Short communication: influence of transglutaminase on the heat stability of milk.

Skim milk powders were prepared from control and transglutaminase-treated skimmed milk. The heat stability of reconstituted transglutaminase-treated skimmed milk (9.0% total solids) was markedly increased in the pH region of minimum stability (pH 6.8 to 7.1) compared with control milk, while the heat stability of reconstituted concentrated transglutaminase-treated skimmed milk (22.5% total solids) increased progressively as a function of pH relative to control milk. The effect of transglutaminase treatment on the heat stability of skimmed milk may have commercial applications, but extensive research is necessary to gain a better understanding of the mechanism by which transglutaminase improves heat stability.

Nucleosides and nucleotides: natural bioactive substances in milk and colostrum.

Nucleotides, nucleosides and nucleobases belong to the non-protein-nitrogen (NPN) fraction of milk. The largest amounts of ribonucleosides and ribonucleotides--ribose forms only were considered in this review--were measured directly after parturition in bovine milk and other ruminants as well as in the milk of humans. Generally, concentrations of most of the nucleos(t)ides tend to decrease gradually with advancing lactation period or nursing time. The species-specific pattern of these minor constituents in milk from different mammals is a remarkable property and confirms, at least, the specific physiological impact of these minor compounds in early life. The physiological capacity of these compounds in milk is given by the total potentially available nucleosides. The main dietary sources of nucleos(t)ides are nucleoproteins and nucleic acids which are converted in the course of intestinal digestion into nucleosides and nucleobases the preferred forms for absorption in the intestine. Thus, nucleosides and nucleobases are suggested to be the acting components of dietary and/or supplemented nucleic acid-related compounds in the gut. They are used by the body as exogenous trophochemical sources and can be important for optimal metabolic functions. Up to 15 % of the total daily need for a breast-fed infant was calculated to come from this dietary source. Concerning their biological role they not only act as metabolites but are also involved as bioactive substances in the regulation of body functions. Dietary nucleotides affect immune modulation, e.g. they enhance antibody responses of infants as shown by a study with more than 300 full-term healthy infants. Dietary nucleos(t)ides are found to contribute to iron absorption in the gut and to influence desaturation and elongation rates in fatty acid synthesis, in particular long-chain polyunsaturated fatty acids in early stages of life. The in vitro modulation of cell proliferation and apoptosis has been described by ribonucleosides, in particular by modified components using human cell culture models. Due to the bio- and trophochemical properties of dietary nucleos(t)ides, the European Commission has allowed the use of supplementation with specific ribonucleotides in the manufacture of infant and follow-on formula. From the technochemical point of view, the ribonucleoside pattern is influenced by thermal treatment of milk. In addition ribonucleosides are useful indicators for quantifying adulterations of milk and milk products.

Apoptosis induced by modified ribonucleosides in human cell culture systems.

The in vitro modulation of apoptosis and cell proliferation by modified in comparison with non-modified ribonucleosides was investigated for the first time using peripheral blood lymphocytes, HL-60 cells and Caco-2 cells as human cell culture models. Modulating effects of several ribonucleosides were found in the range of 10(-7)-10(-3) mol/l. The following ribonucleosides induced significant apoptosis of HL-60 cells: adenosine, N6-dimethyladenosine, N6-(2-isopentenyl)-adenosine, N2-dimethylguanosine. A significant apoptotic effect on PBL was found with N6-dimethyladenosine and N6-(2-isopentenyl)-adenosine. N6-Dimethyladenosine, N6-(2-isopentenyl)-adenosine and guanosine had a pronounced inhibitory effect on Caco-2 cell apoptosis. Regarding the known function of ribonucleosides as pathobiochemical marker molecules for cancer, the possibility of a selective apoptotic effect against malignant cells is discussed.

Crosslinking of sodium caseinate by a microbial transglutaminase.

Sodium caseinate is an effective substrate for transglutaminase. Crosslinking takes place at fast reaction rates resulting in high degrees of crosslinking. The polymers can be hydrolysed by trypsin, but the proteolysates contain residual portions of non- or partlyhydrolysed aggregates. Crosslinking of hydrolysed sodium caseinate decreased the number of free amino groups, but leads only to a very small increase in molecular weight of the peptides.

[Characterization of pancreatic casein plasteins]. / Charakterisierung pankreatischer Casein-Plasteine.

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of beta-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from alpha s-casein, were determined.

Characterization of trypsin immobilized on oxirane-acrylic beads for obtaining phosphopeptides from casein.

The aim of the study was to characterize the proteolytic properties of immobilized trypsin for obtaining phosphopeptide-rich fractions from casein. Trypsin was covalently bound to oxirane-acrylic beads. After incubation for 48 h immobilization degrees of about 85% were achieved. 20% of the immobilized enzyme exhibited no activity towards the substrate N-benzoyl-L-arginine ethyl ester. Compared with homogeneous catalysis with the soluble enzyme a 25% lower degree of proteolysis was calculated and a modified peptide pattern of the resulting proteolysates established. A caseinophosphopeptide (CPP) from alpha s1-CN (59-79) was not detectable after incubation with the carrier-bound enzyme. At a substrate concentration (S) of 15% (w/w) substrate saturation of the enzyme (E) was achieved. Increasing the substrate concentration to 20% (w/w) decreased the conversion rates (content of soluble amino-N) and the liberation of CPPs. Proteolysis of small (1% w/w) and partly also large (20% w/w) substrate concentrations (E/S = 1/100) is subject to changed kinetic conditions. The same was true for small and large enzyme concentrations (S = 5% w/w). Compared with enzyme saturation (E/S = 1/50), lack of enzyme (E/S = 1/800) led to a disproportional decrease in the proteolysis rate and to a markedly decreased content of hydrophobic peptides in the resulting proteolysates. Increasing the pH from 7.8 to 8.8 and the temperature from 37 degrees to 47 degrees C caused only a slight increase in conversion rates, but an overproportional liberation of CPPs (pH 8.8 = + 47%, 47 degrees C = +89%), in particular from beta-casein. Repeated use of immobilized trypsin resulted after nine runs in a loss in proteolytic activity and in CPP yields of approximately 25%, while the peptide pattern of the proteolysates remained qualitatively unchanged. Light microscopy shows that the oxirane-acrylic beads disintegrate to a large extent after nine repetitions.

Ribonucleosides in human milk. Concentration profiles of these minor constituents as a function of the nursing time.

Ribonucleosides are secreted as products of cellular RNA and ribonucleotide metabolism into physiological fluids such as blood, milk and urine. Unmodified and modified ribonucleosides have been detected in the micromolar range as minor constituents in the milk of different mammals. In addition to the common nucleosides adenosine, cytidine, guanosine, inosine and uridine, modified components such as 1-methyladenosine, 1-methylguanosine, 1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoyl-L-threonyladenosine, pseudouridine and 5-aminoimidazole-4-carboxamide-N-ribofuranoside (AICAR) have been identified and most of them quantified in samples of human and/or bovine and/or goat's milk. From these investigations it is known that nucleosides, in analogy to nucleotides, show a typical species-specific pattern. Longitudinal studies have been carried out to determine the concentration profiles of the individual ribonucleosides in the milk of humans as a function of the nursing time.

Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects.

The primary function of dietary proteins is to supply the body adequately with indispensable amino acids and organic nitrogen. Little attention has been paid up to date to milk proteins, in particular caseins, that are currently the main source of biologically active peptides, although other animal as well as vegetable proteins are known to contain potentially bioactive sequences. Such regulatory peptides can be released by enzymatic proteolysis of caseins in vitro and in vivo and may act as potential physiological modulators of metabolism during the intestinal digestion of the diet. It has been proved that bioactive peptides derived from caseins, such as beta-casomorphins and phosphopeptides, can be released during gastrointestinal passage. It is also evident that peptides originating from food proteins should be taken into account as potential modulators of various regulatory processes in the body. The possible regulatory effects concern nutrient uptake (phosphopeptides, casomorphins), postprandial hormone secretion (casomorphins), immune defense (immunopeptides, casokinins, casomorphins) and neuroendocrine information transfer (casokinins). The advances in the research field of bioactive peptides are driven by a molecular understanding of biological processes, and analytical techniques are a critical component of this understanding. Different up-to-date methods, including peptide synthesis and immunochemistry, have been applied to the chemical characterization of bioactive peptides. Especially casein derived peptides have already found interesting applications, both as dietary supplements (phosphopeptides) and as pharmaceutical preparations (phosphopeptides, beta-casomorphins). The question of 'what kinds of bioactive peptides are beneficial and desirable as food constituents or as drugs' should be always carefully examined. However, the possibilities for the design of dietary products and 'natural' drugs look promising.

Heat-induced changes in casein-derived phosphopeptides.

Phosphopeptides derived from casein may function as carriers for calcium and trace elements. In regard to such specific nutritive effects, the heat-induced changes in tryptic phosphopeptides liberated from bovine sodium caseinate as a model system were investigated. Both microwave and oven heating resulted in a marked loss of peptide-bound phosphorous (dephosphorylation) and a decrease of casein-phosphopeptides in the soluble part of the tryptic hydrolysate. It is concluded that hydrolysis of phosphoseryl to seryl residues was the prevailing degradation step to soluble proteolytic products, whereas lysinoalanyl-casein is claimed to be present almost exclusively in the pH 4.6-insoluble part of the tryptic digest.

Ribonucleosides as minor milk constituents.

Ribonucleosides are minor milk constituents and show a typical pattern which is assumed to be species-specific. As well as the unmodified components adenosine, cytidine, guanosine, inosine, and uridine, modified compounds such as Nl-methyladenosine and N6-carbamoylthreonyladenosine--products of the transfer RNA catabolism--have been identified and quantified in individual and bulk herd (race: German black pied) milk samples throughout a whole lactation period. The results of our longitudinal study have shown that--with the exception of the colostral phase--the levels of these minor constituents vary only slightly throughout lactation. These findings imply that ribonucleosides are useful for characterizing milk of different species and technological treatment. Ribonucleosides were determined and balanced, for example, in the course of the churning process, showing that the pattern of these minor milk constituents is useful as a "fingerprint" that allows differentiation between the three butter types defined in the German Federal Butter Ordinance.

[Ribonucleosides in milk: characterization and determination of the concentration profile of these minor components throughout a lactation period]. / Ribonucleoside in Milch: Charakterisierung und Bestimmung des Konzentrationsprofils dieser minoren Komponenten über eine Laktationsperiode.

The ribonucleosides adenosine, cytidine, guanosine, inosine and uridine as well as the modified components N1-methyladenosine and N6-carbamoylthreonyladenosine were characterized and determined quantitatively as minor constituents in raw bovine milk by use of an automated high performance liquid chromatography system. The studies have shown that except for the colostral phase the ribonucleoside levels are constant throughout the whole lactation period. That means, there is a typical ribonucleoside pattern which is assumed to be species-specific.

Biologically active peptides in milk proteins.

Bioactive peptides have been identified as digestion products of several food proteins. All the bioactive sequences are hidden in an inactive state inside the polypeptide chain of the larger protein. Milk proteins are a rich source of biologically active peptides such as exorphins (casomorphins), phosphopeptides and immunopeptides. Such peptides are released during intestinal digestion of caseins and whey proteins. They may be involved in regulation of nutrient entry and influence the postprandial metabolism via stimulation of the secretion of hormones. Furthermore, they may exert a stimulating effect on the immune system. These findings offer new aspects for evaluating the nutritive value of food proteins. Moreover, bioactive peptides have already found interesting applications as dietary supplements and as pharmaceutical preparations.

On-line sample processing and analysis of diol compounds in biological fluids.

We developed a coupled dual column system with an optional post-column derivatization for on-line sample processing, trace enrichment and analysis of aromatic 1,2-diol and aliphatic cis-diol biomolecules (e.g. catecholamines, ribonucleosides). The fully automated high-performance liquid chromatography analyzer tolerates the direct injection of proteinaceous fluids by use of a unique bonded-phase precolumn material which allows the simultaneous performance of covalent affinity and size-exclusion chromatography.

A minimum structured adenine nucleotide for ADP/ATP transport in mitochondria.

An ADP analogue, i.e. 3H- or 14C-labeled 5'-diphosphate of 6-(beta-D-ribofuranosyl-methyl)-4-pyrimidinamine, was synthesized, the structure of which was deduced from structure-activity studies on the substrate specificity of the nucleotide-binding center of the inner mitochondrial membrane integrated ADP/ATP carrier protein. Bearing only the minimal but substrate-essential recognition structures, minimum structured ADP (msADP) is bound to the cytosol-facing active center and transported by the highly specific carrier system across the inner mitochondrial membrane.

Direct clean-up and analysis of ribonucleosides in physiological fluids.

We describe the group-selective separation and quantification of unmodified, modified and hypermodified ribonucleosides in physiological fluids (urine, serum) by on-line multidimensional high-performance affinity chromatography (HPAC)-reversed-phase liquid chromatography (RPLC). The excretion levels and patterns of ribonucleosides such as N1-methyladenosine, N1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoylthreonyladenosine and 2-pyridone-5-carboxamido-N-ribofuranoside were determined in urines from a control group and from patients with different diseases. The HPAC-RPLC method applied represents a powerful tool, e.g. as a non-invasive screening test, a method to investigate disorders in ribonucleoside and/or RNA metabolism, a method for drug monitoring during nucleoside chemotherapy, and a method to study renal ribonucleoside reutilization.

Development of a chromatographic method for the quantitative determination of minor ribonucleosides in physiological fluids. Characterization and quantitative determination of minor ribonucleosides in physiological fluids, Part I.

We describe an on-line multi-column high performance liquid chromatographic method for the selective clean-up and analysis of major and minor ribonucleosides in physiological fluids. Quantitative data obtained for the determination of some methylated ribonucleosides in human urines are compared with those obtained with the traditional off-line method. The on-line technique developed in our laboratory is distinguished from the off-line method by the following features: Sample clean-up and analysis of the target-compounds can easily be automatized, Total time of analysis, for example of urinary ribonucleosides, is decreased to 35 minutes, Laborious and error-prone evaporation and redissolution steps are avoided, Reliability of the overall analytical system can be controlled with ease, Small sample-volumes can be applied directly, Sensitive samples can be processed very rapidly under mild conditions, Results obtained with the on-line and off-line-techniques compare well.

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei.

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.