| Buckwheat is a kind of grain with medicinal use,which is rich in many kinds of nutrients,including protein,dietary fiber,flavonoids and mineral elements.The unique amino acid composition of tartary buckwheat protein determined the higher nutritional value,compared with that of soybean,wheat,corn and other crops.Previous studies had already proved buckwheat proteins and peptides with antimicrobial,antitumor,antioxidant,lowering cholesterol and other physiological activity,therefore buckwheat proteins had prospective application as feed additives.In this study,buckwheat protein was extracted and its in vivo and in vitro biological activities and function were investigated.The study have four parts:1 Extraction,isolation and purification of tartary buckwheat proteinTartary buckwheat proteins were extracted by ammonium sulfate salting out method,thenwere further purified on DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration.The molecular weight of tartary buckwheat protein extracts and separated peaks were determined by SDS polyacrylamide gel electrophoresis,and result demonastrated that the molecular weight of tartary buckwheat protein extracts ranged from 11 KDa to 60 KDa,while the molecular weight of WSBWP was 38 KDa.2 Structural characteristics of tartary buckwheat seed globulinThe primary structure of tartary buckwheat salt soluble protein(WSBWP)was analyzed by Edman sequencing and MALDI-TOF mass spectrometry,according to the sequenced results and the Mascot protein database searched results,the protein precursor consisted of 393 amino acids,including a 22-amino signal peptide and 373-amino mature peptide.The sequence of WSBWP was identical to the N-terminal sequence of the a-subunit of buckwheat 13S globulin,indicated that WSBWP might probably identified as the acidic subunit of 13S globulin of buckwheat storage seed proteins.The three dimensional structure of WSBWP was homologously modeled on SWISS-MODEL workspace,using amaranth seed 11S globulin as template with 43.65%sequence similarity,and the 3Dstructure of WSBWP was very similar to the structure soybean 11Sglobulin,therefore might be a member of soybean 11S globulin superfamily.3 In vitro activity study of tartary buckwheat globulin and peptidesThe in vitro antibacterial activity,bile salts binding activity,free radical scavenging activity and tumor cell proliferation inhibiting activity of tartary buckwheat globulin and its hydrolyed peptides(Pep-1-4)were detected.Antibacterial activity study indicated that the growth of Escherichia coli and Staphylococcus aureus were inhibited by tested samples.WSBWP,Pep-1 and Pep-3 were proved to be able to bind the sodium cholate, sodium taurocholate and sodium deoxycholate bile salts.The antioxidant activity was applied using OHˇfree radical scavenging test,and WSBWP,Pep-1 and Pep-3 was proved to have OHˇscavenging activity.The cell proliferation of human breast cancer cell line MCF-7,human gastric carcinoma cell line MGC80-3 and human colon cancer Caco-2 were examined by MTT method,results showed that the cell proliferation inhibitory effect on MCF-7 cell inhibitory effect was much more obvious than those on MGC80-3 cells and Caco-2 cells.4 Preliminary study of the tartary buckwheat protein application in pig husbandary1)Preliminary study of buckwheat protein application on weaned piglet productionThe average daily weight gain of weaned piglets with buckwheat protein added in basal diets increased 36.84%compared with control group,while the feed to gain ratio decreased 33.33%and the diarrhea rate decreased 41.6%.These results showed that consumption of tartary buckwheat protein improved the growth performance and disease resistance of weaned piglets.Serum biochemical analysis showed that the level of total cholesterol(TC)in group piglets reduced 16.75%and level of triglyceride(TG)reduced 39.73%,however the level of the high density lipoprotein cholesterol(HDL-C)increased and the ratio of HDL-C to LDL-C increased by 32.94%,indicated that the blood of lipid metabolism of weaned piglets was significantly improved by buckwheat protein diet.In addition,the piglets serum alanine aminotransferase(GOT)level of tartary buckwheat protein group was 32.64%higher than the control group,and the level of aspartate aminotransferase(GPT)was 28.07%higher than the control group,while the level of alkaline phosphatase(AKP)was also higher than the control group.In all,the blood metabolism of buckwheat protein group was much more vigorous,therefore the piglets grew much more rapidly.The average pH of gastric juice of buckwheat protein group(3.69)was found to be lower than the control group(4.28),indicated that tartary buckwheat protein could increase the secretion of gastric acid and improved the food digesting ability of piglets.At the same time,the relative stomach weight of tartary buckwheat protein group increased 20.44%compared with the control group,the liver relative weight than the control.Compared with the control group,the stomach relative weight of tartary buckwheat protein group increased 20.44%,while the liver relative weight decreased 19.31%,indicated that buckwheat protein could increase the feed intake of weaned piglets without increasing the liver metabolism burden.The micoorgnism diversity of fecal samples from weaned piglets was analysed by Miseq high-throughput sequencing,Firmicutes,Bacteroidetes and Proteobacteria were certified to be the dominant bacteria at the phylum level analysis.According to the analysis on genus level,the content of probiotics in fecal samples of buckwheat protein group increased obviously,such as Lactobacillus,Ruminococcus and Blautia,however,the content of some pathogenic bacteria such as Prevotella and Streptococcus decreased significantly.Results showed that the average daily weight gain of weaned piglets with buckwheat protein added in basal diets increased,while the feed to gain ratio and the diarrhea rate decreased.In addition,the blood biochemical indexes such as cholesterol,triglycerides of experimental group piglets was proved to be superior to those of control group,and the intestinal flora composition have been improved.2)The amplified application of buckwheat protein on finishing swine productionAfter the propective results of tartary buckwheat protein achieved on weaned piglets,80 fattening pigs with body weight of 80-90 kg were tested for amplified application.The tested finishing pigs were randomly divided into control group and experimental group with 40 pigs in each group,and the diet of the experimental group add 5 percent buckwheat protein while the diet of the control group was added equal amount of soybean protein.The feeding time lasted one month and the pig growth were observed and recorded every day.At the beginning and the end of the test,the fattening pigs were fasted 12 hours before weighing.The feed consumption was recorded to calculate average daily gain(ADG),feed conversion ratio(F/G)and slaughter rate.The amplified application of buckwheat protein on finishing swine production showed that he average daily weight gain of experimental group pigs increased by 22.22%than the control group,while the feed to gain ratio decreased by 14.29%than the control group(p<0.05).Therefore the datas revealed that buckwheat protein additives could obviously improve the growth performance of finishing swine and could increase the feed conversion rate. |
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