【作者】 刘芸；

【导师】 仇农学；

【作者基本信息】 陕西师范大学 ， 植物学， 2011， 博士

【摘要】 随着苹果种植业和加工业的迅速发展,苹果渣的排放量也日趋增加,目前对苹果加工的废弃物——果渣还没有实现有效地综合利用,这不仅影响了苹果产业的可持续发展,也给产区环境造成了极大的污染。苹果渣富含多种营养物质,是良好的发酵原料。本论文以陕西省内苹果园和苹果加工工厂为采集地点,采集不同生长阶段的苹果成熟植株的茎、叶、花、幼果,同时采集了果园土壤、加工厂区土壤、生产线用水、堆积果渣等样本以分离环境微生物,从以上种植、加工贮藏、销售的环境中筛选出纤维素酶活性高的菌株,以及从常见食用菌中优选出能够高效利用苹果渣生长的菌种,以提高苹果渣固态发酵蛋白产量为目的,解决以往研究得到的发酵苹果渣存在的发酵不完全、蛋白含量不高、产物异味严重等问题,并且系统的研究了苹果渣单一菌种发酵和混合菌种发酵、自然发酵和人工发酵等的差异,考察了不同来源的氮源、无机盐等对发酵产物蛋白含量的影响,同时研究了原料配比、温度、接种量、接种比例等因素对产物的影响,确定了最佳苹果渣固态发酵的原料配方和工艺条件；在摇瓶发酵的基础上,研究30L固态罐的发酵条件,同时对固态发酵剂进行了研究。1.2008年2月至2008年10月期间,从陕西省关中及渭北地区3个县6个苹果种植园中采集了不同生长阶段的苹果成熟植株的茎、叶、花、幼果(品种为红富士)及根系土壤样本共计236份,采用组织分离法及土壤稀释平板分离法分离组织表面、体内及土壤中的微生物,经单孢分离及纯化培养后,共得到470个菌株,其中细菌268株,真菌202株。2.从市售苹果表皮、果汁加工厂的生产线、商品浓缩苹果汁和果渣堆积环境中采集土壤、水、果汁和果渣样本120份,采用经典的分离和纯化方法共得到207个菌株,其中细菌121株,真菌86株。3.对上述677株纯化后的菌株通过透明圈法进行初筛后,对筛选得到的76株菌株进行以纤维素酶活为指标的复筛,得到高产纤维素酶的菌株5株,经形态学初步鉴定,其分属于镰孢属Fusarium sp.,酵母属Saccharomyces sp.,木霉属Trichoderma sp.和曲霉属Aspergillus sp.。通过发酵苹果渣试验,对产品进行蛋白含量、感官指标等评价,确定菌株T23,T55,T62为优势菌株,均具有生长对数期较长,发酵周期内纤维素酶活性较高等优势。经过显微形态及ITS序列分析、同源性比较,依次鉴定为酿酒酵母Saccharomyces cerevisiae,产朊假丝酵母Candida utilis和黑曲霉Aspergillus niger,依次命名为酿酒酵母Saccharomycescerevisiae Y1N4,产朊假丝酵母Candida utilis Y2N3和黑曲霉Aspergillus nigerH1N3。4.对苹果干渣的主要组成成分进行了定量分析,其中粗纤维含量约为14.7%,粗蛋白含量约为4.2%,作为培养基原料在生产中需要添加氮源。5.通过单因素试验确定了苹果渣固态发酵的最佳摇瓶工艺：外加氮源为10%(质量含量)麸皮；灭菌采用常规方式,在实际生产中也可采用100℃,30min蒸煮灭菌；含水量为60%。本研究采用的黑曲霉、酿酒酵母、产朊假丝酵母,单菌发酵和混菌发酵均能显著提高产物蛋白含量,以混菌发酵苹果渣效果最佳,在发酵周期内蛋白含量、纤维素酶活性都有明显增长。本研究还考察了菌种的混合比例、混合种类、接种量及分段发酵对产物蛋白含量的影响,同时研究了温度和发酵时间的影响,得到以下结论：采用黑曲霉,酿酒酵母,产朊假丝酵母等量混合的方式接种,接种量为12%(培养基的质量百分比浓度),先接入黑曲霉发酵48h后接入两种酵母菌,得到的发酵苹果渣蛋白含量最高,可达34.9%。在摇瓶发酵的基础上,研究30L卧式固态发酵罐的发酵条件,当选择温度为27℃,通气速率为0.65 N.m3/h,接种量为600 mL(每5kg培养基),搅拌速度为15rpm时,产物蛋白质含量为最大值31.6%。发酵苹果渣饲料中氨基酸组分齐全,赖氨酸等必需氨基酸含量较高,同时富含酶类、核酸、维生素、无机盐和促进动物生长因子、生物学价值优于植物蛋白饲料,可以替代豆粕和鱼粉。另外,添加稻壳能够明显改善培养基的通气状况,添加纤维素酶也能明显增加原料粗纤维的降解效率,提高发酵产物的质量。6.培养四种食用菌凤尾菇Pleurotus sajor-caju、白灵菇Pleurotus nebrodensis、猴头菇Hericium erinaceus、茶树菇Agrocybe chaxinggu的试验结果表明,食用菌菌丝在苹果渣上生长旺盛,麸皮是苹果果渣发酵生产食用菌菌丝的理想氮源,优于尿素、硝酸铵和硫酸铵等合成氮源。发酵后苹果渣的蛋白含量由发酵前的4%左右提高到20%以上。以生长最为旺盛的凤尾菇为例,添加少量无机盐溶液(KH2PO4, MgSO4, ZnSO4),发酵处理能够进一步提高苹果渣的蛋白含量。MgSO4, K2HPO4和ZnSO4的含量分别为18g/kg、3g/kg和0.03g/kg时,接种凤尾菇的苹果渣发酵产物蛋白含量最高,可达26.178%。7.从六种供试物(甲基纤维素,B-环糊精,可溶性淀粉,壳聚糖,魔芋粉和麸皮)中,筛选最适合用于苹果渣固态发酵的发酵剂载体。试验结果表明,壳聚糖对酿酒酵母具有最强的吸附力,且发酵力的大小与六种载体对酿酒酵母吸附量的大小呈正相关,其次为β-环糊精,综合考虑成本及产品外观形状等,选择B-环糊精最为发酵剂的理想载体材料。考察温度、pH、时间、媒介等因素对B-环糊精与酵母吸附能力的关系,确定最佳吸附条件为：温度25℃,pH为6.5,吸附时长为12 h,溶液介质为0.01M的CaSO4时,冻干后其吸附力达到8.2×106 CFU/g,为工业化发酵生产提供理论依据。更多还原

【Abstract】 The apple is the pomaceous fruit of the apple tree, species Malus domestica in the rose family (Rosaceae) and is a perennial. It is one of the most widely cultivated tree fruits, and the most widely known of the many members of genus Malus that are utilized by humans. Apple pomace is the solid residue that remains following extraction of juice from ground apples. With the development of apple planting and processing industry, the production of apple pomace has been increased in a large scale. About 1.1 million metric tons of apple pomace is produced per year in China. With the development of the world’s apple juice concentrate industry, the proportion of China’s apple juice concentrate increased and export enlarged. But the pomace’s disposal is a serious problem being highly biodegradable. Since it is a rich source of acids, carbohydrates, fibers, minerals and vitamin C, its disposal as a waste in the environment is a huge loss of precious natural resources. Production of fermentation based products from apple pomace (AP) is one of the attractive options due to the supply of substrates almost at a throwaway price, minimum land requirements and manufacture independent of weather conditions.In this thesis, apple orchards, apple juice concentrate processing plants and a supermarket in Shaanxi province are the collection sites, for acquisition of different cultivates apple skin sample, different growth stages of apple tree stems, leaves, flowers, young fruit, and orchard soil in three county towns, as water and soil from factory environment, and fresh apple pomace. Microorganisms with high activity of cellolase have been selected from these samples; strains which can use apple pomace efficiently between same ordinary edible fungi have been selected too, for the purpose of increasing protein content in fermentation products, and solving the difficult problem encountered before, such as incomplete fermentation procedure, low content of protein and serious peculiar smell.In this thesis, fresh apple pomace is studied as a test material. Three species, which selected from plants and environmental samples, were used in the solid state fermentation (SSF) processing. Then the researches are carried out for the topics of single fungus and mixed fungi co-fermentation, single factor test and perpendicular tests, different nitrogen sources, natural fermentation and man-controlled fermentation, etc. After ascertaining conditions of flask fermentation,30L solid fermentation machine has been used for large scale fermentation. In addition, solid state fermentation agent has been studied for the first time in apple pomace fermentation.1. From February to October in the year of 2008,236 pieces of samples were collected from different growth stages of Fuji apple trees’ stems, leaves, flowers, young fruit, and orchard soil in three county towns, methods of tissue isolation and soil dilute plate have been used to isolate microorganisms from epiphytic and endophytic of the plant tissues and soil samples.570 isolates were investigated after single spore isolating and purifying, including 268 bacterial isolates and 202 fungal ones.2.120 pieces of samples were collected from different cultivated apples, apple juice concentrates production line, and apple juice concentrates which were expanding bags, both and places where apple pomace piled.207 isolates were investigated after isolating and purifying from the soil, water, apple pomace samples, including 121 bacterial isolates and 86 fungal ones.3. After 677 isolates above-mentioned selected preliminary by clear zone method (CZM),76 isolates were obtained and among them,5 strains were the most prospective in biological production of cellulase, which had high enzyme activities. Morphological characteristics show that these strains belong to four species, Fusarium sp., Saccharomyces sp., Trichoderma sp. and Aspergillus sp., respectively. Strain T23, T55, T62 were defined as dominant strains for their long Logarithmic growth phase and high cellulase activity in fermentation period by protein content and sensory index. Morphological characteristics and internal transcribed spacer (ITS) sequences show that strains were Saccharomyces cerevisiae, Candida utilis, Aspergillus niger, and were named as Saccharomyces cerevisiae Y1N4, Candida utilis Y2N3 and Aspergillus niger H1N3, respectively.4. Determine the main components of apple pomace, the results showed that the content of crude fiber was 14.7%, the content of crude protein was 4.2%. Apple pomace was not good enough as a fermentation material for its lack of nitrogen.5. The best fermentation condition in flasks was determined by single factor test. The results showed as follows:nitrogen was 10% content of bran, normal Sterilization method and the condition of 100℃,30min was good enough for kill all the microbes and spores, and content of water was 60%. Saccharomyces cerevisiae Y1N4, Candida utilis Y2N3 and Aspergillus niger H1N3 used in this study can increase protein content in fermentation medium by single fungus fermentation and mixed fungi fermentation. In fermentation period, both protein content and cellelase activity increase obviously. The study include mix ratio of fungi, species of fungi, Vaccination quantity and section fermentation, as well as the influence of temperature and fermentation time. The results showed that:Saccharomyces cerevisiae Y1N4, Candida utilis Y2N3 and Aspergillus niger H1N3 were mixed equally for inoculum, the vaccination quantity was 12%, Aspergillus niger H1N3 can be inoculum in first, Saccharomyces cerevisiae Y1N4 and Candida utilis Y2N3 were inoculum after 48h, the yield of protein reached 34.9%. On basis of fermentation by flasks,30L Horizontal automatic solid state fermenter was used to investigate the optimum conditions in large scale fermentation. The results showed that:when temperature was 27℃, aeration speed was 0.65 N-m3/h, inoculum was 600mL, and agitation speed was 15rpm, protein content reached optimum value,31.6%. Fermented apple pomace had a complete amino acids component, rich in Lys and other essential amino acids. There also were plenty of enzymes, nucleic acids, vitamins, inorganic salt and growth factors, which were superior to same plant source protein feed, and can instead of soybean meal and fish meal. In addition, rice husk can improve the aeration situation in medium, cellulase can increase degradation of fiber in material, all these would improve the quality of fermented products.6. Cultivating four kinds of edible fungi Pleurotus sajor-caju, Pleurotus nebrodensis, Hericium erinaceus and Agrocybe chaxinggu in apple pomace medium, the results showed that all these fungi grew well, and nitrogen bran (nearly 10% content) was better than urea, ammonium nitrogen, and ammonium sulfate. Protein content in apple pomace increased from 4% (before fermentation) to 20% (after fermentation). Chose Pleurotus sajor-caju as material to evaluate the influence of protein content with inorganic salts (KH2PO4, MgSO4 and ZnSO4), the result showed that inorganic salts can improve the content of protein in fermentation products. The optimal yield of protein reached26.178% when KH2PO4, MgSO4 and ZnSO4 were 18g/kg,3g/kg and 0.03g/kg, respectively.7. Six tested materials including methyl cellulose,β-circle dextrin (CD), soluble starch, chitosan, konjac flour and bran, were investigated for screening the best property solid state fermentation vector used in apple pomace fermentation. Previous studies on SSF vectors have not yet been reported. The results showed that the optimum vector of the highest adsorption ability was chitosan. Fermentation ability (FA) was positive relation with the adsorption power for yeast cells, andβ-CD was the second optimum vector.β-CD was defined as the most ideal vector in SSF for the reason of costs and appearance of productions. The physical properties of the adsorbent were evaluated to better understand the adsorption process. The factors temperature, pH, time and medium were evaluated and the optimal conditions for Saccharomyces cerevisiae Y1N4 adsorption with vectors contained temperature of 25℃, pH of 6.5, adsorption time of 12h and medium of 0.01M CaSO4,β-CD adsorption ability of 8.2 X 106 CFU/g can be achieved.更多还原