Free Amino Acids and Phosphorylethanolamine in Milk Whey of Cow

Free amino acids in the milk of cow were investigated in comparison with those in the plasma. The concentrations of most free amino acids in the milk except for a few amino acid were lower than those in the plasma. It appears that the percentage of each amino acid in the milk against the corresponding amino acid in the plasma is the reflexion of casein synthesis in the mammary gland. Nutritional alteration influenced on the level of some amino acids in the milk. Free phosphorylserine, glycerylphosphorylethanolamine, and phosphorylethanolamine were observed in the milk. Phosphorylethanolamine was present in significantly high concentration in one animal as control, whereas was almost absent in another animal as experimental.     

Effect of the peptide and vitamin composition of casein hydrolysates on the β-galactosidase activity of Kluyveromyces bulgaricus cells

Instead of yeast extracts, casein hydrolysates from several suppliers were added to culture media and analyzed with respect to their ability to stimulate β-galactosidase activity in Kluyveromyces bulgaricus cells. Four enzymatic casein hydrolysates caused a significantly higher stimulation of enzyme activity, while acid casein hydrolysates clearly reduced the enzyme activity. Enzymatic casein hydrolysates, inducing high and low lactase activity, were analyzed with respect to average peptide length (APL), vitamins (niacin, pathothenate) as well as free and bound amino acids. The molecular weights of these casein hydrolysates were estimated by gel filtration. No correlation was found between the degree of enzyme stimulation and the vitamin contents, the APL values and the free amino acid contents of the casein hydrolysates. Casein hydrolysates stimulating the lactase activity were less soluble in water and, in a gel filtration column, they showed three peaks with slightly lower molecular weights than the three peaks seen in hydrolysates which had no effect on activity. APL values of alcoholic precipitates of casein hydrolysates showed an inverse correlation to lactase activity. The molecular weights of alcoholic precipitates of lactase stimulating digests were also lower compared to non-stimulating ones. Alcoholic precipitates with lactase-stimulating activity were more hydrophilic, as was shown by a smaller proportion absorbed in a C18 column and by amino acid analysis. Our results sugest that alcoholic precipitates could probably be important in the lactase stimulation and their composition should be further investigated. The mechanism is nevertheless complex and may be caused by various simultaneous factors.     

N-ε-fructosyllysine and N-ε-carboxymethyllysine,but not lysinoalanine,are available for absorption after simulated gastrointestinal digestion

Food processing leads to a variety of chemical modifications of amino acids in food proteins. Recent studies have shown that some modified amino acids resulting from glycation reactions can pass the intestinal barrier when they are bound in dipeptides. In this study, we investigated as to what extent modified amino acids are released from post-translationally modified casein during simulated gastrointestinal digestion. Casein was enriched with N-ε-fructoselysine, N-ε-carboxymethyllysine, and lysinoalanine, in different degrees of modification. The casein samples were subjected to a two-step proteolysis procedure, simulating gastrointestinal digestion. The digestibility of modified casein as measured by analytical size-exclusion chromatography (SEC) decreased with increasing degree of modification especially after enrichment of fructoselysine and lysinoalanine. Semi-preparative SEC of digested casein samples revealed that fructoselysine and carboxymethyllysine are released bound in peptides smaller than 1,000 Da, which is comparable to native amino acids. The glycation compounds should, therefore, be available for absorption. Lysinoalanine as a crosslinking amino acid, however, is mostly released into longer peptides of at least 30–40 amino acids which should strongly impair its absorption availability.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Influence of Protein Source on Growth-depressing Effect of Excess Amino Acids in Young Rats

The influence of protein quality on the growth-depressing effect of excessive amount of 12 individual essential and semiessential amino acids was examined. Growing rats were fed for 3 weeks diets containing either 10.5% egg albumin or 11.6% wheat gluten (equivalent to the protein content of a 10% casein diet) supplemented with 5% of each of the l-amino acids. In general, the pattern of growth depression produced by the addition of excess amino acids to the egg albumin or the wheat gluten diet was similar to that of the case of casein diet obtained previously under the same experimental conditions. However, the extent of these effects was dependent not only upon the kind of amino acid supplemented with but also upon the source of protein used, and the depressing effect of each of excess amino acids added to the wheat gluten diet was usually severer than those added to casein and egg albumin diets. No evidence was noted of any striking changes in the liver protein and nucleic acid concentrations by either diets, but total liver protein, RNA and DNA contents were decreased in some amino acid groups of the egg albumin diet and in all amino acid groups of the wheat gluten diet except the lysine addition. The free amino acid level in plasma generally showed extreme elevation for the amino acid supplemented in excess in the diet, and in most cases the extent of the elevation was correlated with the growth depression.     

Effect of heating in the presence or absence of glucose on digestive and metabolic utilization of amino acids of casein]

The effect of heating casein with and without glucose was studied with growing rats. Apparent digestibility of dry matter, organic matter, crude protein and amino acids was recorded. The utilization of amino acids was studied by estimating food consumption, growth and blood and muscle free amino acids. Heating casein with glucose at 90 degrees C during 24 hours resulted in significant loss of lysine and affected food intake, protein efficiency ratio and protein digestibility. Threonine and lysine availabilities were lowered.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

Investigations on the Influence of a Low Casein Diet on the Free Fatty Acids in Liver Homogenate by a Method with Some Devices in Purification Procedure Using KOH

Previously, some changes were noticed in energy metabolism of rats fed a low casein diet. In connection with these phenomena, influence of a low casein diet on the composition and amounts of free fatty acids in liver homogenate after autolyzing for a few hours was investigated. For the measurement of free fatty acids, they were purified by a method with some devices in purification procedure using KOH. It was found that amounts of free fatty acids in liver homogenate after autolyzing for a few hours were lower in rats fed a low casein diet.     

Effect of hydrophobicity of utilization of peptides by ruminal bacteria in vitro.

When mixed ruminal bacteria were incubated with a pancreatic casein hydrolysate and free amino acids of a similar composition, rates of ammonia production were much greater for peptides than for amino acids. The pancreatic digest of casein was then fractionated with 90% isopropyl alcohol. Hydrophobic peptides which dissolved in alcohol contained an abundance of phenolic and aliphatic amino acids, while the hydrophilic peptides which were precipitated by alcohol contained a large proportion of the highly charged amino acids. The Km values of the mixed ruminal bacteria for each fraction were similar (0.88 versus 0.98 g/liter), but the Vmax of the hydrophilic peptides was more than twice that of the hydrophobic peptides (18 versus 39 mg of NH3 per g of bacterial protein per h). Pure cultures of ruminal bacteria had a similar preference for hydrophilic peptides and likewise utilized peptides at a faster rate than free amino acids. Since peptide degradation rates differed greatly, hydrophobicity is likely to influence the composition of amino acids passing unfermented to the lower gut of ruminant animals.     

Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.     

The solubilization and degradation of pumpkin seed globulin during germination

Pumpkin seed globulin decreased 88% during the first 4 daysof germination. This decrease was concomitant with a 2.5 foldincrease in water soluble protein which arose directly fromthe water insoluble globulin. The sequence of solubilizationand breakdown of the globulin was followed through 12 days ofgermination. Pumpkin seed globulin was determined to have subunitsof 56,000 daltons, while the new water soluble protein consistedof two proteins, as determined by sodium dodecyl sulfate polyacrylamideelectrophoresis; one having a molecular weight of 42,000 daltonsand the other 28,000 daltons. Trypsin mimicked the first stepof the breakdown by solubilizing pumpkin seed globulin to yieldidentical digestion products as were obtained in vitro. Maximumproteolytic acitvity, as measured by the release of ninhydrinpositive, materials occurred at 6 days of germination, at whichtime both the concentration of free amino acids and the incorporationof ¹⁴C into amino acids increased rapidly. A second proteolyticenzyme system which solubilized the pumpkin seed globulin butdid not act on hemoglobin, casein, or bovine serum albumin reachedits maximum activity at two days of germination. (Received April 17, 1978; )     

Long-lived radicals of amino acids induced by X-ray radiation are the source of hydrogen peroxide in aqueous medium

Formation of long-lived radicals in solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after irradiation, which indicates generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolution of irradiated dry amino acids capable of luminescing over a long time causes formation of hydrogen peroxide in the aqueous medium.     

Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture

Quantitative analyses of the utilization of amino acids by Lactococcus lactis subsp. cremoris FD1 in yeast extract medium (YE) and in casein peptone medium (CP) have been performed. Both free and peptide-bound amino acids were measured. In the CP most amino acids are peptide-bound and some amino acids are virtually only present in peptides. Thirty-six per cent of all peptide bonds in CP are hydrolysed during fermentation (6·3 mmol peptide bonds per gram biomass formed) and there is a transition of the growth rate related ATP consumption Y _xATP (mmol ATP g biomass^-1) from 25 mmol g^-1 to 71 mmol g^-1 coincident with a decrease of the peptide consumption. In YE most of the amino acids are on the free form and only 26% of the peptide bonds are hydrolysed during fermentation (1·5 mmol peptide bonds per gram biomass formed). A constant Y _xATP= 38 mmol g^-1 prevails throughout the fermentation in YE.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Long-lived radicals of amino acids induced by X-ray-radiation are the source of hydrogen peroxide in aqueous medium

The formation of long-lived radicals in the solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after the irradiation, which indicates the generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolving irradiated dry amino acids capable of luminescing over a long time gives rise to the formation of hydrogen peroxide in aqueous medium.     

A Simple Gasometric Method for Determination of Free Amino Acid Contents in Biological Fluids

A simple method for the determination of total amino acid contents in biological fluids which contain various amino acids, peptides and proteins is described. This method is based on the determination in a Gilson differential respirometer of CO₂ gas evolved by the reaction of chloramin-T with α-amino-carboxylic acids in 5% trichloroacetic acid. The use of the trichloroacetic acid greatly suppressed interference by amines and ammonia, thus making this method appreciable to the determination of free amino acids in biological fluids without prior separation. Samples containing 1~20 μmoles of free amino acids can be assayed with accuracy of ±3%. This method was applied to the study of proteolytic digestion of casein.     

Suitability of animals' purified milk caseins and their subunit kappa-caseins as substrates for subtilisin and trypsin.

Acid casein and kappa-casein were purified from different species of animal's milk, such as cow, sheep, goat, and water buffalo. These caseins were used as substrates for commercially available subtilisin and trypsin. It was established that, when acid caseins were used as a substrate for subtilisin, cow acid casein was found to be a better substrate for the enzymes, compared to other animals' milk casein. It was suggested that this acid casein has significantly more aromatic amino acids, as compared to arginine and lysine. K(M) and Vmax values, which were obtained for cow kappa-casein, showed that cow kappa-casein was a better susbstrate for trypsin than the others, suggesting that cow kappa-casein has a rich content of lysine, arginine, and aromatic amino acids by comparison with the others. The calculated C/N ratio also supports this suggestion.     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

Relationship between relative protein value and somein vitro indices of protein quality

Besides amino acid composition of a protein, their bioavailability is an important determinant of the protein quality. In view of the observations over the last decade or two, implicating the small peptide uptake by the mammalian intestine as a major route of protein absorption, a few animal and plant proteins were subjected to sequential enzymatic digestionin vitro with pepsin, pancreatin + trypsin and erepsin and the release of amino acids as small (including dipeptides) and large peptides and free amino acids, was determined. The relative protein values of α-lactalbumin, egg whites, casein, gluten, zein and protein isolates of soyabeans and groundnuts was determined using rat growth method. It was observed that relative protein value were positively correlated with the essential amino acid index of protein, quantity of essential amino acids released as small peptides and the dipeptide content of enzymatic digests, while there was a negative correlation between relative protein value and essential amino acid content of large peptide fraction.