| In this study,lactose-glycated caseinate was prepared via the Maillard reaction between caseinate and lactose.Both caseinate and lactose-glycated caseinate were digested with trypsin to generate respective caseinate digest and lactose-glycated caseinate digest.Meanwhile,caseinate and oligochitosan using the transglutaminase(TGase)-type glycation were used to prepare oligochitosan-glycated caseinate.Using trypsin digested caseinate and oligochitosan-glycated caseinate to obtain respective caseinate digest and oligochitosan-glycated caseinate digest.The aim of this study was to analyze precise glycation sites of this two type glycated modifications,and evaluate the effects of the two glycated caseinate digests on the potential immune activities in vitro and in vivo as well as barrier function of rat intestinal epithelial cells.It provides theoretical basis for potential risk assessment in dairy processing and potential benefits of new protein ingredients.The main results are as follows:(1)Precise glycation sites for two glycation modificationsIt was detected that the lactose-glycated caseinate digest contained lactose at 10.58 ± 0.10 g/kg protein.Compared with caseinate digest,lactose-glycated caseinate digest showed lower amount of 4 amino acids,which is Lys,His,Tyr,and Val.Based on LC/MS-MS analysis,a total of 5 peptide sequences were identified in this digest,and practical glycation sites of this digest were all provided by the lysine residues.These identified peptides were regarded to be produced by Alpha-S1-casein,Beta-casein,and Kappa-casein.Moreover,it was measured that oligochitosan-glycated caseinate digest contained glucosamine at 5.7 ± 0.1 g/kg protein.The amino acid content did not significantly different between caseinate digest and oligochitosan-glycated caseinate digest.A total of 3 peptide sequences were identified in this digest.The practical glycation sites of this digest were all provided by the glutamine residues.These identified peptides were regarded to be produced by Alpha-S1-casein and Alpha-S2-casein.(2)In vitro immune activity of two glycated caseinate digestsTwo glycated caseinate digests had in vitro immune-mediatory.Overall,compared to caseinate digest,the lactose-glycated caseinate digest reduced Con A-induced spleen lymphocyte proliferation values by 5.1%-10.8%,reduced macrophage phagocytosis values by 4.8%-6.5%,and decreased NK cell activity of 8.2%-19.6%.Modification of lactose glycation reduced the ability of caseinate digest to promote the secretion levels of splenocytes cytokine(IL-2 and IFN-?)and the secretion levels of macrophage cytokine(IL-1?,IL-6,and TNF-?).Compared with caseinate digest,the oligochitosan-glycated caseinate digest increased Con A-induced spleen lymphocyte proliferation values by 1.6%-3.9%,increased the macrophage phagocytosis values by 4.6%-7.0%,and increased the NK cell activity of 8.6%-17.3%.Modification of TGase-type glycation increased the ability of caseinate digest to promote the secretion levels of splenocytes cytokine(IL-2 and IFN-?)and the secretion levels of macrophage cytokine(IL-1?,IL-6,and TNF-?).Therefore,the oligochitosan precise glycation via the TGase-type of caseinate digest improved immune-mediatory in vitro,while the Maillard reaction of caseinate digest showed adverse effect on immune-mediatory in vitro.(3)In vivo immune activity of two glycated caseinate digestsTwo glycated caseinate digests had in vivo immune-mediatory.Compared with physiological saline-treated mice group,the results showed that the digest-treated mice group was significantly improved the immune status of the mice by increasing the values of spleen and thymus indices,regulating the hematological parameters,enhancing the secretion levels of immunoglobulin(Ig M,Ig A,and Ig G),and promoting the lymphocyte proliferation as well as NK cell activity(P<0.05)..However,caseinate digest exhibited higher activity than that of lactose-glycated caseinate digest,while oligochitosan-glycated caseinate digest showed higher activity than that of caseinate digest.In addition,compared with physiological saline-treated normal mice,the results indicated that administration of cyclophosphamide significantly decreased above evaluation indices of the suppressed mice in the immuno-suppression mice groups(P<0.05).The digests alleviated these index decreases of the mice caused by the cyclophosphamide.The caseinate digest exhibited higher immuno-enhancing efficacy than that of lactose-glycated caseinate digest,while oligochitosan-glycated caseinate digest showed higher immuno-enhancing efficacy than that of caseinate digest.(4)Effects of two glycated caseinate digests on the barrier function of normal small intestinal epithelial cellsTwo glycated caseinate digests could improve barrier function of normal small intestinal epithelial cells.Compared with the cells treated with caseinate digest,the cells treated with lactose-glycated caseinate digest decreased the trans-epithelial electrical resistance values;while the cells treated with oligochitosan-glycated caseinate digest increased the trans-epithelial electrical resistance values.After incubation for 48 h,the flux values of the cells treated with caseinate digest were decreased from 100% to 64.0%-78.2%,and the flux values of the cells treated with lactose-glycated caseinate digest were decreased to 72.1%-83.4%,while the flux values of the cells treated with oligochitosan-glycated caseinate digest were decreased to 64.1%-72.4%.The cells treated with lactose-glycated caseinate digest via the Maillard reaction had weaker effect of increasing antibacterial activity and reducing the translocation of bacterial;and the cells treated with oligochitosan-glycated caseinate digest via the TGase-type had stronger effect of increasing antibacterial activity and reducing bacterial translocation.In addition,the cells treated with lactose-glycated caseinate digest showed less increased tight junction protein(ZO-1,occludin,claudin-1)expression than caseinate digest;the cells treated with oligochitosan-glycated caseinate digest showed more increased tight junction protein(ZO-1,occludin,claudin-1)expression than caseinate digest.Overall,Maillard reaction of proteins showed an adverse effect to improve the the barrier function of intestinal epithelial cells.Precise glycation of proteins via the TGase-type has beneficial effects to strengthen the barrier function of intestinal epithelial cells.(5)Protective effects of two glycated caseinate digests on the barrier function of injured intestinal epithelial cellsAcrylamide at 2.5 mmol/L was used to injure the cells.Two glycated caseinate digests exhibited the protective effects toward injured-IEC-6 cells.Compared with caseinate digest,the lactose-glycated caseinate digest exhibited weaker effect to increase the trans-epithelial electrical resistance values,while the oligochitosan-glycated caseinate digest showed more effect to increase the trans-epithelial electrical resistance values.When the cells were pretreated with three digests for 48 h,treatement with caseinate digest,lactose-glycated caseinate digest,and oligochitosan-glycated caseinate digest of the cells could decrease the flux values to 76.6%-88.1%,79.3%-90.4%,and 73.0%-84.7%,respectively.This result indicated that oligochitosan-glycated caseinate digest exhibited more strengthened effect on decreasing the paracellular permeability than caseinate digest,while the lactose-glycated caseinate digest had weaker effect on decreasing the paracellular permeability.In addition,treatment with lactose-glycated caseinate digest of the cells showed less increased tight junction protein(ZO-1,occludin,claudin-1)expression than caseinate digest,and treatment with oligochitosan-glycated caseinate digest of the cells showed more increased tight junction protein(ZO-1,occludin,claudin-1)expression than caseinate digest.Therefore,the lactose glycation modification had adverse effect on protection of caseinate digest of the injured cell membrane integrity,while the precise glycation modification via the TGase-type had strong effect on protection of caseinate digest of the injured cell membrane integrity.In summary,precise glycation via the TGase-type had a beneficial effect of proteins on immune-mediatory and barrier function of intestine epithelial cells,while lactose glycation via the Maillard reaction had an adverse effect of proteins on immune-mediatory and barrier function of intestine epithelial cells. |
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