Soy protein hydrolysate grafted cellulose nanofibrils with bioactive signals for bone repair and regeneration.

TEMPO oxidized cellulose nanofibers (T-CNF) were prepared from cellulose pulp which is extracted from bagasse. Soy protein hydrolysate (SPH) was grafted on T-CNF via amidation of carboxylic groups. Biomineralization was, then, assessed via calcium phosphates (CaP) precipitation in twice-simulated body fluid until formation of a new bioactive material. Protein was efficiently grafted without alteration of morphology and nanofibrils packing as reported by Fourier Transform infrared analysis /X Ray Diffraction /Scanning and Transmission Electron Microscopy / Atomic Force Microscopy. Highly crystalline calcium phosphate deposits - ca. 22.1% - were detected, with a Ca/P ratio equal to 1.63, in agreement with native bone apatite composition. In vitro response of human Mesenchymal Stem Cells confirmed the biocompatibility. No significant differences in terms of cell adhesion were recognized while a significant increase in cell proliferation was detected until 7 days. The presence of calcium phosphates tends to cover the nanofibrillar pattern, inducing the inhibition of cell proliferation and promoting the ex-novo precipitation of mineral phases. All the results suggest a promising use of these biomaterials in the repair and/or the regeneration of hard tissues such as bone.

Toxicity study and blood pressure-lowering efficacy of whey protein concentrate hydrolysate in rat models, plus peptide characterization.

We evaluated the acute (single-dose) and subacute (repeated-dose) oral toxicity of alcalase-hydrolyzed whey protein concentrate. Our acute study revealed no death or treatment-related complications, and the median lethal dose of whey protein concentrate hydrolysate was >2,500 mg/kg. In the subacute study, when the hydrolysate was fed at 3 different concentrations (200, 400, and 800 mg/kg), no groups showed toxicity changes compared with controls. Then, whey protein concentrate hydrolysate was orally administered to spontaneously hypertensive rats. Results revealed significant reductions in blood pressure in a dose-dependent manner, and dosing at 400 mg/kg led to significant blood pressure reduction (-47.8 mm Hg) compared with controls (blood pressure maintained) and the findings of previous work (-21 mm Hg). Eight peptides-RHPEYAVSVLLR, GGAPPAGRL, GPPLPRL, ELKPTPEGDL, VLSELPEP, DAQSAPLRVY, RDMPIQAF, and LEQVLPRD-were sequentially identified and characterized. Of the peptides, VLSELPEP and LEQVLPRD showed the most prominent in vitro angiotensin-I converting enzyme inhibition with half-maximal inhibitory concentrations of 0.049 and 0.043 mM, respectively. These findings establish strong evidence for the in vitro and in vivo potential of whey protein concentrate hydrolysate to act as a safe, natural functional food ingredient that exerts antihypertensive activity.

Anti-inflammatory and antioxidant activities, functional properties and mutagenicity studies of protein and protein hydrolysate obtained from Prosopis alba seed flour.

Prosopis species are considered multipurpose trees and shrubs by FAO and their fruit constitute a food source for humans and animals. According to the "Código Alimentario Argentino", "algarrobo flour" is produced by grinding the whole mature pod, but in the traditional process most of the seeds are discarded. In this paper, the flour from seed was obtained. Then, the proteins were extracted and enzymatic hydrolysis was carried out. According to their amino acid profile and chemical score (>100%), the Prosopis alba proteins, are not deficient in essential amino acids considering the amount of amino acid necessary by adults. The protein isolate showed a good solubility (pH 7.4-9), emulsificant capacity, oil binding capacity and water adsorption capacity. The antioxidant ability of proteins was significantly increased with hydrolysis (SC50 values: 50-5µg/mL, respectively). Inhibitory activity of pro-inflammatory enzymes (lipoxygenase and phospholipase) was described. The mutagenicity/antimutagenicity of proteins and protein hydrolysates from seed flour were also analysed. The results suggest that P. alba cotyledon flour could be a new alternative in the formulation of functional foods not only for its high protein content but also by the biological and functional properties of its proteins and protein hydrolysates.

Toxicity study of isolated polypeptide from wool hydrolysate.

The cytotoxicity of wool polypeptide has been evaluated by both cell and animal models. Wool was dissolved in sodium hydroxide solution, the pH value of the solution was adjusted to 5.55 and the precipitate was harvested as wool polypeptide. The spray-dried polypeptide was collected as powders and characterized by SEM, FTIR and TG-DSC. The cell culturing results showed that wool polypeptide had no obvious negative effect on cell viability in vitro. Both acute oral toxicity and subacute 30-day oral toxicology studies showed that wool polypeptide had no influence on body weight, feed consumption, blood chemistry, and hematology at any dose levels. There were no treatment related findings on gross or detailed necroscopy, organ weights, organ/body weight ratios and histology. Our study indicated the absence of toxicity in wool polypeptide and supported its safe use as a food ingredient or drug carrier.

In vivo and in vitro evaluation of the residual allergenicity of partially hydrolysed infant formulas.

Hypoallergenic infant formulas are commonly used for genetically predisposed children and infants diagnosed with cow's milk allergy. This study describes both in vitro and in vivo approaches to assess residual allergenicity of partially hydrolysed infant formulas. Electrophoretic patterns indicated that ß-lactoglobulin and other whey proteins were largely degraded. For safety reasons, according to the European commission-guidelines, it is required that the sensitizing capacity of hypoallergenic formulas is tested in an animal model. In contrast to whey sensitization, no elevated levels of whey-specific IgE, anaphylactic reactions or drop in body temperature were observed in sensitized mice exposed to whey hydrolysates. This indicates that the whey hydrolysates lost their putative sensitizing capacity in a mouse model using oral sensitization, which is highly relevant in relation to the human situation. In combination with the lost capacity of hydrolysed infant formulas to cross-link human IgE antibodies on RBL-huFcɛRI in vitro, both the sensitization and the challenge phase of the allergic response were studied. This combination of assays is proposed as a strategy for the screening of new hypoallergenic formulas aimed at preventing sensitization in atopic children and avoiding clinical symptoms in infants suffering from cow's milk allergy.

Imaging analysis of the gliadin direct effect on tight junctions in an in vitro three-dimensional Lovo cell line culture system.

Tight junctions play a pivotal role in maintaining the integrity of the intestinal barrier. Their alteration is involved in the pathogenesis of celiac disease. Our aim was to investigate the gliadin effect on the tight junction proteins in an in vitro three-dimensional cell culture model through imaging analyses. Lovo multicellular spheroids were treated with enzymatically digested (PT) gliadin 500 µg/mL and its effect on actin, occludin and zonula occludens-1, was evaluated by means of confocal laser microscopy, transmission electron microscopy and image capture analysis. Compared to untreated spheroids, PT-gliadin-treated ones showed enlargement of the paracellular spaces (9.0±6.9 vs. 6.2±1.7 nm, p<0.05) at transmission electron microscopy and tight junction protein alterations at confocal microscopy and image analyses. In untreated cell cultures thickness of the fluorescence contour of actin, zonula occludens-1 and occludin appeared significantly larger and more intense than in the treated ones. In occludin planimetric analysis the lengths of the integral uninterrupted cellular contour appeared longer in untreated than in PT-gliadin treated spheroids (71.8±42.8 vs. 23.4±25.9 µm, p<0.01). Our data demonstrated that tight junction proteins are directly damaged by gliadin as shown by means of quantitative imaging analysis.

Acute and repeated dose (4 weeks) oral toxicity studies of two antihypertensive peptides, RYLGY and AYFYPEL, that correspond to fragments (90-94) and (143-149) from alpha(s1)-casein.

The Lowpept is a powdered casein hydrolysate containing the antihypertensive peptides RYLGY and AYFYPEL, two sequences that correspond to alpha(s1)-casein f (90-94) (RYLGY) and alpha(s1)-casein f (143-149) (AYFYPEL). To support the safety, Lowpept has been examined in an acute and in a 4-week repeated dose oral toxicity studies in rats. Powdered casein hydrolysate administered in a single oral gavage dose of 2000 mg/kg resulted in no adverse events or mortality. Also, casein hydrolysate administered as a daily dose of 1000 mg/kg for 4 weeks by gavage resulted in no adverse events or mortality. No evidence or treatment-related toxicity was detected during both studies. Data analysis of body weight gain, food consumption, clinical observations, blood biochemical, haematology, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. It is concluded that the casein hydrolysate containing the peptides RYLGY and AYFYPEL orally administered to rats was safe and that not treatment-related toxicity was detected even at the highest doses investigated in both acute (2000 mg/kg of body weight) and repeated dose (4 weeks) oral (1000 mg/kg of body weight) toxicity studies.

Casein hydrolysate as a rapid and/or enteric dissolving additive for oral drugs.

Two types of casein hydrolysates, casein A (mean peptide length 3.3) and casein B (mean peptide length 17.4) were prepared by the enzymatic hydrolysis of casein, and their effects on in vitro dissolution rates and oral bioavailability of drugs were evaluated. The in vitro dissolution behavior of the kneaded mixture of three drugs (diclofenac acid, diazepam, and prednisolone) with caseins A and B were significantly improved compared to the drugs alone, even at 1:1 weight ratio of drug and casein hydrolysate, even though casein A and casein B did not interact with drug molecules in the kneaded mixture. Only diclofenac, an acidic drug, showed an increased dissolution rate with added casein hydrolysates, and a more rapid dissolution with casein A than with casein B was observed. When the dissolution of prednisolone from kneaded mixture was compared at pH 1.2 and 6.8, the dissolution rate of prednisolone from the casein A kneaded mixture was considerably higher than that of prednisolone powder at both pHs, and the rate from the casein B kneaded mixture was higher only at pH 6.8. The plasma concentration-time profile showed that prednisolone was completely and rapidly absorbed from the casein A kneaded mixture as well as the prednisolone solution. In addition, prednisolone in the kneaded mixture with casein B was more difficult to absorb up to 1 hr after administration in comparison to prednisolone powder. The slow and lowered absorption of prednisolone by casein B might be explained by conversion of casein B to a shorter soluble peptide in the gastrointestinal tract and by the slow dissolution of prednisolone at acidic conditions. The toxicological tests revealed that casein hydrolysate is a safe drug carrier. Consequently, casein hydrolysates might be safely used to control the dissolution rate and bioavailability of a variety of drugs, depending on the peptide length of the casein fragments.

[Experimental studies on protalmine]. / Eksperimentalin prouchvaniia na protalmin.

The effect of protein-hydrolysate preparation protalmine was studied on physical endurance and body weight of sexually mature rats. It was established that the preparation in a dose of 500 mg/kg of body weight increased static physical endurance and stability to electric stimuli. The effect was more manifested after oral administration of larger dose (1000 mg/kg of body weight). The body weight was not changed considerably in animals of the control group after one month treatment. It was established in other series of experiments performed on sexually mature rats that protalmine increased physical endurance, while Stark protein, used for comparison, did not affect this parameter considerably. Protalmine did not influence atrophy of seminal vesicles and ventral prostate in castrated young rats, e.g. it had no androgenic effect. After venous administration a slight decrease in arterial blood pressure and retardation of heart rate was manifested within the first 10-15 min. The results from these experiments showed that the protein-hydrolyzate preparation protalmine had marked stimulating action.

Excitotoxic food additives--relevance of animal studies to human safety.

Evidence is reviewed supporting the view that excitotoxic food additives pose a significant hazard to the developing nervous system of young children. The following points are stressed: (1) although blood-brain barriers protect most central neurons from excitotoxins, certain brain regions lack such protection (a characteristic common to all vertebrate species); (2) regardless of species, it requires only a transient increase in blood excitotoxin levels for neurons in unprotected brain regions to be "silently" destroyed; (3) humans may be at particularly high risk for this kind of brain damage, since ingestion of a given amount of excitotoxin causes much higher blood excitotoxin levels in humans than in other species; (4) in addition to the heightened risk on a species basis, risk may be further increased for certain consumer sub-populations due to youth, disease or genetic factors; (5) despite these reasons for maintaining a wide margin of safety in the use of excitotoxins in foods, no safety margin is currently being observed, i.e., a comparative evaluation of animal (extensive) and human (limited) data supports the conclusion that excitotoxins, as used in foods today, may produce blood elevations high enough to cause damage to the nervous system of young children, damage which is not detectable at the time of occurrence but which may give rise to subtle disturbances in neuroendocrine function in adolescence and/or adulthood.

[Pharmacological and clinico-pharmacological studies of a combination preparation for calves having antianemia and general tonic action]. / Kombiniran preparat za teleta s protivoanemichno i obshtoukrepvashto deistvie--farmakologichni i kliniko-farmakologichni izsledvaniia.

A combined preparation, biofer, was studied, defining its clinical and pharmacological capacity. Featuring in its composition are: normal bovine gammaglobulin, 8.0 g; ferridextran (dextrofer-100), 32 cm2 (= 3.2 g Fe); cuprum sulfuricum, 0.32 g (= 0.08 Cu); Co chloride, 0.18 g (= 0.08 Co); cyancobalamin, 0.0032 g; and protein hydrolysate up to 100 cm3, at pH = 7.0-7.2. The local and total tolerance of animals for biofer was studied along with the acute toxicity, absorption, and retention in the body of test animals and calves as well as the antianemic action in albino mice and calves. It was found that at 4 degrees C to 8 degrees C the shelf life of biofer was 2 years. Its LD50 at subcutaneous injection to albino rats was 11.7 cm3/kg body mass. At the rate of 0.6 cm3/kg (i/m) rabbits did not manifest local and total intolerance; at 1.8 cm3/kg there was no local inflammation, however, a transient drop of appetite was seen; at 3 cm3/kg rabbits manifested intoxication with exitus. At i/m introduction to rabbits and calves biofer was more slowly absorbed than dextrofer-100. The liver and spleen of animals injected with biofer showed higher values for copper. In i/m application to anemic albino rats biofer showed a better antianemic effect than dextrofer-100. In calves it activated to a better extent both erythropoiesis and leukopoiesis.

[Pharmacological properties of a gelatin hydrolysate and its effect on some skin metabolic processes]. / Farmakologicheskie svoistva gidrolizata zhelatiny i ego vliianie na nekotorye metabolicheskie protsessy kozhi

Experiments have shown that gelatin hydrolysate is non-toxic, does not possess any allergic and local irritating properties. It stimulates the regenerative capacity of the epidermic cells, tends to improve the blood- and lymph-circulation, intervenes in the protein and water-electrolyte metabolism, intensifies oxidative processes and normalizes the acid-base equilibrium of the skin.