Tryptophan determination of food proteins by h.p.l.c. after alkaline hydrolysis

The alkaline hydrolysis of proteins prior to tryptophan analysis has been evaluated, and a method for the measurement of tryptophan by reverse phase h.p.l.c. after alkaline hydrolysis has been proposed and compared with published methods. Hydrolysis with either LiOH or NaOH gave similar results. Tryptophan values and the recovery of added 5-methyltryptophan were similar when hydrolysis was made under tap-water vacuum (1500 Pa) or at high vacuum (2 Pa). Partially-hydrolysed starch reduced the losses of tryptophan during hydrolysis better than thiodiglycol. High levels of indolyl-3-propionic acid or 5-methyltryptophan did not further improve tryptophan recovery. 5-Methyltryptophan is the preferred internal standard; its recovery after hydrolysis more closely resembled that of protein-bound tritiated tryptophan from goat casein than did the recoveries of α-methyltryptophan or free ¹⁴C-tryptophan. Under some conditions, the recovery of protein bound tryptophan was lower than the recovery of free tryptophan. The stability of tryptophan in hydrolysates was improved by using a phosphate buffer pH 7.0 containing 0.02% sodium azide.     

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.     

Tryptophan degradation during heat treatments: Part 2—Degradation of protein-bound tryptophan

The stability of free or protein-bound tryptophan was determined during acid (4 m methane sulfonic acid) or alkaline (4·2 m sodium hydroxide) hydrolysis at 110°C under nitrogen. The influence of time and of glucose, starch or amino acid addition was studied. It was found that hydrolysis with methane sulfonic acid caused tryptophan losses when glucose or starch was present.The thermal degradation of protein-bound tryptophan was studied using either glycyl-l-tryptophyl-glycine or casein. Analysis of tryptophan was carried out after acid or alkaline hydrolysis, through high performance liquid chromatography and UV detection.Heat treatment of tripeptide (9·4 mM, pH 8, 125°C, 3 to 48 h) in the presence of oxygen, air or nitrogen resulted in peptide hydrolysis, with the formation of the corresponding dipeptides and of free tryptophan. After acid or alkaline hydrolysis, tryptophan loss was seen to be higher when the initial heat treatment was performed in the presence of oxygen than in the presence of air.Thermal treatment of 4 or 5% w/v casein solutions (pH 7 or 8) was carried out at 125°C, under oxygen, air or nitrogen. A marked loss of tryptophan was found to occur after 24 h at 125°C when oxygen or air was present. Under nitrogen, protein-bound tryptophan was heat stable, even in the presence of glucose or starch. It is therefore unlikely that the indole ring of protein-bound tryptophan may react with reducing carbohydrates through Maillard-type reactions. Oxidative degradation of protein-bound tryptophan is also unlikely to reach any significant extent during the heat processing of foods, unless catalysts or strong oxidizing agents are present.     

Comparison of acid and amyloglucosidase hydrolysis for estimation of non‐structural polysaccharides in feed samples

Enzymatic methods (amyloglucosidase) and methods based on acid solutions (0.1, 0.2 and 0.3 M H₂SO₄ for 1, 2 and 3 h at 100 °C) for the hydrolysis of non‐structural carbohydrates from different feed samples were compared. The monomeric units resulting from the enzymatic and acid hydrolysis were determined by the glucose oxidase and reducing sugar methods. There was a significant effect of acid concentration and of time of hydrolysis on the glucose and reducing sugar values in the hydrolysate. Glucose values were similar for both the amyloglucosidase method and the most intense conditions of hydrolysis (0.3 M for 3 h) for some samples. Under these conditions, however, the reducing sugar values were higher. No acid hydrolysis method was found to estimate correctly the total non‐structural carbohydrates, but α‐linked glucose polymers in biological samples may be determined by sample hydrolysis with a 0.3 M H₂SO₄ solution for 3 h at 100 °C since the glucose in the hydrolysate is determined by the glucose oxidase method and the sucrose content of the sample is negligible. © 1999 Society of Chemical Industry     

Quantifying phosphine penetration through the bark of pine (Pinus radiata D.Don) logs

Phosphine (PH₃) is used as an in-transit phytosanitary treatment (10-d fumigation period) for pine (Pinus radiata D.Don) logs exported from New Zealand to China. The ability of PH₃ to penetrate through the bark of the logs is not well known. We designed equipment to accurately quantify PH₃ penetration into the bark of wooden blocks (100 × 100 × 50 mm; n = 12) cut from the upper and lower trunk of recently harvested pine trees fumigated at 15 or 25 °C. Fumigations simulated commercial conditions consisting of two phases; phase I with 2.0 g m⁻³ of PH₃ for the first 5 days (1–120 h) and 1.5 g m⁻³ of PH₃ in phase II for the next 5 days (121–240 h). During the 10-d schedule, we achieved the required commercial CT (concentration × time) of ≥48,000 ppm h at both temperatures. Bark thickness (i.e., trunk location) did not significantly affect fumigant penetration. Phosphine penetration through the bark of the wooden blocks was greatest after each application, then the penetrated concentrations diminished over time. Greater penetration occurred at 15 °C than at 25 °C. Further studies are required to better understand the dynamics of PH₃ penetration particularly at lower temperatures and through insect-infested blocks.     

USE OF FLEXIBLE POLYMER MATERIAL FOR PACKAGING FRESH PEACHES

Fresh peaches (Prunus persica) were overwrapped in trays with 1 of 3 formulations of flexible polyvinyl chloride (PVC) film that differed in gas transmission rate or they were held in nonwrapped trays (controls). The CO₂ transmission rate at 0°C for PVC type III film was 280 mL/m^2. h (1 atmos); that of type II was 4 times greater and that of type I, about 5 times greater. The peaches were stored either 14 days at 0° or 7.5°C, or 7 days each at 0° and 7.5°C plus 2 days at 20°C to simulate retail display. The mean CO₂ levels were 10, 7.2 and 4.7% in packages that were wrapped with PVC III film and held at 7.5°, 0°/7.5° and 0°C, respectively. CO₂ in packages wrapped with PVC I or II was below 3% at each storage temperature. O₂ concentration remained about 4% in all packages. Weight loss was less and fruit was firmer among those packaged in PVC III than among nonwrapped controls at each of the 3 storage temperatures. Storage temperature had no effect on weight loss or of fruit held in PVC III film. External appearance of fruit packaged with the 3 types of film was significantly better than that of the controls. Internal appearance of the peaches was unaffected by any of the treatments. A microatmosphere favorable for fresh peaches can be maintained within packages overwrapped with polymer films that are selectively permeable to respiratory gases.     

Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are K_M 30 µM and k_cat 356 s⁻¹ at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D /L -l(1,2,3,4,5) P₅. © 1999 Society of Chemical Industry     

Fate of aflatoxin B(1) in contaminated corn gluten during acid hydrolysis

BACKGROUND: Aflatoxins are a group of mycotoxins that cause serious chronic disease outbreaks and contaminate several food products such as corn and its by‐product, corn gluten. The aim of the current study was to evaluate the effect of hydrochloric acid (HCl) on aflatoxin B₁ (AFB₁) degradation in contaminated corn gluten under different HCl concentrations, hydrolysis temperatures and hydrolysis times. RESULTS: During the wet milling process the highest AFB₁ level (45.68 µg kg^?1) (37.86%) was found in corn gluten fraction. Treatment with 1 mol L^?1 HCL at 110 °C resulted in degradation of AFB₁ by 27.6% (33.07 µg kg^?1) after 4 h and reached 42.5% (26.26 µg kg^?1) after 8 h. Increasing HCl concentration from 1 to 3 mol L^?1 HCl resulted in increased degradation of AFB₁, while complete degradation occurred in the presence of 5 mol L^?1 HCl after 4 h at 110 °C. Meanwhile, half‐life time of AFB₁ was recorded after 2 h at 100 °C and was < 2 h at 110 °C in the presence of 3 mol L^?1 HCl. CONCLUSION: It could be demonstrated that the manufacture of hydrolyzed vegetable protein is a suitable method for decontamination of aflatoxin in highly contaminated grains, especially gluten fractions. The hydrolysis reaction could be considered in terms of first‐order reaction kinetics of AFB₁ degradation. Copyright © 2010 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

Extraction of polyphenols and hydrolysis of birdproof sorghum starch

The presence of polyphenols in the testa and pericarp of birdproof sorghum has an inhibitory effect on enzyme activity during hydrolysis of sorghum starch. In this communication the effect of different NaOH concentrations at various temperatures on the extraction of polyphenols from birdproof sorghum is described. It was found that a 0.25 M NaOH solution extracted all the polyphenols within 75 min at 22°C. The enzymic hydrolysis of birdproof sorghum starch (without polyphenols) was also investigated. A recovery of up to 92±0.6% glucose from the available starch was obtained. The change in sugar composition during hydrolysis was monitored by h.p.l.c.     

Functions of Different Yak Bone Peptides

Nine kinds of oligopeptide were obtained from the yak bone-marrow protein via the hydrolysis of some single or mixed proteases, and the functions of various yak bone peptides were compared. The results indicated that the peptide from yak bone hydrolyzed by Alcalase plus Flavourzyme at 50°C for 4 h showed the strongest antioxidant activity in a H₂O₂ system and chelating activity to Cu²⁺; Yak bone peptide derived from the hydrolysis by papain PSM500 plus Peptidase R presented the most efficient radical-scavenging activity to 2,2-diphenyl-1-picrylhydrazyl free radicals; Yak bone peptide hydrolyzed by Protamex single had the best calcium binding activity and the peptide from the hydrolysis of yak bone by trypsin displayed the most ideal chelating activity on iron (II). Taken together, yak bone peptide hydrolyzed by Alcalase plus Flavourzyme at 50°C for 4 h had the best integration function. Therefore, it might be useful for the application in functional foods.     

Determination of tryptophan by high-performance liquid chromatography of alkaline hydrolysates with spectrophotometric detection

A procedure for quantitation of tryptophan in feedstuff is described. It consist of hydrolysis in sodium hydroxide at 100 °C for 4 h, neutralization of the resulting hydrolysate to pH 7, dilution with sodium borate buffer (pH 9), and analysis by reverse-phase high-performance liquid chromatography with spectrophotometric determination of tryptophan at 280 nm. The recovery of tryptophan from lysozyme, added to some feedstuff before hydrolysis, ranges from 98.6 to 100%. A reduction of the time of analysis has been achieved as compared to previous methods for analysis of tryptophan.     

USE OF IMMOBILIZED LACTASE IN PROCESSING CHEESE WHEY ULTRAFILTRATE

Enzymatic hydrolysis of lactose in cottage cheese whey ultrafiltrate was investigated. Lactase of A. niger was immobilized on an alumina-silica catalyst support by the linking agent tolylene-2, 4-diisocyanate. The resulting immobilized enzyme preparation had an activity of 3 standard international units of lactase per gram at pH 4 and 37 °C. The optimum pH and temperature for hydrolysis of lactose by immobilized lactase were 3.5 and 50°C, respectively. Immobilization of the enzyme resulted in reductions of 1.1 pH units and 15°C in optimum pH and temperature, respectively. The two constants of the simple Michaelis-Menten rate expression were obtained from Lineweaver-Burk plots of the initial reaction rate data obtained at 37°C. Estimated values for V_max and the apparent K_m were 7.8 (μmoles/min-g) and 0.26 (M), respectively. Inhibition by the product galactose was measured by studying the hydrolysis reaction in a batch reactor. The inhibition constant K_i was estimated from batch reactor data to be 0.005 and 0.053 (M) at 35 and 50°C, respectively. Activation energies of 8.1 and 6.4 (kcal/gmole) were obtained for the immobilized and soluble enzyme reactions, respectively. The behavior of the batch reactor as measured in terms of a plot of conversion versus time was essentially the same for both conventional and deionized whey ultrafiltrate.     

Multiple α-galactosidases from <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> ZU-G1: purification,characterization and application in soybean milk hydrolysis

α-Galactosidase had applied in food and feed industries for hydrolyzing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to purify the α-galactosidase of Aspergillus foetidus ZU-G1 and compared the biochemical and hydrolytic properties of three major α-galactosidase forms (α-gal I, α-gal II and α-gal III). The molecular mass of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 106.3, 49.7 and 109.9 kDa, respectively. Its optimum reaction temperature was 60 °C and stable below 50 °C. The optimum pH of α-gal I and α-gal III was 5.0 and α-gal II was 4.0. Under 28 °C conditions for 24 h, α-gal I was stable at pH 4.0, α-gal II was stable at pH 6.0, and α-gal III was pH 5.0. α-Galactosidase was completely inhibited by Ag⁺. CuSO₄·5H₂O and SDS were powerful inhibitors of α-gal I and α-gal III but had little effect to α-gal II. EDTA did not strongly affect α-gal I and α-gal III, while strongly affect α-gal II. CaCl₂·2H₂O, MgSO₄·7H₂O and MnSO₄·7H₂O were activation for α-gal I, α-gal II and α-gal III. No significant inhibition of enzymes activity was observed in the presence of raffinose, lactose as well as other sugars tested. Synthetic substrate p-nitrophenyl-α-d-galactopyranoside was not preferentially hydrolyzed than natural substrates, such as melibiose, stachyose and raffinose. Under 40 and 50 °C incubation for 1–5 h, the stachyose of soybean milk was degraded by α-gal I, α-gal II and α-gal III and strongly hydrolyzed by α-gal II, and the raffinose of soybean milk was completely hydrolyzed by α-gal II and weakly hydrolyzed by α-gal I and α-gal III. The distinct hydrolytic and biochemical properties of α-gal I, α-gal II and α-gal III further signify the α-galactosidase of A. foetidus ZU-G1 was propitious to soybean milk and related food industry.     

Methionine Sulfoxide Determination After Alkaline Hydrolysis of Amino Acid Mixtures, Model Protein Systems, Soy Products and Infant Formulas

Two previously reported methods (2M NaOH, 18 hr, 100°C; 3M NaOH, 16 hr, 110°C) for alkaline hydrolysis of proteins containing methionine sulfoxide (MetSO) were compared in free amino acid and model protein systems. Recoveries of MetSO from amino acid mixtures after 2M NaOH hydrolysis and ion-exchange chromatography were higher than after 3M NaOH hydrolysis. Recoveries of methionine (Met), MetSO and methionine sulfone (MetSO₂) from model proteins after 2M NaOH hydrolysis suggested destruction of Met, no production of MetSO₂ and, in the presence of glucose, possible production of small amounts of MetSO. Except for one soy isolate, measured MetSO was ≦ 7% of total methionine (oxidized plus unoxidized) in soy products. In milk- and soy-based infant formulas, measured MetSO ranged from 7 - 32% of totalmethionine.     

Physical and Molecular Properties of Re-extruded Starches as Affected by Extruder Screw Configuration

Amylomaize V, amylomaize VII, wheat and rice starches were extrusion cooked in a single-screw extruder at 140°C barrel temperature and 140 rpm screw speed. Extrudates were ground and re-extruded through a 3 mm cylindrical die nozzle using screws with no mixing (NM), 1 mixing (1M) or two mixing (2M) elements. Overall expansions were the highest and bulk densities were lowest with 1M. Radial expansions for amylomaize V, amylomaize VII, and wheat starches were the highest with 1M and lowest with 2M. Amylopectin contents of starches decreased with corresponding increases of gel permeation chromatograph peak II fractions when number of mixing elements was increased. λ_max values of peak I and peak II decreased upon re-extrusion of starches with increasing number of mixing elements.     

Kinetic of the hydrolysis of pectin galacturonic acid chains and quantification by ionic chromatography

These experiments were designed to develop a rapid, repeatable and accurate analysis method for the quantification of galacturonic acid of pectins. Different pectin hydrolysis procedures (chemical and enzymatic) were carried out with H₂SO₄, TFA and HCl at different acid concentrations (0.2, 1 and 2 M) and temperatures (80 and 100 °C). Enzymatic and combined chemical and enzymatic hydrolysis of pectin were also studied. A acid hydrolysis under drastic conditions (100 °C) is insufficient for complete hydrolysis and results in low recovery of galacturonic acid residues. Mild chemical hydrolysis (0.2, 1 and 2 M H₂SO₄ 72 h at 80 °C) is also insufficient for complete depolymerization. Its main advantage is cleavage of the galacturonic acid chains into oligomeric forms without any degradation within 72 h of hydrolysis. However, enzymatic hydrolysis with VL9 for 2 h at 50 °C and combined chemical and enzymatic hydrolysis (0.2 M TFA at 80 °C for 72 h and VL9) give high recovery of this acid. Analysis of the liberated sugar residue by HPAEC allows us to determine the galacturonic acid composition of this polysaccharide accurately with high selectivity and sensitivity in one assay without the need for derivatization.     

Physicochemical Properties and Storage Stability of Lutein Microcapsules Prepared with Maltodextrins and Sucrose by Spray Drying

The purpose of this study was to determine the physicochemical properties of lutein microcapsules. Nine types of lutein microcapsules were prepared in order to determine their encapsulation efficiency and yield. Results show that lutein microcapsules with maltodextrin M040 and sucrose at the weight ratio of 3:1 (designated as M040:1) had the highest encapsulation efficiency (90.1%) among the lutein microcapsules, as well as a higher encapsulation yield (90.4%). The onset glass transition temperatures (T_gi) and the surface dents of the lutein microcapsules decreased as the dextrose equivalent value of maltodextrin and the weight ratio of sucrose increased. Enthalpy relaxation experiments were conducted for the lutein microcapsules M040:1 at (T_gi – 5) , (T_gi – 10), and (T_gi – 15) °C, and the obtained data were fitted to the Kohlrausch–Williams–Watts model. Results show that the mean relaxation time (τ) (316 h) of M040:1 lutein microcapsules aged at (T_gi – 15) °C was greater than the τ (161 h) at (T_gi – 10) °C and τ (60.5 h) at (T_gi – 5) °C. Effects of temperature and oxygen transmission rates for package film on the storage stability of M040:1 lutein microcapsules were also investigated. Findings show that rates of lutein degradation and color change increased by an order of magnitude as storage temperature (4 to 97 °C) and oxygen transmission rate of the package film (0.018 to 62.8 cc/m² day) increased. These results suggest that lutein is highly unstable and susceptible to thermal and oxidative degradations. However, microencapsulation with appropriate wall materials of higher relaxation time and high oxygen barrier packaging can increase the storage life.     

Controlled Atmosphere and Ethylene Effects on Quality of California Canning Apricots and Clingstone Peaches

Storage at 2% O₂ plus 5% CO₂ at 1.1°C maintained higher flesh firmness and lower pH and retarded decay more effectively than air storage of immature (M1) and over-mature (M3) Patterson and Tilton apricot fruits. CA storage of fruits picked at the optimum maturity stage (M2) produced little benefit over air storage, however. -Treatment with 100 ppm ethylene for 48 hours accelerated softening and color change at 20°C compared to ripening in air and may potentially be used to prepare immature apricot fruits for canning in the shortest possible time. Large differences in storageability and canned quality following storage were found among the five clingstone peach cultivars tested. Loadel and Carolyn: peaches, if in sound condition at harvest, can be stored for up to 4 wk under 2% O₂+ 5% CO₂ at 1.1°C. Andross, Klamt and Halford peaches should be stored for shorter storage periods only. Fruits ripened at 20°C with ethylene (100 ppm for 48 hr) were similar to those ripened without it in appearance, texture, and flavor.