Sulfur Amino Acid Stability. Hydrogen Peroxide Treatment of Casein, Egg White, and Soy Isolate

Egg white solids (EWS), soy protein isolate (SPI), and casein were treated with differing levels of H₂O₂ at both 40 and 90°C. Total and reactive cysteine/cystine (Cys), total methionine, methionine sulfoxide and sulfone contents were determined for all samples. At 40°C methionine in all samples was readily converted to its sulfoxide, with little sulfone or cysteic acid being formed. At 90°C Cys was rapidly converted to the stable cysteic acid and methionine to its stable sulfone form. The production of the less stable methionine sulfoxide at 90°C was minimal. Of the three proteins studied, the methionine and Cys residues in EWS were least stable under oxidizing conditions, whereas those present in SPI and casein were more stable.     

响应面优化硝普钠法测定发酵液中的蛋氨酸含量

为了优化硝普钠法(sodium nitroprusside method,SNP)测定发酵液中的蛋氨酸(Met)含量的实验条件,利用紫外可见分光光度计分析不同氨基酸中加入各SNP反应试剂、反应不同时间对反应液吸收峰的影响。在甘氨酸(Gly)添加量、NaOH加入量、SNP加入量、H₃PO₄加入量、各试剂加入后的反应时间等单因素实验的结果上,利用响应面法优化SNP法。结果表明:与SNP试剂反应后,除了组氨酸(His)外,其它氨基酸不会对Met的测定产生影响。反应试剂H₃PO₄的加入对513 nm处吸收峰的形成起决定性作用;发酵液中各成分SNP反应后,最大吸收峰无明显变化。利用单因素和响应面法优化SNP法,得出较佳测定方案:发酵液1500×g离心15 min;5 mL上清液中加入0.74 mL 5 mol/L NaOH和0.15 mL 10% SNP溶液,摇匀试管,放置30 min;将2 mL H₃PO₄逐滴加到混合物中,振荡试管,反应5 min,在513 nm处测吸收值。当Met浓度为1.0 g/L时,最佳测定条件下吸收值为1.952。通过光谱分析确认Gly的加入会降低Met和His反应的吸收峰值,可通过分析最大吸收峰波长是否在513 nm处,判断样品中是否有组氨酸影响测定。实验获得的SNP法可用于迅速筛选蛋氨酸高产菌。     

Combined chemical and enzymic treatments of corn husk lignocellulosics

The potential for using corn (Zea mays L) husk residues (carbohydrates 827 g kg^?1, lignin 66 g kg^?1 DM) as a carbohydrate source for the production of soluble sugars by combined chemical pretreatment and enzymic hydrolysis was assessed. Comparative investigations of acidic and alkaline pretreatments on corn husk have shown that pentose-containing carbohydrates comprised 86–93% of the solubilised fraction. While pretreatments with 1.25 M NaOH at 25.85° C resulted in preferential extraction of hemicellulose having DP; > 12, acid pretreatments 0.51 M H₂SO₄, 0.51 M H₃PO₄ at (85° C) resulted in extensive depolymerisation of this polysaccharide. Xylose and low molecular weight carbohydrates were identified as the major products. Subsequent hydrolysis of the solubilised fraction with crude hemicellulase preparations yielded 40.90% fermentable sugars. When NaOH (0.02–1.25 M), H₂SO₄ and H₃PO₄ (0.02–0.51 M) were used as pretreatment solvents (25–85° C, 2 h), NaOH was the most effective in increasing the susceptibility of the residual husk towards enzymes, yielding 83–96% reducing sugars. This solvent solubilised up to 60.6% of the lignin and appeared to disintegrate the fibrillar structure of husk. The crystallinity of husk residues increased following the chemical pretreatments and was positively correlated with cellulose content. Enzymic hydrolysis with commercial cellulase preparations proceeded in two stages: a rapid breakdown of amorphous cellulose after which the hydrolysis rate levelled off. Similar biphasic patterns were observed for the pyrolysis temperature of cellulose. Under the most optimal conditions for husk saccharification (pretreatment with 1.25 M NaOH, 25° C, 2 h, followed by enzymic hydrolysis using a mixture of cellulase and cellobiase), 96% of the cellulose-enriched residues was hydrolysed to reducing sugars. A cellulase preparation from Trichoderma reesei exhibited substantial hemicellulolytic activity and could, therefore, be used as the sole saccharifying enzyme preparation for husk lignocellulosics.     

Enzymatic Release of Peptides, Methionine and Cystine from Dry Beans Following Various Heat Treatments

Ground Great Northern and pinto bean (Phaseolus vulgaris) samples were processed under varying conditions of time, temperature and pressure and then hydrolyzed in vitro using a mixture of trypsin, chymotrypsin and amino peptidase. The hydrolyzed samples were ultrafiltrated and the peptides (< 10,000 daltons) were collected and analyzed for their sulfur amino acid content (methionine, cysteine, and cystine). From 48% to 100% of the cysteine/cystine residues in the protein were released after 6 hr of enzymatic hydrolysis, whereas only 10% to 40.2% of the methionine residues were released during the same time period. The maximum rate of peptide release occurred at 3 hr of proteolysis, while the maximum rate of methionine and cysteine release occurred at 2 hr of proteolysis.     

Characteristics of Acid Hydrolysate from Defatted Peanut Flour

We hypothesized that aflatoxin-contaminated defatted peanut flour could be acid hydrolyzed to produce an aflatoxin-free seasoning sauce. Physical and chemical properties and amino acid composition of the flour hydrolyzed at 120°C for 4 hr with 5N HCI and destruction of aflatoxin B, during hydrolysis were investigated. The pH (5.72), specific gravity (1.20), total solids (402 mg mL-¹), and reducing sugar (2.9 mg mL^-1) and total nitrogen (17.5 mg mL-¹) contents of the peanut hydrolysate were similar to those of commercial soy sauces or seasoning sauces made by chemical processes. Glutamic acid, aspartic acid, arginine and glycine were the major amino acids in the hydrolysate and should contribute substantially to enhancing flavor. Aflatoxin B, was totally destroyed during acid hydrolysis at 100 or 120°C.     

Kinetics of Racemization of Amino Acid Residues in Casein

Exposing casein to alkali (0.1N NaOH at 65°C) partly racemized the amino acid residues. A plot of D/L ratios for seven amino acids versus time of treatment shows fast initial racemization rates that decrease after about 1 hr. A linear free energy relationship exists between racemization rates and inductive effects of amino acid side chains or R-substituents (σ*). Kinetic studies over the temperature range 25–75°C yields activation energies (in Kcal/mole) for aspartic acid (20.8), phenylalanine (28.7), glutamic acid (32.5), and alanine (32.4). Racemization rates increase with hydroxide ion concentration but not with protein concentration. Acetylation of amino groups prevents lysinoalanine formation but not racemization, permitting studies which distinguish between effects of these two alkali-induced reactions. Understanding principles that govern racemization should help in designing food processes that minimize undesirable, deteriorative changes in food proteins.     

Rapid in vitro enzymic predictive model for the in vivo digestibility of food proteins

The absorbance of the deep orange colour produced by incubating protein hydrolysates with trinitrobenzene sulphonic acid was used to follow the liberation of free amino groups. Five animal and five plant proteins were hydrolysed with pepsin-pancreatin and papain-pronase E and aliquots were taken every 4 hr. To facilitate proteolysis the proteins were presolubilized in 0.05 M HCl at 70°C for 2 hr with 1.0 ml 0.8% mercapto-ethanol added to reduce disulphide bonds. An increase in absorbance occurred in all enzymatic digests between 8-20 hr incubation. No further hydrolysis took place after 20-28 hr incubation. The extent of the hydrolysis in vitro correlated with in vivo measurements of digestibility and so may serve as a more rapid predictor of digestibility.     

The methionine precursor dl-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function

dl-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H₂O₂ or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for dl-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.     

Microwave digestion of proteins

 The time needed for a conventional acidic digestion of peptide bonds in proteins with 6 N HCl was reduced from 18–24 h to 2 h when microwaves were applied. The rate-determining step of the splitting of the peptide bonds depends on the amino acid composition of the proteins. Myoglobin, lysozyme and bovine serum albumin were tested as pure model proteins. The amino acid contents determined after ion-exchange HPLC and postcolumn derivatisation with ninhydrin correlated well with values obtained on the basis of known amino acid sequences. Tryptophan, methionine as well as cysteine/cystine were partly or completely destroyed under the hydrolysis conditions applied. Almost 95% of the thermolabile amino acids (based on theoretical values) could be recovered. The conditions were further applied for the hydrolysis of food proteins like casein, commercially available whey proteins as well as pea proteins, and a comparison with the results of the conventional hydrolysis (24 h) was made. Received: 26 February 1998 / Revised version: 20 March 1998     

Microwave digestion of proteins

 The time needed for a conventional acidic digestion of peptide bonds in proteins with 6 N HCl was reduced from 18–24 h to 2 h when microwaves were applied. The rate-determining step of the splitting of the peptide bonds depends on the amino acid composition of the proteins. Myoglobin, lysozyme and bovine serum albumin were tested as pure model proteins. The amino acid contents determined after ion-exchange HPLC and postcolumn derivatisation with ninhydrin correlated well with values obtained on the basis of known amino acid sequences. Tryptophan, methionine as well as cysteine/cystine were partly or completely destroyed under the hydrolysis conditions applied. Almost 95% of the thermolabile amino acids (based on theoretical values) could be recovered. The conditions were further applied for the hydrolysis of food proteins like casein, commercially available whey proteins as well as pea proteins, and a comparison with the results of the conventional hydrolysis (24 h) was made. Received: 26 February 1998 / Revised version: 20 March 1998     

HPLC Method for Cyst(e)ine and Methionine in Infant Formulas

Cyst(e)ine and methionine were converted into cysteic acid and methionine sulfone by oxidizing with performic acid. The oxidized samples were then subjected to acid hydrolysis (6N HCl, 105–110°C/24 hr). After derivatization with phenylisothiocyanate, reverse phase HPLC separation was carried out at 48°C and with UV detection. Different gradients and pH values in the eluent were assayed to determine the best resolution. Analytical parameters, detection and quantification limits, linearity, precision and accuracy, were determined. The method was reliable and accurate for measuring cyst(e)ine and methionine in infant formulae.     

AVAILABILITY OF AMINO ACIDS IN SARCOPLASMIC FISH PROTEINS COMPLEXED WITH SODIUM HEXAMETAPHOSPHATE

SUMMARY– Comparisons were made on the availability (enzyme digestion) of 4 amino acids (lysine, methionine, tryptophan, and threonine) in sarcoplasmic fish proteins and sarcoplasmic fish proteins complexed with sodium hexametaphosphate. The protein complexes were formed by reacting 1% solutions of sarcoplasmic fish proteins at pH values ranging from 2–4 and at phosphate concentrations ranging from 0.005–0.05 M. Uncomplexed sarcoplasmic protein was obtained by precipitation from 70% isopropanol. The samples were digested with the enzyme pronase and the available amino acids were measured with a bio-assay system. No significant differences in the availability (enzyme digestion) of amino acids were found between complexed and noncomplexed samples. Assays of samples dried at 75°C showed that nearly 100% of the methionine, threonine and tryptophan and 85% of the lysine were available. When the samples were subjected to moist heat (50% moisture, 100°C) for up to 24 hr, availability of lysine, methionine, tryptophan and threonine was reduced by 35, 22, 39 and 34%, respectively. Results of this study indicate that the nutritional characteristics of fish proteins are unimpaired when they are complexed with sodium hexametaphosphate.     

Recent progress of soybean protein foods: Chemistry,technology, and nutrition

Abstract

The most important chemical reactions during the process of soybean protein foods are the intermolecular reactions among the residues exposed on the surface of the protein molecules through the denaturation process. In native soybean protein molecules, most amino acid residues responsible for the reactions—such as cysteine (‐SH), cystine (S‐S), and hydrophobic amino acid residues—are buried in the inside region of the molecule, inaccessible to water. These residues become reactable with each other through the exposure from the inside by heat denaturation during processing. The unique textures of soybean protein foods, such as tofu, kori‐tofu, yuba, and texturized products produced by extruder, etc., are the results of both the intermolecular interchange reaction between the exposed ‐SH and S‐S groups and the intermolecular hydrophobic reaction among the exposed hydrophobic amino acid residues. The exposure of amino acid residues is also important for the hydrolysis of soybean proteins by enzymes, through which soy sauce is produced, because the cleavage of the peptide bonds is carried out after binding between the active sites of the enzymes and the enzyme‐specific amino acid residues exposed through denaturation. These facts indicate the importance of the three‐dimensional structures of soybean protein molecules in the technology of soybean protein foods. Recently great progress has been made in the manufacturing techniques of soybean protein foods, such as soy milk, tofu, abura‐age, textured protein products, and soy sauce. The quality of soy milk and tofu was very much improved by controlling the action of the biologically active substances such as lipoxygenases and β‐glucosidases which are contained in soybeans and responsible for the production of off‐flavor. A new abura‐age, whose texture does not deteriorate during frozen storage or drying, was developed by using soybean protein isolate and oil as materials. A new type of textured protein product was also developed: a deep‐fat‐fried nugget with unique texture and flavor. This product is textured through a twin‐type extruder. For soy sauce manufacturing new biotechnology has been applied on the pilot‐plant scale. This is a system of continuous fermentation through bioreactors with the immobilized whole cells of microorganisms, by which the fermentation term is shortened strikingly. New and important discoveries were made on the nutrition of soybean proteins. According to recent experiments using human beings, the amino acid score of soybean proteins is 100 for persons more than 2 years old, indicating that the nutritive value of soybean proteins is equal to animal proteins. Further, it was elucidated that soybean proteins have cholesterol‐lowering action. A discussion is presented on the future of the soybean protein foods.     

Lactobacillus salivarius for Conversion of Soy Molasses into Lactic Acid

The feasibility of conversion of soy molasses, a low value by-product of soy protein production, to lactic acid by fermentation with Lactobacillus salivarius was investigated. The basic environmental parameters affecting growth and lactic acid production were determined. Lactic acid production in soy molasses was optimal at pH 5.6 and 42°C. Addition of 0.5% yeast extract to soy molasses reduced fermentation time from 36 to 10 hr and increased lactic acid production by 30%.     

Response of milk fatty acid composition to dietary supplementation of soy oil, conjugated linoleic acid, or both

Thirty-six Holstein cows were blocked by parity and allotted by stage of lactation to 6 treatments to evaluate the effects of dietary soy oil, conjugated linoleic acid (CLA; free acid or calcium salt), or both, on CLA content of milk. Diets were fed for 4 wk and are as follows: (1) control, (2) control + 5% soy oil, (3) control + 1% CLA, (4) control + 1% Ca(CLA)₂, (5) control + 1% CLA + 4% soy oil, and (6) control + 1% Ca(CLA)₂ + 4% soy oil. Rumen volatile fatty acid -concentrations, blood fatty acid concentrations, milk yield, and milk composition were measured weekly or biweekly. Dry matter intake and milk yield were recorded daily. Dietary supplementation of soy oil or CLA had no effect on daily milk yield, milk protein concentration and production, or milk lactose concentration and production. Supplementation of unsaturated fatty acids as soy oil, CLA, or Ca(CLA)₂ increased total fatty acid concentration in plasma, decreased milk fat concentration and production, and had no effect on rumen volatile fatty acid concentrations. The weight percentage of CLA in milk was increased from 0.4 to 0.7% with supplementation of 1% CLA, to 1.2% with supplementation of soy oil, and to 1.3% with supplementation of 1% CLA plus soy oil. Supplementation with Ca(CLA)₂ or Ca(CLA)₂ + soy oil increased the CLA content of milk fat to 0.9 and 1.4%, respectively. In summary, adding 5% soy oil was as effective as supplementing CLA, Ca(CLA)₂, or a combination of 1% CLA (free acid or calcium salt) + 4% soy oil at increasing CLA concentrations in milk fat. Feeding CLA as the calcium salt resulted in greater concentrations of CLA in milk fat than did feeding CLA as the free acid. Dietary supplementation of 5% soy oil or 4% soy oil + 1% CLA as the free acid or the calcium salt increased the yield of CLA in milk.     

Amino acid composition,molecular weight distribution and antioxidant activity of protein hydrolysates of soy sauce lees

The proteins of soy sauce lees (SSLP) were hydrolysed by Alcalase in the presence of ultrasound or traditional water bath to obtain hydrolysates S2–S6. The analysis of protein content indicated that enzymatic hydrolysis could significantly improve the extraction efficiency of proteins. By determination of molecular weight distribution, >10 and 5–10 KDa fractions of native SSLP (S1) decreased during hydrolysis, whilst 3–5 KDa fraction increased. Gradual increases of free, total and antioxidant amino acids were observed for S1–S4, and the differences between S4 and S6 were slight. Tyrosine was the major free amino acid of S1–S6, whilst glutamic acid had the highest amount in total amino acid composition. S2–S6 showed stronger DPPH radical scavenging activities in a dose-dependent manner than S1. All the results suggested that ultrasound treatment showed an inhibition behaviour on the enzymatic hydrolysis of SSLP.     

Determination of sulfur-containing amino acids in food products

To define the most accurate method for determination of sulfur-containing amino acids in foods, the possibility was tried of using a method suggested by Moore. The method is based on preliminary oxidation of cystine and methionine by performic acid and transformation of these substances to acid hydrolysis resistant cysteic acid and methionine sulphone. Model experiments and use of foods (wheat flour, grade II, and protein part of chicken egg) demonstrated that exposure of proteins to performic acid brings about oxidation of sulfur-containing amino acids to the hydrolysis-resistant forms which may be defined by chromatography with a greater accuracy. Experiments without preliminary oxidation by performic acid showed that foods experience an appreciable destruction (by 30-60%) of cystine and methionine. Therefore, it is advisable that during studies into amino acids contained by proteins, additional hydrolysis may be performed with preliminary oxidation of the samples by performic acid, which enables the determination of the content of methionine and cystine with a greater accuracy.     

Enzymatic Lactose Hydrolysis for Prevention of Lactose Crystallization in a Whey Spread

The enzymatic lactose hydrolysis for elimination of sandiness was studied in a whey-buttermilk spread of previously optimized composition (18% fat, 12% protein, 17% lactose). Soluble enzyme preparations of either the acid (Aspergillus) or the neutral (Kluyveromyces) type were suitable for the minimum 30% hydrolysis required to prevent the lactose crystallization. Two types of acid enzymes used at either 1 mg or 2 mg per g hydrolyzed mixture produced the desired effect after 2 hr of hydrolysis at 30°C. Similar results were obtained with two types of neutral enzymes at 2 mg per g hydrolyzed mixture after either 2 or 4hr of hydrolysis at 30°C.     

Gallic acid oxidizes Met residues in peptides released from bovine β-lactoglobulin by in vitro digestion

Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of β-lactoglobulin (β-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of β-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of β-LG was lower in the presence than in the absence of GA. The peptides released from β-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of β-LG in the presence of GA. Met(7), Met(24) and Met(145) in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues.     

A study of chemical characteristics of soy sauce and mixed soy sauce: chemical characteristics of soy sauce

Mixed soy sauce is one among the Korean traditional fermented soy sauces consumed as condiment for cooking in Korea, especially mixed soy sauce is made of the fresh seafood and the meat different from the soy sauce. Therefore, mixed soy sauce provides the strong and complicated umami taste, which is stronger than that of soy sauce. The objective of this study was to investigate for the chemical characteristics changes of mixed soy sauce in comparison with soy sauce during fermentation process (0, 120, 240 and 360 days) at different fermentation temperatures (4 and 20 °C), change in moisture, total nitrogen, crude fat, ash, pH, titratable acidity, total acidity, salt content and reducing sugar of mixed soy sauce in comparison with soy sauce. Especially, histidine, arginine, threonine, methionine, phenylalanine, and lysine, essential amino acid and among adenosine triphosphate (ATP)-related compounds, inosine (HxR) and hypoxantine (Hx) were prominent in mixed soy sauce at 20 °C. Otherwise, 2,4-dinitrophenylhydrazine (DNPH) derivative and 3DG content were prominent in soy sauce at 20 °C. The content of 2,4-DNPH derivative and 3DG in soy sauce is correlated with its browning intensity.