Detection Of Gastrointestinal Hydrolysis From Milk Casein And Its Absorption Through CACO-2 Monolayer Cell Model

Casein accounts for about 80% of total milk protein. It has not only the important nutrition value, but also some physiological functions. In vitro, a series of physiological active peptides were found in the hydrolysis of casein, such as the antioxidativepeptide, ACE inhibitory peptides, opioid peptides, etc. But whether the casein digested within the physiological environment can produce similar physiological active peptides, and whether these peptides can be intactly absorbed in the body and play the corresponding physiological role remains to be proven.The in vitro and in vivo gastrointestinal hydrolysis of bovine casein was studied. We analyzed the degree of hydrolysis and some physiological functions (such as antioxidant and antibacterial activity). The peptides distribution in the hydrolysate was obtained by SDS-PAGE, Tricine-SDS-PAGE, CE, RP-HPLC, at the same time evidenced by mouse gastrointestinal digestion. Caco-2 cell model was established to evaluate its effects on intestinal cells metabolism and monolayer cell absorption of casein hydrolysate. The main results are as follows.(1) The results of in vitro gastrointestinal hydrolysis are as follow. The free amino acid content and the degree of hydrolysis were continuously increasing along with the casein hydrolysis, but the rate of increase was falling. The change of free amino content showed that hydrolysis degree of the gastrointestinal was greater than the intestine hydrolysis and gastric hydrolysis. The degree of gastric hydrolysis could reach 12.9% for 5h. The degree of gastrointestinal hydrolysis could reach 72%. The DPPH free radical clearance rate of casein hydrolysate was 29.2% at 1h-degestion. The degree of hydrolysis was continuously increasing, to achieve the maximum of 46.4% at 2h, gradually reduced to 34.4% at 5h. Casein hydrolysate had inhibitory effects on both Taphylococcus aureus and E.coli. The inhibition effect on Taphylococcus aureus was greater than E.coli and reached highest with 2h digestion hydrolysate. The bacteriostatic circle diameter was 36.72mm and 19.50mm for Taphvlococcus aureus and E.coli, respectively.(2) Through SDS-PAGE analysis, it was found that the main molecular weight distribution in the solution decreased from 24-26 kDa to below 17 kDa after 5h gastric hydrolysis. Furtherly, Tricine-SDS-PAGE analysis found that the molecular weight was mainly distributed at about 7 kDa and 3.5 kDa stripe after 5h intestinal hydrolysis. The results of the CE and RP-HPLC analysis showed in single peak, good peak shape and no trailing phenomenon. CE image illustrated the whole hydrolysis process including the casein decomposing to the small molecular peptides and free amino. HPLC could clarify the scope of the hydrophilic-hydrophobic distribution, which could prove that the polypeptide of the poles could be priority digestive hydrolysis, and could produce many hydrophilic peptides. The hydrophilic peptide could keep more polypeptide chain is not affected by the destruction of the intestinal digestion. Compare in vivo and in vitro hydrolysate by CE and HPLC, it proved that the simulation in vitro gastrointestinal hydrolysis method had a certain similarity in retention time, molecular size or hydrophilic-hydrophobic distribution, which was analyzed by computer aided similarity evaluation system and showed the similarity of more than 80%.(3) Cell culture conditions were optimized and monolayer Caco-2 cell model was established. When the cell density of 1 X 105/mL cultured in 25cm2 cell culture bottle, the passage time was 4 to 5days, and the passage ratio 1:3, and the digestion time of Caco-2 cells was 4min. Cultivation for 21d in Transwell plate, the Caco-2 cell monolayer film can be observed. Compared with blank group, the cell membrane was very tight and no apparent fracture. Through the determination results of TEER, where in the 21 d TEER value was 1579Ω·cm2, which was greater than the minimum of 500oΩ·cm2. Determined by fluorescent yellow standard curve and Papp formula of the fluorescent yellow transport from the film, we could get the value of Papp of 0.27 X 10-6cm/s, less than 0.5 X 10"6cm/s, and the average transmittance of 0.37%. Three indexes of membrane permeability reached the requirements. The established monolayer film was very tight and complete.(4) The result of the hydrolysate absorption by the cell monolayer was carried out. The CE and RP-HPLC was used to detect apical and basolateral polypeptide solution. The result of CE showed that small molecule polypeptide less than 6 kDa, could be successfully absorbed into single layer basolateral side after14 minutes electrophoresis. The small polypeptide and amino acid were more likely to be absorbed quickly. The result of HPLC showed that the hydrophilic-hydrophobic peptide could be successfully absorbed into single layer underside. But hydrophobic peptides were easy to be degraded or blocked.This study preliminarily explored the characteristics of milk casein hydrolysis in vivo and in vitro and established four kinds of methods to detect peptides patterns. At the same time the established Caco-2 cell absorption model was used to testify the absorption mechanism of hydrolysis derived from bovine milk casein. This thesis provides a foundation for the further research of casein absorption mechanism in gastrointestinal and promotes the active polypeptide mechanism research.