Production Of NSP Enzymes With Apple Pomace And Cottonseed Powder In Solid State Fermentation

Apple pomace and cottonseed powder are produced in huge quantity every year in China, but they are still not put into full use now, which not only is an economic waste of resources, but also causes the pollution of environment when they are deposited randomly and rotted naturally. On the other hand, the price of conventional materials in fermentation industry, such as carbon source including starch, glucose and sucrose, nitrogen source including wheat brean, soybean meal and peptone, increased markedly in recent years. As a result, the production cost increased in fermentation industry and many enterprises were running in the red. In this paper, apple pomace and cottonseed powder are adopted as raw material to produce NSP enzymes instead of conventional materials, which can reduce the production cost as well as benefit the full use of these solid residues.1,Effect of gossypol on the growth of microorganisms and the bio-detoxification of free gossypolThe effect of gossypol on the growth of microorganism and the residual level of free gossypol in the production should be taken into account when cottonseed powder was used as raw material in fermentation industry. The germination of spores of fungi such as Aspergillus niger and Aspergillus oryzae can be restrained by gossypol at the initial stage. When the free gossypol level was 10μg/mL in the substrate, the germination efficiency of spores of Aspergillus oryzae F5 was only 36.4% of that of the control. However, the restraining effect was no longer significant at a later stage. This indicated that gossypol can only restrain the germination of spores, but can not kill them. The growth of the mycelium can be restrained slightly when the fungi were cultured in the medium containing 10μg/mL gossypol, but the restraining effect was not significant.All the microorganisms adopted in this study can detoxify free gossypol more or less. But the detoxification efficiency varied observably between different strains, and Geotrichum candidum G07 had the highest detoxification efficiency. Plackett–Burman design and response surface methodology were adopted to optimize the fermentation medium for Geotrichum candidum G07 to detoxify free gossypol in cottonseed powder by the solid-state fermentation. Results showed that the optimum medium was composed of 1.63% (w/w) (NH₄)₂SO₄, 0.10% (w/w) KH₂PO₄, 0.05% (w/w) MnSO₄, 0.1% (w/w) MgSO₄, 0.2% (w/w) CaCl₂ and 62.19% (w/w) initial moisture content. The residual free gossypol level decreased from 144.0μg/g to 30.42μg/g after incubating at 30°C for 48h in the optimized conditions and the detoxification efficiency reached 78.9%, which indicated that G. candidum G07 was a useful strain to detoxify free gossypol in cottonseed powder and the statistical methods adopted in this paper were effective and powerful. This study can contribute towards decreasing the residual level of free gossypol in cottonseed powder, and can benefit the full use the agricultural residue.2,Production of pectinase from apple pomace and cottonseed powder by Aspergillus niger HG-1 in solid state fermentation.Monofactorial and orthogonal experiments were adopted to optimize the culture medium and fermentation conditions of Aspergillus niger HG-1 to produce pectinase from apple pomace in solid state fermentation. The activity of pectinase reached 22248 U/g when Aspergillus niger HG-1 was cultured at 30℃for 48h in the optimized medium containing 10g apple pomace, 10g cottonseed powder, 0.2g (NH₄)₂SO₄ and 0.06g KH₂PO₄ at the optimized conditions including moisture content of solid substrate 60% (w/w) and 20g dry substrate /250mL flask. The optimal temperature and pH of pectinase reaction were at 45℃and pH 5.0. The prime characteristic of the pectinase was also studied. Pectinase was stable below 50℃and between pH 3.0 and pH 6.0. Ca²⁺, Mg²⁺ and Fe²⁺ increased pectinase activity whereas Ba²⁺, Mn²⁺, Zn²⁺ obviously inhibited it.3,Production of pectinase, cellulase and proteinase from apple pomace and cottonseed powder by Aspergillus niger F1 and F3 in solid state fermentation.The objectives of this work were to produce multienzyme bio-feed, biodegrade the anti-nutritional factors such as pectin and tannins in apple pomace, and obtain the nutritional enrichment of the fermented substrate. The mixture of apple pomace and cottonseed powder (1:1, w/w), supplemented with 1% (w/w) (NH₄)₂SO₄ and 0.1% (w/w) KH₂PO₄, was proved to be the optimum medium for the mixed strains of Aspergillus niger F1 and F3 (2:1, w/w). The activities of pectinase, proteinase and cellulase achieved 21168 U/g, 3585 U/g and 1208U/g, and the biodegradation efficiency of pectin and tannins reached 99.0% and 66.1%, respectively, when 0.4% (w/w) of the test fungi were inoculated and incubated at 30℃for 48h in solid state fermentation. The utilization of apple pomace in the paper can be served as a model for the similar waste recycling.4,Production ofβ-mannanase, xylanase and cellulase from apple pomace and cottonseed powder by Aspergillus niger SL-05 in solid state fermentation.Plackett–Burman design and response surface methodology were adopted to optimize the fermentation medium for Aspergillus niger SL-05 to produce extra-cellularβ-mannanase, xylanase and cellulase in solid state fermentation with apple pomace and cottonseed powder as the main raw material in the medium. The optimal medium forβ-mannanase production contained apple pomace and cottonseed powder (1:1, w/w) as carbon and nitrogen sources, 2% (w/w) urea, 2% (w/w) glucose, 0.12% (w/w) KH₂PO₄ and 60% (w/w) initial moisture content. The optimal medium for xylanase production contained apple pomace and cottonseed powder (1:1, w/w) as carbon and nitrogen sources, 2% (w/w) urea, 2% (w/w) glucose, 0.06% (w/w) KH₂PO₄ and 65% (w/w) initial moisture content. The optimal medium for cellulase production contained apple pomace and cottonseed powder (1:1, w/w) as carbon and nitrogen sources, 2% (w/w) urea, 2% (w/w) glucose, 0.09% (w/w) KH₂PO₄ and 62% (w/w) initial moisture content. Under optimized conditions,β-mannanase production of 296 Units/g (U/g) dw, xylanase production of 6347 U/g dw and cellulase production of 66032 U/g dw can be achieved, which were improved 61%, 49% and 53% compared with that of the initial medium, respectively.The growth kinetics of Aspergillus niger SL-05 was investigated. The results showed that the optimal fermentation time for the tree enzymes production under the optimized conditions would be 48 h. As spores were produced after 48 h, the enzymes production tended to be slower. The major enzymes secretion was observed during 24 48 h of inoculation with high cellular metabolism activities. In this period, the amount of total sugar and reducing sugar decreased dramatically, the pure protein increased rapidly, the pH value of the medium decreased, and the dry weight loss rate increased distinctly. Then, small increase in enzyme secretion was found. Compared with the amino acid content before fermentation, the content of most amino acid increased after fermentation, especially limiting amino acids: Lys, Met and His, which were improved 38%, 85%, 69% compared with that of the initial medium, respectively.The characteristics of the three enzymes were also extensively studied.β-mannanase, xylanase and cellulase were acidic enzymes and the optimal pH was 5.0, 5.0, and 4.5, respectively. Three enzymes all remained above 85% of the initial activity after incubated at pH 3.5⁶.0. The optimal reaction temperature was 80℃, 55℃and 75℃, respectively. Thermal stabilities ofβ-mannanase and cellulase were high. They remained above 80% of initial activity afterβ-mannanase incubated for 5 h at 50℃and cellulase incubated for 30 min at 60℃. But xylanase remained only 17.35% after incubated for 30min at 60℃. The Km and Vmax of theβ-mannanase were obtained, which were 0.83 g/l and 166.67μmol/min, respectively. The activity of the mannanase was inhibited greatly by Cu²⁺ (91%) and was activated by Fe²⁺ and Mg²⁺, especially Fe²⁺ (127%). The activity of mannanase was inhibited by 0.5 mmol/L Ca²⁺; however it was activated at 1.0 mmol/L Ca²⁺.All the studies above showed that it was technically and economically feasible to produce pectinase, cellulase,β-mannanase, xylanase and other NSP enzymes from apple pomace and cottonseed powder by Aspergillus niger or other fungi in solid state fermentation. It was also an effective way to make the best of apple pomace, an agricultural waste produced in larger quantities in China.