The Hydrolyzing Efficiency Of Isoflavone By β-glucosidase And Effect Of The Enzyme On The Growth Performance Of Male Broiler

In this research, high-yield P-glucosidase strain was screened, and the fermentation properties and enzymatic characteristics of Aspergillus niger was studied. The condition of soybean isoflavone hydrolyzed by P-glucosidase was optimized, and cloning and sequencing of P-glucosidase gene was performed. Avian broilers cocks were used to study the effect of P-glucosidase on its growth performance, absorption and metabolism of soybean isoflavone, meat quality, fat metabolism, anti-oxygenic capability and hormone levels to approach of the mechanism of growth promoting effects ofβ-glucosidase.1. Strain screening and identificationSix strains selected from nature were performed on their fermentation of P-glucosidase. The results indicated that the solid state fermentation and fluid state fermentation of zju2 had the highest ability to produce P-glucosidase, which demonstrated that zju2 was the most suitable strain to produceβ-glucosidase. The genome of zju2 was extracted and fungal universal primers were used to perform PCR amplification of 18SrDNA, followed by gene cloning and sequencing. Homology comparison was carried out by using BLAST procedure and GenBank data.The results indicated that the homology of this strain and Aspergillus niger (D63697) was as high as 99%, and the their distance of Phylogenetic tree was the nearest. Meanwhile, according to its colony and sporus morphology, this strain was identified as Aspergillus niger zju2.2. Fermentation properties of Aspergillus niger zju2The solid culture and fermentation conditions of Aspergillus niger zju2 to produce P-glucosidase was optimized. The results revealed that it was suitable for Aspergillus niger zju2 to produceβ-glucosidase when wheat bran was used as the major culture and the proportion of corncob powder was less than 20% or rice bran was less than 10%. In comparison with the former, wheat bran alone as the solid culture was more suitable for Aspergillus niger zju2 to grow and produceβ-glucosidase. The addition of carbon source had not significant effects onβ-glucosidase level, and addition of 2% inorganic nitrogen (ammonium sulfate, ammonium nitrate, ammonium chloride and urea) could increase the enzyme activity, while addition of more than 2％could obviously reduce the level ofβ-glucosidase, and addition of 2-6% peptone,4% yeast extract,2% soybean meal and 4% cottonseed meal respectively could obviously increase the activity ofβ-Glucosidase. The optimized conditions for Aspergillus niger zju2 to produceβ-glucosidase were 70% water content culture, inoculation amount of 107 cfu/g culture, pH6.0 culture, fermentation temperature of 28℃and culture time of 72 h. After optimization, the enzyme activity was as high as 508U/g.3. Enzymologic properties ofβ-Glucosidase from Aspergillus niger zju2β-Glucosidase was purifed by using the produce of ammonium sulfate precipitation, Sephadex G-25 desalination and Sephadex G-100. The properties ofβ-glucosidase including its molecular weight, the optimal reaction temperature, the optimal pH, thermal stability, pH stability and Michaelis constant were performed by enzymologic methods. The results indicated thatthe optimal reaction temperature was and pH was70℃and 4.5 respetively. The enzyme was stable under 40℃,50℃and 60℃,but not stability above 80℃. It was not stable in the buffer when pH were 3,7,8, 9, while stability in the buffer when pH were 4,5,6, and the best was 5. The enzyme molecular weight was 65.2kDa and its Km was 41.67mM, Vmax was 23.81U/L.4. Hydrolysis efficiency in vitro ofβ-Glucosidase from Aspergillus niger zju240% content Soybean isoflavone (the isoflavonal glycoside conjugates accounts for 86.33%) andβ-Glucosidase from Aspergillus niger zju2 was used tooptimize the hydrolysis condition including hydrolysis time, temperature and enzyme amount. Three factors and five levels quadratic general rotary unitized design was adopted in this study. The judgment criterion was enzymolysis product free aglycones. The results indicated that a mathematical model was established among free aglycones amount (y) and reaction time, reaction temperature and the enzyme amount:y= 0.876+0.029 X1-0.071 X2+0.072 X3-0.048X,X2+0.015 X1X3+0.017X2X3-0.035 X12-0.074 X22-0.091 X32, the multiple correlation coefficient of the model was 0.86, which met the extreme significant level and this model was effective. In the levels selected in this model, the factor and their effect degrees on hydrolysis rate from higher to lower were enzyme amount, temperature and time. After optimization, the optimal parameters forβ-glucosidase from Aspergillus niger zju2 to hydrolyze soybean isoflavone were 5.3h (time),55.4℃(temperature) and 68.7 U/g (enzyme level).5. Cloning and sequence analysis ofβ-Glucosidase gene from Aspergillus nigerTotal RNA was extracted by using Trizol method, and PCR amplification was performed using cDNA obtained from reverse transcription as template, then sequencing and structure prediction were performed. The results indicated that the total length of Aspergillus niger zju2β-Glucosidase gene was 2583bp, encoding 860 amino acid. The theoretical molecular weight was 93.2kDa, and the theoretical isoelectric point was 4.45. Structure predictions indicated that the signal peptide cleavage site located between the 19th and 20th amino acid residue. Theβ-Glucosidase mature peptide fragment had 841 amino acids and its theoretical molecular weight was 91.2 KDa. The mature peptide had 15 potential glycosylation sites. Secondary structure predictions indicated that this protein had 568 random coils,165α-helixes, 108 sheets, and their proportions accounted for 67.54%,19.62% and 12.84%, respectively. This enzyme had similar three-dimensional conformation to lexl in protein library, and the homology was 23.7%.6. Effect of P-Glucosidase on the growth performance of cock and its mechanismOne day old male Avine broilers were randomly allocated to four treatment groups and each with four pens of 15 chicks. All birds were fed with the same basal diet withβ-glucosidase added at the amount of 0% (as the control group),0.2%,0.4%, and 0.6%, respectively. The result indicated that:(1)β-glucosidase could improve the bird growth performance:daily weight gain of broiler chicken increased by 8.26% (p<0.05) by adding 0.2%β-glucosidaseto (0.6U/g feed). Feed-weight ratio decreased by 5.8%(p<0.01),2.68%(p<0.01) and 2.23%(p<0.05) by adding 0.2 %,0.4%,0.6% ofβ-Glucosidase, respectively. (2) The diet adding 0.2% ofβ-glucosidase could improve utilization rate of soybean isoflavone:β-Glucosidase could significantly influence absorption and metabolism of soybean isoflavone, and therefore the isoflavonal glycoside conjugates decreased by 68.02% in the serum of broiler. (3)β-Glucosidase could improve meat quality:dripping loss of broiler decreased by 22.00%(p<0.05), and the L value of breast muscle decreased by 11.17%(p<0.01), the a value of breast muscle increased by 41.81%(p<0.01). (4) P-Glucosidase could improve digestion function:pancrease weight rate decreased by 18.08%(p<0.05) by adding 0.2% ofβ-glucosidase and the liver weight rate decreased by 8.19%(p>0.05). The activity of duodenal amylase increase by 30.43 %(p<0.05) and the activity of lipase and parenzyme increase by 12.40%(p>0.05) and 57.93 (p>0.05) respectively. Digestion rates of crude protein and crude fat increased by 9.02%(p<0.05)and 7.40%(p<0.01)respectively. (5)β-Glucosidase did not significantly influence the skeletal growth and development:it did not significantly influence the growth of femur and tibia of broiler, and skeletal growth and development related serum indexes such as calcium, phosphorus, alkaline phosphatase. (6)β-Glucosidase could influence some indexes of fat metabolism:β-Glucosidase did not significantly influence abdominal fat accumulation, fat accumulation among muscles, tail fat accumulation, HSL and LPL in fat tissue, triglyceride, free fatty acids and total cholesterol levels. However high-density lipoprotein cholesterol significantly increased by 15.10%(p<0.05) and low-density lipoprotein cholesterol decreased by 36.43%(p<0.01). (7)β-Glucosidase could obviously improve the anti-oxidation capability of brioler:It significantly increase the activity of superoxide dismutase in serum of broiler by 16.45%(p<0.05) and the malondialdehyde content decreased by 18.16%(p<0.05). The activity of Glutathione Peroxidase Glutathionereductase in liver increased by 33.00%(p<0.05) and 14.88(p<0.05) respectively. (8)β-Glucosidase could increase hormone secretion: testosterone, estradiol secretion, IGF-I increased by 51.43%(p>0.05) and 14.59% (p>0.05) and 57.78%(p<0.05) respectively.This study indicated thatβ-Glucosidase could hydrolyzes the isoflavonal glycoside conjugates in digestive tract of broiler, improve utilization rate of soybean isoflavone and broiler growth performance.