Endogenous lysine flow at the distal ileum of the protein-fed rat: Investigation of the effect of protein source using radioactively labelled acetylated lysine or lysine transformed to homoarginine

The study aimed to determine if the source of purified dietary protein influenced endogenous lysine flow at the terminal ileum of the growing rat. Partially guanidinated gelatin and isolated soya bean protein based diets, containing chromic oxide as an indigestible marker, were given to 225-g liveweight rats. The endogenous flow of lysine at the terminal ileum was then determined indirectly, based on the absorption of homoarginine, after sampling of ileal contents at slaughter. Endogenous lysine flow was also determined by reference to chromium levels in sampled ileal digesta, after feeding rats either gelatin or isolated soya bean protein or casein based diets in which some of the lysine had been acetylated with [¹⁴C]-acetic anhydride. The [¹⁴C] acetylated lysine was used to distinguish between lysine of dietary and endogenous origin. Mean endogenous ileal lysine flows were 426 and 442 μ g^?1 dietary freeze dry matter intake for the guanidinated gelatin (n=6) and soya bean (n = 6) treatments, respectively, and there was no significant (P>0.05) effect of protein source on endogenous flow. Nor did there appear to be any effect of dietary protein source on endogenous ileal lysine flow when measurement was based on the radio-actively labelled acetylated lysine. The data, considered together, indicate no effect of the source of dietary protein per se on endogenous ileal lysine flow in the rat when purified proteins were given over a short (8-h) time period. The mean true ileal digestibility coefficients for dietary lysine (0.897, 0.904, 0.926, 0.971, 0.916; acetylated gelatin, guanidinated gelatin, guanidinated soya bean, acetylated soya bean, acetylated casein, respectively) indicated a high degree of absorption of lysine before the end of the ileum in the growing rat.     

Effects of oxidised linoleic acid on the formation of Nε‐carboxymethyl‐lysine and Nε‐carboxyethyl‐lysine in Maillard reaction system

This study investigated the effects of oxidised linoleic acid (18:2) on N^ε‐carboxymethyl‐lysine (CML) and N^ε‐carboxyethyl‐lysine (CEL) formation in Maillard reaction systems. Model systems of lysine/glucose (L/G), lysine/18:2 (L/18:2), lysine/18:2/glucose (L/18:2/G), myofibrillar protein/glucose (MFP/G), MFP/18:2 and MFP/18:2/G were maintained at 37 °C for 6 weeks. The results showed that CML/CEL contents in L/G (6.99 and 0.96 mmol mol^?1 lysine, respectively) were significantly higher than those in L/18:2/G (1.43 and 0.41 mmol mol^?1 lysine, respectively), and there is a small amount of CML/CEL generation in L/18:2. However, the CML/CEL levels in MFP/G (197.2 and 83.8 ng mg^?1 protein, respectively) were markedly lower than those in MFP/18:2/G (283.2 and 118.5 ng mg^?1 protein, respectively). 18:2 favours the formation of CML/CEL in MFP/18:2/G, not in L/18:2/G. All these findings indicated that the role of 18:2 on CML/CEL formation in Maillard reaction system was complex, and depended on CML/CEL formation rate and substrate types (lysine or lysine residue in protein).     

An isotopic method for determining chemically reactive lysine based on succinylation

A method is described for the routine succinylation of proteins using [¹⁴C]succinic anhydride as a means of measuring free ?-amino groups. Maximal succinylation was achieved using 6M guanidine hydrochloride as protein solvent and an 80-fold molar excess of succinic anhydride relative to total lysine residues. Treatment with hydroxylamine (pH 13, 25°C, 5 min) removed unwanted O-succinyl esters. Succinylated protein was precipitated with trichloroacetic acid, residual label washed out with ethanol and the extent of labelling measured in a scintillation counter. The method gave close to theoretical values (n.s., P >0.05) for lysyl residues in egg white lysozyme, bovine haemoglobin, ovalbumin and bovine serum albumin but gave a low value with β-lactoglobulin and an overestimate with insulin attributable to the relatively high contribution of α- relative to ?-amino groups in this low molecular weight protein. The method gave good results for 12 soya protein samples and was shown to be very sensitive to ‘isopeptide-type’ heat damage in these samples. The results correlated well (r = 0.91) with those obtained by the well established dye-bound lysine difference procedure whereas, as could be expected, both these methods correlated poorly with total lysine determinations (r=0.69 for succinic anhydride and r=0.77 for the dye-binding method). The proposed method shows promise as a rapid procedure for the estimation of available lysine, but further studies are necessary to test its ability to measure nutritionally available lysine in all categories of heat damage.     

DIFFERENT ARRANGEMENT OF ɛ-(γ-GLUTAMYL)LYSINE CROSS-LINKING IN ALASKA POLLOCK (THERAGRA CHALCOGRAMMA) SURIMI PROTEINS BY STREPTOVERTICILLIUM AND ENDOGENOUS TRANSGLUTAMINASES DURING SUWARI PROCESS

The objective of the present study is to compare the protein cross‐linking reaction in Alaska pollock surimi that is catalyzed by a commercially available microbial transglutaminase and by endogenous Alaska pollock transglutaminase. The endogenous transglutaminase was inhibited by EGTA and activated by CaCl₂ The microbial transglutaminase was added to the salted surimi with and without EGTA and CaCl₂. These surimi pastes were incubated at 25C up to 24 h followed by cooking at 90C. The resultant gels were fractionated into soluble and insoluble (aggregate) fractions by SDS‐urea extraction. Compositional analysis revealed that the aggregate consisted predominantly of cross‐linked myosin heavy chain. The distribution of ?‐(γ‐glutamyl)lysine isopeptide in the soluble and aggregate fractions andpeptide mapping analyses of the aggregate fraction demonstrate that the formation of isopeptide cross‐links in Alaska pollock surimi proteins during suwari process differs when catalyzed by the microbial transglutaminase and endogenous transglutaminase.     

Comparative study of gelation and cross-link formation during enzymatic texturisation of leguminous proteins

Isopeptide bonds that resulted from protein cross-linking, catalysed by a microbial transglutaminase (MTG), substantially contributed to the physicochemical modification of leguminous proteins. For the development of texturised vegetable protein (TVP)-based foodstuffs using MTG, valid methods for an efficient control of the gelation process are a prerequisite. Formation of ε-(γ-glutamyl)lysine cross-links in a simple food model system, containing proteins from soy [Glycine max (L.) Merr.] or pea (Pisum sativum L.), was monitored by quantification of the ε-(γ-glutamyl)lysine bond via HPLC-MS and by determining the depletion of free amino groups during cross-linking spectrophotometrically after derivatisation with o-phthaldialdehyde (OPA). Increasing gel strengths during incubation with MTG were measured via texture analysis. The OPA method proved too unspecific for controlling the enzymatic gelation process of leguminous proteins. Specifically for each substrate, the levels of isopeptide cross-links, detected via HPLC-MS analysis, correlated well with the gel strength of the texturised proteins (R² = 0.961–0.994). Rapidly measurable, gel strength was shown to be a reliable command variable for managing MTG-induced gelation. Its use also allowed indirect estimation of the degree of feasible cross-linking.Industrial relevanceLeguminous proteins represent a valuable alternative to animal proteins for the manufacture of texturised foodstuffs. However, due to the poor gelling properties of the native proteins, their potential is still unexploited. For the development of TVP using MTG, simple gel strength measurements were shown to be a valid tool for the control of the gelation process. For this purpose, unlike OPA determination of free amino groups and LC-MS analysis of isopeptide bonds, tedious sample preparation is not required.     

Spectrophotometric assay using o-phtaldialdehyde for the determination of transglutaminase activity on casein

In this work, the possibility of using a simple and quick method was tested for determining transglutaminase activity on casein using a spectrophotometric assay. The enzyme activity was estimated on the basis of the decrease of o-phthaldialdehyde reactive ε-amino groups of lysine following the formation of isopeptide bonds. The lysine residues involved in the formation of isopeptide bonds when the reaction reaches its plateau are equal to 0.126 μmol per mg of casein. This value results as equal to 0.205 μmol per mg of casein when N-carbobenzoxy-glutaminyl-glycine is added to the reaction medium as a small size acyl group donor. The electrophoretic analysis of the reaction products emphasised a different kinetic formation of casein polymers with the two substrate solutions used. This proposed method has resulted as accurate, with a mean coefficient of variation of 4.6%.     

Nε-carboxymethyllysine is formed during heating of lysine with ketoses

In a model system with lysine monohydrochloride and various reducing sugars, the contents of N^ε-carboxymethyllysine (CML) were measured after heat treatment at 98°C for 3–24 h. The results show for the first time that CML could be formed by the reaction of lysine and fructose or lysine and sorbose. The formation of CML also shows time-dependent differences between aldoses and ketoses. After heating up to 6–12 h, the reaction of lysine with ketoses leads to similar amounts of CML as the reaction with aldoses, whereas after longer heating significantly more CML was produced by the reaction with aldoses. The highest CML contents (2380 mg CML kg^?1 lysine) were measured in the galactose model system after heating for 24 h.     

Comparison of immunohistochemical,histochemical and immunochemical methods for the detection of wheat protein allergens in meat samples and cooked,dry, raw and fermented sausage samples

Nowadays is it a common practice to add vegetable protein in the production of meat products. Because of the possible substitution of high-quality raw meat with vegetable protein without the labelling the product package and because of the allergenic potential of many vegetable proteins, it is important to develop accurate methods for its detection. The objective of the study was to compare histochemical, immunochemical (ELISA, ALERT gliadin screening test) and immunohistochemical methods for the detection of wheat protein in meat samples and sausages. Histochemical methods were useful for the detection of flour in meat samples, but the immunohistochemical method was better for the detection of wheat protein. ALERT gliadin screening test detected gliadin from 10?mg?kg^?1, while an immunohistochemical method detected wheat protein concentrations from 1?g?kg^?1 and an ELISA method detected wheat protein concentrations from 4?g?kg^?1. ALERT gliadin screening test showed results within 1 day, whilst an ELISA detection method took 2 days, and an immunohistochemical procedure took 5 days at the soonest, all including sample preparation. This study also focused on optimisation of an immunohistochemical method for samples of cooked sausage. In addition, three samples were sufficient for wheat protein detection at a concentration of 1?g?kg^?1 (and greater) with a confidence level greater than 95%.     

Chemical activation of piperidine by formaldehyde and formation of lysine-specific Maillard reaction products

Piperidine is considered as a lysine-specific Maillard reaction product that can be formed from free lysine through decarboxylation and deamination reactions or through cyclization of pent-4-en-1-amine, the counterpart of acrylamide from lysine. Due to the importance and reactivity of piperidine its further interaction products in glucose/lysine model system was investigated. A useful strategy based on Py-GC/MS analysis was developed using an isotope labelling technique to identify reaction products incorporating piperidine moieties. Products simultaneously possessing five lysine carbon atoms (C2′–C6′) and the Nε-amino group from lysine in addition to glucose carbon atoms were targeted using specifically labelled precursors such as [¹⁵Nα]Lysine·2HCl, [¹⁵Nε]Lysine·2HCl, [U-¹³C₆]Lysine·2HCl, [¹³C-6]Lysine·2HCl and [U-¹³C₆]Glucose. Detailed labelling studies using specifically ¹³C-enriched sugars have shown that the piperidine can form reactive 1-methylidenepiperidinium ion with formaldehyde, which is able to undergo further aldol addition reactions to form compounds, such as 3-(piperidin-1-yl)propanal and 3-(pyridin-1(4H)-yl)propanal. Furthermore, these studies have also demonstrated that oxidation of piperidine into di- and tetrahydropyridine derivatives can generate reactive eneamine moieties capable of nucleophilic attack at carbonyl groups and formation of pyridine derivatives.     

Studies on fish muscle protein. Nutritional consequences of the changes occurring during frozen storage

Samples of cod, whiting, herring, lemon sole and skate muscle were frozen and stored at ? 8°C for up to 20 months. Samples of cod muscle were also stored at ? 30°C. During storage at ?8°C the decrease in the solubility of the muscle proteins in 5% NaCl (denaturation) was most rapid in whiting followed in order by skate, cod, herring and lemon sole. Little change was detected in the NaCl solubility of cod muscle protein stored at ? 30°C. An increase in the pH was found to occur in all the species examined. The relationship between these changes and the toughening that occurs in fish muscle during frozen storage is considered. No nutritionally significant decrease in the lysine availability or the in vitro digestibility of the muscle proteins was found to occur in any of the species examined. In whiting, lemon sole and skate very small, statistically significant, increases in the available lysine content were detected after 7 months of storage at ?8°C and this may indicate that unfolding of the protein chains had occurred.     

Available lysine,protein digestibility and lactulose in commercial infant formulas

Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.7±9.5 mg g⁻¹ protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.0±8.3 mg g⁻¹ protein). In vitro protein digestibility values ranged 85.5–88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow's milk. However, all values were below the limit of 600 mg L⁻¹ recommended for UHT milk.     

Digestible reactive lysine in selected milk-based products

Reactive lysine contents, true ileal reactive lysine digestibility, and true ileal digestible reactive lysine contents were determined in a wide range of processed milk products. A previously validated assay based on determining reactive lysine in both food and ileal digesta, after reaction of these materials with O-methylisourea, was applied. Semisynthetic diets containing milk products as the sole sources of protein and including chromic oxide as an indigestible marker were fed to growing rats. Digesta from the terminal ileum were collected posteuthanasia and, with samples of the diets, analyzed for reactive lysine (homoarginine) contents. True reactive lysine digestibility was determined after correcting for endogenous lysine loss at the terminal ileum of rats fed an enzyme hydrolyzed casein-based diet, followed by ultrafiltration (5000 Da) of the digesta. Digestible total lysine (determined using conventional methods) was also determined. The true ileal reactive lysine digestibility was high (>91%) in all the milk products tested, but was highest in the UHT milk (100%) and lowest in the infant formulas (91 to 93%). Total lysine digestibility (conventional measurement) significantly underestimated reactive lysine digestibility for all the products tested. The mean underestimation ranged from 1.3 to 7.1% units. The mean digestible total lysine content was significantly different from the available lysine content for most of the products examined. In some cases this difference was small (<3%), but for a number of the products (evaporated milk, whole milk protein, lactose hydrolyzed milk powder, and a sports formula) the difference was greater (6.5 to 14%). This would suggest firstly that total lysine and total lysine digestibility determined using conventional methods were inaccurate when applied to some milk-based foods, and secondly that some of the milk products have undergone lysine modification. In general, milk proteins are a highly digestible source of amino acids and lysine.     

Studies on rumen metabolism. VII. Effect of lipids (in vitro) on the digestion of grass fibre and cellulose

Production of propionate from grass fibre, by rumen micro-organisms, in closed systems increased and acetate decreased over 48–72 h incubation periods when lipid was added. Similar effects of lipid on [¹⁴C] propionate and [¹⁴] Cacetate production were obtained when [¹⁴C] cellulose was added to the substrate as a marker. Total volatile fatty acid (VFA) or total [¹⁴C] VFA production from grass fibre was not affected significantly by lipid. Cod liver oil was more effective than either linseed oil or peanut oil in producing these effects. Glycerol liberated from lipid by lipolytic activity would seem to favour acetate formation, rather than propionate formation. When grass fibre was fermented in an artificial micro-rumen for 24 h, addition of oil stimulated the production of total VFA's largely due to significant increases in acetate. Relative rates of formation of the individual short chain fatty acids did not change over a 24 h period. On the other hand, less total [¹⁴C] VFA was produced from [¹⁴C] cellulose in the dialysate of the artificial micro-rumen when oil was added. The specific activities of the individual VFA's were higher without oil but maximum specific activities were obtained slightly earlier when oil was added.     

The In Vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X-100. Biochemical analysis indicated that this protein, which showed cross-reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore-forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [¹⁴C]sucrose/[³H]dextran leakage ratios. Furthermore, the generation of a ΔΨ by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a ΔΨ generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.     

Nutritional Evaluation of the Protein in Oilseed Products Heated with Sugars

Defatted flours and protein isolates from glandless cottonseed, peanut, and soybean were complexed with glucose or sucrose in a ratio of 1:1 by weight and heated at 100°C for 0, 2, or 6 hr. Quality changes by the heat treatment were assessed by determining the extent of browning (browning index), in-vitro protein digestibility, available lysine total amino acids, and computed protein efficiency ratio (C-PER). Whereas the sucrose-containing complexes changed very little in all the quality parameters determined, the counterparts containing glucose decreased substantially in protein quality along with the increased intensity of browning. Before heating, protein digestibility of the complex containing defatted flour of an oilseed was lower than that of the isolate counterpart. When heated, however, the isolate-glucose complexes decreased more in protein digestibility than the flour-glucose complexes. The available lysine content decreased by 76-83% for the complexes with glucose in contrast with 2-10% for those with sucrose. As to total amino acids, marked losses in arginine, lysine, tryptophan, and histidine were found in the complexes containing glucose, with the arginine loss as high as the lysine loss. Lysine-rich soy proteins lost lysine in greater percentages than lysine-poor peanut and cottonseed proteins. C-PER decreased by 63-86% in the glucose-containing complexes, with the highest decrease shown for soy flour-glucose. In-vitro protein digestibility, available lysine, and C-PER were all highly correlated (P < 0.01) with browning index. Available lysine was also highly correlated with C-PER.     

Effect of cooking on protein quality of chickpea (Cicer arietinum) seeds

Amino acid composition and in vitro protein digestibility of cooked chickpea were determined and compared to raw chickpea seeds. Heat treatment produced a decrease of methionine, cysteine, lysine, arginine, tyrosine and leucine, the highest reductions being in cysteine (15%) and lysine (13.2%). Protein content declined by 3.4% and in vitro protein digestibility improved significantly from 71.8 to 83.5% after cooking. The decrease of lysine was higher in the cooked chickpea seeds than in the heated protein fractions, globulins and albumins. The structural modification in globulins during heat treatment seems to be the reason for the increase in protein digestibility, although the activity of proteolytic inhibitors in the albumin fraction was not reduced. Results suggest that appropriate heat treatment may improve the bioavailability of chickpea proteins.     

Oral absorption,tissue distribution and excretion of a radiolabelled analog of a milk-derived antihypertensive peptide,Ile-Pro-Pro,in rats

Milk-derived bioactive peptides have been shown to attenuate the development of hypertension in rats and reduce blood pressure in humans. The aim of the present study was to examine the absorption, tissue distribution and excretion of intact Ile-Pro-[¹⁴C]Pro in rats. Radioactivity was determined in blood and tissues. After oral administration, Ile-Pro-[¹⁴C]Pro was absorbed from the intestine and the peak level was observed after 2 h. Considerable amounts of radioactivity were found by liquid scintillation in tissues, e.g., in the liver, kidneys, adrenals and aorta. Chromatographic and mass spectrometric analyses of plasma samples proved that Ile-Pro-[¹⁴C]Pro was present in all samples analysed. The majority of the radioactivity was associated with plasma proteins and in vitro tests showed that Pro-Pro attaches to plasma proteins (over 98%) whereas Ile-Pro-Pro is not bound. In conclusion, Ile-Pro-[¹⁴C]Pro is partly absorbed intact and the distribution of radioactivity in tissues may be related to the blood-pressure-lowering effect of the peptide.     

Protein quality of germinated Phaseolus vulgaris

Advantages of seed germination consist of an increase in the bioavailability of proteins as well as the change in the antinutritional factors which limit their utilization. Throughout this work, the effects of germination and cooking after germination on the protein in black and white beans (Phaseolus vulgaris) were evaluated. Antinutritional factors that affect the utilization of such protein were also assessed. The amounts of protein, available lysine, tannins, PER, and protein digestibility in vitro and apparent, of beans germinated and germinated-cooked were quantified. The germination significantly (p≤0.05) increased the content of proteins, inactived trypsin inhibitors, and raised the available lysine. Germination and cooking completely inactivated the trypsin inhibitors, which became lysine less available, decreased tannins, and increased protein digestibility and PER value. Differences between black and white beans were observed and attributed to variations in structure, composition, and varieties, among other factors. Cooking complements the effect of germination by improving the protein quality of P. vulgaris and increasing its bioavailability for the human consumption.     

Nutritional Properties and Metabolic Studies of Acylated Beef Heart Myofibrillar Proteins

Beef heart myofibrils were acylated with several concentrations of acetic (AA) and succinic (SA) anhydride, and digestibility and utilization of the myofibrillar proteins were determined. Results for in vitro hydrolysis of the untreated and acylated proteins varied, depending on the enzyme(s) used in the analysis. Rat protein efficiency ratios and net protein ratios for untreated (PER = 2.83, NPR = 116) and acetylked (PER = 2.55, NPR = 110) proteins were greater than for casein (PER = 2.50, NPR = 100), whereas values for succinylated proteins (PER = 2.36, NPR = 87) were less than for casein. Most of the radioactivity recovered after 24 hr from rats fed ¹⁴C-acylated myofibrillar proteins was in expired CO₂; 62.8% for ¹⁴C-acetylated and 45.8% for ¹⁴C-succinylated proteins. Rats acclimated to an acylated protein diet for 28 days showed improved metabolism of ¹⁴C-acetylated protein and decreased metabolism of ¹⁴C-succinylated proteins; 75.7% and 38.1% recovery as expired CO₂, respectively.