用水杨酸和三氯乙酸除蛋白后苯胺硫甲酰基衍生物降解严重。结论除胱氨酸外,其余16种氨基酸的热稳定性均较好,且生成的苯胺硫甲酰基衍生物比较稳定,可以通过异硫氰酸苯酯衍生进行定量分析。相比于水杨酸和三氯乙酸沉淀法, C_(18) SPE柱是游离氨基酸除杂方法的首选,而无机盐影响异硫氰酸苯酯与氨基酸衍生,因此此方法不适用于含盐样品的衍生。     

Free Amino Acids and Related Compounds in Seeds and Sprouts of Winged Bean Psophocarpus tetragonolobus (L.) DC)

Free amino acids and related compounds in the nonprotein nitrogen (NPN) fraction of winged bean seeds and sprouts were quantitatively determined. The proportion of NPN to total nitrogen was low in the seeds (9%) and in the cotyledons of the sprouts (11%), and relatively high in the epicotyls of the sprouts (54%). About 6% and 40% NPN were attributable to free amino acid nitrogen in the seeds and the sprouts, respectively. The free amino acid profile of seeds was characterized by the presence of sizable amounts of γ-L-glutamyl-L-tyrosine and γ-L-glutamyl-L-phenylalanine, indicating similarities to that of soybean seeds. Accumulation of free amino acids, particularly nonprotein amino acids such as α-aminoadipic acid and γ-aminobutyric acid, were demonstrated in the sprouts. No canavanine was detected in either seeds or sprouts.     

响应面法优化郫县豆瓣游离氨基酸的提取工艺及呈味特性分析

本文优化了郫县豆瓣中游离氨基酸提取工艺并对其呈味特性进行研究。以游离氨基酸提取量作为指标,在单因素实验的基础上,采用响应面试验优化游离氨基酸提取工艺,并通过氨基酸自动分析仪测定6种郫县豆瓣中氨基酸含量。采用味道强度值(Taste activity value,TAV)确定主要呈味物质及贡献,并利用滋味感官评价对郫县豆瓣进行呈味评定。结果表明,最佳提取工艺参数:液料比27:1(mL/g),超声温度35℃,超声时间30 min,氨基酸提取量18.51 mg/g。6种郫县豆瓣样品中呈鲜酸味氨基酸(谷氨酸(Glu)、天冬氨酸(Asp)、天冬酰胺(Asn))含量最高,总量平均达6.380 mg/g;其次为甜鲜味氨基酸(苏氨酸(Thr)、丝氨酸(Ser)、甘氨酸(Gly)、丙氨酸(Ala)、脯氨酸(Pro)),总量平均达5.540 mg/g,呈苦味和苦甜味氨基酸含量最低,6种样品呈鲜味氨基酸总量平均达11.920 mg/g。丙氨酸、谷氨酸、天冬氨酸、赖氨酸、组氨酸、精氨酸和缬氨酸TAV大于1,是郫县豆瓣呈味主要贡献者。滋味感官评价结果显示6种郫县豆瓣鲜、咸口感最为突出。通过响应面优化郫县豆瓣游离氨基酸的最佳提取条件,该工艺方便可行;6种郫县豆瓣中游离氨基酸组成和含量存在一定差异,其中谷氨酸鲜味贡献作用最大。     

Bestimmung der Folsäure-Gehalte von Getreide,Getreideprodukten, Backwaren und Hülsenfrüchten mit Hilfe der Hochleistungsflüssigchromatographie (HPLC)

The folic acid content of grain, cereal products (including beer), bakery products and legumes was determined by means of high-performance liquid chromatography (HPLC). Free folate (monoglutamate forms) and total folate (monoglutamate + polyglutamate forms) were differentiated. Of the grain analysed, rye, with a mean value of 143 μg/100 g, contained more total folate than wheat (mean=91 μg/100 g). The total folate content of bakery products ranged from 14 μg/100 g (whole grain rye bread) to 88 μg/100 g (crispbread). Beer had a very low total folate content (mean=3 μg/100 ml). The mean of the free folate portion was 76.5% in grain and cereal products and 65.6% in bakery products. Of the legumes analysed, beans (mean=128 μg/100 g) had the highest content of total folate, followed by lentils (103 μg/100 g) and peas (57 μg/100 g). The mean value of the free folate portion in legumes (73.1%) was comparable with the values of grain, cereal products and bakery products. In addition to tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF, pteroylglutamic acid (PteGlu) and 10-formyl-PteGlu were determined in all products (except beer). Their proportion (PteGlu+10-formyl-PteGlu) of the total folate content ranged from 23.5% to 44.4%.     

毛细管气相色谱法测定卷烟中的游离氨基酸

本试验描述了一种用于烟草中游离氨基酸测定的毛细管气相色谱方法,这种方法基于氯甲酸乙酯衍生化。1g烟样用75%乙醇经超声波振荡提取后,游离氨基酸用阳离子交换树脂纯化,然后在水溶液中用氯甲酸乙酯(ECF)衍生化。该方法简单、衍生化可在水溶液中进行且速度快(常温下仅需5秒)、衍生物较稳定、重现性和回收率实验结果均令人满意。用该法对加料前后的烟草和卷烟中的游离氨基酸进行了分析,结果发现,加料后游离氨基酸尤其是脯氨酸含量呈减少趋势。23个品牌的国内外卷烟分析表明,云南产的卷烟中游离氨基酸平均含量最高,国外卷烟略低,安徽、上海等华东地区生产的卷烟含量最低。其中云南卷烟所含脯氨酸比其它卷烟要高得多,而国外卷烟的天冬氨酸、天冬酰氨含量较高。      

A Procedure for Gas Chromatographic Analysis of Free Amino Acids in Meats

SUMMARY— A gas liquid chromatography (GLC) procedure was developed to quantitatively measure the free amino acid content of meat and meat products. The amino acids were extracted from the meats with water and purified using dialysis and ion exchange chromatography. n-Butyl N-trifluoro-acetyl amino acid derivatives were prepared and analyzed on the gas chromatograph. Factors used for quantitative purposes were calculated using the peak areas and concentrations of known amino acid derivatives.
The isolation and purification procedures were able to remove all carbohydrates, proteins, and 98% of all inorganic salts from the amino acid solutions. Eleven of the common amino acids were completely resolved using the GLC condition described in this investigation. In addition, three GLC peaks contained two amino acids each. The procedure was able to detect free amino acid in sausage in concentrations as low as .01mg/g.     

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。     

Free amino acid profiles from ‘Pinot noir’ grapes are influenced by vine N-status and sample preparation method

This study examined the impact of extraction method on ammonia, free amino acids, and yeast assimilable nitrogen (YAN) concentrations in ‘Pinot noir’ berries obtained from a vine nutrition study (altered supply of N, P, or K). Berries were either juiced or exhaustively extracted as whole berries prior to analysis. Extracts, compared to juice samples, had a significantly higher level of ammonia–N, assimilable amino acid–N, and YAN. For example, juice YAN values were approximately 50% of extract YAN values, when both were expressed in the same units. Free amino acid profiles and relative concentrations of individual amino acids were different in juice versus extracts, depending on how well the skin fraction was extracted prior to analysis. Lowering N supply reduced free amino acids, with arginine being reduced more than the other 20 free amino acids identified in ‘Pinot noir’ berries. This was true in both juice and extracts. Since berry skin contributed to actual YAN, wineries that determine YAN from mainly the pulp fraction (juice) may underestimate YAN and as a result add more (artificial) N supplement than is required for the healthy fermentation of red winemaking (whole berry fermentations). Extraction procedure should be taken into consideration when comparing grape YAN.     

Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions

The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications.     

Taste of Water-Soluble Extracts Obtained from Over-Fermented Tempe

Over-fermented tempe, known as tempe semangit, is popular in Indonesian culture, especially in Java, as an umami seasoning in traditional foods. The objective of this study was to characterize taste-active compounds in water soluble extracts of over-fermented tempe. Over-fermented tempe was prepared from fresh tempe in which the fermentation was prolonged (0 to 96 h). Free amino acids in the water soluble extract were analyzed and characterized for their taste activity values. Water soluble extracts contained umami and bitter tasting free amino acids at relatively high concentrations. Their umami and bitter taste activity values were higher than the taste threshold concentration. Water soluble extract from 72 h over-fermentation had a higher umami taste activity value than bitter taste activity, exhibiting the highest umami taste dilution factor. The high-performance liquid chromatography profile of water soluble extract fractions obtained using Sephadex G-25 gel filtration chromatography demonstrated that fractions having higher umami taste intensity had more hydrophilic components than hydrophobic components.     

酸浆豆腐后熟过程中风味物质的形成途径分析

采集不同后熟阶段的酸浆豆腐为样本，对其游离氨基酸、有机酸、挥发性风味物质进行测定。结果显示，游离氨基酸和有机酸是滋味物质的主要来源，其中酸味氨基酸是主要的呈味物质，在不同的后熟阶段占总氨基酸的33%～35%。后熟过程中乳酸、乙酸和柠檬酸3 种有机酸是主要呈味物质且含量最高，其总量约占有机酸总量的50.08%～65.00%。后熟过程中共检测到84 种风味化合物，包括醛类、醇类、酚类、呋喃类等化合物，共同赋予酸浆豆腐独特的酵香风味。