Production of NSP Enzymes with Apple Pomace and Cottonseed Powder in Solid State Fermentation
|School||Shandong Agricultural University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Pomace Cottonseed meal Solid-state fermentation NSP enzyme Response surface analysis|
Pomace and cottonseed production is huge, but has yet to be fully comprehensive utilization, which is not only a waste of resources, but also brought environmental pollution problems. Commonly used in the fermentation industry other conventional materials such as starch, glucose, sucrose, carbon, soybean meal, wheat bran, such as a nitrogen source peptone, in recent years a large gap soaring prices, resulting in rising costs fermented products, many added values ??than low product loss. Therefore, this paper intends to carbohydrate-rich protein-rich apple pomace and cottonseed meal as carbon and nitrogen sources, partially replace conventional fermentation raw materials, production NSP enzymes to reduce production costs, a partial solution to the fermentation industry competing with food , compete with livestock feeding problems, these residues to enhance the value of the resources, but also conducive to environmental protection. 1, gossypol on microorganisms and biological detoxification with cottonseed meal as the raw material for fermentation, the fermentation process must consider the impact of gossypol and gossypol product residues. The results show that gossypol Aspergillus oryzae F5, Aspergillus niger HG-1, Aspergillus niger F1, Aspergillus niger F2, Aspergillus niger F3, F4, etc. Aspergillus oryzae fungal spore germination has a certain extent. In the early stages of spore germination, inhibitory effect is more obvious in the 6h medium containing gossypol 10μg/mL Aspergillus oryzae F5 control spore germination rate was only 36.4%, but with time, this inhibitory effect no longer apparent, indicating that gossypol can inhibit spore germination, but does not kill spores. Gossypol on the growth of fungal hyphae have a certain impact, but did not reach a significant level, gossypol content of 10μg/mL medium without mycelial growth was significantly inhibited. The microorganisms used in the experiments have some cottonseed meal detoxification effect, but different strains as well as between different strains of the same species of gossypol detoxification capabilities vary greatly, including detoxification Geotrichum G07 strongest. Using Plackett-Burman experimental design and response surface experimental design dialogue Geotrichum G07 solid-state fermentation cottonseed detoxification process was optimized, the results indicate, the best medium consisted of 1.63% (w / w) (NH 4 < / sub>) 2 SO 4 , 0.10% (w / w) KH 2 PO 4 , 0.05% (w / w) MnSO 4 , 0.1% (w / w) MgSO 4 , 0.2% (w / w) CaCl 2 , initial a water content of 62.19% (w / w). In the best medium, 30 ° C cultured 48h, free gossypol content of the matrix by the 144.0μg / g down to 30.42μg / g, which indicates Geotrichum G07 has a high gossypol detoxification ability. On the dry weight of the weight loss and gossypol residues regression analysis showed that in 0 ~ 48h period, gossypol detoxification rate and cell growth was highly linear correlation. Vitro digestion experiments showed that the binding of biological samples gossypol detoxification than FeSO 4 virus sample with gossypol in vitro digestion process is more stable than the sample is biological detoxification FeSO 4 virus samples higher security. Biological nutritional value of cottonseed meal after detoxification has been enhanced with protamine crude protein content increased significantly, up 12.6 percent, respectively, and 19.7%; amino acids and essential amino acid content also increased significantly, by 17.0% and respectively 36.4% methionine, lysine and threonine were increased by 46.9%, 21.0% and 59.3%. 2, Aspergillus niger HG-1 solid-state fermentation production of pectinase apple pectin content is high, can induce microorganisms secrete large amounts of pectinase, is the production of pectinase good raw materials. This selection of Aspergillus niger pectinase higher yield for the production of HG-1 strains, using single factor and orthogonal experiments to optimize its solid state fermentation process, the results showed that the optimal medium for enzyme production pomace 10g, cotton Meal 10g, (NH 4 ) 2 SO 4 0.2g, K 2 HPO 4 0.06g, initial moisture content of 60%; optimum loading capacity per bottle 20g dry material, 30 ℃ incubation 48h, pectinase enzyme activity of up to 22248 U / g. Meanwhile, the enzymatic properties of pectinase produced a preliminary study, the results show that the optimum temperature for the enzyme enzymatic reaction 45 ℃, the optimum pH was 5.0; below 50 ℃, pH 3.0 ~ 6.0 when stability good; Ca 2 sup>, Mg 2 sup>, Fe 2 sup> activation of the enzyme, while Ba 2 sup>, Mn 2 sup>, Zn 2 sup> inhibition. 3, Aspergillus niger F1 and F3 solid mixed fermentation production of pectinase, protease and cellulase compared with a single strain fermentation, mixed fermentation has more advantages, multi-strain in the substrate utilization capabilities complement each other, you can at low prices, nutritional composition of complex matrices with good growth. Two strains used in this study has a different enzyme of Aspergillus niger, Aspergillus niger F1 Although cellulosic material has a strong ability to decompose with cottonseed meal as nitrogen source in a fermentation system, the relatively weak proteolytic ability to become the growth limiting factor; contrast, Aspergillus niger F3 decomposition of the protein although strong, but in pomace as a carbon source in the fermentation system, a relatively weak ability to decompose cellulose-based substance as limiting factor for its growth; both for mixed fermentation, the enzymes complementary roles, the two sides can get better growth, enzyme composition of fermentation products is more reasonable, both cellulosic substances hydrolases, hydrolases also contains proteins, while higher levels of enzyme activity. The results showed that the optimal mixed fermentation medium composed of pomace and cottonseed 1:1 (w / w), (NH 4 ) 2 SO 4 1% (w / w), KH 2 PO 4 0.1% (w / w), Aspergillus niger best ratio of F1 and F3 2:1 (w / w), inoculum 0.4% (w / w), 30 ℃ incubated for 48h; pectinase, protease, cellulase activity, respectively, and 21168 U / g, 3585 U / g, and 1208U / g. In addition to lower protein content, the apple pectin, tannin and other higher levels of anti-nutritional factors restricting its feeding value is the limiting factor. During the fermentation process, pectin and tannin degradation of anti-nutritional factors were 99.0% and 66.1%, to eliminate the anti-nutritional effect. In addition, the fermentation process is the use of low molecular weight sugars, protein and amino acid content was significantly increased to obtain an overall improvement in the nutritional value. 4, SL-05 Solid Aspergillus niger fermentation xylanase, cellulase and β-mannanase addition pectinase, cellulase outside, β-mannanase enzyme with xylanase enzymes other NSP apple pomace may also be used as the main raw materials for production. In this paper, pomace as carbon source by single factor experiments and statistical experimental design, the use of regression analysis of Aspergillus niger SL-05 solid-state fermentation of β-mannanase, xylanase and cellulase process conditions, identified the strain SL-05 Fermentation best medium: cottonseed meal and pomace ratio 1:1 (w / w), urea, 2% (w / w), glucose 2% (w / w), KH < sub> 2 PO 4 0.12% (w / w, β-mannanase), 0.06% (w / w, xylanase), 0.09% (w / w , cellulose), moisture content 60% (w / w, β-mannanase), 65% (w / w, xylanase), 62% (w / w, cellulase). Optimum cultivation time 48 h, no sporulation time, activity values ??have reached a high level. Under optimal conditions, β-mannanase, xylanase and cellulase enzyme activities were 296 U / g (dry yeast), 6347 U / g (dry yeast) and 66032 U / g (dry song), were increased than before by 61%, 49%, 53%. After fermentation, most of the amino acid content increased, especially several common limiting amino acid, lysine, methionine and histidine were increased by 38%, 85%, 69%. For β-mannanase, xylanase and cellulase enzymatic properties Preliminary studies indicate that three enzymes are acidic enzymes, respectively 5.0,5.0,4.5 optimal reaction pH in the range pH 3.5 ~ 6.0 processed within 6 h and 1 h of residual enzyme activity were maintained at above 85%; three optimum temperature of the enzyme were 80 ℃, 55 ℃, 75 ℃, β-mannanase and cellulase thermal stability than Good, β-mannanase 50 ℃ treatment 6 h, 60 ℃ cellulase residual activity after 60 min treatment are maintained at above 80%, while 60 ℃ xylanase treatment of residual activity after 60 min only 17.35%. β-mannanase enzyme reaction Km and Vmax were 0.83 g / L, 166.67μmol/min. Experiments also studied metal ions on β-mannanase, the results show that, Fe 2 sup> and Mg 2 sup> on the enzyme activity was activated, in which Fe 2 sup> activation of the most significant, up 127%; Cu 2 sup> for β-mannanase activity significantly inhibited (91%); Ca 2 sup> at a concentration of 0.5 mmol / L mannose enzymes when activating effect, but at a concentration of 1.0 mmol / L when the performance of inhibition. These studies show that: the pomace and cottonseed meal substitute wheat bran, soybean meal, starch and other conventional materials as pectinase, cellulase, xylanase, β-mannanase enzyme NSP enzyme technically raw materials feasibility of generating a variety of hydrolytic enzymes, increased protein content, while also degrade pectin, tannins and other anti-nutritional factors, improve feed feeding value.