Simultaneous determination of lysine, lysinoalanine, furosine, and pyridosine in food and feed (author's transl)]

A method for the simultaneous separation and determination of lysinoalanine (LAL), lysine, furosine and pyridosine with an automatic amino acid analyzer using a single Column 4 buffer- and 3 temperature-programme is described. The procedure needs 160 Minutes analysis time and allows a separation and quantitative determination of LAL, when present in amounts of more than 100 ppm (semiquantitative above 10 ppm). All amino acids found in food proteins and all other ninhydrinpositive compounds tested until now do not interfere with the determination.     

A simple method for the preparation of furosine and pyridosine reference material

After heatingN -acetyllysine and glucose for 4 h at 90 °C in the dry state and subsequent acid hydrolysis with 7.8 N HCl, preparative fractionation of the dihydrochlorides of furosine and pyridosine was achieved by cation-exchange chromatography. The lysine derivatives could be prepared with high yield and sufficient purity for the use as reference material.

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Ein einfaches Verfahren zur Darstellung von Furosin- und Pyridosin-Standards

Zusammenfassung Nach dem Erhitzen vonN -Acetyllysin und Glucose für 4 h bei 90 °C in trockenem Zustand und nachfolgender Hydrolyse mit 7.8 N HCl erfolgte eine präparative Fraktionierung der Dihydrochloride von Furosin und Pyridosin durch Kationenaustauschchromatographie. Die Lysinderivate wurden in guter Ausbeute und in für Referenzmaterial ausreichender Reinheit erhalten.

     

Effect of carbohydrate DE on blocked lysine and furosine in a liquid nutritional product

A controlled, quantitative study of Maillard browning vs. dextrose equivalents (DE) was performed on a liquid nutritional product. The early stage Maillard markers furosine and available lysine were determined in retort-sterilised, pilot scale batches formulated with carbohydrate systems with DE variations of 2, 4, 10, and 20. Both markers varied proportionately with DE; every DE increase of 2 units resulted in the blockage (glycation) of an additional 1% of total lysine. When DE 20 maltodextrin was replaced with an 80/20 blend of DE 5 maltodextrin and sucrose (blend DE = 4), lysine blockage decreased by 831 mg/100 g protein, which was 8.25% of the total lysine, and the mole equivalent of 1.75 g of fructoselysine. The quantitative browning/DE relationships enable reliable projections of the nutritional benefits which may be attained through the use of low-DE maltodextrins.     

Kinetics of hydroxymethylfurfural,lactulose and furosine formation in milk with different fat content

In the context of the general applicability of hydroxymethylfurfural (HMF), lactulose and furosine as time-temperature integrators (TTIs) for thermal processing of milk, the influence of milk fat content was studied. Formation kinetics were analysed for milk with fat content of 4.0 +/- <0.1%. In previous experiments, it was observed that, under isothermal and non-isothermal heating conditions, formation of the three chemical compounds could be described by pseudo-zero order kinetics. Since the kinetic model was known, the experimental design could be simplified. Data were analysed by a non-linear regression procedure and results were evaluated by construction of joint confidence regions and temperature time tolerance (TTT-) diagrams. Formation kinetics of HMF and lactulose was not affected by milk fat content. Regarding furosine, significant differences were observed between kinetic parameters in whole, semi-skimmed and skimmed milk. The observed differences however were negligible in the context of process impact evaluation.     

Evaluation of the furosine and homoarginine methods for determining reactive lysine in rumen-undegraded protein

Three samples of soybean meal (SBM), 3 samples of expeller SBM (SoyPlus, West Central Cooperative, Ralston, IA), 5 samples of distillers dried grains with solubles (DDGS), and 5 samples of fish meal were used to evaluate the furosine and homoarginine procedures to estimate reactive Lys in the rumen-undegraded protein fraction (RUP-Lys). One sample each of SBM, expeller SBM, and DDGS were subjected to additional heat treatment in the lab to ensure there was a wide range in reactive RUP-Lys content among the samples. Furosine is a secondary product of the initial stages of the Maillard reaction and can be used to calculate blocked Lys. Homoarginine is formed via the reaction of reactive Lys with O-methylisourea and can be used to calculate the concentration of reactive Lys. In previous experiments, each sample was ruminally incubated in situ for 16 h, and standardized RUP-Lys digestibility of the samples was determined in cecectomized roosters. All rumen-undegraded residue (RUR) samples were analyzed for furosine and Lys; however, only 9 of the 16 samples contained furosine, and only the 4 unheated DDGS samples contained appreciable amounts of furosine. Blocked RUP-Lys was calculated from the furosine and Lys concentrations of the RUR. Both the intact feed and RUR samples were evaluated using the homoarginine method. All samples were incubated with an O-methylisourea/BaOH solution for 72 h and analyzed for Lys and homoarginine concentrations. Reactive Lys concentrations of the intact feeds and RUR were calculated. Results of the experiment indicate that blocked RUP-Lys determined via the furosine method was negatively correlated with standardized RUP-Lys digestibility, and reactive RUP-Lys determined via the guanidination method was positively correlated with standardized RUP-Lys digestibility. Reactive Lys concentrations of the intact samples were also highly correlated with RUP-Lys digestibility. In conclusion, the furosine assay is useful in predicting RUP-Lys digestibility of DDGS samples, and the guanidination procedure can be used to predict RUP-Lys digestibility of SBM, expeller SBM, DDGS, and fish meal samples.     

赖氨酸/葡萄糖美拉德反应产物对丙烯酰胺生成的抑制作用

建立天冬酰胺/葡萄糖模式反应体系,运用高效液相色谱法、自由基清除法等手段研究赖氨酸/葡萄糖美拉德反应产物对该模式体系中丙烯酰胺生成量及体系抗氧化性的影响。结果显示:高、低分子量的赖氨酸/葡萄糖美拉德反应产物(H-LGP和L-LGP)均能显著降低模式反应体系中的丙烯酰胺生成量,其中H-LGP和L-LGP的添加量为10 mg/mL时,对丙烯酰胺生成的抑制率最大,分别为32.0%和31.4%。H-LGP和L-LGP(均为10mg/mL)的添加还可显著提高反应体系清除自由基DPPH和ABTS的能力。结论:赖氨酸/葡萄糖美拉德反应产物(LGP)能够显著抑制天冬酰胺/葡萄糖模式反应体系中丙烯酰胺的生成,且高浓度的LGP能够显著提高反应体系的抗氧化性。     

Effect of Amadori rearrangement products on the non-enzymic browning in model systems

The Amadori product was prepared from D-glucose and L-alanine after Hashiba. The browning process was investigated by determining the absorbance at 520 nm, in aqueous solutions at 110 °C for 5–8 h. Because of the limited access of oxygen and its low solubility in the reaction medium, the browning proceeded after zeroth order kinetics with the maximum browning rate at pH = 8–9. The browning rate remained unaffected by additions of sodium sulphite, rutin. propyl gallate (except when present at high levels), iron(III)chloride or copper(II)chloride but decreased in presence of L-cysteine or iron(II)chloride. Hydrogen peroxide bleached the pigment but did not inhibit the subsequent browning of reaction products. Under experimental conditions the solution of Amadori product did not darken with substantially greater rate than the solution of D-glucose and L-alanine. Lower additions of D-glucose to the solution of Amadori product moderately increased the reaction rate while additions of L-alanine or L-hydroxyproline moderately decreased the browning rate.     

Studies on the formation of lysinomethylalanine and histidinomethylalanine in milk products

From reaction mixtures consisting ofN-acetyldehydroaminobutyric acid methyl ester andN -acetyl-l-lysine orN -acetyl-l-histidine, respectively, distinct amounts of the cross-link amino acidsN -(2-amino-2-carboxy-l-methylethyl)-l-lysine (lysinomethylalanine, LMeAL) andN -(2-amino-2-carboxy-1-methyl-ethyl)-l-histidine (histidinomethylalanine, HMeAL) were isolated via preparative ion-exchange chromatography and identified by¹H- and¹³C-nuclear magnetic resonance. In the amino acid chromatogram, both compounds eluted clearly separated from other basic amino acids. However, neither LMeAL nor HMeAL could be detected in numerous acid hydrolysates of a range of milk products. In model studies, threonine showed a significantly lower tendency for an alkali-induced -elimination reaction compared to serine. The reactivity of the resulting dehydroaminobutyric acid towards nucleophiles was more than tenfold lower as compared to dehydroalanine. Thus, the formation of LMeAL as well as of HMeAL during food processing is negligible.

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Studien zur Bildung von Lysinomethylalanin und Histidinomethylalanin in Milchprodukten

Zusammenfassung Aus Reaktionsansätzen bestehend ausN-Acetyldehydroaminobuttersäurenthylester undN -Acetyl-l-lysin beziehungsweiseN -Acetyl-l-histidin konnten die Crosslink-AminosäurenN -(2-Amino-2-carboxy-1-methyl-ethyl)-l-lysin (Lysinomethylalanin, LMeAL) undN -(2 amino-2-carboxy-2 -methyl-ethyl)-l-histidin (Histidinomethylalanin, HMeAL) durch präparative Ionenaustauschchromatographie isoliert und anschließend durch 1H-und¹³C-NMR identifiziert werden. Das Aminosäurechromatogramm zeigte eine eindeutige Trennung beider Aminosäurederivate von anderen basischen Aminosäuren. Dennoch konnten in einer großen Zahl von Säurehydrolysaten der verschiedensten Milchprodukte weder LMeAL noch HMeAL nachgewiesen werden. In Modelluntersuchungen erfolgte die -Elimination an Threonin im Vergleich zu Serin in einem deutlich geringeren Maße. Die Reaktivität der dabei entstehenden Dehydroaminobuttersäure gegenüber Nucleophilen war mehr als zehnfach geringer als die von Dehydroalanin. Damit kann die Bildung sowohl von LMeAL als auch von HMeAL bei der Verarbeitung von Nahrungsmitteln als vernachlässigbar gering eingeschätzt werden.

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Dedicated to Prof. Dr. W. Baltes on the occasion of his 65th birthday     

盐酸处理对壳聚糖的降解作用研究

研究以壳聚糖为原料,针对壳聚糖因水溶性差,反应物浓度小的难题,采用醋酸溶解和添加2%的盐酸,于90℃保温搅拌6h,可以使此壳聚糖体系的黏度从3.00Pa.s降至0.266Pa.s,溶液流动性好,同时可使壳聚糖浓度提高到10%。该处理方法不仅使壳聚糖浓度增加,而且又避免了采用高温加热法易产生单糖的缺陷。     

鲣鱼双酶酶解工艺优化及其产物动态变化研究

以鲣鱼为对象,优化了胰蛋白酶(trypsin)与木瓜蛋白酶(papain)复合酶解鱼肉蛋白的条件,研究了酶解过程中酶解液中游离氨基酸组成和含量、肽分子质量分布以及呈味核苷酸二钠含量的动态变化规律。结果表明:木瓜蛋白酶和胰蛋白酶双酶酶解鲣鱼最优条件为:最适比例1:3,总酶添加量3 000 U/g,底物浓度为25%。酶解过程中,酶解液中游离氨基酸总量不断增加,酶解6 h后,呈苦味氨基酸含量有所减少,而呈鲜味氨基酸大幅增加;对氨基酸呈味强度值(taste activity value,TAV)的分析表明,Glu、Met、Ile和His等几种氨基酸对酶解液的呈味特性起了关键作用。随着酶解时间的增加,大分子质量蛋白和肽类组分逐渐减少而小分子质量肽类组分逐渐增多,酶解6 h后分子质量小于0.5 ku的组分含量达到80.17%。呈味核苷酸二钠含量随酶解时间缓慢增加。     

Analysis of dithiocarbamate fungicides. Reaction products of the thiuram disulphide fungicide thiram (TMTD) during acid hydrolysis

The acid hydrolysis products (CS₂, COS, and H₂S) of thiram (tetramethylthiram disulphide, TMTD) absorbed in a methanolic amine reagent (ethylenediamine, piperidine) were investigated by second derivative UV spectroscopy. When the hydrolysis temperatures are below the boiling point the formation of the side products COS and H₂S at the expense of CS₂ is unavoidable. Failure to carefully scrub the liberated gases with a lead acetate solution leads to the absorption of H₂S also in the amine reagent and, like COS, causes an absorption at 230 nm that is erroneously attributed to COS. With the second-derivative technique it has been undoubtedly proved that, under the conditions of the Deutsche Forschungsgemeinschaft method S 15, thiram liberates CS₂ and COS in a molar ratio of 1.92 to 0.06 (average of eight determinations) but not in a molar ratio of 1.5 to 0.5, as previously reported by some workers. Under these conditions it is impossible, contrary to earlier claims, to differentiate thiram from other dithiocarbamates based on the ratio of the hydrolysis products in residue analysis.

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Analytik der Dithiocarbamat-Fungicide. Reaktionsprodukte des Thiuramdisulfid-Fungicids Thiram (TMTD) während der Säurehydrolyse

Zusammenfassung Mittels Derivativ-UV-Spektroskopie wurden die Produkte der Säurehydrolyse (CS₂, COS und H₂S) des Fungicids Thiram (TMTD) nach Absorption in einem methanolischen Aminreagens (Ethylendiamin, Piperidin) ermittelt. Bei Hydrolysetemperaturen unter dem Siedepunkt ist die Bildung der Nebenprodukte COS und H₂S auf Kosten von CS₂ unvermeidlich. Erfolgt kein sorgfältiges Waschen der hydrolytisch freigesetzten Gase mit einer Bleiacetatlösung, so wird auch H₂S im Aminreagens absorbiert, das wie COS eine UV-Absorption bei 230 nm verursacht und die COS-Bestimmung verfälscht. Mit der Derivativspektroskopie konnte eindeutig gezeigt werden, daß Thiram unter den Bedingungen der DFG-Methode S 15 zu CS₂ and COS im molaren Verhältnis von 1,92 zu 0,06 (8 Messungen) hydrolysiert wird und nicht, wie von anderen Autoren angenommen, im molaren Verhältnis von 1,5 zu 0,5. Daher ist es, den gehegten Hoffnungen entgegen, nicht möglich, auf der Basis der Verteilung der Hydrolyseprodukte Thiram von anderen Dithiocarbamaten rückstandsanalytisch zu unterscheiden.

     

植物油脂水解工艺及不饱和脂肪酸组成研究

研究了碱性水解小桐子油脂制备脂肪酸的较佳工艺条件：油水比（g/g）为2：1,乙醇-水比（v/v）为2：1,皂化时间1.5h、温度70℃,在该条件下脂肪酸得率、酸值和碘值的平均值分别为：98.25%、203.67mgKOH/g和109.89g/100g。采用桐油、光皮油、棕榈油和大豆油等植物油脂考察了该工艺的原料适用程度,该水解工艺条件下各种油脂制备所得的脂肪酸得率和酸值都接近98%和199mgKOH/g。采用GC-MS分析五种植物油脂脂肪酸组成和含量,其不饱和脂肪酸含量依次为：桐油（94.88%）〉大豆油（84.07%）〉光皮油（81.90%）〉小桐子油（77.84%）〉棕榈油（77.19%）;其中多价不饱和脂肪酸含量次序为桐油（85.36%）〉大豆油（58.01%）〉棕榈油（56.81%）〉光皮油（55.31%）〉小桐子油（42.16%）。大豆油、棕榈油、光皮油和小桐子油四种植物油脂脂肪酸成分基本相同,主要是亚油酸、7,10-十八碳二烯酸、油酸。桐油中脂肪酸组成与其他植物油脂有较大区别,含大量三价不饱和脂肪酸,其主要成分有油酸（9-十八烯酸）13.83%、8,11-十八碳二烯酸24.37%、亚麻酸（9,12,15-十八碳三烯酸和6,9,15-十八碳三烯酸）60.01%。     

Transformation of oleuropein and its hydrolysis products during Spanish-style green olive processing

Products obtained from elenolic acid glucoside hydrolysis during the storage of Spanish-style green olive brines were elenolic acid and glucose. The disappearance of elenolic acid glucoside from brines followed a first-order kinetics rate and was influenced by the storage temperature. Hydrolysis of this glucoside also occurred in brines ultrafiltered at 10000 Da pore size, indicating that no enzymatic action was needed to break the glycoside bond. Inoculation experiments with microorganisms obtained from a Spanish-style green olive brine demonstrated that this microflora was able to use the glucose formed from elenolic acid glucoside hydrolysis, although in our experiments only yeasts could grow in the assayed brines. It needs to be stressed that the glucose liberated by elenolic acid glucoside hydrolysis may represent about 20% of the total initial glucose in brines at the start of the fermentation step in Spanish-style green olive processing. © 1998 SCI.     

OCC稀酸水解液发酵制取乙醇的研究

在硫酸质量分数4%、温度190℃、时间40min、液固比15（ml/g）、助催化剂（FeSO4）1%情况下以二次纤维（OCC）为原料制取水解液。在温度28℃、转速150r/min、接种量10%条件下,选取酿酒酵母（CICC1001）对水解液发酵制取乙醇进行了研究,考察了菌种驯化、水解液过中和处理以及助催化剂（FeSO4）对乙醇产量的影响。结果表明：驯化酿酒酵母可以提高OCC水解液发酵速率以及乙醇产量;以Ca（OH）2过中和处理后产乙醇量明显优于中和处理,最高乙醇产量比中和处理可高出85.20%;加入FeSO4的水解液与未加入FeSO4水解液发酵规律一致,而未加入FeSO4水解液发酵产乙醇量滞后于加入FeSO4的水解液。     

Fluorometric determination of chemically available lysine: adaptation, validation and application to different milk products

A spectrophotometric method based on the reaction between available lysine and ortho-phthaldialdehyde (OPA) was adapted and validated for fluorometric determination of the chemically available lysine contents in milk matrices (UHT and conventional in-bottle sterilized cow milk, milk-based infant formulas and infant formula ingredients). The values of the analytical parameters show its usefulness as a routine method (linearity, r = 0.9992; detection limit, 0.0066 mg/mL assay; accuracy, 99-108%; precision, intra-day 2.1-5.9% and inter-day 3.5 10.2%). No statistically significant differences (p < 0.05) were found between the values obtained with the adapted method and those obtained applying the 1-fluoro-2,4-dinitrobenzene (FDNB) (Carpenter) technique. The OPA method was used to measure the chemically available lysine contents in UHT and sterilized milk marketed in Spain, to study the evolution of chemically available lysine during the shelf-life of UHT milks, and finally the quality of name- and store-brand UHT milks was also compared. No statistically significant differences (p < 0.05) were found between either the available lysine contents of the same type of UHT or sterilized milk or between store- and name-brand UHT milks. Statistically significant differences (p < 0.05) were found between the chemically available lysine contents in UHT and sterilized milk. Losses of chemically available lysine ranging from 2.7 to 29% were obtained during the shelf-life of UHT milk.     

肉制品中苯甲酸、山梨酸检测的前处理方法研究

依据苯甲酸、山梨酸易溶于水这一特性,用蒸馏水作为提取剂,将样品中的苯甲酸、山梨酸提取出来,然后选用适宜的蛋白沉淀剂去除提取液中的大量水溶性蛋白质。该方法经济、方便、快捷,是检测肉制品中苯甲酸、山梨酸较为理想的前处理方法。     

低值鱼酶解工艺的研究

为了运用高新技术开发利用低值鱼,创造经济效益,防止环境污染,研究了梅鱼在不同蛋白酶作用下的水解情况.结果表明,中性蛋白酶的效果最好,最佳工艺条件为:固液比为1:1,加酶量150 u/g鱼蛋白,温度为45℃,反应时间为4 h.水解液经真空浓缩后,得到一种含有多种氨基酸和多肽的高营养价值的产物.