Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein.

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

Effect of protein fortification on heat damage and occurrence of ß-casomorphins in (un)digested donor human milk intended for nutrition of preterm infants.

Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides ß-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.

Alkali extraction of rice residue protein isolates: Effects of alkali treatment conditions on lysinoalanine formation and structural characterization of lysinoalanine-containing protein.

The influence of alkali extraction conditions on the formation of lysinoalanine (LAL) and the structural characterization of lysinoalanine-containing protein in rice residue protein isolates (RRPI) were explored in this study. It was found that LAL content increased from 0.256 to 13.079 g/kg as NaOH concentration increased from 0.03 to 0.09 M and then decreased to 1.541 g/kg at 0.13 M NaOH. The extraction temperature and time were found to have a positive correlation with LAL content. The highest LAL content (25.679 g/kg) was observed with alkali extraction using 0.09 M NaOH at 75 °C for 120 min. The comparative structural analysis results showed that alkali treatment could degrade cystine, lysine, threonine and arginine to generate LAL; increasing alkali content would cause variations in secondary structure and micropore appearance on the surface of lysinoalanine-containing protein, whereas increasing alkali treatment temperature and time could enlarge the surface particle size of the protein.

Alkali solution extraction of rice residue protein isolates: Influence of alkali concentration on protein functional, structural properties and lysinoalanine formation.

This study evaluated the nutrient property and safety of the rice residue protein isolates (RRPI) product (extracted by different alkali concentrations) by exploring the protein functional, structural properties and lysinoalanine (LAL) formation. The results showed that with the rising of alkali concentration from 0.03M to 0.15M, the solubility, emulsifying and foaming properties of RRPI increased at first and then descended. When the alkali concentration was greater than 0.03M, the RRPI surface hydrophobicity decreased and the content of thiol and disulfide bond, Lys and Cys significantly reduced. By the analysis of HPLC, the content of LAL rose up from 276.08 to 15,198.07mg/kg and decreased to 1340.98mg/kg crude protein when the alkali concentration increased from 0.03 to 0.09M and until to 0.15M. These results indicated that RRPI alkaline extraction concentration above 0.03M may cause severe nutrient or safety problems of protein.

Effect of basic alkali-pickling conditions on the production of lysinoalanine in preserved eggs.

During the pickling process, strong alkali causes significant lysinoalanine (LAL) formation in preserved eggs, which may reduce the nutritional value of the proteins and result in a potential hazard to human health. In this study, the impacts of the alkali treatment conditions on the production of LAL in preserved eggs were investigated. Preserved eggs were prepared using different times and temperatures, and alkali-pickling solutions with different types and concentrations of alkali and metal salts, and the corresponding LAL contents were measured. The results showed the following: during the pickling period of the preserved egg, the content of LAL in the egg white first rapidly increased and then slowly increased; the content of LAL in the egg yolk continued to increase significantly. During the aging period, the levels of LAL in both egg white and egg yolk slowly increased. The amounts of LAL in the preserved eggs were not significantly different at temperatures between 20 and 25ºC. At higher pickling temperatures, the LAL content in the preserved eggs increased. With the increase of alkali concentration in the alkali-pickling solution, the LAL content in the egg white and egg yolk showed an overall trend of an initial increase followed by a slight decrease. The content of LAL produced in preserved eggs treated with KOH was lower than in those treated with NaOH. NaCl and KCl produced no significant effects on the production of LAL in the preserved eggs. With increasing amounts of heavy metal salts, the LAL content in the preserved eggs first decreased and then increased. The LAL content generated in the CuSO4 group was lower than that in either the ZnSO4 or PbO groups.

Detection of milk powder and caseinates in Halloumi cheese.

Halloumi cheese is traditionally manufactured from fresh milk. Nevertheless, dried dairy ingredients are sometimes illegally added to increase cheese yield. Lysinoalanine and furosine are newly formed molecules generated by heating and drying milk protein components. The levels of these molecular markers in the finished Halloumi have been investigated to verify their suitability to reveal the addition of skim milk powder and calcium caseinate to cheese milk. Because of the severe heating conditions applied in curd cooking, genuine Halloumi cheeses (n=35), representative of the Cyprus production, were characterized by levels of lysinoalanine (mean value=8.1 mg/100g of protein) and furosine (mean value=123 mg/100g of protein) unusual for natural cheeses. Despite the variability of the values, a good correlation between the 2 parameters (R=0.975) has been found in all cheeses, considering both the fresh and mature cheeses as well as those obtained from curd submitted to a prolonged cooking following a traditional practice adopted by a very small number of manufacturers. Experimental cheeses made by adding as low as 5% of skim milk powder, or calcium caseinate, or both, to cheese milk fell outside the prediction limits at +/-2 standard deviation of the above-reported correlation regardless of curd cooking conditions or ripening length. This correlation may be adopted as a reliable index of Halloumi cheese genuineness.

Liquid infant formulas: technological tools for limiting heat damage.

In a study considering 15 commercial samples of liquid milk-based infant formulas (MBF) from different manufacturers, the levels of selected molecules, that is, furosine (FUR), galactosyl-beta-pyranone (GAP), lactulose (LCT), and lysinoalanine (LAL), have been measured to provide estimation of the heat damage in these products. The ranges of the studied markers were as follows: FUR=153-600 mg 100 g(-1) of protein, GAP=0.5-4.3 mg L(-1), LCT=226-1511 mg L(-1), and LAL=1.0-16.1 mg 100 g(-1) of protein. The highest levels were found in MBF intended for the youngest babies. Experimental samples were produced in an industrial plant to evaluate the relative contribution of individual technological aspects to the final heat damage. About 90% of both GAP and LCT contents was due to the ultrahigh-temperature sterilization process itself. This effect was more than halved when the pH of the ingredient mixture was adjusted from 7.2 to 6.9 before sterilization or when the product recirculated in the plant was discarded. Up to 60 and 20%, respectively, of the FUR and LAL levels in the finished product were already present in protein ingredients (whey powder, whey protein concentrate). Accurate optimization of processing conditions and scrupulous selection of raw materials proved to be effective means to minimize heat damage in such special food products.

Dehydroalanine and lysinoalanine in thermolyzed casein do not promote colon cancer in the rat.

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.

Synthesis of two peptide mimetics as markers for chemical changes of wool's keratin during skin unhairing process.

The sheep skins unhairing process with preliminary alkaline treatment of the wool leads to two unnatural dipeptide mimetics lysinoalanine (Lys(*) - Ala) and ornithinoalanine (Orn(*)- Ala) obtaining. They are result from the keratin hydrolysis process. The changes of wool keratin make it resistant to sulphide degradation. We synthesized and characterized these unnatural dipeptides under the experimental conditions. The structures and mechanism of Lys(*) - Ala and Orn(*)- Ala obtaining were elucidated. The using of newly synthesized products as markers for control of wool's keratin changes during skin unhairing process was demonstrated. The developments have also been the result of economic and environmental pressures to meet environmental regulations.

Simultaneous analysis of lysine, Nepsilon-carboxymethyllysine and lysinoalanine from proteins.

Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.

A GC-FID method for analysis of lysinoalanine.

Lysinoalanine (LAL) is an unwanted byproduct, which is formed during the processing of protein and protein-containing foods and feeds. A GC method for the quantitative analysis of LAL under conventional chromatographic conditions has been developed. The method was applied to the analysis of pure standard substances, boiled eggs, commercial caseinates, fresh cheese, fresh cheese made from milk supplemented with caseinate, and fresh cheeses adulterated with caseinate after cheese making process. Results demonstrated the reliability of the GC capillary chromatography for the analysis of LAL in protein containing foods. LOD and LOQ of 50 and 152 ppm of LAL in protein, respectively, were achieved. Range of linearity, precision, and accuracy of the method, measured using diaminopimelic acid as internal standard, were satisfactory for quantification purpose. The method might also be suitable for the quantitative analysis of other amino acids such as lysine and arginine. Results also indicated the utility of this methodology for detecting protein quality of egg products and caseinates as well as fresh cheese adulterations.

Dose-dependent utilisation of casein-linked lysinoalanine, N(epsilon)-fructoselysine and N(epsilon)-carboxymethyllysine in rats.

During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.

The absorption and metabolism of modified amino acids in processed foods.

The chemical reactions involved in the modifications of amino acids in processed food proteins are described. They concern the Maillard reaction, reaction with polyphenols and tannins, formation of lysinoalanine during alkaline and heat treatments, formation of isopeptides, oxidation reaction of the sulfur amino acids, and isomerization of the L-amino acids into their D-form. Information on the digestion, absorption, and urinary excretion of the reaction products obtained by using conventional nutritional tests is given. The studies that have been made on the metabolism of these molecules by using a radioisotopic approach to follow their kinetics in the organism after ingestion are also reviewed. This approach provides unique data on the quantitation of the metabolic pathways and on the kinetics of the metabolic processes involved.

Dehydroalanine crosslinks in human lens.

This study was conducted to develop a methodology for the purification and detection of histidinoalanine, lanthionine and lysinoalanine in the lens tissue. Cataractous and aged human lens proteins were hydrolysed and fractionated by using anion-exchange chromatography. The fraction containing the bulk of dehydroalanine crosslinks was derivatized with dansyl chloride and then separated and quantified by means of RP-HPLC. The spectral and chromatographic properties of all three substances purified and quantified in this study were identical to those of their synthesized counterparts. Histidinoalanine and lanthionine were the most abundant dehydroalanine crosslinks in both water-soluble and water-insoluble lens proteins. Histidinoalanine levels in water-soluble proteins from the cataractous lenses of Indian origin were 6.2-fold higher than those in water-soluble proteins from normal lenses (1.68+/-0.75 vs 0.26+/-0.06 nmol/mg protein; p<0.001). In water-insoluble proteins, they were 2.2-fold higher in cataractous lenses compared with normal lenses (1.59+/-0.76 vs 0.73+/-0.17 nmol/mg protein; p<0.01). Lanthionine levels were significantly higher in water-insoluble proteins of cataractous lenses when compared to non-cataractous lenses (2.5+/-1.68 vs 0.95+/-0.08 nmol/mg protein; p<0.03). Unlike histidinoalanine, this crosslink appears to accumulate in relatively high concentrations in water-soluble lens proteins; its concentration was 9-fold higher than histidinoalanine from the same proteins (0.26+/-0.06 HAL vs 2.34+/-0.76 LAN nmol/mg protein; p<0.0004). The concentration of lysinoalanine was in the picomolar range and in cataractous lens proteins only.

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.

Changes of amino acid composition and lysinoalanine formation in alkali-pickled duck eggs.

Duck eggs were pickled in alkali for 20 days to prepare Pidan. The extent of the degradation of compositional amino acids, the formation of lysinoalanine (LAL) in Pidan, and the relationship between the formation of LAL and the racemization values of D-serine and D-aspartic acid in Pidan albumen during the pickling period were investigated. Results showed that the remaining percentages of Cys, Arg, Lys, Ser, and Thr in albumen were much lower than that of the corresponding amino acid in yolk. The formation of LAL in albumen in the first stage was due to the speedy increase in the pH and the abundant formation of dehydroalanine (DHA) from cysteine. However, the formation of LAL in the later pickling period was related much more to the alkali-treating time than to the pH factor. Among the amino acids, cysteine was observed to be the most sensitive to alkaline and contributed mostly to the formation of LAL throughout the pickling period.

Detection and identification of the cross-linking amino acids N tau- and N pi-(2'-amino-2'-carboxy-ethyl)-L-histidine ("histidinoalanine", HAL) in heated milk products.

An unknown ninhydrin positive compound, X, was detected in acid hydrolysates of heated skim milk samples by amino acid analysis, eluting between phenylalanine and pyridosine in the chromatogram. The formation of X correlated with heating time and temperature. preparative ion-exchange chromatography enabled the isolation of X and a second minor compound from a milk protein hydrolysate and from a model mixture consisting of N alpha-acetylhistidine and methyl-2-acetamidoacrylate (acetyldehydroalaninemethylester), in a relative abundance of 8 to 1. By 1H-NMR spectroscopy, the two compounds could be identified as the N tau- and N pi-isomers of N-(2'-amino-2'-carboxy-ethyl)-L-histidine (histidinoalanine), a cross-link amino acid that has not been described in food proteins up to now. In a number of foods containing milk protein, the N tau-histidinoalanine contents were between 50 and 1800 mg/kg protein, which is in a concentration range comparable to the potential nephrotoxic cross-link lysinoalanine, which was determined simultaneously.

Direct analysis of the disulfide content of proteins: methods for monitoring the stability and refolding process of cystine-containing proteins.

So far, the cystine (disulfide) content of proteins has been routinely determined by indirect methods, i.e., cystines are first converted to more stable half-cystines prior to analysis. We present a study which demonstrates that the cystine content can be directly and accurately analyzed at low picomole to femtomole levels. The method involves (a) the employment of a vacuum during sample hydrolysis which permits quantitative recovery of cystine, and (b) the dabsyl chloride precolumn derivatization method which yields stable DABS-cystine that is subsequently analyzed by HPLC. Direct analysis of the cystine residue is important in numerous areas of protein research. It allows, by a single analysis, simultaneous determination of the sulfhydryl (cysteine, as S-carboxymethyl derivative) and disulfide (cystine) contents. The technique can be used to follow the refolding process of fully reduced, cystine-containing proteins. Direct analysis of the cystine content also serves to monitor the extent of inactivation of cystine-containing proteins caused by alkaline pH and heat treatments. In this mode of protein inactivation, cystines are selectively destroyed and converted to lanthionine and lysinoalanine. Both the decrease of cystine and the recoveries of lanthionine and lysinoalanine can be simultaneously evaluated by the proposed method. Examples of these applications are presented here.

Rapid method for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine and the amino sugars in proteins and tissues.

A rapid and sensitive chromatographic method is described for determining desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine, glucosamine and galactosamine at picomole levels in protein and tissue hydrolysates. This method uses either an automated amino acid analyser with a 17.5 X 0.28 cm microcolumn packed with 6.0 +/- 0.5 micron spherical resin, thermostated at 52 degrees C, one buffer system (0.21 M sodium citrate, pH 5.125) and 3-nitrotyrosine as the internal standard, or conventional instruments using the same system but with larger diameter columns and resins (11.0 +/- 1.0 micron). This method should be especially valuable for determining collagen and elastin in tissue hydrolysates from the amounts of 5-hydroxylysine, and desmosine or isodesmosine present, respectively, and for studying protein hydroxylation, glycosylation, cross-linking formation, and the turnover rates of collagen and elastin in normal and diseased tissues.