Preparation, Optimization, and Anti-Pulmonary Infection Activity of Casein-Based Chrysin Nanoparticles.

Introduction: Chrysin has a wide range of biological activities, but its poor bioavailability greatly limits its use. Here, we attempted to prepare casein (cas)-based nanoparticles to promote the biotransfer of chrysin, which demonstrated better bioavailability and anti-infection activity compared to free chrysin. Methods: Cas-based chrysin nanoparticles were prepared and characterized, and most of the preparation process was optimized. Then, the in vitro and in vivo release characteristics were studied, and anti-pulmonary infection activity was evaluated. Results: The constructed chrysin-cas nanoparticles exhibited nearly spherical morphology with particle size and ζ potential of 225.3 nm and -33 mV, respectively. These nanoparticles showed high encapsulation efficiency and drug-loading capacity of 79.84% ± 1.81% and 11.56% ± 0.28%, respectively. In vitro release studies highlighted a significant improvement in the release profile of the chrysin-cas nanoparticles (CCPs). In vivo experiments revealed that the relative oral bioavailability of CCPs was approximately 2.01 times higher than that of the free chrysin suspension. Further investigations indicated that CCPs effectively attenuated pulmonary infections caused by Acinetobacter baumannii by mitigating oxidative stress and reducing pro-inflammatory cytokines levels, and the efficacy was better than that of the free chrysin suspension. Conclusion: The findings underscore the advantageous bioavailability of CCPs and their protective effects against pulmonary infections. Such advancements position CCPs as a promising pharmaceutical agent and candidate for future therapeutic drug innovations.

Solid self-emulsifying casein carrier for the improvement on the oral bioavailability of simvastatin.

Simvastatin (SV) is a statin drug that can effectively control cholesterol and prevent cardiovascular diseases. However, SV is water-insoluble, and poor oral bioavailability (<5 %). Solid self-emulsifying carrier system is more stable than liquid emulsions, facilitating to improve the solubility and bioavailability of poorly soluble drugs. In the present study, a solid self-emulsifying carrier stabilized by casein (Cas-SSE) was successfully used to load SV to improve its solubility in water, by formulation selection and emulsification process optimization. Compared with oral tablets, the release of SV from Cas-SSE was significantly enhanced in artificial intestinal fluid. Furthermore, everted gut sac experiments indicated some water-soluble dispersing agents such as hydroxyethyl starch (HES), were not conducive to drug absorption. Pharmacokinetic studies suggested Cas-SSE without dispersing agent has much higher relative bioavailability (184.1 % of SV and 284.5 % of simvastatin acid) than SV tablet. The present work suggests Cas-SSE is a promising drug delivery platform with good biocompatibility for improving oral bioavailability of poorly water-soluble drugs.

Real ileal amino acid digestibility of pea protein compared to casein in healthy humans: a randomized trial.

BACKGROUND: It is necessary to propose plant alternatives to animal proteins that are of good nutritional quality. Pea is a good candidate owing to its high protein content and its well-balanced amino acid (AA) profile. OBJECTIVES: This study aimed to assess the real ileal AA and nitrogen digestibility (RIDAA and RIDN) of pea protein isolate as compared to milk casein in humans. It also aimed to evaluate their nutritional quality through calculation of the digestible indispensable amino acid score (DIAAS) and to determine the net postprandial protein utilization (NPPU). METHODS: Fifteen healthy volunteers were included in a randomized, single-blinded, 2-arm, parallel-design trial. They were equipped with a naso-ileal tube. They ingested the test meals, which consisted of 9 successive portions of mashed potatoes containing either pea protein or casein, intrinsically labeled with nitrogen 15. Ileal content, plasma, and urine samples were collected regularly over an 8-h postprandial period. RESULTS: The mean RIDAA values were 93.6% ± 2.9% for pea protein and 96.8% ± 1.0% for casein, with no difference between the sources (P = 0.22). Leucine, valine, lysine, and phenylalanine were significantly less digestible in pea than in casein. The RIDN values were 92.0% ± 2.7% and 94.0% ± 1.7% for pea protein and casein, respectively, and were not different (P = 0.11). The DIAAS was 1.00 for pea protein and 1.45 for casein. The NPPU was 71.6% ± 6.2% and 71.2% ± 4.9% for pea protein and casein, respectively (P = 0.88). CONCLUSIONS: Although some AAs are less digestible in pea protein than in casein, the real ileal digestibility and the NPPU were not different. The DIAAS of 1.00 obtained for pea protein demonstrated its ability to meet all AA requirements. This study shows the potential of pea isolate as a high-quality protein. This study was registered at clinicaltrials.gov as NCT04072770.

HPLC with fluorescence detection for the bioanalysis and pharmacokinetic study of Doxorubicin and Prodigiosin loaded on eco-friendly casein nanomicelles in rat plasma.

A rapid, efficient, and sensitive liquid chromatographic assay hyphenated to fluorometric detector (HPLC-FLD) was developed and validated for the determination of doxorubicin (DXR) and prodigiosin (PDG) in rat plasma. The sample pre-treatment involves a protein precipitation with acetonitrile with satisfying extraction efficiency (98% and 85% for DXR and PDG, respectively). The chromatographic separation was accomplished using stationary phase: Agilent Zorbax Eclipse plus-C18 analytical column (250 × 4.6 mm, 5 µm) and gradient eluting mobile phase of ammonium acetate (pH = 3), acetonitrile and methanol with programmed fluorescence detection. As the proposed method has been validated, it was subsequently implemented to evaluate DXR and PDG loaded on novel eco-friendly Casein nano drug delivery system after intravenous injection in healthy rats. A comparative pharmacokinetics' study was carried out in rats for DXR in free form, DXR alone entrapped in the nanomicelle and DXR with PDG entrapped in the nano micelle. After testing the differences in pharmacokinetic parameters of the different formulations using ANOVA, the results showed insignificant differences among the tested parameters. This indicates that the presented nanomicelle delivery system has succeeded to incorporate PDG and DXR in a hydrophilic, safe, and potent formulation. This novel nanomicelle has negligible effect on the distribution and elimination of DXR.

Improved estimation of in vitro protein digestibility of different foods using size exclusion chromatography.

While the harmonized INFOGEST model provides a physiologically relevant platform for simulated digestion, it needs to be combined with adequate analytical methods to enable quantification and comparison of protein digestibility in different food matrices. We have shown that size exclusion chromatography (SEC) can be used to estimate the proportion of small peptides potentially available for uptake. Combined with determination of total dissolved protein, the % of small peptides per total protein was calculated as a physiologically relevant estimate of protein digestibility (DSEC). Values for DSEC differed for casein (87.6%), chicken mince (72.6%), heated pea protein concentrate (67.8%), bread (63%), beef entrecote (57.7%) and pea protein concentrate (57.8%). In contrast to existing methods (TCA soluble protein, free NH2-groups), the proposed SEC based method gives separate insight into the two fundamental processes during protein digestion (solubilization and break-down), while maintaining the ability to rank digestibility of very different food proteins.

Multidomain drug delivery systems of ß-casein micelles for the local oral co-administration of antiretroviral combinations.

The antiretroviral (ARV) cocktailrevolved the treatment of the human immunodeficiency virus (HIV) infection. Drug combinations have been also tested to treat other infectious diseases, including the recentcoronavirus disease 2019 (COVID-19) outbreak. To simplify administration fixed-dose combinationshave been introduced, however, oral anti-HIV therapy still struggles with low oral bioavailability of many ARVs.This work investigated the co-encapsulation of two clinically relevant ARV combinations,tipranavir (TPV):efavirenz (EFV) anddarunavir (DRV):efavirenz (EFV):ritonavir (RTV),within the core of ß-casein (bCN) micelles. Encapsulation efficiency in both systems was ~100%. Cryo-transmission electron microscopy and dynamic light scattering of the ARV-loaded colloidaldispersions indicatefull preservation of the spherical morphology, and x-ray diffraction confirm that the encapsulated drugs are amorphous. To prolong the physicochemical stabilitythe formulations were freeze-driedwithout cryo/lyoprotectant, and successfully redispersed, with minor changes in morphology.Then, theARV-loaded micelles were encapsulated within microparticles of Eudragit® L100, which prevented enzymatic degradation and minimized drug release under gastric-like pH conditionsin vitro. At intestinal pH, the coating polymer dissolved and released the nanocarriers and content. Overall, our results confirm the promise of this flexible and modular technology platform for oral delivery of fixed dose combinations.

Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data.

Correlation and validation of the results of simulated gastrointestinal digestion of food compounds towards in vivo data is essential. The objective of this work was to monitor the digestion of milk micellar casein in the porcine upper intestinal tract and to match the outcome with the gastric in vitro digestion following the Infogest harmonized protocol. In pig duodenum, small amounts of intact caseins were present in all samples, while caseins were observed up to 60 min of gastric in vitro digestion. The peptide profile generated after in vitro and in vivo digestion showed clear similarities with specific overrepresented regions rich in proline and other hydrophobic residues. The statistical comparison of the in vivo and in vitro peptidome resulted in satisfactory correlation coefficients, up to 0.8. Therefore, the in vitro protocol used was a robust and simple model that provides a similar peptide profile than that found in porcine duodenum.

Casein Protein Processing Strongly Modulates Post-Prandial Plasma Amino Acid Responses In Vivo in Humans.

Micellar casein is characterized as a slowly digestible protein source, and its structure can be modulated by various food processing techniques to modify its functional properties. However, little is known about the impact of such modifications on casein protein digestion and amino acid absorption kinetics and the subsequent post-prandial plasma amino acid responses. In the present study, we determined post-prandial aminoacidemia following ingestion of isonitrogenous amounts of casein protein (40 g) provided as micellar casein (Mi-CAS), calcium caseinate (Ca-CAS), or cross-linked sodium caseinate (XL-CAS). Fifteen healthy, young men (age: 26 ± 4 years, BMI: 23 ± 1 kg·m-2) participated in this randomized cross-over study and ingested 40 g Mi-Cas, Ca-CAS, and XL-CAS protein, with a ~1 week washout between treatments. On each trial day, arterialized blood samples were collected at regular intervals during a 6 h post-prandial period to assess plasma amino acid concentrations using ultra-performance liquid chromatography. Plasma amino acid concentrations were higher following the ingestion of XL-CAS when compared to Mi-CAS and Ca-CAS from t = 15 to 90 min (all p < 0.05). Plasma amino acid concentrations were higher following ingestion of Mi-CAS compared to Ca-CAS from t = 30 to 45 min (both p < 0.05). Plasma total amino acids iAUC were higher following the ingestion of XL-CAS when compared to Ca-CAS (294 ± 63 vs. 260 ± 75 mmol·L-1, p = 0.006), with intermediate values following Mi-CAS ingestion (270 ± 63 mmol·L-1, p > 0.05). In conclusion, cross-linked sodium caseinate is more rapidly digested when compared to micellar casein and calcium caseinate. Protein processing can strongly modulate the post-prandial rise in plasma amino acid bioavailability in vivo in humans.

Modification of protein structures by altering the whey protein profile and heat treatment affects in vitro static digestion of model infant milk formulas.

Heat treatments induce changes in the protein structure in infant milk formulas (IMFs). The present study aims to investigate whether these structural modifications affect protein digestion. Model IMFs (1.3% proteins), with a bovine or a human whey protein profile, were unheated or heated at 67.5 °C or 80 °C to reach 65% of denaturation, resulting in six protein structures. IMFs were submitted to in vitro static gastrointestinal digestion simulating infant conditions. During digestion, laser light scattering was performed to analyze IMF destabilization and SDS-PAGE, OPA assay and cation exchange chromatography were used to monitor proteolysis. Results showed that, during gastric digestion, α-lactalbumin and ß-lactoglobulin were resistant to hydrolysis in a similar manner for all protein structures within IMFs (p > 0.05), while the heat-induced denaturation of lactoferrin significantly increased its susceptibility to hydrolysis. Casein hydrolysis was enhanced when the native casein micelle structure was modified, i.e. partially disintegrated in the presence of lactoferrin or covered by heat-denatured whey proteins. The IMF destabilization at the end of the gastric digestion varied with protein structures, with larger particle size for IMF containing native casein micelles. During intestinal digestion, the kinetics of protein hydrolysis varied with the IMF protein structures, particularly for IMFs containing denatured lactoferrin, exhibiting higher proteolysis degree (67.5 °C and 80 °C vs. unheated) and essential amino acid bioaccessibility (67.5 °C vs. unheated). Overall, the protein structures, generated by modulating the whey protein profile and the heating conditions, impacted the IMF destabilization during the gastric phase and the proteolysis during the entire simulated infant digestion.

Formation of bioactive peptides during simulated gastrointestinal digestion is affected by αs1-casein polymorphism in buffalo milk.

The polymorphism of buffalo αs1-casein has been reported, but little is known about their effect on the biological properties. The objective of this study was to investigate the effect of αs1-casein polymorphism on the digestive properties and bioactivities of buffalo milk protein in simulated gastrointestinal digestion. In this study, two variants of αs1-casein, with one amino acid substitution of Leu193 (AA) â†’ Ser193 (BB), were used. Under acidic gastric conditions, the particle size of αs1-casein variant BB was smaller and showed higher digestibility compared to variant AA. A total of 154 and 149 peptides were identified, respectively, from simulated gastrointestinal digestion of variants AA and BB; of three peptides have been previously reported to exert ACE-inhibition, anticancer, antioxidant, and anxiolytic activities. Our study demonstrated that αs1-casein polymorphism affects the digestive properties and the formation of bioactive peptides.

Amino Acid Availability of a Dairy and Vegetable Protein Blend Compared to Single Casein, Whey, Soy, and Pea Proteins: A Double-Blind, Cross-Over Trial.

Protein quality is important for patients needing medical nutrition, especially those dependent on tube feeding. A blend of dairy and vegetable proteins (35% whey, 25% casein, 20% soy, 20% pea; P4) developed to obtain a more balanced amino acid profile with higher chemical scores, was compared to its constituent single proteins. Fourteen healthy elderly subjects received P4, whey, casein, soy, and pea (18 g/360 mL bolus) on five separate visits. Blood samples were collected at baseline until 240 min after intake. Amino acid availability was calculated using incremental maximal concentration (iCmax) and area under the curve (iAUC). Availability for P4 as a sum of all amino acids was similar to casein (iCmax and iAUC) and whey (iCmax) and higher vs. soy (iCmax and iAUC) and pea (iCmax). Individual amino acid availability (iCmax and iAUC) showed different profiles reflecting the composition of the protein sources: availability of leucine and methionine was higher for P4 vs. soy and pea; availability of arginine was higher for P4 vs. casein and whey. Conclusions: The P4 amino acid profile was reflected in post-prandial plasma levels and may be regarded as more balanced compared to the constituent single proteins.

Fabrication and Characterization of Zein Composite Particles Coated by Caseinate-Pectin Electrostatic Complexes with Improved Structural Stability in Acidic Aqueous Environments.

Zein composite particles coated with caseinate-pectin electrostatic complexes (zein-caseinate-pectin particles) were fabricated using an electrostatic deposition and liquid-liquid dispersion method without heating treatment. Compared to zein particles coated only with caseinate, the acidic stability of zein-caseinate-pectin particles was greatly improved, and the particle aggregation was suppressed at pH 3-6, especially at pH values near the isoelectric point of caseinate (pH 4-5). Besides, desirable long-term storage stability and re-dispersibility were observed. Under different zein to curcumin (Cur) feeding ratios (10:1, 20:1, 30:1 and 40:1, w/w), the Cur-loaded zein-caseinate-pectin particles had a spherical shape with an average diameter ranging from 358.37 to 369.20 nm, a narrow size distribution (polydispersity index < 0.2) and a negative surface charge ranging from -18.87 to -19.53 mV. The relatively high encapsulation efficiencies of Cur (81.27% to 94.00%) and desirable re-dispersibility were also achieved. Fluorescence spectroscopy indicated that the encapsulated Cur interacted with carrier materials mainly through hydrophobic interactions. The in-vitro release profile showed a sustained release of Cur from zein-caseinate-pectin particles in acidic aqueous environment (pH 4) up to 24 h, without any burst effect. In addition, the encapsulation retained more ABTS•+ radical scavenging capacity of Cur during 4 weeks of storage. These results suggest that zein-caseinate-pectin particles may be used as a potential delivery system for lipophilic nutrients in acidic beverages.

Inhibition of amyloid beta toxicity in zebrafish with a chaperone-gold nanoparticle dual strategy.

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders, yet no major breakthroughs have been made in AD human trials and the disease remains a paramount challenge and a stigma in medicine. Here we eliminate the toxicity of amyloid beta (Aß) in a facile, high-throughput zebrafish (Danio rerio) model using casein coated-gold nanoparticles (ßCas AuNPs). ßCas AuNPs in systemic circulation translocate across the blood brain barrier of zebrafish larvae and sequester intracerebral Aß42 and its elicited toxicity in a nonspecific, chaperone-like manner. This is evidenced by behavioral pathology, reactive oxygen species and neuronal dysfunction biomarkers assays, complemented by brain histology and inductively coupled plasma-mass spectroscopy. We further demonstrate the capacity of ßCas AuNPs in recovering the mobility and cognitive function of adult zebrafish exposed to Aß. This potent, safe-to-use, and easy-to-apply nanomedicine may find broad use for eradicating toxic amyloid proteins implicated in a range of human diseases.

Casein-derived peptides as an alternative ingredient for low-phenylalanine diets / Péptidos derivados de la caseína como ingrediente alternativo para las dietas bajas en fenilalanina

Introduction: casein-derived peptides can be liberated both in vivo via normal digestion of casein, as well as in vitro via enzymatic hydrolysis. These peptides were suggested to have biological activity. Objectives: the aim of this study was to describe the production and characterization of casein peptides and to explore the potential of these peptides as an option for low-phenylalanine diets. Methods: peptides were produced by tryptic hydrolysis of sodium caseinate and acid precipitation with HCl, followed by precipitation with ethanol or aggregation of CaCl2 or ZnSO4. Results: the amino acid analysis revealed a significant reduction in the amount of phenylalanine from the original protein. Conclusion: casein-derived peptides could be a future alternative of short chain peptides to low-phenylalanine formulations

Introducción: los péptidos derivados de la caseína se pueden liberar tanto in vivo, a través de la digestión normal de la caseína, como in vitro a través de la hidrólisis enzimática. Se sugirió que estos péptidos tenían actividad biológica. Objetivos: el objetivo de este estudio fue describir la producción y caracterización de péptidos de caseína y explorar el potencial de estos péptidos como una opción para las dietas con bajo contenido de fenilalanina. Métodos: los péptidos se produjeron por hidrólisis tríptica de caseinato de sodio y precipitación ácida con HCl, seguido de precipitación con etanol o agregación de CaCl2 o ZnSO4. Resultados: el análisis de aminoácidos reveló una reducción significativa en la cantidad de fenilalanina de la proteína original. Conclusión: los péptidos derivados de la caseína podrían ser una alternativa futura de los péptidos de cadena corta a las formulaciones con bajo contenido de fenilalanina

Optimization of Chitosan-α-casein Nanoparticles for Improved Gene Delivery: Characterization, Stability, and Transfection Efficiency.

Among non-viral vectors, the cationic polymer chitosan has gained attention as a gene delivery system. We hypothesized that the addition of casein into the nanoparticle's structure would facilitate a proper gene transfer. The work herein presented aimed to optimize the production method of chitosan-casein nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system. ChiCas NPs formulation optimization was carried out by analyzing several characteristics such as NP size, zeta potential, and chitosan and casein incorporation efficacy. The best formulation developed presented small and homogenous particle size (around 335 nm) and positive zeta potential (≈ + 38 mV), and showed to be stable for 34 weeks both, at 4°C and 20°C. The particles were further used to entrap or to adsorb DNA and form NPs-DNA complexes. In vitro transfection studies, carried out in COS-7 cells, suggested a low transfection efficiency of the different NPs:DNA ratios tested, comparatively to the positive control. Nonetheless, we could observe that the complexes with larger sizes presented better transfection results than those with smaller diameters. To conclude, ChiCas NPs have great technological potential since the preparation process is very simple, and the DNA incorporation efficacy is very high and shows to be physically very stable. The NPs:DNA ratio still needs to be optimized with the aim of achieving better transfection results and being able to anticipate a high gene expression on DNA-based vaccination studies.

Interactions Between Flavonoid-Rich Extracts and Sodium Caseinate Modulate Protein Functionality and Flavonoid Bioaccessibility in Model Food Systems.

With growing interest in formulating new food products with added protein and flavonoid-rich ingredients for health benefits, direct interactions between these ingredient classes becomes critical in so much as they may impact protein functionality, product quality, and flavonoids bioavailability. In this study, sodium caseinate (SCN)-based model products (foams and emulsions) were formulated with grape seed extract (GSE, rich in galloylated flavonoids) and green tea extract (GTE, rich in nongalloylated flavonoids), respectively, to assess changes in functional properties of SCN and impacts on flavonoid bioaccessibility. Experiments with pure flavonoids suggested that galloylated flavonoids reduced air-water interfacial tension of 0.01% SCN dispersions more significantly than nongalloylated flavonoids at high concentrations (>50 µg/mL). This observation was supported by changes in stability of 5% SCN foam, which showed that foam stability was increased at high levels of GSE (≥50 µg/mL, P < 0.05) but was not affected by GTE. However, flavonoid extracts had modest effects on SCN emulsion. In addition, galloylated flavonoids had higher bioaccessibility in both SCN foam and emulsion. These results suggest that SCN-flavonoid binding interactions can modulate protein functionality leading to difference in performance and flavonoid bioaccessibility of protein-based products. PRACTICAL APPLICATION: As information on the beneficial health effects of flavonoids expands, it is likely that usage of these ingredients in consumer foods will increase. However, the necessary levels to provide such benefits may exceed those that begin to impact functionality of the macronutrients such as proteins. Flavonoid inclusion within protein matrices may modulate protein functionality in a food system and modify critical consumer traits or delivery of these beneficial plant-derived components. The product matrices utilized in this study offer relevant model systems to evaluate how fortification with flavonoid-rich extracts allows for differing effects on formability and stability of the protein-based systems, and on bioaccessibility of fortified flavonoid extracts.

Physicochemical characterization of mineral (iron/zinc) bound caseinate and their mineral uptake in Caco-2 cells.

Milk proteins (especially caseins) are widely accepted as good vehicle for the delivery of various bioactive compounds including minerals. Succinylation is one of the most acceptable chemical modification techniques to enhance the mineral binding ability of caseins. Addition of minerals to succinylated proteins may alter their physicochemical and biochemical properties. Physicochemical characteristics of succinylated sodium caseinate (S.NaCN)-mineral (iron/zinc) complexes were elucidated. Chromatographic behaviour and fluorescence intensity confirmed the structural modification of S.NaCN upon binding with minerals. The bound mineral from protein complexes showed significantly higher (P < 0.05) in vitro bioavailability (mineral uptake) than mineral salts in Caco-2 cells. Also, iron bound S.NaCN showed higher cellular ferritin formation than iron in its free form. These mineral bound protein complexes with improved bioavailability could safely replace inorganic fortificants in various functional food formulations.

Consumption of Whey in Combination with Dairy Medium-Chain Fatty Acids (MCFAs) may Reduce Lipid Storage due to Urinary Loss of Tricarboxylic Acid Cycle Intermediates and Increased Rates of MCFAs Oxidation.

SCOPE: The aim of the paper is to investigate whether changes in the metabolome could explain observed changes in body composition in overweight adults after consumption of butter with high level of medium-chain fatty acids (MCFAs) in combination with casein or whey. METHODS AND RESULTS: With GC-TOF and LC-Q/MS, metabolites in plasma and urine from a 12-week randomized double-blinded human intervention including 52-abdominally overweight adults were analyzed. The participants consumed 63 g per day of milk fat (high or low in MCFAs) and 60 g per day of protein (whey or casein). Urinary loss of the tricarboxylic acid cycle metabolites and a concomitantly increase of glycerol in blood were observed in the whey + high-MCFAs group, indicating potential lower anabolic processes, such as lipogenesis, by draining substrates. High intake of MCFAs resulted in elevated level of urinary adipic (independently of protein type) and plasma sebacic acid (with whey), indicating a potential increase in oxidation of MCFAs, which might lead to energy loss. CONCLUSION: The type of protein showed highest effect on the overall metabolic profiles, but ω-oxidation of MCFAs in the liver seemed to be the main reason for the observed reduction in body fat mass after consumption of high MCFAs, independent of type of protein.

Toxicity and biodistribution of orally administered casein nanoparticles.

In the last years, casein nanoparticles have been proposed as carriers for the oral delivery of biologically active compounds. However, till now, no information about their possible specific hazards in vivo was available. The aim of this work was to assess the safety of casein nanoparticles when administered orally to animals through a 90 days dose-repeated toxicity study (OECD guideline 408), that was performed in Wistar rats under GLP conditions. After 90 days, no evidences of significant alterations in animals treated daily with 50, 150 or 500 mg/kg bw of nanoparticles were found. This safety agrees well with the fact that nanoparticles were not absorbed and remained within the gut as observed by radiolabelling in the biodistribution study. After 28 days, there was a generalized hyperchloremia in males and females treated with the highest dose of 500 mg/kg bw, that was coupled with hypernatremia in the females. These effects were related to the presence of mannitol which was used as excipient in the formulation of casein nanoparticles. According to these results, the No Observed Adverse Effect Level (NOAEL) could be established in 150 mg/kg bw/day and the Lowest Observed Effect Level (LOEL) could be established in 500 mg/kg bw/day.

Effect of molecular weight on the transepithelial transport and peptidase degradation of casein-derived peptides by using Caco-2 cell model.

The transepithelial transport routes of casein-derived peptides with different molecular weights (MWs) were investigated using a Caco-2 cell monolayer. The peptidase hydrolysis during transport was also studied. The results indicate that the paracellular route was the main pathway for F1 (1600-1300Da) and F2 (1000-500Da), and the bioavailabilities were 10.66% and 9.54%, respectively. Peptidase hydrolysis results reveal that brush-border peptidases (BBPs) as well as some other peptidases were responsible for peptide degradation in the paracellular route. The maximum hydrolysis rate of the former was 6.91 and 5.59µM Gly/min for the latter. However, PepT1 was involved in the transport of F3 (<500Da) and its bioavailability was 16.23%. BBPs were the main peptidases involved in the PepT1 transport and the maximum hydrolysis rate was 11.4µM Gly/min. Furthermore, we found that the amino acid sequence of di- and tripeptides might affect their bioavailabilities significantly.