Quantitation of lysinoalanine in dairy products by liquid chromatography–mass spectrometry with selective ion monitoring

The unnatural amino acid lysinoalanine (LAL) has been identified in milk and cheese products by liquid chromatography mass spectrometry (LC/ESI/MS) with selective ion monitoring (SIM) of the 9-fluorenyl-methylchloro-formate (FMOC) derivative. LAL is not present in raw milk or derived from Mozzarella cheese; however, high amounts of LAL are found in calcium caseinate and milk powder. As expected, milk fortified with caseinate or whey protein powder produces cheese with higher LAL content. Our analytical procedure is based on the simultaneous detection of specific ion masses of the FMOC–LAL derivative and the N-ε-methyl-lysine internal standard. A linear relationship was observed within the 0.2–20 ppm concentration range, in addition to a high correlation coefficient and ∼3% relative standard deviation.     

D-Lysine,alloisoleucine and lysinoalanine in supplementary proteins with different lysine availabilities

D -Lysine, alloisoleucine and lysinoalanine were determined in 16 commercial protein supplements on which lysine availability had been measured by slope-ratio assay with pigs and by chemical dinitrophenylation. About 2.5% of lysine was racemised by protein hydrolysis in 6 M HCl. Only three of 10 samples with poor lysine availability by slope-ratio assay contained significantly more D -iysine than control proteins (P < 0.01). D -Lysine was not significantly correlated with lysine availability by either method; nor did it improve the poor correlation between the slope-ratio assay and dinitrophenylation. The highest level of alloisoleucine was less than 14% that of isoleucine. In all proteins except skim milk powder lysinoalanine occurred at 0.3% or less of the corresponding lysine level. Neither alloisoleucine nor lysinoalanine was related to lysine availability.     

Effects of alkali treatment on the in-vitro digestibility of proteins and the release of amino acids

Three protein sources, casein, soya bean and rapeseed concentrates, were subjected to alkali treatment (0.2 M, 60° C) for 2 or 6 h. The impact of these treatments on protein digestibility and on release of amino acids, especially lysinoalanine, was evaluated by an in-vitro enzymic digestion method with simultaneous dialysis of digestion products. The impairment of digestibility was higher for casein and soya bean concentrate than for rapeseed concentrate. Whatever the amount of lysinoalanine produced in each protein, it was poorly released by proteolytic enzymes. The rate of release of other amino acids was reduced by the treatments, but to different levels for each protein. Arginine and lysine were particularly affected. As can be inferred from the release of the target amino acids, the hydrolytic capacity of chymotrypsin was not specifically impaired, in contrast to that of trypsin for casein and of elastase for all protein sources. This technique was useful to evaluate quickly the effects of processing on the digestibility of proteins.     

An analytical approach to reveal the addition of heat-denatured whey proteins in lab-scale cheese making

A simple analytical procedure for the detection of self-aggregated heat-denatured whey proteins (HDWP) in model cheeses was developed. The principle of the approach lies in the solubilization of the cheese matrix by a sodium citrate solution (0.2 M, pH 7.0) resulting in the dissociation of the casein micelles and the insolubilization of HDWP aggregates, which are collected in the pellet after a centrifugation step. The reliability of the procedure was tested in lab-scale cheeses from peroxidase-positive pasteurized milk with different protein-based ingredients (microparticulated whey protein concentrate, milk protein concentrate, whey protein isolate and Ricotta cheese) at concentrations ranging from 0.2 to 1.2% protein (w/v on cheese milk). A linear relationship between the amount of the HDWP added to cheese milk and that recovered from model cheeses was observed. Heat-damage indicators, furosine and lysinoalanine, showed levels in the experimental cheese samples not related with added HDWP, but represented a source of information on the ingredients other than liquid milk. Overall, in the model cheeses, the proposed method was an easy-to-apply and reliable tool for the evaluation of the presence of HDWP-based products. Further investigation is required for the application to real cheeses and for the evaluation of possible interferences from proteolysis during ripening.     

Effects of Sulfhydryl Compounds,Carbohydrates, Organic Acids,and Sodium Sulfite on the Formation of Lysinoalanine in Preserved Egg

To identify inhibitors for lysinoalanine formation in preserved egg, sulfhydryl compounds (glutathione, L‐cysteine), carbohydrates (sucrose, D‐glucose, maltose), organic acids (L‐ascorbic acid, citric acid, DL‐malic acid, lactic acid), and sodium sulfite were individually added at different concentrations to a pickling solution to prepare preserved eggs. Lysinoalanine formation as an index of these 10 substances was determined. Results indicate that glutathione, D‐glucose, maltose, L‐ascorbic acid, citric acid, lactic acid, and sodium sulfite all effectively diminished lysinoalanine formation in preserved egg albumen and yolk. When 40 and 80 mmol/L of sodium sulfite, citric acid, L‐ascorbic acid, and D‐glucose were individually added into the pickling solution, the inhibition rates of lysinoalanine in the produced preserved egg albumen and yolk were higher. However, the attempt of minimizing lysinoalanine formation was combined with the premise of ensuring preserved eggs quality. Moreover, the addition of 40 and 80 mmol/L of sodium sulfite, 40 and 80 mmol/L of D‐glucose, 40 mmol/L of citric acid, and 40 mmol/L of L‐ascorbic acid was optimal to produce preserved eggs. The corresponding inhibition rates of lysinoalanine in the albumen were approximately 76.3% to 76.5%, 67.6% to 67.8%, 74.6%, and 74.6%, and the corresponding inhibition rates of lysinoalanine in the yolk were about 68.7% to 69.7%, 50.6% to 51.8%, 70.4%, and 57.8%. It was concluded that sodium sulfite, D‐glucose,L‐ascorbic, and citric acid at suitable concentrations can be used to control the formation of lysinoalanine during preserved egg processing.     

Implications of antinutritional components in soybean foods

There are a number of components present in soybeans that exert a negative impact on the nutritional quality of the protein. Among those factors that are destroyed by heat treatment are the protease inhibitors and lectins. Protease inhibitors exert their antinutritional effect by causing pancreatic hypertrophy/hyperplasia, which ultimately results in an inhibition of growth. The lectin, by virtue of its ability to bind to glycoprotein receptors on the epithelial cells lining the intestinal mucosa, inhibits growth by interfering with the absorption of nutrients. Of lesser significance are the antinutritional effects produced by relatively heat stable factors, such as goitrogens, tannins, phytoestrogens, flatus‐producing oligosaccharides, phytate, and saponins. Other diverse but ill‐defined factors appear to increase the requirements for vitamins A, B₁₂, D, and E. The processing of soybeans under severe alkaline conditions leads to the formation of lysinoalanine, which has been shown to damage the kidneys of rats. This is not generally true, however, for edible soy protein that has been produced under milder alkaline conditions. Also meriting consideration is the allergenic response that may sometimes occur in humans, as well as calves and piglets, on dietary exposure to soybeans.     

食品加工过程中有害物质-赖丙氨酸研究进展

食品加工过程中产生的有毒有害物质是目前食品安全研究的一个热点。含蛋白质的食品在高温或碱处理时,赖氨酸易和胱氨酸、丝氨酸、磷酸丝氨酸或脱氢丙氨酸发生交联,生成一种新的氨基酸--赖丙氨酸。本文对赖丙氨酸的产生条件、机理、对实验动物和人体的危害及控制等方面的研究进展进行介绍,也对赖丙氨酸未来的研究方向作出展望。     

Lysinoalanine content of formulas for enteral nutrition

Casein and caseinates are the main ingredients of formulas for enteral nutrition. Their manufacturing procedure and the thermal treatments necessary to assure microbiological stabilization and satisfactory shelf-life of the end-products are particularly favorable for the formation of lysinoalanine (LAL), a cross-linked amino acid that is considered a useful marker of the thermal damage and reduced digestibility of proteins. The lysinoalanine content of 18 different kinds of formulas for enteral nutrition was determined by HPLC after derivatization. The liquid formulas have an average value of 528 microg/g protein LAL, ranging from 160 to 800 microg/g protein (average content of formulas for pediatric use 747 microg/g protein). These values are rather high considering that the average value detected in UHT-treated drinkable milk is 117 microg/g protein. In principle, the preparation of caseinates and the thermal stabilization of the end products are the two steps more favorable for the formation of LAL. The fact that the five samples stabilized by an UHT-treatment have an average value of 512 microg/g protein suggests that the LAL content depends more on the quality of the starting ingredients than on the sterilization process. A better selection of the starting ingredients should improve the quality of formulas for enteral nutrition, which is very desirable when formulating foods for consumers with very high nutritional demands.     

赖丙氨酸特性及检测

赖氨酸是食品中限制性氨基酸,在碱处理和加热时很易与蛋白质分子内其它氨基酸形成交联键,生成某些新氨基酸,如赖丙氨酸等。赖丙氨酸可由赖氨酸残基ε–氨基与脱氢丙氨酰残基缩合而成,后者可能通过胱氨酸残基碱降解、脱硫后生成,也可来自丝氨酸或磷酸丝氨酸残基分解。赖丙氨酸是一种金属螯合剂,因此,可使一些含有金属的酶,如羧肽酶A和羧肽酶B失去活性;且赖丙氨酸对小白鼠具有肾毒性。尽管至今为止,赖丙氨酸对人体毒性尚不明确,但在婴儿食品中,赖丙氨酸含量需严格控制;赖丙氨酸含量可作为蛋白质加工破坏程度和消化性能一个指标。常用的赖丙氨酸分析方法有气相色谱法、液相色谱法、氨基酸分析仪法和薄层层析法等。