【作者】 孙中涛；

【导师】 杜金华；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 苹果渣与棉粕产量巨大,但目前尚未对其进行充分的综合利用,这不仅是资源的浪费,也带来了环境污染问题。另一方面发酵工业普遍采用的常规原料,如淀粉、葡萄糖、蔗糖等碳源,豆粕、麸皮、蛋白胨等氮源,近年来缺口很大,价格飞升,导致发酵产品成本上升,很多附加值较低的产品出现亏损。因此,本文拟将碳水化合物含量丰富的苹果渣与蛋白质含量丰富的棉粕用作碳源与氮源,部分取代常规发酵原料,生产NSP酶,以降低生产成本,部分解决发酵工业与人争粮、与畜争饲的问题,提升这些残渣资源的价值,同时也有利于环境保护。1、棉酚对微生物的影响及其生物脱毒以棉粕作为发酵原料,必须考虑棉酚对发酵过程的影响与产品中棉酚的残留问题。研究结果表明,棉酚对米曲霉F5、黑曲霉HG-1、黑曲霉F1、黑曲霉F2、黑曲霉F3、米曲霉F4等真菌孢子的萌发具有一定的抑制作用。在孢子萌发的初期,抑制效应比较明显,在6h时,含棉酚10μg/mL的培养基中米曲霉F5孢子的萌发率仅为对照的36.4%,但随着时间的延长,这种抑制效应不再明显,这说明棉酚只能抑制孢子的萌发,但不能杀死孢子。棉酚对真菌菌丝的生长具有一定的影响,但未达到显著水平,棉酚含量为10μg/mL的培养基中菌丝生长没有受到明显的抑制。实验所采用的微生物对棉粕脱毒都有一定的效果,但不同菌种以及同一菌种的不同菌株之间棉酚脱毒能力相差很大,其中白地霉G07的脱毒能力最强。采用Plackett–Burman实验设计与响应面实验设计对白地霉G07固态发酵棉粕脱毒的工艺进行了优化,结果标明,最佳培养基组成为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,初始含水量为62.19%（w/w）。在上述最佳培养基中,30°C培养48h,基质中自由棉酚的含量由144.0μg/g下降至30.42μg/g,这表明白地霉G07具有较高的棉酚脱毒能力。对干重减重与棉酚残留量的回归分析表明,在0～48h期间,棉酚脱毒率与菌体生长呈高度线性相关。体外消化实验表明,生物脱毒样品中的结合棉酚比FeSO₄脱毒样品中的结合棉酚在体外消化过程中更稳定,也就是生物脱毒样品比FeSO₄脱毒样品的安全性要高。生物脱毒后棉粕的营养价值得到了提高,粗蛋白与精蛋白含量提高显著,分别达12.6%与19.7%;氨基酸总量与必需氨基酸含量也大幅度提高,分别达17.0%与36.4%,蛋氨酸、赖氨酸与苏氨酸含量分别提高46.9%,21.0%与59.3%。2、黑曲霉HG-1固态发酵生产果胶酶苹果渣中果胶含量高,可诱导微生物分泌大量的果胶酶,是生产果胶酶的良好原料。本文选用果胶酶产量较高的黑曲霉HG-1为生产菌种,采用单因子实验和正交实验对其固态发酵工艺进行优化,结果表明,最适产酶培养基为苹果渣10g、棉粕10g、（NH₄）₂SO₄ 0.2g、K₂HPO₄ 0.06g、初始水分含量60%;最佳装料量为每瓶20g干料,30℃恒温培养48h,果胶酶酶活力可达22248 U/g。同时,对所产果胶酶的酶学性质进行了初步研究,结果表明,该酶酶促反应最适温度为45℃,最适pH为5.0;在50℃以下,pH 3.0～6.0时稳定性良好;Ca²⁺、Mg²⁺、Fe²⁺对该酶有激活作用,而Ba²⁺、Mn²⁺、Zn²⁺有抑制作用。3、黑曲霉F1与F3固态混菌发酵生产果胶酶、蛋白酶与纤维素酶与单一菌种发酵相比,混菌发酵具有更多的优势,多菌种在基质利用能力上互相补充,可以在价格低廉、营养组成复杂的基质上良好生长。本文所采用的两株黑曲霉具有不同的酶系,黑曲霉F1虽然对纤维素类物质的具有很强的分解能力,但在以棉粕为氮源的发酵体系中,相对较弱的蛋白质分解能力成为其生长的限制性因素;与此相反,黑曲霉F3虽然对蛋白质的分解能力很强,但在以苹果渣为碳源的发酵体系中,相对较弱的纤维素类物质的分解能力成为其生长的限制性因素;二者进行混菌发酵时,酶系作用互补,双方均可得到更好的生长,发酵产物的酶系组成更为合理,既有纤维素类物质的水解酶类,也含有蛋白质的水解酶类,同时,酶活水平更高。研究结果表明,混菌发酵的最佳培养基组成为苹果渣与棉粕1:1（w/w）,（NH₄）₂SO₄ 1%（w/w）,KH₂PO₄ 0.1%（w/w）,黑曲霉F1与F3最佳配比为2:1（w/w）,接种量0.4%（w/w）,30℃恒温培养48h;果胶酶、蛋白酶、和纤维素酶活力分别达到21168 U/g,3585 U/g和1208U/g。除蛋白质含量较低外,苹果渣中果胶、单宁等抗营养因子含量较高也是制约其饲用价值的限制性因素。在发酵过程中,果胶与单宁等抗营养因子的降解率分别为99.0%和66.1%,可消除其抗营养作用。另外,发酵过程中低分子糖类被利用,蛋白质与氨基酸含量显著提高,营养价值得到全面的改善。4、黑曲霉SL-05固态发酵生产木聚糖酶、纤维素酶与β-甘露聚糖酶除果胶酶、纤维素酶外,β-甘露聚糖酶与木聚糖酶等其他NSP酶也可以采用苹果渣为主要原料进行生产。本文以苹果渣为碳源,通过单因素实验和统计学实验设计,采用回归分析研究了黑曲霉SL-05固态发酵产β-甘露聚糖酶、木聚糖酶和纤维素酶的工艺条件,确定了菌株SL-05发酵产酶的最佳培养基:棉粕和苹果渣比例1:1（w/w）、尿素2%（w/w）、葡萄糖2%（w/w）、KH₂PO₄ 0.12%（w/w,β-甘露聚糖酶）、0.06%（w/w,木聚糖酶）、0.09%（w/w,纤维素酶）,含水率60%（w/w,β-甘露聚糖酶）、65%（w/w,木聚糖酶）、62%（w/w,纤维素酶）。最佳的培养时间为48 h,此时未见孢子生成,酶活值已经达到了较高水平。在最佳条件下,β-甘露聚糖酶、木聚糖酶与纤维素酶的酶活力分别为296 U/g（干曲）、6347 U/g（干曲）与66032 U/g（干曲）,比优化前分别提高了61%、49%、53%。发酵后大部分氨基酸含量增加,特别是几种常见限制性氨基酸,赖氨酸、蛋氨酸与组氨酸分别提高了38%、85%、69%。对β-甘露聚糖酶、木聚糖酶和纤维素酶酶学性质的初步研究表明,三种酶均为酸性酶,最佳反应pH分别为5.0、5.0、4.5,在pH 3.5～6.0范围内处理6 h和1 h残余酶活均保持在85%以上;三个酶的最适反应温度分别是80℃、55℃、75℃,β-甘露聚糖酶和纤维素酶热稳定性较好,β-甘露聚糖酶50℃处理6 h、纤维素酶60℃处理60 min后剩余酶活都保持在80%以上,而木聚糖酶60℃处理60 min后残余酶活只剩下17.35%。β-甘露聚糖酶酶促反应的Km和Vmax分别是0.83 g/L、166.67μmol/min。实验还研究了金属离子对β-甘露聚糖酶的影响,结果表明,Fe²⁺与Mg²⁺对酶活有激活作用,其中Fe²⁺的激活作用最为显著,可达127%;Cu²⁺对β-甘露聚糖酶的酶活有明显的抑制作用（91%）;Ca²⁺在浓度为0.5 mmol/L时对甘露糖酶具有激活作用,但在浓度为1.0 mmol/L时表现为抑制作用。以上研究表明:以苹果渣与棉粕代替麸皮、豆粕、淀粉等常规原料作为果胶酶、纤维素酶、木聚糖酶、β-甘露聚糖酶等NSP酶的生产原料在技术上具有可行性,在产生多种水解酶类、提高蛋白质含量的同时,还可降解果胶、单宁等抗营养因子,改善饲料的饲用价值。更多还原

【Abstract】 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.更多还原