| Casomorphins (CM) are thought as important bioactive peptides from milk released during enzymatic hydrolysis of casein in gut-gastric tract and, as lumenal signal molecules, influence endocrine and immunity of gut-gastric tract. We ascertained the condition, under which casein were hydrolyzed by pepsin in vitro to produce CM , established the methods of HPLC to analyze the CM and determined the bioactivity of CM by using NG108-15 hybrid tumor cell. RT-PCR method were developed to study the transcription and expression of gastrin gene in G cell of 21 day's weaning piglet after ted CM and casein hydrolysates. This observation was proved by the immunohistochemical experiment. The enhancement of the cellular and humoral immunity in 21 day's weaning piglet after fed CM and casein hydrolysates were also observed in vitro. The research consisted of six sections.1 Establishment of Reversed-Phase High Performance Liquid Chromatography (HPLC) for CasomorphinsTo develop a HPLC method for assaying similar peptides, A acetonitrile:H2O (3:1) was used as mobile phase, 0.1% phosphate acid used as acidic modifier. Gradient elution was adopted with Kromasil Cg column (5 urn, 4.6 mmx 150 mm) and ultraviolet detector at 215 nm. Three casmorphins, which were B-CM-7,B-CM-5 and B-CM-3, had different chromatography profile. The retention of B-CM-7 ,B-CM-5 and B-CM-3 were 21.913min , 9.517 min and 11.038min .The sample mixed with three Casomorphins was analyzed and different chromatography profile can be obtained. And a good linear relationship exits between peak area and B-CM-7 , B-CM-5 concentrations ranged from 0.125 to 1.0 mg/mL. The RSD of purity and migration time of them were 4.83% and 5.19%. The recovery of them were 97.6% and 96.6%. respectively This provides a simple and feasible method to analyze CMs.2 Analysis on Casein Digested by Pepsin , Cell-Biological Determination of Casein Hydrolysates and Concentration assay of Casomorphin by HPLCCasein dissolved was treated with porcine pepsin(30:l w/w)in acidy water, pH1.4 for 120min at 37C. Its hydrolysate was centrifuged and then the supernatant was separated bysephadexG-15 gel filtration chromatography and four fractions were obtained.Samples together with NG108-15 hybrid tumor cell, which own a great deal of 6 opioid receptor, were incubated and the effect on adenylate cyclase activity of this hybrid tumor cell was examined in the presence and absence of naloxone by 125I labeled cAMP RIA kit. The results showed that P -CM-5, P -CM-7, casein hydrolysate, fraction 3# and fraction 4# inhibited adenylate cyclase activity of NG108-15 hybrid tumor cell and decreased the intracellular level of cAMP. Their inhibitions were reversed by naloxone. It was demonstrated that they were opioid activity materials. The opioid activity of P -CM-5 was higher than that of P -CM-7. Its inhibition ratio was 77.6%. P -CM-3, fraction 1# andfraction 2 ,casein itself were not opioid-like materials.B -CM-5 and B -CM-7 in 2hr casein hydrolysate were detected by using HPLC, the average concentration were 37.73ug/mL and 134.27ug/mL, respectively. And the P -CM-7 concentration of 3# fraction and 4# fraction ,Which derived from SephadexG-15 in column, were 44.79ug/mL and 18.90ug/mL, respectively. B -CM-5 and B-CM-7 in the 1* fraction and 2# fraction were not found. The results detected by HPLC were in accordance with that of cell biological analysis, which further proved that HPLC can used in the determination of peptides analysis.3 Detection of Casomorphins in piglets' blood, stomach and jejunum digesta by means of HPLC and determination of their opioid activity24 male piglets were divided into 3 groups randomly: control group (weaning in 28 day's old), Experimental I group (fed with B -CM-7) and Experimental II group (fed with casein hydrolysate ). Experimental groups were wean on 21 day and fed with casein hydrolysate or B-CM-7( 2 times every day, 10mL every time ). On 32 day 6 chosen piglets from each group were killed for determination of CMs in stomach , jejunum digesta and blood.T...
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