Aminopeptidase N gene expression and abundance in caprine mammary gland is influenced by circulating plasma peptide

This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation.     

氨肽酶脱苦效果的研究

目的研究从豆腐乳中筛选到的一种枯草芽孢杆菌所产的氨肽酶脱除大豆蛋白质水解物苦味的效果。方法用高效液相色谱法和感官评定。结果1%的大豆蛋白质溶液经胰蛋白酶水解,水解液呈苦味,添加氨肽酶可较好地脱苦,水解液中游离氨基酸的总量增加了28.8%,疏水氨基酸的量也明显增加。结论枯草芽孢杆菌氨肽酶具有较好的脱苦效果。     

氨肽酶脱苦效果的研究

目的研究从豆腐乳中筛选到的一种枯草芽孢杆菌所产的氨肽酶脱除大豆蛋白质水解物苦味的效果。方法用高效液相色谱法和感官评定。结果1％的大豆蛋白质溶液经胰蛋白酶水解，水解液呈苦味，添加氨肽酶可较好地脱苦，水解液中游离氨基酸的总量增加了28．8％，疏水氨基酸的量也明显增加。结论枯草芽孢杆菌氨肽酶具有较好的脱苦效果。     

Debittering Mechanism in Bitter Peptides of Enzymatic Hydrolysates from Milk Casein by Aminopeptidase T

The bitter peptide fraction present in casein hydrolysates obtained by using three proteases (subtilisin, papain and trypsin) was treated with aminopeptidase T from Thermus aquaticus YT-1. The bitterness of the bitter peptide fraction could be decreased, and it sometimes disappeared completely, with an increase in free amino acids. The percentages of total free amino acids released from each bitter peptide fraction (subtilisin, papain and trypsin) by aminopeptidase digestion for 20 hr were approximately 11%, 8.7%, and 6.5%, respectively. Bitter peptide (α_s1-CN f91-100) was isolated from a tryptic hydrolysate of casein by HPLC, its threshold value of bitterness being 2.9 ppm (w/v). The peptide (α_s1-CN f96-100) obtained from the amino peptidase digestion of this bitter peptide showed no bitterness.     

乳酪蛋白源免疫调节肽

酪蛋白是乳中最主要的蛋白质 ,除了提供氨基酸和能量的营养功能外 ,酪蛋白还是生物活性肽的重要来源。这些生物活性肽本身以非活性状态存在于蛋白质氨基酸序列之中 ,当用适当的蛋白酶进行体外水解或在胃肠道消化过程中以及食品加工过程中 ,它们的活性就被释放出来 ,并可作为生理功能的重要调节剂。大量证据表明乳酪蛋白中存在大量的免疫调节肽。本文对乳酪蛋白来源的免疫调节肽的序列结构及其对免疫系统的调节机能做一综述。     

Amino Acid Absorption in Portal Blood After Duodenal Infusions of a Soy Protein Hydrolysate Prepared by a Novel Soybean Protease D3

ABSTRACT:  The intestinal absorption of amino acids from decapeptide was investigated in rats under unrestrained conditions. The soy protein hydrolysate utilized in the experiment was produced by a novel soybean protease D3. The enzymatic features of protease D3 showed high homology with cathepsin L and cathepsin K and the average molecular weight of D3 hydrolysate is approximately 1200. We compared the intestinal absorption of D3 hydrolysate in portal blood with that of an amino acids mixture and soy protein with the same amino acid composition by determining the concentration of individual amino acids after a single administration of a nitrogen source. The absorptive velocity and intensity of each amino acid were calculated from its rate of elevation in the portal blood. And in most cases, these were higher in the D3 hydrolysate than in amino acids mixture and protein. The proportion of the amount of each amino acid absorbed in portal blood from D3 hydrolysate was much more like the composition of the administrated amino acids than like that from the amino acids mixture. The result of in vitro digestion assay indicated that D3 hydrolysate was hydrolyzed easier than the hydrolysates produced by microbial proteases. This is the first report to demonstrate that the D3 hydrolysate, which contains decapeptide as a dominant fraction, was more rapidly utilized than the amino acids mixture and protein as is the case with di-, tripeptides. This suggested that this hydrolysate could be available for nutraceutical use as well as use in nutritious foods for athletes and patients.     

Partially Hydrolyzed Soy Protein Shows Enhanced Transport of Amino Acids Compared to Nonhydrolyzed Protein across an Intestinal Epithelial Cell Monolayer

Consumption of protein hydrolysates has been proposed to stimulate muscle anabolism more than intact (nonhydrolyzed) proteins via accelerated delivery of amino acids for muscle protein synthesis (MPS). We evaluated whether the rate of amino acid uptake and transport across intestinal cells was enhanced for soy protein hydrolysates versus nonhydrolyzed soy protein. Intact and partially hydrolyzed proteins were subject to simulated gut digestion and applied to the apical surface of Caco‐2 monolayers. Basolateral media was harvested after 3 h and quantitatively analyzed for free amino acids using ion‐exchange chromatography and comparison to an included reference standard. Basolateral concentrations of all amino acids were higher (mean 32%) for hydrolyzed versus nonhydrolyzed protein with the greatest differences in histidine, lysine, and valine. Scale‐up production of the soy protein hydrolysate did not diminish its enhanced absorption properties. These data support the hypothesis that hydrolyzed soy protein may provide dietary amino acids that are more rapidly transported across the intestinal epithelium versus intact soy protein. This would be important under conditions where rapid and increased levels of amino acids are needed such as in the stimulation of MPS.     

Enzymes of the small intestine of the calf: effect of dietary protein source on the activities of some enzymes in the small intestinal mucosa and digesta

The objective of this study was to examine the effect of dietary protein source on the morphology and enzyme activities in the small intestinal mucosa and digesta of preruminant calves. The control diet contained skim milk powder. Plant proteins from soy concentrate (unhydrolysed or partially hydrolysed) and potato concentrate were incorporated into experimental diets. Duodenal, jejunal and ileal mucosal biopsies and digesta were collected from calves fitted with simple cannulae and continuously infused with milk replacers into the abomasum. Biopsies were used for morphology measurements. The activity of dipeptidylpeptidase IV, aminopeptidases, lactase and alkaline phosphatase was estimated in biopsies and digesta. Incorporating plant protein had a limited effect on the intestinal mucosa. A partial villus atrophy and crypt hyperplasia were noted with the soy concentrate only. Decreases in the activity of dipeptidylpeptidase IV with the potato concentrate diet and of aminopeptidase N and lactase with the hydrolysed soy concentrate diet, were also observed in ileal biopsies. Mucosal enzymes were released in the lumen and most of them accumulated in digesta of the distal small intestine. The activity of aminopeptidase N and lactase in ileal digesta was higher in calves fed the soy and potato concentrate diets than in those fed the control diet. © 2002 Society of Chemical Industry     

OXIDATION OF METHIONINE RESIDUES OF CASEIN BY HYDROGEN PEROXIDE. Effects on In Vitro Digestibility

Analytical methods were adapted to the determination of methionine sulfoxide and methionine sulfone in casein. Direct determination by alkaline hydrolysis appears to be more accurate than indirect determination by the carboxymethylation-performic oxidation method. Proteolysis of casein with Pronase was adopted as an in vitro digestibility test. In the conditions chosen, 41% of the methionine residues of casein were released as free methionine. A solution of casein was treated with up to 0.12M hydrogen peroxide. No methionine sulfone was formed. Most of the methionine residues were oxidized into methionine sulfoxide. When milk was treated with 0.018M hydrogen peroxide, 51% of its methionine residues was transformed into methionine sulfoxide. Hydrogen peroxide-oxidized casein samples were submitted to the Pronase digestibility test. Release of free methionine was inversely related to the methionine sulfoxide content. No free methionine sulfoxide was released from casein. Release of other amino acids was little affected by hydrogen peroxide treatment. Similar results were found with milk.     

Microstructure and Texture of Process Cheese,Milk Curds,and Caseinate Curds Containing Native or Boiled Soy Proteins

Scanning electron microscopy was used to examine the internal microstructure of process cheese, process cheese containing native soy protein, process cheese containing boiled soy protein, rennet coagulated milk curd, milk curd containing native soy protein, milk curd containing boiled soy protein, rennet coagulated caseinate curd, caseinate curd containing native soy protein, and caseinate curd containing boiled soy protein. Textural characteristics were determined with an Instron Food Testing System. Soy proteins caused the fine network microstructure of processed cheese to change to coarse porous structure. Hardness of cheese was lowered, but cohesiveness was increased by adding soy protein to process cheese. In milk curd, particles of soy protein were clustered in groups in micrographs. Cohesiveness of milk curd was lowered by native soy protein and even more by boiled soy protein. Hardness and springiness were lowered by boiled soy protein. Boiled soy protein was more extensively aggregated than was native soy protein. Soy proteins were not clearly distinguishable from casein in the micrograph of caseinate curds. Soy proteins reduced hardness and cohesiveness of caseinate curd but not as drastically as in milk curd.     

Effects of dietary protein degradability and casein or amino acid infusions on production and plasma amino acids in dairy cows

Responses to daily abomasal infusions of 400 g sodium caseinate, 400 g hydrolyzed casein, or 11.3 g L-methionine plus 30.1 g L-lysine were compared in eight Holstein cows fed diets with estimated ruminal protein degradabilities of 70 and 60.%. Basal diets contained corn silage and corn with either soybean meal or 66.7:33.3 soybean meal:corn gluten meal added. Infusion with Methionine plus lysine increased milk protein content when cows fed either diet but increased milk fat content and yield only when the soybean meal diet was fed. Sodium caseinate increased milk and milk protein production and decreased milk fat percentage. Concentration of total essential amino acids, branched chain amino acids, and urea cycle amino acids were increased by the infusion of both casein sources. Methionine-lysine infusion increased plasma lysine and taurine, a metabolite of methionine, suggesting that absorbed methionine was extensively metabolized. Results demonstrate an impact of both ruminal degradability of dietary protein and form of infused protein on amino acid nutrition of lactating daily cows.     

Hygienic characteristics of food hydrolysates made from small ocean fish and krill

A study was made of the biological value of acid and enzymatic hydrolysates from capelin, luminous anchovy and krill. Hydrolysates were obtained with the use of protosubtilin G-10-X or hydrochloric acid. The products were found to contain 39 to 64% of "crude" protein, with about 40% of total nitrogen belonging to non-protein one, 0.47-2.07% of lipids, 29.7-54.3% of mineral substances including 26.6-52.4% of sodium chloride. All the hydrolysates were limited in tryptophan, the deficiency being more demonstrable in acid hydrolysates. Enzymatic hydrolysate from luminous anchovy was rich in sulfur-containing amino acids (score 112%), whereas the remaining products were marked by their deficiency (score 53-90%). The products were rich in lysine, leucine, isoleucine, and aromatic amino acids. The anabolic efficacy was discovered to be the highest for enzymatic hydrolysate from luminous anchovy, exceeding the analogous characteristics for casein. The biological value of hydrolysate from capelin and krill was lower than that of casein. This was supported by the amino acid analysis data. The assimilability of all hydrolysates was established as fairly high. Hydrolysates are employed for manufacturing broth bricks and pastes.     

Microstructure and chemical changes in Twarog cheese made from ultrafiltrated milk and from lactose-hydrolysed milk

Twarog cheese was manufactured from fresh milk, lactose-hydrolysed milk and retentate. Microstructure, chemical composition, protein breakdown, amino acid distribution and organoleptic properties were studied during ripening.Inoculation of the milk with β-galactosidase, before cheese processing, reduced the clotting time, enhanced the protein breakdown and enhanced amino acid accumulation during ripening. Concentrating the milk, by ultrafiltration, prolonged the coagulation time and resulted in a cheese with greater amounts of moisture, fat, total nitrogen and higher pH than other cheeses.Essential free amino acids are considered to constitute more than half of the total free amino acids of Twarog cheese and glutamic acid, leucine and phenylalanine are the major free amino acids in the ripened cheese.Quality of the cheese was improved either by ultrafiltration or by lactose hydrolysis and the acceptable flavour was more pronounced in β-galactosidase-treated cheese.Microstructure of the protein in young cheeses does not differ in all treatments. The disintegration and fusion of casein advanced during ripening in the outer protein matrix more than internally. The appearance of the cheese matrix was similar in both control and ultrafiltrated cheeses while the casein in β-galactosidase-treated cheese fused faster than in other treatments.     

Ussing chamber results for amino acid absorption of protein hydrolysates in porcine jejunum must be corrected for endogenous protein

BACKGROUND: Ussing chambers have been used extensively as an ex vivo model to investigate intestinal nutrient absorption. In this study Ussing chambers were used to investigate the absorption of amino acids and peptides in pig jejunal tissue using a highly hydrolysed casein hydrolysate (PeptoPro®), casein and whey protein isolate after they had been digested with pepsin and pancreatin to simulate digestion products in the jejunum. Jejunal tissue was collected from three pigs and mounted into Ussing chambers, equilibrated, and the luminal chamber loaded with one of three test nitrogen sources. Luminal solution samples were taken every 10 min over a 90 min incubation period and the amino acid concentration determined. To determine the endogenous amino acid contribution of the tissue to the luminal solution, Ussing chambers containing no test N source (blanks) were prepared and treated similarly to the Ussing chambers containing the test N sources. RESULTS: The endogenous amino acid contribution was 0.5 mg in the luminal solution at time 0 and increased to 1 mg after 90 min. The mean amino acid absorption after 10 min incubation for the pre‐digested highly hydrolysed casein hydrolysate (16.6%) was significantly (P < 0.05) higher than for the pre‐digested casein (7.6%) and whey protein isolate (6.7%). CONCLUSION: Endogenous amino acids were a quantitatively significant portion of the luminal solution amino acids present in the Ussing chambers containing the test N sources. Ussing chambers may be a suitable tool for studying amino acid absorption of protein hydrolysates but correction for the endogenous amino acid contribution from the intestinal tissue must be made. Copyright © 2009 Society of Chemical Industry     

Effects of insulin and postruminal supply of protein on use of amino acids by the mammary gland for milk protein synthesis

We examined the relationships between amino acid supply, net utilization of amino acid by the mammary gland, and milk protein yield, in investigations that utilized a hyperinsulinemic-euglycemic clamp. A two-way crossed factorial design was employed. There were two 12-d periods involving abomasal infusions of either water or a mixture of casein (500 g/d) plus branched-chain amino acids (88 g/d), with a hyperinsulinemic-euglycemic clamp during the last 4 d of each period. During the clamp, insulin was infused at 1.0 microg x kg BW(-1) x h(-1) to increase circulating levels fourfold, and euglycemia was maintained by infusion of glucose. The insulin clamp treatments increased milk protein yield by 15 and 25% during abomasal infusion of water or casein plus branched-chain amino acids, respectively. Circulating concentrations of essential amino acids were reduced (33%) during insulin clamp treatments, especially branched-chain amino acids (41%). Arteriovenous difference of essential amino acids across the mammary gland was linearly related to their arterial concentrations. However, milk protein yield was not related to either arterial concentration or arteriovenous difference, for any of the essential amino acids. During insulin clamp treatments, the mammary gland was able to support the increased milk protein yields by increasing extraction efficiency of essential amino acids, mammary blood flow, and glucose uptake. Furthermore, a positive mammary balance of total amino nitrogen and carbon was maintained for all treatments. These adaptations demonstrate the unique ability of the mammary gland to adjust local conditions to allow for an adequate nutrient supply.     

Identification of an Angiotensin I-converting Enzyme Inhibitory Peptides from Protein Hydrolysates by a Soybean Protease and the Antihypertensive Effects of Hydrolysates in 4 Spontaneously Hypertensive Model Rats

ABSTRACT: Our previous study demonstrated that, because of its substrate specificity, protein hydrolysates by protease D3, which is originated from soybean, exhibited the prominent property of being less bitter than other enzymatic hydrolysates. In the 1st experiment in this series, angiotensin I-converting enzyme (ACE) inhibitory peptides from soy protein hydrolysate by D3 were identified by the establishment of a novel and effective peptide identification method. The amino acid sequences of candidate ACE inhibitory peptides were determined by electrospray ionization mass/mass spectrometry (MS/MS) analysis after rough purification of the samples with gel filtration chromatography and reverse-phase chromatography. Some of the candidate peptides had amino acid sequences that showed homology with those of the reported ACE inhibitory peptides. Then, 8 types of novel candidate peptides were synthesized according to a solid-phase method, and their ACE inhibitory activity was confirmed as the IC₅₀ value. The most potent inhibitor was NWGPLV (IC₅₀= 21 μ M ). In the 2nd experiment, the antihypertensive activity of protein hydrolysates by D3 was investigated in spontaneously hypertensive model rats (SHRs). The dose-dependent antihypertensive effect of soy protein hydrolysate was confirmed, and systolic blood pressure was significantly reduced after the oral administration of doses exceeding 100 mg/kg. Casein hydrolysate was found to have the most potent effects on suppressing blood pressure as well as ACE inhibitory activity among the various food protein hydrolysates studied because of the primary structure of casein. These results indicate that hydrolysates by D3 could be a useful food ingredient because it has the physiological function (antihypertensive activity).     

The effects of casein on the metabolism of fatty acids,methyl ketones and secondary alcohols by Penicillium roqueforti in buffered solutions

The metabolism of fatty acids and methyl ketones by Penicillium roqueforti was studied in phosphate buffered solutions and buffered solutions with added soluble casein. It was found that casein enhanced the metabolism of fatty acids and the quantity of methyl ketones produced. Methyl ketones were also found to be metabolised by P. roqueforti in phosphate buffered solutions. The addition of fatty acids inhibited the metabolism of the ketones. The inhibitory effects of the fatty acids were nullified in the presence of casein hydrolysate.     

大豆蛋白水解物促进乳酸发酵的作用

通过研究添加大豆蛋白水解物对保加利亚乳杆菌培养过程中体系的酸度、粘度、细菌生长量的影响，验证了大豆蛋白水解产物对保加利亚乳杆菌增殖的促进作用。     

高盐稀态牡蛎酿造酱油的开发与品质分析

以豆粕、小麦及牡蛎酶解液为原料,采用高盐稀态发酵工艺,研发具有甜香风味的功能性牡蛎酱油,并将其与对照酱油的感官品质、氨基酸含量及风味成分进行了分析比较。结果表明,在牡蛎酶解液添加量为25%时,牡蛎酱油的氨基态氮含量达1.2 g/100 mL。牡蛎酱油较对照酱油在海鲜风味方面更为突出,同时在鲜度、香气方面以及整体评价也更佳。牡蛎酱油和对照酱油均鉴定出6种鲜味氨基酸,但牡蛎酱油的鲜味氨基酸含量（41.8%）高于对照酱油（40.5%）;牡蛎酱油中牛磺酸含量高达36.7 mg/100 g,约为对照酱油的13.6倍。牡蛎酱油的挥发性风味物质中醇类、呋喃类相对含量较高,分别为59.5%、20.7%,从而赋予其香浓而独特的风味。     

九龙牦牛奶渣氨基酸含量和蛋白质组成分析

本研究的目的是分析家庭自制的九龙牦牛奶渣中氨基酸含量和蛋白组成特点。结果显示,九龙牦牛奶渣中总氨基酸含量约为36.5%,含量高的氨基酸主要包括谷氨酸、脯氨酸、亮氨酸、天冬氨酸、赖氨酸。必需氨基酸占总氨基酸的比例约为36%。电泳分析显示,牦牛奶渣蛋白质组分以酪蛋白为主,其比例约为82%。实验观察到牦牛酪蛋白的初步降解。研究结果表明,九龙牦牛奶渣的生化组成与普通牦牛奶渣接近。