Effect of Myoinositol Phosphate Esters on In Vitro and In Vivo Digestion of Protein

Effects of phytate and its hydrolysis products (myo-inositol phophate esters) on protein digestion were investigated by in vitro and in vivo procedures. Tri-, tetra-, penta-, and hexa-phosphate ester fractions, isolated from phytate hydrolysates, inhibited pepsin digestion of casein and bovine serum albumin in vitro. The inhibition ranged from 0% for the mono- and diphosphate esters to 9–14% with the hexa-phosphate ester (phytate). The phosphate ester fractions did not significantly affect trypsin digestion of the proteins. In the in vivo study, rats were fed phytate and hydrolysates thereof (mixtures of the esters above) at levels of 1–3.5% of the diet. Under the conditions of this study, neither phytate nor its hydrolysates significantly affected protein utilization or weight gain in rats.     

Effect of Phytate and Other Myo-Inositol Phosphate Esters on Lipase Activity

The effects of phytate and other myoinositol phosphate esters (containing one or more phosphate groups) on lipase activity were evaluated by an in vitro procedure. Porcine pancreatic lipase was treated with four concentrations of phytate (0–4 mM) and myo-inositol-2-monophosphate (0–12 mM). The lipase was also treated with six myoinositol phosphate ester fractions (4 mM) isolated from hydrolyzed phytate. The enzyme was incubated (0.5 hr) with the esters prior to addition of substrate and measurement of fatty acid production. Phytate at 4 mM and 1-2-MP at 12 mM caused significant inhibition of lipase, 14.5% and 8.2%, respectively. The isolated inositol phosphate ester fractions caused significant decreases in lipase activity. The decrease in enzymatic activity was highly correlated (r = 0.980) with degree of inositol phosphorylation.     

Effect of Phytate and Other Myo-inositol Phosphate Esters on α-Amylase Digestion of Starch

The effects of phytate and other myo-inositol phosphate esters (containing one or more phosphate groups) on α-amylase digestion of soluble potato starch were evaluated by an in vitro procedure. Human salivary or Bacillus subtilisα-amylase was treated with either 2 mM or 5 mM phytate, myo-inositol-2-monophosphate (l–2-MP), or phytate hydrolyzed to various degrees, and then incubated at 37°C with the starch at pH 4.15 or 6.90. Starch digestion varied with degree of phosphorylation of inositol, inositol phosphate ester concentration, pH and enzyme source. At pH 4.15, phytate (2 mM) and I-2-MP (2 mM) reduced starch digestion by salivary α-amylase to 8.5 and 78.3%, respectively, of the control.     

Effect of Lectins and the Mixing of Proteins on Rate of Protein Digestibility

The rate of digestibility of protein in raw bean extract (RBE), heat-treated bean extract (HBE), casein and bovine serum albumin (BSA) was determined. The pepsin and/or pancreatin hydrolysis of RBE which contains lectins or hemagglutinins was less than that of other proteins. Addition of lectins at the same concentration present in RBE decreased the rate of digestion of HBE, casein and BSA to levels close to that of RBE. In comparison with the respective single proteins, mixtures of RBE or HBE with casein have lower digestibilities while a mixture of casein and BSA, higher digestibilities. The results suggest that lectins can affect the activity of digestive enzymes and that mixing of proteins has an effect on digestibility which is unpredicted by amino acid composition.     

Enzymatic Conditions of an In Vitro Method to Study Protein Digestion

An in vitro digestion method was proposed for studying protein digestion. Protein (casein) was submitted to peptic proteolysis in a closed system followed by hydrolysis with pancreatic enzymes in a “digestion cell” with continuous elimination of digested products by dialysis. The peptic digestion was performed with a pepsin source of high specific activity (3152 units/mg protein) with an enzyme:substrate (E:S) ratio of 1: 250. The second-step of proteolysis was carried out with pancreatin for 6 hr at an E:S ratio of 1:25. A 10 mM sodium phosphate buffer, pH 7.5, was used as the circulating dialysis buffer.     

酪蛋白抗诱变水解物生化特性的研究

采用Ams鼠伤寒沙门氏菌实验研究酪蛋白的胃蛋白酶的水解物的抗诱变性，采用超滤和等电点法分离水解物，证明水解产物比酪蛋白具有更强的抗诱变性，经快速液相色谱FPLC和质谱法分析表明具体抗诱变性短肽是由9到11个氨基酸构成的，分子量在1000到2000道尔顿范围内。     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

Phytate Hydrolysis by Phytase in Cereals; Effects on In Vitro Estimation of Iron Availability

Phytate (inositol hexaphosphate) hydrolysis by endogenous and exogenous phytases was studied for their effect on increasing iron availability in cereals. Wheat bran and whole meal flours of rye and oats were soaked at optimal conditions for phytase activity (55°C, pH 5) for different time intervals. Phytate and its degradation products were determined by HPLC and related to iron solubility under simulated physiological conditions. Small amounts of phytate (< lμmol/g) had a strong negative effect on iron solubility. When inositol hexa- and pentaphosphates of wheat bran and rye flour were completely hydrolyzed by activating endogenous phytase, iron solubility under simulated physiological conditions increased from 3 to 53% (wheat) and 5 to 21% (rye). Addition of wheat phytase to uncooked oatmeal increased iron solubility from 4 to 11 and in precooked to 18%, while endogenous phytase of uncooked oatmeal had less effect on phytate digestion and iron solubility.     

Iron-binding properties, amino acid composition, and structure of muscle tissue peptides from in vitro digestion of different meat sources

ABSTRACT:  Background: The enhancing effect of meat on nonheme iron bioavailability in humans is thought to be due to the release of low-molecular-weight (LMW) iron-binding peptides during digestion. Objective: To better characterize the LMW iron-binding peptides from meat digests. Methods: Cooked beef, chicken, cod, lamb, and pork myofibrillar or sarcoplasmic protein extracts, casein, and egg albumin were digested in vitro with pepsin or pepsin/pancreatin. Ultrafiltrates were analyzed for N and iron and further characterized by gel filtration with added ⁵⁹Fe, amino acid analysis, and LC-MS. Results: 84% to 98% of total iron in enzymic digests was associated with soluble LMW peptides (< 10 kDa) of the myofibrillar proteins compared to only 2% to 20% in the corresponding sarcoplasmic protein digests. Pepsin digestion alone of the myobrillar proteins generated > 80% soluble LMW iron, compared to < 5% with casein and egg albumin. Iron-binding peptides from myofibrillar protein with an estimated 2 kDa molecular mass were separated by gel filtration. Peptides in this fraction were enriched in aspartic and glutamic acid residues and included potential peptide fragments of myosin. Conclusion: LMW (< 10 kDa) peptides in enzyme digests of myofibrillar proteins were the major facilitators of iron solubility. Unlike with casein, egg albumin, and most sarcoplasmic proteins, these LMW peptides were generated on pepsin digestion. One group of iron-binding peptides had a mass of approximately 2 kDa and was enriched in glutamic and aspartic acids. Such early generation of a multitude of LMW iron-binding peptides could explain the enhancing effect of muscle tissue on iron absorption.     

Phytate Reduction in Oats during Malting

To reduce phytate in oat products and thereby increase mineral bioavailability, optimal conditions for phytate degradation in oats were investigated. The effects of malting and incubation on phytate reduction in oats were determined and compared with phytate degradation in wheat, rye and barley. Studies of incubation temperatures showed an optimum for phytate reduction in oats between 37–40°C which differed from that in wheat (55°C). Malting of oats for 5 days at 11°C and subsequent incubation for 17 hr at 37–40°C reduced phytate by 98%. Addition of malted rye also reduced the phytate content of oats to low levels.     

Phytate phosphorus intake and disappearance in the gastrointestinal tract of high producing dairy cows

Thirty Holstein cows were fed diets of 50% grain and 50% corn silage (dry matter basis) through the first 18 wk of lactation. Grain mixes were supplemented with inorganic calcium sources (aragonite, calcite flour, or albacar) to provide .6 or .9% dietary calcium (dry matter basis). Phytate phosphorus intake averaged 38.3 and 42.6 g/d in digestion trials conducted during wk 4 and 10 of lactation, respectively. Even though large quantities of feed and significant amounts of grain were consumed, 98% of dietary phytate phosphorus was hydrolyzed to inorganic phosphorus. Neither calcium source nor calcium quantity affected the hydrolysis of phytate phosphorus.     

Interactions Between Phytate, Protein, and Minerals in Whey Fractions of Black Gram

Whey fractions cor taining proteins (albumins), phytate, and minerals were prepared from black gram (Phaseolus mungo L.) cotyledons and were employee to study the interactions between protein, phytate, and minerals at pH 2.80, 6.40, and 8.40. Black gram cotyledons contained 1.7% phytate, of which 88.7% existed in watersoluble form. Phytate phosphorus represented for about 89% of total phosphorus in black gram cotyledons. Recovery of phytate in fraction I (pH 2.80), fraction II (pH 8.40), and fraction III (pH 6.40) was 45%, 69%, and 4%, respectively, after 48 hr dialysis. At pH 2.80, complexation occurred between phytate and proteins. Complexation between phytate and proteins at pH 8.40 was mediated by divalent cations such as calcium, magnesium, and zinc. Fraction II had higher concentrations of divalent cations (calcium, magnesium, and zir c) than the other two fractions I and III.     

Interactions Among Calcium, Zinc and Phytate with Three Protein Sources

Various combinations of calcium (4.94 mmol), zinc (0.0071 mmol) and phytate (0.284 mmol) were added either to soy concentrate, casein or torula yeast to determine effects of their interaction on in vitro solubility of protein, calcium, zinc, and phytate (PA). Two Ca sources, calcium carbonate (CaCO₃) and calcium-citrate-malate complex (CCM) were used. Two pH levels, 2.0 and 5.5, were used to simulate gastrointestinal pH conditions. An increase in pH significantly reduced (P<0.01) Zn solubility in all treatments with all protein sources. The solubility of Ca and PA were significantly decreased (P< 0.01) when both components were present probably due to formation of insoluble Ca-PA complexes. At pH 5.5, with casein and yeast proteins, Zn was significantly more (P< 0.01) soluble in samples with CCM, in the absence of PA, than in those with CaCO₃.     

Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis)

Pepsins 1 and 2 from the stomach of skipjack tuna (Katsuwonus pelamis) were purified to homogeneity by using a series of chromatographic purification involving DEAE-cellulose, Sephadex G-50 and Sephadex G-75 with increase in purity of 246-fold and 213-fold, respectively. Molecular weights of pepsins 1 and 2 were estimated by SDS–PAGE to be 33.9 and 33.7 kDa, respectively. The N-terminal amino acid sequences of the first 20 amino acids of both isoenzymes were YQDGTEPMTNDADLSYYGVI. The optimal pH and temperature for pepsin 1 were 2.5 and 50 °C, respectively, while pepsin 2 showed optimal activity at pH 2.0 and 45 °C. The activity of two pepsins was stable in the pH range of 2–5 and at temperatures up to 50 °C. The activity of purified pepsins was strongly inhibited by pepstatin A in a dose-dependent manner. SDS and cysteine showed inhibitory effects toward both pepsins. Activity of pepsin 2 was slightly activated by NaCl, but NaCl had no effect on pepsin 1. Pepsins 1 and 2 had high affinity and hydrolytic activity toward hemoglobin with K _m of 54 and 71 μM, respectively. k _cat of pepsins 1 and 2 were 38.1 and 44.3 s⁻¹, respectively. Both pepsins effectively hydrolyzed bovine serum albumin, egg white, natural actomyosin from brownstripe red snapper muscle and acid-solubilized collagen from arabesque greenling skin. Nevertheless, the hydrolytic activity was slightly less than that of pepsin from porcine stomach.     

Enzymatic Reduction of Phytate in Whole Wheat Breads

The presence of phytate in flour may be responsible for reduced bioavailability of iron, magnesium, zinc, and calcium from bread. The effect of various concentrations of commercial phytase or phosphatase added to whole wheat flour-yeast doughs on their phytate and nonphytate phosphorus content has been investigated. By using 2.0% (flour basis) of phytase and 0.11% phosphatase the initial phytate phosphorus concentration of the dough was reduced to 1/8 and 1/12 of its initial values, respectively. Storage of the whole wheat breads for up to 96 hr at room temperature showed further significant reduction of phytate phosphorus. The phytate phosphorus content of yeast leavened whole wheat breads decreased during 2 hours of dough fermentation, baking and the subsequent 48 hours of storage at room temperature from 24 mg/100g dough (dry matter) to 1.7 mg/100g bread (dry matter); the phytate phosphorus continued to decrease and after 96 hours storage it was 0.6 mg/100g bread.     

分步酶解酪蛋白制备小分子ACE抑制肽

通过模拟胃肠道消化,采用单酶和复合酶分步水解酪蛋白获得小分子的血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制肽。首先通过胃蛋白酶水解条件的优化获得具有高ACE抑制活性肽。然后以此为底物通过胰蛋白酶和胰凝乳蛋白复合酶水解条件优化获得具有高ACE抑制活性的小分子肽。结果表明：第一步的胃蛋白酶水解最优条件为：[E]/[S]=6%、[S]=0.015g/mL、pH=1.8、t=37℃、t=2h,水解产物稀释10倍后ACE抑制率为84.5%,分子质量集中在2000D以下;第二步的复合酶水解最优条件为：m胰蛋白酶(6%):m胰凝乳蛋白酶(3%)=2:1、pH=7.8、t=48℃、t=5h,水解产物稀释10倍后ACE抑制率为85.9%,分子质量集中在500D以下。研究表明,通过分步酶解选择合适的酶解条件可以获具有较高ACE抑制活性的小分子肽。     

Dialysis Cell for the In Vitro Measurement of Protein Digestibility

A dialysis cell was devised to study in vitro digestion of proteins. The inner reaction vessel (surrounded by a tubular membrane with molecular weight cutoff of 1000) was fixed into a cylindrical outer compartment where buffer circulation was provided. The dimensions of compartments, membranes and buffer flow rate were determined with labelled amino acids in order to ensure the most efficient diffusion pattern. For the digestion assay, casein was first hydrolyzed with pepsin (pH 1.9) for 30 min. The mixture was then made alkaline (pH 7.5) and poured into the dialysis tube with pancreatin. Nitrogenous material collected with the sodium phosphate buffer was analyzed without further fractionation for the direct measurement of hydrolysis kinetic or of protein digestibility.     

Lipolytic activity of poppy seed

Crude lipase emulsion from poppy seed hydrolyses its endogenous substrate at an optimum pH of 4.9 and optimum temperature of 22-25°C. The lipase emulsion is stable up to 50°C. The enzyme is activated by low concentration of calcium ions; at higher concentrations a partial activity drop occurs. The addition of sodium chloride decrease the lipase activity, whereas casein, whey and bovine serum albumin stimulate its activity. The addition of sucrose does not affect the activity. During the enzymic reaction triglycerides are hydrolyzed yielding diglycerides, free fatty acids and to a lower extent monoglycerides as well.     

Zinc and Phytate Distribution in Peas. Influence of Heat Treatment, Germination, pH, Substrate, and Phosphorus on Pea Phytate and Phytase

The largest proportions of zinc and phytate, 88.7 and 97.1%, respectively, were in the Garfield pea cotyledon; the greatest concentrations were in the germ. Cooking peas by two different methods resulted in 13% phytate reduction. Peas incubated 6.5 hr from 25 to 80°C yielded maximum phytate loss (25%) at 60°C due to phytase activated hydrolysis. Germination (10 d) decreased pea phytate 75% and increased phytase activity 12-fold. Semi-purified germinated pea phytase showed temperature optimum at 45°C, pH optimum of 5.2, 30% inhibition by 1 mM inorganic P, and substrate preference for pyrophosphate. Incubation of early germinated peas at optima pH and temperature is suggested for maximum phytate reduction.