Formyline, a new glycation compound from the reaction of lysine and 3-deoxypentosone

A previously uncharacterized glycation compound was isolated from a reaction mixture of N-α-Boc-lysine and 3-deoxypentosone by semi-preparative ion-exchange chromatography and was identified by nuclear magnetic resonance (NMR) spectroscopy as 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline). 3-Deoxypentosone and pentoses like ribose, arabinose, or xylose were identified as the predominant precursors of the new glycation compound, but formyline can also be formed from lysine and degradation products of disaccharides and glucuronic acid. The Amadori products lactuloselysine and ribuloselysine, which were synthesized by improved methods and characterized by NMR spectroscopy, were shown not to form formyline when heated in dry state without lysine added, indicating that reactions between the lysine side chain and dicarbonyl compounds are the main pathways for formyline formation rather than transformation of lysine containing Amadori products. Finally, it was shown by HPLC analysis after enzymatic digestion that peptide-bound formyline can be formed during incubation of casein and 3-deoxypentosone under conditions comparable to food processing.     

RAPID HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETECTION OF FUROSINE (ε‐N‐2‐FUROYLMETHYL‐L‐LYSINE) IN YOGURT AND CHEESE MARKETED IN TURKEY*

Furosine (ε‐N‐2‐furoylmethyl‐L‐lysine) content determination in the yogurt and different cheese types (pickled white, kasar, processed, canned tulum, blue‐veined and mozzarella cheeses) marketed in Turkey was performed using ion‐pair reversed‐phase high performance liquid chromatography (RP‐HPLC). Calibration study (R² = 0.9999), analytical method validation and recovery studies gave satisfactory results. The lowest furosine values were observed in pickled white cheeses (5.35 ± 0.01 to 7.28 ± 0.02 mg/100 g protein). All cheeses except pickled white showed furosine values between 182.16 ± 0.12 (canned tulum) and 261.32 ± 0.10 mg/100 g protein (ripened kasar). The highest content of furosine was observed in whole yogurt (316.47 ± 0.17 mg/100 g protein) which could be because of severe heat treatment and the addition of milk powder during the manufacturing process. The method provides a rapid, reproducible and accurate determination of this Amadori compound (ε‐deoxy‐fructosyl‐lysine) in yogurt and cheese samples.     

Forty years of furosine - forty years of using Maillard reaction products as indicators of the nutritional quality of foods

The Maillard reaction products (MRPs) most widely used as markers of the nutritional quality of foods are furosine, N(epsilon)-carboxymethyllysine (CML), hydroxymethylfurfural, pyrraline, pentosidine and pronyl-lysine. One of the MRPs identified first was furosine, which was quantified in foods 40 years ago as a chemical indicator of the Amadori compound N(epsilon)-fructoselysine. Since then, furosine has gained broad attention by food chemists and biomedical researchers, as its formation upon heat treatment is well characterised. Moreover, it represents the Amadori products from early Maillard reactions in which amino acids react with reducing carbohydrates, resulting in a loss of their availability. This is of importance for the essential amino acid lysine, which is also the limiting amino acid in many proteins. In order to evaluate the nutritional quality of a protein, the concomitant analysis of free - and nutritionally available - lysine and the amount of lysine reacted to form the respective MRP is essential, even for mildly processed foods. The other chemical markers of heat treatment such as CML, pyrraline, pentosidine or pronyl-lysine seem to be useful markers of the advanced stages of Maillard reactions. Compared to the conditions in which furosine is formed, these compounds are generated under more severe conditions of heat treatment. However, the concentrations analysed are significantly lower than those of furosine. Therefore, the nutritional evaluation of a food protein should include not only furosine, but also other chemical markers of heat treatment such as, for example, CML, pyrraline and pentosidine.     

Effect of red sweet pepper dehydration conditions on Maillard reaction,ascorbic acid and antioxidant activity

In this study we evaluated the application of low pressure-controlled temperature drying in the production process of dehydrated sweet red pepper and contrast the quality of this dehydrated vegetable in terms of the concentration of some heat liable components (ascorbic acid), decrease on the antioxidant activity and formation of furosine (Amadori compound of lysine formed during the Maillard reaction). Analysis of data showed that furosine is a good indicator for controlling the dehydration process of red pepper. In addition, the stability of the antioxidant activity and proteical damage (measured as furosine formation) in red pepper is a function of the dehydration conditions: Higher temperatures exert a strong influence on the kinetics of degradation, accelerating the rate of decomposition of antioxidant compounds and the loss of proteical quality.     

Furosine as an index of heat treatment intensity in meat products: Its application to cooked ham

Furosine, a product of acid hydrolysis of Amadori compounds, has been proposed as an index of the heat treament intensity in various food products. In this paper we suggest furosine as an index of heat treatment in pork-meat products as well. Furosine is not detectable in fresh raw pork muscle and in injected tumbled pork muscle, the latter being used for the production of cooked ham. Activation energy of furosine formation in raw muscle and tumbled muscle, in the temperature range of 70 °-90 °C, was 79.2 kJ/mole and 81.7 kJ/mole, respectively. Furosine concentration was assessed in cooked hams whose time-temperature profiles, with reference to the cooking and cooling processes, were well known, thus enabling the verification of the feasibility of its use in the evaluation of heat damage. The good correlation between the values found during this investigation and the values foreseen by kinetic calculation confirms that furosine can be used as an index of heat treatment intensity in the production of cooked ham.     

赖氨酸Amadori和Heyns化合物热裂解产物对烟草风味的影响研究

  目的  比较赖氨酸Amadori和Heyns化合物的热解产物及对其烟草风味的影响。  方法  以赖氨酸和葡萄糖或果糖为原料,以偏重亚硫酸钠为催化剂、甲醇和冰醋酸为溶剂,分别合成赖氨酸的Amadori和Heyns化合物,采用在线热裂解-气相色谱/质谱联用技术(Py-GC/MS)对比了2种化合物的热解产物差异,并通过感官评吸实验比较了二者对卷烟风味的影响。  结果  （1）赖氨酸Amadori和Heyns化合物热解均能产生醛酮类、呋喃类、吡啶类、吡嗪类及吡咯类香味物质,同一类别产物的相对含量有较大差异。（2）赖氨酸Heyns化合物热解得到的风味成分数量更多。（3）赖氨酸Amadori化合物和Heyns化合物对卷烟的主要作用为增香、增浓和降低刺激,并使烟气更加流畅顺滑,赖氨酸Heyns化合物增加了卷烟白肋烟特征。  结论  赖氨酸Amadori化合物和Heyns化合物的热解产物不同,对卷烟风味的影响存在差异。      

Modelluntersuchungen zu Bedingungen der Bildung vonN ε-Carboxymethyllysin in Lebensmitteln

In model experiments with equimolar mixtures of lysinemonohydrochloride and glucose [88% (w/v) water content, 100° C heating temperature] the influence of several conditions (hydrolysis, pH) and ingredients (iron, phosphate, and nitrite) on the formation ofN ^ε-carboxymethyllysine (CML) were evaluated. CML was analysed using a reversed-phase HPLC-method after derivatisation witho-phthaldialdehyde. CML, which is an oxidative derivative of fructoselysine, is also formed during the acid hydrolysis applied for amino acid determination in food products. In model mixtures without hydrolysis only 8–21% CML compared to that in hydrolysed samples was found. Therefore, in food products all hydrolyses for CML must be performed after borohydride reduction in order to destroy fructoselysine. This can be controlled by the determination of furosine. The pH of the model mixtures considerably influenced the formation of CML. At pH 4.0 only 70 mg, at pH 7.0 370 mg, and at pH 9.0 3170 mg CML/kg lysine were determined. The CML concentration also clearly increased with higher concentrations of iron, phosphate, and nitrite. This is explained by a promoting effect on the oxidation of fructoselysine to CML.     

Quantification of Amadori products in cheese

To obtain basic information about the extent of the early Maillard reaction in commercially available cheese varieties of varying degrees of maturity, Amadori products (APs) formed between reducing sugars and the ε-amino group of lysine (ε-APs) or the α-amino of selected amino acids (α-APs) were analyzed. APs were determined as the corresponding N-(2-furoylmethyl) amino acids (FMAAs) after acid hydrolysis using RP–HPLC with UV-detection as well as mass spectroscopy. Identification of the FMAAs resulting from the α-APs of lysine, valine, leucine, and isoleucine was achieved for the first time for cheese samples. The content of furosine, which is a hallmark for ε-APs, ranged from to 3–75 mg/100 g protein corresponding to 24–591 μmol ε-AP/100 g protein. ε-AP levels declined during ripening in all investigated cheeses, depending on the cheese type and stage of maturation. The mean content of four α-APs ranged from 138 to 681 μmol/100 g protein. Lowest concentrations were found in long-term ripened cheese samples, indicating a putative degradation of α-APs and ε-APs during advanced stages of maturation. The ratio of α-APs to ε-APs might be a useful indicator of cheese ripening.     

奶及奶制品中糠氨酸检测方法的研究进展

糠氨酸源于美拉德反应的早期产物,并最终经酸水解产生。它间接反映了美拉德反应的程度、赖氨酸营养功能的损失程度和食品受热程度等,被广泛应用于食品的品质评价。虽然糠氨酸在酸水解时的产率仅有30~40%,但在相同的检测条件下,该产率是稳定的。近40年来,糠氨酸的检测方法研究主要集中于色谱分离系统和检测器的选择与优化,以及酸水解条件对糠氨酸产率的影响。本文针对奶及奶制品中糠氨酸的检测方法,从高效液相色谱法（high performance liquid chromatography, HPLC）与检测标准的建立、其他色谱技术或检测器的应用、酸水解条件的研究等方面进行综述,并对目前的主要问题和研究方向进行了展望,为进一步开发更高效、更灵敏、更准确和更稳定的新检测方法提供参考。     

Fast and sensitive determination of furosine

Sensitive determination of furosine in acid hydrolysates of foods was achieved by isocratic ion-pair reversed-phase HPLC and direct UV-detection within a run time of 5 minutes and levels lower than 1.5 mg per kg of protein. The formation of furosine during hydrolysis of food samples with hydrochloric acid of varying concentration was studied. Furosine formation increased linearly with acid concentration (4 to 8 mol/L).

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Methode zur schnellen und empfindlichen Bestimmung von Furosin

Zusammenfassung Mittels isokratischer Ionenpaar-RP-HPLC gelang die empfindliche Bestimmung von Furosin in Säurehydrolysaten von Lebensmitteln innerhalb von 5 Minuten Laufzeit und mit einer Nachweisgrenze von unter 1,5 mg pro kg Protein. Die Bildung von Furosin während der Hydrolyse von Lebensmittelproben erwies sich zwischen Konzentrationen von 4 und 8 mol/L als linear von der Salzsäurekonzentration abhängig.

     

KINETICS OF LYSINE LOSS IN COMPRESSED MODEL SYSTEMS DUE TO MAILLARD REACTION1

The kinetics of degradation of the essential amino acid lysine have been determined in compressed lysine-glucose-cellulose models exposed to expected and accelerated storage test temperature extremes. The compressed models were stressed at 40, 50 and 60°C at low water activities in the range of 0.19 to 0.21 for various periods. Six different parameters (color, fluorescence, reducing capacity, furosine, glucose and lysine) were employed to follow the course of the reaction. The t 1/2 values estimated from first order plots for lysine degradation in these models were 370, 13 and 1.5 h at 40, 50 and 60°C, respectively. An Arrhenius plot relating the first order reaction rate constant with the temperature has yielded a high activation energy of 57 kcal/mol, and a high Q10 of 14, which is indicative of large temperature dependence of lysine degradation in these compressed models. There was poor correlation of fluorescence and of color increase with lysine loss. Good correlations were obtained between lysine loss and the increases in reducing capacity and furosine at 50 and 60°C.     

Glycation compounds in peanuts

In the present study, the extent of the Maillard reaction in peanuts was investigated, using N-ε-fructosyllysine (ε-Fru-Lys, determined via furosine) as an indicator for the early stage, and pyrraline and N-ε-carboxymethyllysine (CML) as representatives for advanced glycation. In commercial samples, ε-Fru-Lys ranged between 1.5 and 13.3 mmol/mol lysine. Pyrraline was found in amounts between not detectable (below 0.3 mmol/mol lysine) and 9.0 mmol/mol lysine, and CML between 0.8 and 2.7 mmol/mol lysine. Lysine modification by glycation products was very low in cooked and fried peanuts (below 1%). In laboratory-scale roasting experiments, the amount of ε-Fru-Lys reached maximum values of 12.0 mmol/mol lysine after 20 min at 160 °C, whereas pyrraline increased up to 38.5 mmol/mol lysine after 25 min at 170 °C. Amount of CML was up to 1.8 mmol/mol lysine in peanuts roasted for 25 min at 170 °C. Such high amounts of pyrraline have not yet been described for any other food item. Only about one tenth of the totally observed lysine modification of up to 50% can be explained by the three glycation compounds, indicating that currently unknown reactions occur during peanut roasting. Reactions between proteins and carbonyl compounds, most likely originating from oxidative degradation of lipids, may play an important role for lysine derivatization in peanuts and should be analyzed more in detail.     

Blocked Lysine in Dairy Products: Formation,Occurrence, Analysis,and Nutritional Implications

Lysine residues in milk proteins become “blocked” when they react with reducing sugars, particularly lactose, in the Maillard reaction. The blocked or glycated lysines reduce the biological availability of the lysine to metabolic processes and also hinder hydrolysis of the parent protein by digestive enzymes. Heating and storage of milk and milk products are the major promotants of the Maillard reaction and resulting chemical damage to the proteins. Several methods have been proposed to estimate the extent of this protein damage. Two major compounds, furosine, a product of acid hydrolysis of lactulosyl‐lysine, the 1st stable product of the Maillard reaction, and carboxymethyl‐lysine are used for assessing the early and advanced stages of the Maillard reaction, respectively. In addition, several methods are used for assessing the bioavailability of lysine in a protein; these include chemical, enzymic, and animal‐based methods. This review discusses the Maillard reaction and its significance in milk and dairy products, methods of assessing the extent of the reaction and of the bioavailability of lysine, and the nutritional significance of blocked lysines and associated Maillard reaction products in milk proteins.     

Influence of Antioxidants on Maillard Browning Reaction in a Casein-Glucose Model System

Mixtures of casein and glucose with and without antioxidants were prepared to give a moisture content of 11%, and stored at 37°C. under 60% relative humidity for O-60 days. The effects of antioxidants on the Maillard reaction were monitored by browning, available lysine and furosine level. Browning decreased with the addition of α-tocopherol, BHT, BHA or propyl gallate but not ascorbic acid in mixtures stored 60 days. All antioxidants improved the retention of available lysine and lowered the concentration of furosine compared to the control.     

Development of the Maillard reaction in foods cooked by different techniques. Intake of Maillard-derived compounds

Specific and non-specific Maillard reaction (MR) indices such as CIELab colour, browning measurement, furosine, hydroxymethylfurfural (HMF) and furfural, as well as the nutrient content, were analysed for commonly consumed dishes, to test the effects of different culinary treatment on dishes composed of the same ingredients. In addition, the consumption of early MR products (MRP), Amadori compounds, HMF and furfural from a normal serving of these dishes was calculated. As expected, recipes including frying, apart from their particular composition, led to significantly higher values of furosine and HMF, ranging from 4.40 to 175 and from 0.30 to 22.7 mg/kg, respectively; consequently they provided the highest levels of Amadori compounds and HMF intake (0.42–26.8 and 0.02–2.38 mg/serving, respectively). Even so, MRP intake/serving was not very high in comparison with levels reported in the bibliography on some individual foods typically studied in terms of MR development, suggesting that the culinary treatments used do not make a great contribution to the daily MRP consumption.     

Fluorimetric determination of niacin in foods by high-performance liquid chromatography with post-column derivatization

A method to determine the content of niacin in various foods by liquid chromatography is proposed and includes hydrochloric acid hydrolysis of the sample, pre-column conversion of the different vitamers (NAD, NADP, nicotinamide and nicotinic acid) into nicotinic acid by alkaline hydrolysis, separation on a C18 stationary phase, post-column derivatization by UV irradiation in the presence of copper (II) ions and hydrogen peroxide, and fluorimetric detection. The proposed method leads to a good recovery rate (90–107%) and a satisfactory repeatability (coefficient of variation less than 4%). Owing to its very low quantification limit (0.2 μg g⁻¹) and the excellent resolution of the nicotinic acid peak, it could most probably be applied to the determination of niacin in any foodstuff. ©     

盐酸水解方法对鸡肉中氨基酸含量的影响

摘 要: 目的 研究盐酸水解过程中, 水解时间和添加巯基乙醇和苯酚保护剂对鸡肉中18种氨基酸含量的影响, 确定获得鸡肉氨基酸最高含量的盐酸水解方法。方法 以30个鸡胸肌(3种鸡、每种10个)为实验对象, 设计6个水解时间, 研究盐酸水解时间对鸡肉样品中18种氨基酸含量的影响, 确定获得氨基酸最高含量的盐酸水解时间。在上述研究的基础上, 随机选择6个鸡胸肌(3种鸡、每种2个)为实验对象, 设置3个水解时间, 研究巯基乙醇和苯酚对鸡肉样品中18种氨基酸含量的影响, 最终确定获得鸡肉中18种氨基酸最高含量的盐酸水解方法。结果 缬氨酸(valine, Val)和异亮氨酸(isoleucine, Ile)的最佳水解时间为24或26 h, 两水解时间无显著性差异(P>0.05); 其余16种氨基酸的最高含量均在22 h; Ile的含量在盐酸水解法水解24 h显著高于20 和22 h(P<0.05), 但在苯酚+盐酸水解法水解22h含量最高; 天冬氨酸(aspartate, Asp) 的含量在巯基乙醇+盐酸水解法水解22 h显著高于20 和24 h(P<0.05), 但在盐酸水解法水解22h含量最高; 甘氨酸(glycine, Gly)、丙氨酸 (alanine Ala)、酪氨酸(tyrosine, Tyr) 和苯丙氨酸 (phenylalanine, Phe)的含量在苯酚+盐酸水解法水解22 h显著高于20 h(P<0.05), 但水解22与24 h之间无显著性差异(P>0.05), 其中, Gly在苯酚+盐酸水解法水解22 h含量最高, Ala在盐酸水解法水解22 h含量最高, Tyr和Phe在巯基乙醇+盐酸水解法水解22 h含量最高; Val在盐酸水解法水解24 h含量最高, 脯氨酸(proline, Pro)和赖氨酸 (lysine, Lys)在苯酚+盐酸水解法水解22 h含量最高, 亮氨酸 (leucine, Leu) 在巯基乙醇+盐酸水解法水解22 h含量最高; 其余氨基酸在不同水解方法及水解时间中均无显著性差异(P>0.05), 但都在盐酸水解法水解22 h时含量最高。结论 巯基乙醇和苯酚对鸡肉中的含硫氨基酸无保护作用, 获得鸡肉中18种氨基酸最高含量的盐酸水解方法是盐酸水解法水解22 h。     

Usefulness of some Maillard reaction indicators for monitoring the heat damage of whey powder under conditions applicable to spray drying

The Maillard reaction (MR) rate was observed according to available lysine loss, furosine, hydroxymethylfurfural (HMF), furfural, and brown colour during the heating of freeze-dried nano-filtered whey at 60, 75, and 90 °C and water activities of 0.11, 0.33, 0.43, and 0.73. The physical state of lactose was measured and associated with MR rate. The values obtained for available lysine, furosine, HMF and browning index ranged between, respectively, 11.3 and 1.63 (g 100 g⁻¹ protein), 0.44 and 11.1 (g 100 g⁻¹ protein), not detected and 57.7 (mg 100 g⁻¹ protein) and 0.0104 and 0.1707. The greatest heat damage occurred with low moisture content and high temperature. The MR rate was influenced by the physical state of lactose, heating temperature and the moisture content of the whey. This occurred to a greater extent during the initial and intermediate stages of the MR as opposed to during the formation of coloured compounds.     

Evaluation of the extent of the early Maillard-reaction in milk products by direct measurement of the Amadori-product lactuloselysine.

A new method for the evaluation of the extent of lysine modification caused by the Maillard reaction is presented, based on the direct determination of the Amadori product lactuloselysine plus unmodified lysine after complete enzymatic hydrolysis by ion-exchange chromatography. Using a simple mathematical relation, the amount of modified lysine in milk products can be estimated without knowledge of the initial lysine value before heating or storage.     

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.