| There was still a great challenge to extract the less-abundant soybean proteins (LAPs) from defatted soybean meal, because of their less content in soybean, most of them escape the traditional extraction method and the shield of abundant storage proteins (APs) (glycinin and β-conglycinin). So, we carried out the research for optimization the LAPs extraction and enrichment in defatted soybean meal, and tried to reveal the function of LAPs extracts on the weaning mice without the influence of APs after analization the compositon of the LAPs extrats.1 Extraction and composition identification of LAPsThe aim of our study was to investigate the effect of isopropanol concentration, total extraction time (ultrasonic time+bath-vibrating), ultrasonic power and ultrasonic time on the extraction of LAPs in defatted soybean meal and optimize the these factors, and determined the composition of LAPs extracts of under the optimal conditions by SDS-PAGE, MALDI-TOF-MS, LC and GC. The results showed that:(1) In single experiment,50% ispropanol was beneficial to remove the APs, and 400 W, ultrasound for 10 min, extract 1 h of total time were the optimal single factor for LAPs extraction. (2) The optimal conditions for LAPs extraction and enrichment were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W and 1 h of total extraction time. (3) In the LAPs extracts, some LAPs which couldn’t be extracted by traditional method, such as phospholipase D alpha 1-like isoform X2, β-amylase,16.5 kDa oleosin, uncharacterized protein and unknown soybean proteins were detedcted easily, and no abundant soybean protein (β-conglycinin and glycinin) and soy isoflavone, the residuals of isopropanol and acetonie were only 0.0231% and 0.0430% respectively.2 Effects of LAPs on the growth performance in mice180 wining mice were selected to divide into 5 groups (Included control and four deal groups, D0.5mg, Dlmg, D2mg and D4mg respectively, and the "mg" indicated the content of LAPs in the oral solution) and 3 repeats of each group. The effects of LAPs extracts on the growth performance in mice were studied after 1 week of feeding AIN93G ration for adaption to the environment. Results were as follows:(1) The mice weight of every experimental period and deal group were higher than their controls, the D2mg group was the highest. (2) The ADG, G/F and growth rate at every experimental period and deal group were higher than the control and group D2mg was the highest, except the 1-7 d experimental period. (3) At 7 d, the heart index of group D4mg was significant lower than the control(.P< 0.05). At 28 d, both the heart index and kiney index of the mice in every deal group were higher than the control.In conclusion, the performance of mice was inhibited at 1-7 d for oral LAPs extracts, and then appeared obvious growth promotion at other experiment period, especially the group D2mg appeared the most significant effect.3 Effects of LAPs on the immune function and antioxidant capacity in miceThe research was aimed to determine the effects of LAPs extracts on immune function and antioxidant capacity in mice. The results were:(1) The number of leukocyte, lymphocyte, monocyte and neutrophils of serum were higher than the control in group D2mg and D4mg at 7 d,14 d and 21 d, and the group E was highest. (2) For the index of thymus, there was no significant difference between groups but group D2mg was lower than the control at 7 d and 28 d. There was no significant difference between deal groups and control in the index spleen, and the group D4mg was lower than the control at 7 d,14 d and 21 d. The index of liver in all deal groups was lower than the control at 7 d,14 d and 21 d, but higher at 28 d. (3) The IL-2 content of serum in group D2mg at every experiment period was higher than the control. TNF-a, IL-10 and IgG was no significant difference compared to the control at 7 d and 28 d, group D2mg was higher than other groups. The relative transcript level of IL-2, TNF-a gene in spleen in all deal groups was higher than the control at 7 d and 28 d (except group D2mg). There was no difference between groups in the content of D-lactate and His at 28 d, but group D2mg was lower at 28 d and higher at 7 d than the control. (4) The T-AOC content in serum in all deal groups was higher than the control at 28 d, group D1mg and D2mg were higher than the control at every experiment period. There was no significant difference in the relative transcript level of SOD, CAT and GPX genes in mice liver at all groups at 7 d, group D1mg and D2mg were higher than the control at 28 d.The results indicated that:At the initial stage (7 d), the extracts result in the immune reaction occurred and the intestine harmed, but no significant difference in the antioxidant ability of mice.4 Effects of LAPs on the blood indicator and relative transcript level of genes about digestion and metabolism in miceThe research was to study the effects of LAPs on the digestion and metabolism in mice, the experimental animal and grouping were as the same as 2. The results were:(1) No significant difference between groups in the content of TC and TG of mice serum, and D2mg was the lowest group. The Apo-B content in mice serum was higher in every deal group at 7、14 d and lower (except group D0.5mg at 28 d) at 21,28 d. The relative transcript level of HMG-CoA, ACS, SREBP-1c and SREBP-2 of mice liver in group D2mg were lower than other deal group at 7 d. At 28 d, the HMG-CoA and ACS in group D2mg were the highest and the SREBP-1c and SREBP-2 were the highest in group E. (2) At all groups, there was no significant difference in the GLU content of serum between groups at 7,14 and 21d, but higher than the control at 28 d. The insulin level was higher in group D1mg and D2mg, but lower in group D4mg. The relative transcript level of PEPCK and GCK was higher in group D0.5mg and D1mg, but lower in group D2mg and D 4mg at 7 d, and the PEPCK was the lowest but GCK was the highest in group D4mg in all experimental groups at 28 d. (3) There was no significant difference in the content of ALB, GLB and A/G in serum between deal groups and control at 7 d. No significant difference in the ALB content between deal groups and the control, but the GLB content was higer than the control and the A/G was lower. The relative transcript level of ALB gene was higher than the control at 7 d, but lower in group D4mg than other experimental groups at 28 d. The content of ALT in deal groups was lower at 7 d, but higher at 21 and 28 d than the control (except for ALT of group D0.5mg).In sum, the extracts changed the digestion and metabolism of sugar, lipid and protein by affecting the serum insulin level, the relative transcript level of HMG-CoA, ALB and PEPCK genes, and especially in group D2mg.5 Effects of LAPs on the intestinal microbial flora in miceWe studied the effects of extracts on the structural diversity of bacterial flora and similarity of groups by high-throughput sequencing. The results were:(1) The proper amount extracts changed the intestinal microflora, increased the richness of microorganism and diversity of mice; (2) The Firmicutes, Cyanobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the Streptococcus, Bacillus, Lactobacillus, Lactococcus, Allobaculum, Chloroplast were the dominant bacteria at phylum level and genus level respectively; (3) The Lactobacillus acetotoleran (both Lactobacillus and Lactococcus) was highest in group D2mg, which increased by 25.11% compared to the control. Especially, the percentage of Lactobacillus was significant higher than group D0.5mg and D5mg (P< 0.05). (4) 1620 OTU was shared and 10.53% of total OUT at every experimental group, and there was difference in the microbial flora structure between groups and individuals of the same group by UniFrac analysis. |
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