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    The (193-209) 17-residues peptide of bovine β-casein is transported through Caco-2 monolayer. Regazzo Daniela,Mollé Daniel,Gabai Gianfranco,Tomé Daniel,Dupont Didier,Leonil Joëlle,Boutrou Rachel Molecular nutrition & food research Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β-casein (β-CN, 193-209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β-CN (193-209) peptide across Caco-2 cell monolayer. In addition, we evaluated the possible routes of the β-CN (193-209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β-CN (193-209) peptide resisted the action of brush-border membrane peptidases, and that it was transported through the Caco-2 cell monolayer. The main route involved in transepithelial transport of the β-CN (193-209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. Our results demonstrated the transport of an intact long-chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide. 10.1002/mnfr.200900443
    Effect of sodium caseinate and vitamin A complexation on bioaccessibility and bioavailability of vitamin A in Caco-2 cells. Rana Seema,Arora Sumit,Gupta Chitra,Kapila Suman Food research international (Ottawa, Ont.) Native sodium caseinate-vitamin A (VA) complexes (Sodium caseinate-VA complex, NaCaS-VA) and modified sodium caseinate-VA complexes i.e. Succinylated sodium caseinate-VA complex (SNaCaS-VA), reassembled sodium caseinate-VA complex (RNaCaS-VA) and reassembled succinylated sodium caseinate-VA complex (RSNaCaS-VA) were prepared and evaluated for their in-vitro bioaccessibility and in-vitro bioavailability of VA through Caco-2 cell lines.VA degraded under acidic conditions as the physiological pH during digestion in stomach was highly acidic (1.2-1.8). During in-vitro gastric digestion, sodium caseinate provided protection to VA, hence, higher VA content was retained in digesta as compared to free VA (oily form). Vitamin uptake by Caco-2 cells was significantly different for digested sodium caseinate-VA complexes as compared to free VA. The peptide content of casein and various sodium caseinate-VA complexes was monitored throughout digestion process. Variation in the complex composition had an effect on protein digestibility and peptide distribution. The bioavailability of VA through sodium caseinate-VA complexes was evaluated by exposing Caco-2 cells to the digesta of milk fortified with various complexes. The total uptake of VA by Caco-2 cells was highest for milk fortified with RSNaCaS-VA followed by RNaCaS-VA, control milk, SNaCaS-VA, NaCaS-VA and free VA. During the formation of RNaCaS-VA and RSNaCaS-VA complexes more hydrophobic sites are exposed, leading to the attachment of VA on the interior hydrophobic regions of sodium caseinate molecule. This led to higher stability of VA during gastrointestinal digestion and further resulted in higher bioaccessibility and bioavailability of vitamin A in Caco-2 cells. 10.1016/j.foodres.2019.01.019
    Effect of protein composition of a model dairy matrix containing various levels of beta-casein on the structure and anti-inflammatory activity of in vitro digestates. Rafiee Tari N,Arranz E,Corredig M Food & function An increasing body of evidence demonstrates that differences in protein composition in the food matrix can significantly affect its biological functionality. The present research hypothesized that a matrix containing the same level of dairy protein, but with different composition, even when showing similar properties during digestion, may have a different biological functionality. To test this hypothesis, three matrices, containing 2.8% protein and similar amounts of fat and solid were prepared, either with 100% whey proteins, or with a ratio of caseins to whey protein of 40 : 60, but differing in β-casein ratio. The mixtures were subjected to in vitro digestion, and the digestates were used in uptake experiments using Caco-2 cell monolayers. The basolateral fraction metabolized by the cells was used to stimulate human LPS-stimulated THP-1 macrophages and the concentration of selected cytokines were measured, as an indication of potential differences in biological functionality between the different dairy matrices. All three digestates induced a significant reduction in IL-1β cytokines, with the casein-containing treatments inducing a greater decrease compared to that containing only whey proteins. The matrix containing the highest ratio of β-casein induced the lowest secretion of proinflammatory cytokines TNF-a and IL-6. This study demonstrated that milk protein composition does not only affect the rate of gastric proteolysis and structure of the gastric digestate, but will cause differences in physiological effects. This research stressed the role of milk protein components during digestion, and of β-casein in particular, and their potential to modulate biological functions in the gastrointestinal tract. 10.1039/c8fo01860j
    Short communication: Inhibition of angiotensin 1-converting enzyme by peptides derived from variants of bovine β-casein upon apical exposure to a Caco-2 cell monolayer. Petrat-Melin Bjørn,Le Thao T,Møller Hanne S,Larsen Lotte B,Young Jette F Journal of dairy science This study investigated the consequence of genetically contingent amino acid substitutions in bovine β-casein (CN) genetic variants A, A, B, and I on the structure and bioactive potential of peptides following in vitro digestion. The β-CN variants were digested in vitro using pepsin and pancreatin, and a peptide profile was obtained by liquid chromatography tandem mass spectrometry, revealing among others, the β-casomorphin precursor peptides VYPFPGPIHN and VYPFPGPIPN, derived from variant A/B and from A/I, respectively. These 2 peptides were synthesized and assessed for angiotensin 1-converting enzyme (ACE) inhibitory capacity before and after incubation with a monolayer of Caco-2 intestinal cells. The VYPFPGPIHN was a stronger ACE inhibitor than VYPFPGPIPN, with the concentration needed to reach half-maximal inhibition (IC) of 123 ± 14.2 μM versus 656 ± 7.6 μM. Exposure to a Caco-2 intestinal cell monolayer did not affect ACE inhibition by VYPFPGPIHN, but resulted in an almost 2-fold increase in inhibition by VYPFPGPIPN after incubation. Subsequent tandem mass spectrometric analysis identified the truncated peptide VYPFPGPIP, suggesting hydrolysis by a cell membrane associated peptidase. Thus, genetic variation in bovine β-CN results in the generation of peptides that differ in bioactivity, and are differently affected by intestinal brush border peptidases. 10.3168/jds.2016-11684
    Effect of molecular weight on the transepithelial transport and peptidase degradation of casein-derived peptides by using Caco-2 cell model. Wang Bo,Li Bo Food chemistry 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. 10.1016/j.foodchem.2016.08.106
    Bio-accessible milk casein derived tripeptide (LLY) mediates overlapping anti- inflammatory and anti-oxidative effects under cellular (Caco-2) and in vivo milieu. Sowmya Kandukuri,Mala Dev,Bhat Mohd Iqbal,Kumar Naveen,Bajaj Rajesh Kumar,Kapila Suman,Kapila Rajeev The Journal of nutritional biochemistry Inflammation and oxidative stress are closely linked patho-physiological processes which occur concurrently in many diseased conditions. Recently, interdependence between these two processes explains the antioxidant paradox associated with failure to select appropriate agents required for prevention of diseases known to be induced by oxidative stress. Present study established the overlapping anti-inflammatory and anti-oxidative potential along with bio-accessibility of milk casein derived tripeptide (LLY). Tripeptide exhibited anti-inflammatory response under ex vivo conditions by suppressing (P<.01) mice splenocytes proliferation and modulating their cytokines (IFN-γ, IL-10 and TGF-β) with improved phagocytosis of peritoneal macrophages. Conversely, tripeptide displayed extraordinary radical scavenging ability and cellular anti-oxidative potential using chemical assays and HO induced oxidative stress model on Caco-2 cells. Under cellular assessment, on one hand tripeptide inhibited (P<.01) intracellular ROS generation and reduced MDA and protein carbonyls but on the other also increased (P<.01) the activity of anti-oxidative enzyme, catalase without much effect on SOD and GPx. This anti-oxidative potential was further established by studying relative expression of genes (Nrf-2 and Keap1) and Nrf-2 nuclear translocation associated with anti-oxidative signaling in Caco-2 cells. Bio-accessibility of tripeptide and its intact transport across Caco-2 cell monolayer was also found to be 1.72±0.22% through PepT1 mediated transport mechanism. Besides, tripeptide displayed strong anti-oxidative and anti-inflammatory potential under in vivo conditions in mice against ethanol induced oxidative stress by elevating (P<.01) liver GSH content and by decreasing (P<.01) the activities of anti-oxidative enzymes, MDA along with reduced expression of CYP2E1, PPAR-α, TNF-α and COX-2 genes than ethanol control. 10.1016/j.jnutbio.2018.09.002
    Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers. Cao Yong,Miao Jianyin,Liu Guo,Luo Zhen,Xia Zumeng,Liu Fei,Yao Mingfei,Cao Xiaoqiong,Sun Shengwei,Lin Yanyin,Lan Yaqi,Xiao Hang Journal of agricultural and food chemistry The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P < 0.05). Interestingly, when calcium and magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein. 10.1021/acs.jafc.6b05711
    Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments. Sakurai Nao,Nishio Shunsuke,Akiyama Yuka,Miyata Shinji,Oshima Kenzi,Nadano Daita,Matsuda Tsukasa Journal of biochemistry Casein (CN) is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic CN and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of CN was investigated. Confocal microscopy using component-specific antibodies showed that αs1-CN antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular CN signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein (EEA1) and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4°C. Liquid chromatography coupled with mass spectroscopy analysis of the protein fraction in the basal-side medium identified the αs1-CB fragment including the N-terminal region and the αs2-CN fragment containing the central part of polypeptide at 100-1,000 fmol per well levels. Moreover, β-CN C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that CNs are partially degraded by cellular proteases and/or peptidases and immunologically active CN fragments are transported to basal side of the cell monolayers. 10.1093/jb/mvy034
    Structural Requirement of Casein Peptides for Transcytosis through the Caco-2 Cell Monolayer: Hydrophobicity and Charge Property Affect the Transport Pathway and Efficiency. Yang Yijie,Wang Bo,Li Bo Journal of agricultural and food chemistry Casein is a rich source of bioactive peptides with complete amino acid composition. In this study, the casein peptides identified in our previous study with different hydrophobicities and charge properties were employed to investigate the transport efficiency via the transcytosis pathway across Caco-2 cell monolayers. Results revealed that the apparent permeability coefficient () values of transcytosis exhibited a linear correlation with a pI of positively charged peptides during bidirectional transport. A similar law was found as for the peptides with different hydrophobicities. The transcytosis route of Pep-II to Pep-VII appears to be the clathrin- and caveolin-independent transcytosis pathway as well as caveolae-mediated transcytosis pathway, showing a linear correlation with values, respectively. Additionally, no direct correlation was shown between the hydrophobicity of peptides and clathrin-mediated transcytosis. Our results help to increase the bioaccessibility of peptide drugs across intestinal mucosa by developing strategies to alter the physicochemical properties without changing bioactivity. 10.1021/acs.jafc.9b04831
    Re-assembled casein micelles improve in vitro bioavailability of vitamin D in a Caco-2 cell model. Cohen Yifat,Levi Moran,Lesmes Uri,Margier Marielle,Reboul Emmanuelle,Livney Yoav D Food & function The pandemic of vitamin D (VD) deficiency, and the global rise in obesity stimulate a need for staple low-fat foods and beverages enriched with VD. In light of consumer demand for a clean label, the use of natural endogenous food ingredients as delivery vehicles is of great interest. To this end, re-assembled casein micelles (rCM) have been shown to help retain VD during processing and shelf life and provide high bioavailability in low-fat milk and non-fat yoghurt. This follow-up study focused on the performance of VD-loaded rCM after drying and reconstitution, considering VD retention during simulated digestion, and the subsequent in vitro bioavailability of the vitamin. rCM conferred great protection to VD during simulated digestion with a significant increase in vitamin retention for 1 h under gastric conditions. This observation is believed to arise from the vitamin-casein binding and the system's natural gelation (curd formation) near the casein isoelectric point that seclude the vitamin from environmental stressors and couple its release with digestive proteolysis of the rCM matrix. Vitamin absorption by Caco-2 cells from digested rCM was not significantly different from the absorption of the digested free VD. However, thanks to the highly protective effect of the rCM, against VD gastric degradation, the overall effect of the rCM was a 4-fold higher bioavailability, compared to the free VD. 10.1039/c7fo00323d