Antioxidant mechanisms of enzymatic hydrolysates of beta-lactoglobulin in food lipid dispersions

The antioxidant activities of aqueous phase beta-lactoglobulin (beta-Lg) and its chymotryptic hydrolysates (CTH) were compared in this study. Proteins and peptides have been shown to inhibit lipid oxidation reactions in oil-in-water emulsions; however, a more fundamental understanding of the antioxidant activity of these compounds in dispersed food lipid systems is lacking. CTH was more effective than an equivalent concentration of beta-Lg in retarding lipid oxidation reactions when dispersed in the continuous phase of Brij-stabilized oil-in-water emulsions (pH 7). Furthermore, it was observed that CTH had higher peroxyl radical scavenging and iron-binding values than beta-Lg. Liquid chromatography-mass spectrometry (LC-MS) was used to measure the rate of oxidation of three oxidatively labile amino acid residues (Tyr, Met, and Phe) in certain CTH peptide fragments. Significant oxidation of specific Tyr and Met residues present in two separate 12 amino acid peptide fragments was observed in the days preceding lipid oxidation (39 and 55% of Tyr and Met were oxidized, respectively, by day 4 of the study); however, no significant oxidation of the Phe residue present in a specific 14 amino acid peptide fragment could be observed during the same time period. These data could suggest that Met and Tyr residues are capable of scavenging radical species and have the potential to improve the oxidative stability dispersed food lipids.     

Amino acid analysis of feedstuff hydrolysates by cation exchange high performance liquid chromatography

Corn, peanut meal, and soybean meal samples were either untreated or oxidized with performic acid before hydrolysis; the amino acids were determined by cation exchange high performance liquid chromatography (LC) and conventional cation exchange LC using an amino acid analyzer (AAA). Reproducibility of each procedure was assessed by repeated injections of the same calibration standard solution over a period of several days. LC data were more precise with regard to coefficients of variation for amino acid retention times, but were more variable with regard to peak areas. Although some significant differences between methods were noted, feedstuff amino acid values obtained by LC and AAA compared very well. The only consistent differences observed within each feedstuff were that Phe and Tyr values were significantly lower when analyzed by LC compared with AAA. Results of this study suggest that modular LC instrumentation can be used to accurately and reproducibly analyze amino acids in feedstuff hydrolysates. Advantages of using ninhydrin derivatization for feedstuff analysis, as opposed to using o-phthalaldehyde or dansyl chloride, are discussed.     

1H NMR metabolite fingerprint and pattern recognition of mullet (Mugil cephalus) bottarga

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate data analysis (MVA) was used to investigate the molecular components of the aqueous extract of samples of bottarga, that is, salted and dried mullet (Mugil cephalus) roe, manufactured in Sardinia (Italy) from mullets of known and unknown geographical provenience. Principal component analysis (PCA) applied to the processed (1)H NMR spectra indicated that samples tend to cluster according to their geographical origin and also on the basis of storage and manufacturing procedures. The most important metabolites that characterized grouping of samples are the free amino acids methionine (Met), glutamate (Glu), histidine (His), phenylalanine (Phe), tyrosine (Tyr), and isoleucine (Ile); trimethylamine (TMA) and dimethylamine (DMA), both biomarkers of degradation; nucleotides and derivatives; choline (Cho) and phosphorylcholine (P-cho); and lactate (Lac).     

Changes in the Amino Acid Composition of Decomposing Plant Materials in Soil: Species and Depth Effects

Abstract

This project studied the amino acid composition of samples taken from an experiment of in situ incubation of plant debris, mixed with mineral earth, to identify changes during decomposition. Medicago sativa debris, and Eucalyptus globulus and Pinus halepensis ground litter, were mixed with a OM‐poor mineral earth, and buried in the soil at 5, 20, and 40 cm depth, for 2 years. The total amino acid composition of the final samples was compared with that of the initial ones. The amino acid composition of the several initial mixtures was quite similar. Also, the differences between initial and incubated samples were small, but still large enough to detect consistent changes. The pattern of change was consistent for only nine amino acids: Gly consistently increased, whereas Lys, His, Leu, and (to a lesser extent) Arg, Tyr, Phe, Ser, and Tau consistently decreased. The ratio Gly/(Lys+His+Leu+Arg+Tyr+Phe+Ser) is taken as the amino acid signature (AaS). This ratio increased from about 0.30–0.35 in the undecomposed mixtures, to about 0.55–0.60 after 2 years of field decomposition, and the comparison with data from other authors suggests that it could be a useful indicator of the degree of biochemical evolution of nitrogen compounds in soil samples. In contrast, changes in the traditional groups of amino acids, i.e., acid, basic, neutral, and sulfur (S)‐containing, were hardly detectable.     

氨基酸部分替代硝态氮对小白菜生长、硝酸盐累积及大量元素含量的影响

A hydroponic experiment was carried out to determine the influence of replacing 20% of nitrate-N in nutrient solutions with 20 individual amino acids on growth, nitrate accumulation, and concentrations of nitrogen （N）, phosphorus （P）, and potassium （K） in pak-choi （Brassica chinensis L.） shoots. When 20% of nitrate-N was replaced with arginine （Arg） compared to the full nitrate treatment, pak-choi shoot fresh and dry weights increased significantly （P ≤ 0.05）, but when 20% of nitrate-N was replaced with alanine （Ala）, valine （Val）, leucine （Leu）, isoleucine （Ile）, proline （Pro）, phenylalanine （Phe）, methionine （Met）, aspartic acid （Asp）, glutamic acid （Glu）, lysine （Lys）, glycine （Gly）, serine （Ser）, threonine （Thr）, cysteine （Cys）, and tyrosine （Tyr）, shoot fresh and dry weights decreased significantly （P ≤ 0.05）. After replacing 20% of nitrate-N with asparagine （Asn） and glutamine （Gin）, shoot fresh and dry weights were unaffected. Compared to the full nitrate treatment, amino acid replacement treatments, except for Cys, Gly, histidine （His）, and Arg, significantly reduced （P ≤0.05） nitrate concentrations in plant shoots. Except for Cys, Leu, Pro, and Met, total N concentrations in plant tissues of the other amino acid treatments significantly increased （P ≤ 0.05）. Amino acids also affected total P and K concentrations, but the effects differed depending on individual amino acids. To improve pak-choi shoot quality, Gln and Asn, due to their insignificant effects on pak-choi growth, their significant reduction in nitrate concentrations, and their increase in macroelement content in plants, may be used to partially replace nitrate-N.     

Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study

Reduction of free radicals generated by Fe(II)/cumene-hydroperoxide (CumOOH) by amino acids (Gly, Cys, Met, His, and Trp) and proteins (bovine serum albumin (BSA), beta-lactoglobulin, and lactoferrin) was followed by electron spin resonance spectroscopy using alpha-phenyl-N-tert-butylnitrone (PBN), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps. The radical species detected were mostly carbon-centered radicals from CumOOH fragmentation (methyl/*H3 and ethyl/*H2CH3), although carbon-centered radicals originated from amino acids could be formed in the presence of Cys, Met, His, or Trp. All proteins and amino acids, except Cys, were effective at inhibiting generation of radicals from the Fe(II)/CumOOH system. Trp was the amino acid with the highest antiradical activity, followed by His > Gly approximately Met. Lactoferrin was the protein showing the most efficient inhibition of radical formation from the Fe(II)/CumOOH system, and BSA and beta-lactoglobulin were not significantly different in their antiradical activities. These results suggest that proteins with higher inhibitory activity on lipid oxidation promoted by transition metal catalytic decomposition of hydroperoxides should be those with elevated metal-chelating and radical-scavenging properties as well as low concentration and accessibility of reducing groups from amino acids capable of activating metals, such as sulfhydryl groups.     

Analyzing sulfur amino acids in selected feedstuffs using least-squares nonlinear regression

Five feedstuffs were oxidized using performic acid, and these, along with their unoxidized counterparts, were acid hydrolyzed for multiple times (0-144 h) in degassed and vacuum-sealed glass tubes. The methionine sulfone, cysteic acid, methionine, and cysteine contents were determined for each hydrolysis time. Least-squares nonlinear regression of the sulfur amino acid contents and hydrolysis time was used to predict the actual sulfur amino acid content as well as the hydrolysis and loss rates. Least-squares nonlinear regression estimates for methionine content compared well with those of methionine sulfone for most of the feedstuffs tested. In contrast, the estimates for cysteine agreed poorly with cysteic acid. The loss rates during acid hydrolysis for methionine, methionine sulfone, and cysteic acid were low. Overall, acid hydrolysis in an evacuated sealed tube for 24 h without prior oxidation is suitable for methionine, but not cysteine, quantitation in some complex feedstuffs.     

Effects of Cultivars,Transplanting Patterns,Environment, and Their Interactions on Grain Quality of Japonica Rice

With the increasing scarcity of rural labor, the rice transplanting pattern is encountering a shift from artificial transplanting (AT) to mechanical transplanting (MT) in numerous rice‐growing districts of China. The shift of transplanting patterns combined with altered growing environment during the grain‐filling stage in different years presumably affects rice quality. Nevertheless, related information is currently limited. This study investigated the effects of cultivars, transplanting patterns, environment, and their interactions on appearance, milling, eating, and nutritional qualities of four japonica rice varieties. The significant interactive effects of cultivars, environment, and transplanting patterns on almost all rice quality parameters (except Thr, Met, and Ile) were observed. Cultivars and environment were the main factors influencing rice appearance and milling and eating qualities. Cultivar was the primary factor affecting rice nutritional quality. Among all treatments, environment showed the strongest effect on percentage of chalky kernel, milled rice yield, peak viscosity, breakdown, setback, consistence, amylose, Glu, Tyr, and Met contents. However, Leu and Phe contents were unaffected by environment but only by cultivars and transplanting patterns. In addition to amylose and protein, Glu and Met contents were also involved in determining rice eating quality. Amino acid contents (except Cys, Tyr, and Met) were significantly negatively correlated with head rice yield, showing the function of amino acids in controlling rice milling quality. Percentage of chalky kernel as well as protein and almost all amino acid contents were significantly negatively correlated with the difference of maximum and minimum temperature (DMMT) and positively correlated with relative humidity (RH), whereas head rice yield did it reversely. Amylose content and setback were significantly negatively related to daily maximum temperature (DMAT), daily minimum temperature (DMIT), daily average temperature (DAT), and effective temperature accumulation (ETA). However, peak viscosity, breakdown, and consistence had contrary performances. According to these results, we can infer that DMMT and RH are important environmental factors affecting rice appearance, milling qualities, and nutritional qualities and that DMAT, DMIT, DAT, and ETA are key environmental factors influencing rice eating quality.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

Determination of sulfur amino acids and tryptophan in foods and food and feed ingredients: collaborative study

Samples of 4 foods, 1 animal feed, isolated soy protein, and beta-lactoglobulin were analyzed by 9 laboratories to determine concentrations of cysteine as cysteic acid, methionine as methionine sulfone, and tryptophan. Sulfur amino acids were determined by AOAC method 43.A08-43.A13 for food and feed ingredients, in which samples are oxidized with performic acid before protein hydrolysis with 6N HCl. Tryptophan was determined after protein hydrolysis with 4.2N NaOH. In both methods, free amino acids were separated by ion-exchange or reverse-phase chromatography. Each laboratory was provided with detailed methods and with sealed vials containing solutions of standards. Samples were analyzed in duplicate, and variation between laboratories was determined. Coefficients of variation between laboratories for the 6 samples ranged from 5.50 to 11.8% for methionine as methionine sulfoxide, 8.59 to 17.3% for cysteine as cysteic acid, and 3.87 to 16.1% for tryptophan. Amino acid recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid obtained from amino acid sequence data. The mean recovery of cysteine was 97% with a range of 88-119%. For methionine, mean recovery was 98% (range 89-115%) and for tryptophan, 85% (range 59-102%). Method 43.A08-43.A13 for food and feed ingredients has been adopted official first action for determination of cysteine and methionine in processed foods. The alkaline hydrolysis method has been adopted official first action for determination of tryptophan in foods and food and feed ingredients.     

Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterial gamma-glutamyltranspeptidase.

The tastes of several bitter amino acids and their gamma-glutamyl derivatives were compared. The bitterness of Phe, Val, Leu, and His was reduced, sourness was produced, and preferences were increased by gamma-glutamylization. Because the effect of gamma-glutamylization of bitter amino acids was most obvious for Phe, which is an atypical bitter amino acid, an enzymatic method for the synthesis of gamma-glutamylphenylalanine (gamma-Glu-Phe) involving bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 200 mM Gln, 200 mM Phe, and 0.5 unit/mL GGT, pH 10.4. After 1.5-h of incubation at 37 degrees C, 140 mM gamma-Glu-Phe was obtained, the yield being 70%. gamma-Glu-Phe was purified on a Dowex 1x8 column and then identified by NMR.     

荧光假单胞杆菌RB-42 RB-89的趋化性与烟草根部定殖研究

烟草PGPR(PlantGrowth PromotingRhizobacteria)菌株RB 89、RB 42对烟草根部收集液有较强的趋化性。在稀释浓度为10-5时,RB 89、RB 42的趋化值依次为1 79、1 83。RB 42和RB 89对氨基酸趋化性差异显著,对酪氨酸、脯氨酸、甘氨酸、笨丙氨酸表现出较强的趋化性,对组氨酸、苏氨酸没有表现出趋化性;RB 42和RB 89菌株对有机酸的趋化性没有明显区别,只是RB 42对乳酸的趋化性表现得比对其它有机酸的强些;菌株对糖类物质的趋化性最弱。菌株RB 89、RB 42在烟株根表的定殖受灭菌、趋化剂影响较大,土壤灭菌、趋化剂处理可增加菌株在根表的定殖量。     

Properties of protein powders from arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus) byproducts

Functional, nutritional, and thermal properties of freeze-dried protein powders (FPP) from whole herring (WHP), herring body (HBP), herring head (HHP), herring gonad (HGP), and arrowtooth flounder fillets (AFP) were evaluated. The FPP samples have desirable nutritional and functional properties and contained 63-81.4% protein. All FPP samples had desirable essential amino acid profiles and mineral contents. The emulsifying and fat adsorption capacities of all FPP samples were higher than those of soy protein concentrate. The emulsifying stability of WHP was lower than that of egg albumin but greater than that of soy protein concentrate. Thermal stability of the FPP samples is in the following order: HGP > HBP > WHP > HHP > AFP.     

Amino acid analysis of feeds in The Netherlands: four-year proficiency study

To improve accuracy and precision of amino acid analysis in 12 Dutch feed laboratories, a proficiency study was organized twice annually over a 4-year period. The method used included reflux acid hydrolysis for 22 h followed by evaporation, separation on a cation-exchange resin in an amino acid analyzer, and photometric detection after post-column derivatization with ninhydrin. For the determination of sulfur-containing amino acids, samples were oxidized prior to hydrolysis. For the determination of tryptophan, samples underwent an alkaline hydrolysis excluding oxygen, were separated by liquid chromatography on a Hypersil ODS analytical column, and were assayed by UV or fluorescence detection. The average relative standard deviations within (CVr) and between (CVR) laboratories were 3 and 7%, respectively. For some mixed feed samples, the results from the proficiency study were compared with those obtained by the International Analytical Group. For those samples, the relative standard deviation of the difference between IAG and Dutch groups was only 1.9%. For samples that were analyzed twice during this 4-year period, relative standard deviation of between-series differences was only 2.1%.     

Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope ((13)c/(12)c and (15)n/(14)n) ratios and free amino acid ratios

The stable isotope ratios ((13)C/(12)C and (15)N/(14)N) of casein measured by isotope ratio mass spectrometry (IRMS) and some free amino acid ratios (His/Pro, Ile/Pro, Met/Pro, and Thr/Pro) determined by HPLC in samples of ewes' milk cheese from Sardinia, Sicily, and Apulia were found to be parameters independent of ripening time. Multivariate data treatments performed by applying both unsupervised (principal component analysis and cluster analysis) and supervised [linear discriminant analysis (LDA)] methods revealed good discrimination possibilities for the cheeses according to place of origin. In this respect, particularly significant were the variables Ile/Pro, Thr/Pro, (13)C/(12)C, and (15) N/(14)N ratios on which basis 100% discrimination and classification of the samples by LDA was obtained.     

Nutritional evaluation of ethanol-extracted lentil flours

Lentil flours were extracted with 80% ethanol at 25 and 50 degrees C for 1, 2, or 3 h. The various nitrogen fractions, soluble carbohydrates, three amino acids (Lys, His, and Tyr), available lysine, protein digestibility, and vitamins B(1) and B(2) were analyzed to evaluate the effect of extraction. Extraction resulted in an increase in the total nitrogen content of the extracted flours, with extraction temperature affecting the nature of the nitrogen (protein or nonprotein) content. There was also a large reduction in the oligosaccharides of the raffinose family, although the effect of temperature was appreciable only in the case of stachyose. There was hardly any effect on the concentrations of the amino acids analyzed or on protein digestibility; however, a positive correlation between protein digestibility and the available lysine was recorded in the samples. The vitamin B(1) and B(2) contents underwent variable decreases depending on extraction temperature.     

Mechanism of production of troponin T fragments during postmortem aging of porcine muscle

Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The peptide bonds of His37-Glu38 and Thr51-Ala52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu21-Ala22 and His37-Glu38 sites.     

Effects of the addition of amino acids and peptides on lipid oxidation in a powdery model system

The effects of the addition of amino acids and peptides on the oxidation of eicosapentaenoic acid ethyl ester (EPE) encapsulated by maltodextrin (MD) were investigated. The encapsulated lipid was prepared in two steps, that is, by mixing of EPE with MD solutions (+/-amino acids and peptides) to produce emulsions and freeze-drying of the resultant emulsions. The addition of amino acids and peptides improved the oxidation stability of EPE encapsulated with MD, and the inhibition of lipid oxidation by the amino acids and peptides was more effective at 70% relative humidity (RH). Met, Arg, and Trp were effective amino acids for antioxidation at RH = 10 and 40%, whereas at RH = 70%, His was the most effective amino acid, preventing the oxidation of EPE almost perfectly. Carnosine also exhibited a strong antioxidant effect at RH = 70%, but the effect of anserine was inferior. The addition of Met + Trp or Met + Arg inhibited the oxidation of EPE encapsulated with MD at RH = 40%. Cys accelerated the oxidation of EPE, indicating that the thiyl radical may act as a pro-oxidant. No close relationship was observed between the radical scavenging abilities of amino acids and peptides measured in the aqueous diphenylpicrylhydrazyl solution and their antioxidative effects in the powdery system. It is possible that the radical-scavenging ability of amino acids and peptides detected by ESR in the powder system is responsible for the antioxidative activity of these compounds.     

Regulatory effect of amino acids on the pasting behavior of potato starch is attributable to its binding to the starch chain

The binding of an amino acid, glycine (Gly), alanine (Ala), epsilon-aminocaproic acid (-AC), monosodium glutamate (GluNa), or lysine (Lys), to starch was examined by a biomolecular interaction analyzer (IAsys). A starch sample (ATS) hydrolyzed to an extent of 1% hydrolysis rate with 15% sulfuric acid was used as a model starch for the binding examination. The reducing end of ATS was oxidized by the Somogyi reagent, and the conversion of the reducing end to the carboxyl group of ATS was confirmed by a carboxylic acid fluorescence labeling reagent. The oxidized ATS was immobilized to the amino group of a sensor cuvette by using water-soluble carbodiimide and N-hydroxysuccinimide through an amide bond. The IAsys examination showed that Gly, Ala, and epsilon-AC scarcely bound to the immobilized starch chains but that GluNa and Lys favorably bound with their increasing concentrations. The relative binding index (RBI) of each amino acid was defined by the ratio of the slope of the linear regression equation between the binding response and the concentration for each amino acid to that for Gly. Because the relationships between the RBI and the pasting characteristics (pasting temperature, peak viscosity, breakdown, and swelling index) could each be expressed by a linear regression equation with a high correlation coefficient, it is concluded that the regulation of the pasting behavior of starch with an amino acid is caused by binding of the amino acid to the starch chains.     

Inactivation of staphylococcal enterotoxin-A with an electrolyzed anodic solution.

Electrolyzed anodic NaCl solutions [EW+], prepared by the electrolysis of 0.1% NaCl, have been shown to instantly inactivate most pathogens that cause food-borne disease. Elimination of food-borne pathogens does not necessarily guarantee food safety because enterotoxins produced by pathogens may remain active. We have tested whether EW+ can inactivate Staphylococcal enterotoxin A (SEA), one of the major enterotoxins responsible for food poisoning. Fixed quantities of SEA were mixed with increasing molar ratios of EW+, and SEA was evaluated by reversed-phase passive latex agglutination (RPLA) test, immunoassay, native polyacrylamide gel electrophoresis (PAGE), and amino acid analysis after 30 min incubations. Exposure of 70 ng, or 2.6 pmol, of SEA in 25 microL of PBS to a 10-fold volume of EW+, or ca. 64.6 x 10(3)-fold molar excess of HOCl in EW+, caused a loss of immuno-reactivity between SEA and a specific anti-SEA antibody. Native PAGE indicated that EW+ caused fragmentation of SEA, and amino acid analysis indicated a loss in amino acid content, in particular Met, Tyr, Ile, Asn, and Asp. Staphylococcal enterotoxin-A excreted into culture broth was also inactivated by exposure to an excess molar ratio of EW+. Thus, EW+ may be a useful management tool to ensure food hygiene by food processing industries.