Hepatoprotective action of papain-hydrolyzed buffalo milk protein on carbon tetrachloride oxidative stressed albino rats.

Buffalo skim milk retentate was hydrolyzed with papain for 4 h (enzyme:substrate, 1:200), resulting in a retentate hydrolysate (RH) with a degree of hydrolysis of 23%. We then investigated the potential hepatoprotective activity of RH at 250 and 500 mg/kg of body weight per day on carbon tetrachloride (CCl4)-induced oxidative stress in albino rats. Liver biomarkers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase), kidney biomarkers (urea, creatinine), and serum lipid profile (total lipids and triglycerides) were measured, in addition to histopathological status. Injection of CCl4 significantly increased all liver and kidney biomarkers compared with the negative control. In contrast, CCl4 injection significantly reduced hepatic antioxidant enzyme activities; that is, glutathione peroxidase and superoxide dismutase. Oral administration of RH for 28 d effectively maintained a physiologically normal range of liver and kidney biomarkers compared with the positive control. Furthermore, RH administration significantly increased activities of glutathione peroxidase and superoxide dismutase. Histopathological sections of CCl4-stressed rats treated with RH were different from that of the positive control and were similar to those of the negative control, in a concentration-dependent manner. Our results demonstrated the antihepatotoxic activities of buffalo milk RH and demonstrated that the higher RH concentration (500 mg/kg of body weight per day) could maintain the healthy biological status of the CCl4-injected rats.

Supplementing rabbit diets with butylated hydroxyanisole affects oxidative stress, growth performance, and meat quality.

Butylated hydroxyanisole (BHA) is a synthetic antioxidant analogous of vitamin E. It is used as a preservative to prevent free radical-mediated oxidation in high-fat foods, and this study's objective was to investigate the effects of BHA on oxidative stress and apoptosis in addition to delineating its efficacy as a growth-promoting feed additive. 60 weaned male rabbits (V-line) were randomly divided into four equal groups: BHA0.0 (control), BHA50, BHA100, and BHA150, administered basal diets with 0.0, 50, 100, and 150 mg BHA/kg of feed for 60 days. Animals were examined for growth performance, markers of oxidative stress and apoptosis, and meat characteristics. Compared to the control group, rabbits receiving BHA-supplemented diets exhibited increases in BW and average daily gain (P < 0.01), where BHA50 and BHA100 groups showed increased muscle content of methionine aspartic acid, serine, and glutamine (P < 0.05). These two groups also exhibited elevated catalase and superoxide dismutase activities and diminished malondialdehyde levels in the liver. Butylated hydroxyanisole upregulated fatty acid synthase gene (FASN), especially in BHA100 animals. Bcl-2-associated X/B-celllymphoma-2 (Bax/Bcl-2) ratio significantly increased in animals receiving higher doses of BHA, and the weight of the liver significantly increased following BHA treatment. Supplementing growing rabbits with lower doses of dietary BHA may promote growth performance and meat quality via maintaining the redox balance. Hence, the 50-100 mg/kg may be recommended as a safe and still effective feed additive as well as an oxidative stress attenuator.

Hypolipidemic and antioxidant activity of the aqueous extract from the uneaten pulp of the fruit from Cordia dichotoma in healthy and hyperlipidemic Wistar albino rats.

Hyperlipidemia is a major risk factor for coronary heart disease. Hyperlipidemia increases the incidence of myocardial ischemia and cardiac events. This study evaluated the potential hypolipidemic and antioxidant action of the aqueous extract from the uneaten pulp of the fruit from Cordia dichotoma ("CDNP extract"). In vivo studies were performed for 10 weeks on dietary hyperlipidemic and healthy Wistar albino rat models that received two dose levels of the CDNP extract (0.50 and 1.00 g/kg body weight). Serum lipid profiles were determined for the experimental animals. Dietary hyperlipidemic rats were characterized by an elevated lipid profile compared to the healthy control, i.e., increased levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), and triglycerides (TG), although the level of high-density lipoprotein (HDL-C) was reduced. Levels of antioxidant enzymes, i.e., glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), were significantly higher in the dietary hyperlipidemic rats than in the normal healthy ones, while the level of malondialdehyde (MDA) was significantly lower. Force-feeding hyperlipidemic Wistar albino rats with the CDNP extract at two doses decreased TC, LDL-C, VLDL-C, and TG to normal levels. The risk ratio, which was as high as 870 % for the hyperlipidemic rats was decreased by the treatment to levels close to that calculated for the healthy control rats. Levels of high-density lipoprotein cholesterol (HDL-C) were very low in the hyperlipidemic Wistar albino rats but increased significantly when CDNP extract was adminstered, attaining similar HDL-C levels to those of healthy control rats. Treatment with the CDNP extract also improved the levels of antioxidant enzymes (GR, GST, GPx, SOD, and CAT) in hyperlipidemic Wistar albino rats. Thus, the CDNP extract improves the lipid metabolism of healthy and hyperlipidemic Wistar albino rats and can be employed in the management of dietary hyperlipidemia.

Amino acid grafting of beta-lactoglobulin mediated by phosphorus oxychloride.

beta-Lactoglobulin was phosphorylated with 80 mol of POCl3/mol protein in the presence of triethylamine and amino acids or their esters added at a total molar excess of 6 mol base/mol POCl3. The extent of phosphorylation was reduced when the amino acids replaced triethylamine as the base. Arginine and lysine were grafted to protein molecules in amounts proportional to the beta-lactoglobulin phosphorylation, while histidine grafting was very weak. The electrophoretic patterns of the modified proteins showed increased negative charges, reduced isoionic points and slight dimerization. The emulsifying properties of the modified proteins were improved.

Susceptibility to trypsinolysis of esterified milk proteins.

Methyl-, ethyl- and propyl-esters of beta-lactoglobulin, alpha-lactalbumin and beta-casein were prepared and then hydrolyzed with trypsin in various conditions. Resulting hydrolysates were analysed by SDS electrophoresis and RP-HPLC. The degree of hydrolysis of esterified samples was generally lower than those of the non-modified proteins. The highest degrees of hydrolysis were obtained at pH 7--8 with native and esterified protein samples. beta-Lactoglobulin propyl ester and beta-casein methyl ester yielded the lowest degrees of hydrolysis. Ethyl- and propyl-esters of beta-casein showed high resistance towards tryptic attack, even after 20 h of hydrolysis. SDS electrophoretic patterns of tryptic hydrolysates of native proteins showed bands corresponding to low molecular weights. Tryptic hydrolysates of esterified proteins showed bands with higher sizes. RP-HPLC profiles of tryptic hydrolysates of esterified samples showed peaks with longer elution times than those obtained with native proteins, indicating the presence of more hydrophobic peptide populations. A peptic pre-treatment improved tryptic action on esterified proteins. It resulted in a better resolution of RP-HPLC profiles and in a complete disappearance of the protein after 20 h tryptic hydrolysis.

Esterified milk proteins inhibit DNA replication in vitro.

DNA replication was studied in vitro in the presence of native and esterified milk proteins [alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and beta-casein (BCN)]. Addition of unmodified proteins to the PCR medium did not change the result of the reaction seen by electrophoresis, even at excessive ratios of basic amino acids in proteins:phosphate groups in DNA as high as 100:1. Addition of esterified proteins greatly reduced the intensity of the bands corresponding to the newly synthesized DNA, at ratios as low as 1:1 and 5:1 in case of methylated-BLG and methylated-ALA, respectively. The inhibitory effect of esterified proteins was directly proportional to their extent of esterification and strongly related to their DNA-binding capacity. Generally, inhibition of PCR with esterified proteins was similar to what can be observed with histones. However, stronger inhibition was observed with highly esterified proteins when using a higher ratio of basic:acid residues (1:1) when compared with 0.5:1 ratio in case of histones. Highly esterified BCN did not exert any inhibitory effect because of its relatively lower pI when compared with that of other esterified milk proteins and due to its lower positive net charge at the pH used for PCR. During a second PCR run, only the addition of new DNA template was able to reinitiate the reaction, giving rise to new synthesized DNA. Addition of Taq DNA polymerase did not enhance DNA synthesis, showing that inhibition was performed only by binding of DNA template and not by the inhibition of the polymerase.

Characterization of two safe Enterococcus strains producing enterocins isolated from Egyptian dairy products.

Five bacterial cocci isolates were selected from a wide pool of 503 isolates collected from traditional Egyptian dairy products on the basis of their inhibitory activities against Lactobacillus brevis F145, Lactobacillus bulgaricus 340, Enterococcus faecium HKLHS, Listeria ivanovii ATCC, Listeria innocua CIP 80.11 and Listeria monocytogenes EGDe 107776. These 5 isolates were identified as E. faecium TX1330 and E. faecium E980 by 16S rDNA amplification and sequencing. The antibacterial activity of the two strains was not affected by treatment of the cell free culture supernatant with catalase but their activities disappeared completely when digested with protease K, α-chymotrypsin and trypsin. The antimicrobial substance was stable over a wide range of pH (2-10) and was active after heating at 100 °C for 10 min. Bacteriocin yield in two strains reached a maximum (1,600 AU/ml) at the end of the exponential phase (6 h) and remained stable until the end of 24 h-incubation period when the medium reached pH 5.5. Maximal production of bacteriocin was obtained when growing the bacterial cells at temperatures ranging between 30 and 37 °C. Bacteriocin production was unaffected when the bacterial cells grew under severe conditions of pH (9.6) and in high salt (6.5% NaCl). Thanks to PCR gene amplification the bacteriocins produced by E. faecium TX1330 could be identified as enterocins A and B structural genes, while the bacteriocins produced by E. faecium E980 could be identified as enterocins P and L50A structural genes, which can be classified into two enterocin subclasses (IIa and IIc), respectively. PCR amplification demonstrated that the two studied strains did not contain virulence factors asal, cyl A and B, ace, efaAfs and espfm. These two strains were sensitive to most of the tested antibiotics but were resistant to tetracycline. E. faecium E980 was also resistant to chloramphenicol.

Mineral absorption by albino rats as affected by some types of dietary pectins with different degrees of esterification.

Male albino rats were fed diets contained 6.85% mineral salts for 2 weeks (adaptation condition). Then they were fed the dietary pectin administered diet for 6 weeks to evaluate the effect of administration of pectin on the absorption of some monovalent, bivalent and heavy metals in the serum of rats. The experimental parameters included, monovalent minerals (K, Na), bivalent minerals (Zn, Cu, Ca, Fe), heavy metals (Pb, Cd), serum uric acid and serum creatinine. The obtained results indicated that the serum contents of monovalent minerals were negatively affected by pectin administration. The low degree of esterification of pectin was more effective on the absorption of bivalent minerals. Also, the rat serum levels of lead and cadmium were reduced by pectin administration. Serum total proteins were reduced by pectin administration. The level of rat serum of uric acid and creatinine fed different sources of pectin were within normal levels and were insignificantly lower than that recorded for control samples.

Phosphorylation of beta-lactoglobulin using amino acids as the sole base and nucleophile of the reaction.

beta-Lactoglobulin was phosphorylated with 20, 40, and 80 mol of POCl3/mol protein in the presence of 4, 5, and 6 molar excess of basic amino acid per mol POCl3. Maximal phosphorylation yields of 5 and 3 mol P/mol protein were achieved when the highest stoichiometries of POCl3/arginine and lysine were used. Proportional high amounts of basic amino acids were also grafted to the protein molecule during its phosphorylation through the phosphoamide bond. Modified proteins displayed increased negative charges and reduced isoelectric points and were monomeric. The phosphorylated and phosphoamidated beta-lactoglobulin showed improved functional properties.

Improvement of solubility and of emulsifying properties of milk proteins at acid pHs by esterification.

Three milk proteins (beta-lactoglobulin, alpha-lactalbumin and beta-casein) were esterified to different extents with methanol, ethanol and propanol, then their solubility was studied in the pH-range of 3-10. Their emulsifying properties were also checked in the pH-range of 3-7 by measuring the oil-droplet size using laser light scattering. Solubility of all esterified proteins was improved in the acidic pH range (3-6). The magnitude of improvement was depending on the extent of esterification, the nature of the grafted ester group and the nature of the modified protein. Emulsifying activity and stability of esterified milk proteins in the acidic pH range of 3-5 were generally higher compared to the corresponding native proteins. This improvement was associated with the degree of solubility, the degree of esterification, the type of ester group grafted and the nature of the used protein. The highest emulsifying activity improvement was observed for methyl ester derivatives at pH 5 where the native proteins have poor emulsifying properties.

Biochemical dynamics and hypocholesterolemic action of Hibiscus sabdariffa (Karkade).

Male albino rats were fed on a mixture of cholesterol and cholic acid for 12 weeks to induce hypercholesterolemia, then Karkade was administered at 5 and 10% for 9 weeks to evaluate its hypocholesterolemic and hypolipemic effect. The experimental parameters include total lipids, cholesterol, triglycerides and phospholipids. Liver and kidney functions were also investigated in normal, hypo- and hypercholesterolemic rats administered Karkade. A remarkable progress (lowering effect) in the level of different lipid fractions was noticed in spite of the continued cholesterol and cholic acid loading during the treatment. However, blood phospholipids were increased after Karkade administration. Hypercholesterolemia has resulted in an effect on the activity of liver and kidney functions. Therefore, the measurement of serum enzyme activity has provided a useful tool for hepatic recovery. Although the administration of Karkade at 5 and 10% induced a significant decrease in the activity of serum GOT and serum GPT, alkaline and acid phosphatase as well as total serum protein, the values nearly returned to the initial levels after 9 weeks of Karkade administration.

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy.

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated beta-lactoglobulin and of methylated alpha-lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated ,-casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210-340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210-340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.

Metabolic effects of licorice roots (Glycyrrhiza glabra) on lipid distribution pattern, liver and renal functions of albino rats. MS.

The biochemical activity of licorice roots was investigated by administrating it to hypercholesterolaemic rats at two different doses. High cholesterol level in the serum and most organs of hypercholesterolaemic rats was accompanied by high levels of triglycerides and total lipids but low one of phospholipids. The hypocholesterolaemic action of licorice roots was accompanied by hypolipaemic action but higher level of phospholipids in the rat serum and most organs. The results showed that while the high cholesterol level in the rat serum has impairing effects on liver and renal functions, the licorice administered to these rats has a counteracting action i.e. it can improve the impaired function of both liver and p4f1ey, It has also a hypoglycaemic action.

The biochemical role and hypocholesterolaemic potential of the legume Cassia fistula in hypercholesterolaemic rats.

The hypocholesterolaemic effect of Cassia fistula was investigated using hypercholesterolaemic male albino rats. Hypercholesterolaemia was induced by feeding on a mixture of cholesterol plus cholic acid for a 12 weeks period. Hypercholesterolaemia was characterized by significant increase in the average levels of total lipids, total cholesterol, and triglycerides and significant decrease in phospholipids content. Administration of Cassia fistula significantly reduced blood and liver total lipids. Brain, spleen, kidneys and heart followed nearly the same trend but with moderate effect. Blood, liver, kidneys, spleen and heart total cholesterol was significantly reduced, while that of brain was not affected. The level of triglycerides was markedly improved. There was a moderate rise, however, in phospholipids content in all studied organs. That is to say a marked progress in the correction of lipid metabolism occurred. Also, administration of Cassia fistula induced a significant decrease in the high activities of serum GOT, GPT, alkaline and acid phosphatase and the values nearly returned the initial values. Total serum protein, albumin (A), globulin (G), A/G, free amino acids, uric acid and creatinine were also determined and their values were improved and attained nearly the normal values of the control group.