Study On Effect And Mechanism Of Ultrasound Pretreatment On The Bioavailability Of Plant Protein

Plant protein plays an important role in people's daily life for its high nutritional value,wide source and lower price.However,some protein resources had little bioavailability and wasted because of its special complex structure.Lots of studies found that ultrasound pretreatment could improve the characters and function of the plant proteins greatly,but little is known on the changes of the bioavailability after ultrasound pretreatment.Furthermore,the protein digestion is complex after entering the digestive tract.The whole process in the digestive tract of protein should be explored clearly for designing more and more nutritional protein products.In the present study,we explored the effects of ultrasound pretreatment on the bioavailability of plant protein(rice,oat,corn and soy)through protein structure,molecular weight distribution and gene expression by simulated in vitro and Caco-2 cell model methods.The mainly results were followed as:(1)The in vitro simulated digestion and Caco-2 cell absorption model were established to study the bioavailability of four proteins.The in vitro simulated digestibility of the four proteins(rice,oat,corn and soy)was 88.69%,86.58%,84.63% and 87.67%,respectively.The absorption rate from AP side to BL side was 8.22%,8.73%,10.10% and 5.96%,respectively,and the efflux rate from the BL side to the AP side was 7.91%,7.51%,4.98% and 4.85%,respectively.(2)The optimal ultrasonic conditions for the four proteins were determined by stepwise optimization of single factor experiments.The optimized conditions of rice protein were followed as: ultrasound power density was 500 W/L,ultrasound time was 5 min and intermittent ratio was 1 s/2 s.The optimized conditions of oat protein were followed as: ultrasound power density was 500 W/L,ultrasound time was 10 min and intermittent ratio was 1 s/2 s.The optimized conditions of corn protein were followed as: ultrasound power density was 800 W/L,ultrasound time was 10 min and intermittent ratio was 1 s/1 s.The optimized conditions of soy protein were followed as: ultrasound power density was 500 W/L,ultrasound time was 20 min and intermittent ratio was 1s/1s.After ultrasonic pretreatment,the digestibility of rice,oat,corn and soy protein increased by 9.49%,9.97%,8.19% and 9.84%(P<0.05),respectively,and the absorption rate increased by 5.59%,4.16%,8.28% and 18.69%(P<0.01).(3)The molecular structure,micromorphology and molecular weight distribution of the four proteins before and after ultrasound pretreatment were investigated.The results of CD and fluorescence spectroscopy indicated that the ultrasonic pretreatment changed the content of four secondary structures(?-helix,?-sheet,?-turn and random coil),stretched the tertiary structure,and more hydrophobic residues were exposed.Particle size,AFM and SEM showed that ultrasound reduced the particle size and loosened the structure,which were beneficial for the enzymatic reaction.The results of molecular weight distribution indicated that ultrasound decreased the content of more than 2000 Da peptides and increased the content of the molecular weight distribution at the range of 200~1000 Da peptides.These results indicated that ultrasonic pretreatment can effectively improve the bioavailability of the four proteins.(4)A preliminary research was conducted to study the absorption pathway and transport regulation mechanism of four protein gastrointestinal digests.The results showed that the four protein gastrointestinal digests were all absorbed by paracellular pathway under Caco-2 cell model.QPCR results showed that the expression of Occludin was decreased during protein transport process(P<0.05),while the expression of ZO-1 and Claudin were not significantly changed(P>0.05).Furthermore,after moving off the protein digests and incubating cells with medium for 12 h,the Occludin expression returned to normal,and the ZO-1 and Claudin expression increased significantly(P<0.01).These results indicated that Occludin was involved in the regulation of paracellular transport on Caco-2 cell model,while the normal maintenance of cell paracellular transport required the combined of Occludin,ZO-1 and Claudin.