油茶粕中茶皂素的降解及其利用研究
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摘要
油茶(Camellia Oleifera)盛产于我国南方丘陵地带及东南亚部分地区,在林业及农副产品中具有很高的综合利用价值。油茶籽用于茶油的提炼,经榨油提取后形成大量油茶饼粕。油茶饼粕(Camellia Meal)中含有多种有机化合物及营养物质,因此在化工、轻工、食品及饲料工业中具有很高的利用价值。茶皂素(Tea saponin)是五萜三甙类糖甙化合物,具有许多物理化学性质及生物生理活性,对化工业、农业、牧渔业等的发展均有促进作用。在饲料业应用中,大量的茶皂素刺激鼻粘膜,具有溶血及鱼毒作用,口服少量茶皂素具有抗渗消炎、健脾降脂、抗菌抗过敏、保护肠胃、提高动物生长性能的作用。
益生菌饲料添加剂的生产是对发酵饲料技术的完善。作为功能性饲料,益生菌饲料调节动物肠道菌落平衡,不产生抗药性,适口性好,动物消化率高且功能稳定。枯草芽孢杆菌(Bacillus subtilis)具有典型芽孢杆菌特征,是经美国食品药物管理局(FAD)和美国饲料公定协会(AAFCO)公布的40余种益生菌之一
因此,研究油茶粕中的茶皂素的实用降解技术及利用降解油茶粕发酵生产枯草芽孢杆菌,可为油茶粕在饲料中的应用提供有力的实验依据。为饼粕作为饲料原料在饲料添加剂中的应用,扩大了饲料来源,为饲料业的发展提供的新方向及新思路对突破农副业发展瓶颈具有积极的现实意义。
本文以油茶粕作为研究对象,对其进行营养分析；结合微生物生长特性,研究微生物降解茶皂素的发酵工艺,利用降解油茶粕发酵生产枯草芽孢杆菌,主要结果如下：
利用各种测定方法,确定油茶粕的营养成分含量,结合枯草芽孢杆菌、白腐菌及黑曲霉对油茶粕的利用特性,筛选确定可降解茶皂素的微生物为黑曲霉。
对黑曲霉固态发酵降解油茶粕中的茶皂素进行了研究,运用单因素实验及Box-Behnken实验设计,结合高效液相色谱法(HPLC)检测技术,优化其固态发酵工艺,确定其最佳降解条件为：利用8层纱布控制传质传氧,初始含水量70%,pH4.35,接种2.18×107个单孢子/mL的悬液5mL,发酵温度34.5℃,发酵时间84h,发酵结束油茶粕中茶皂素降为2.04%,降解率达到89.99%。
利用降解油茶粕为原料,以活菌数为指标研究液态发酵生产饲料添加剂枯草芽孢杆菌益生菌。运用单因素实验、Plackett-Burman实验、最陡爬坡实验筛选液态发酵培养基因素,利用正交优化实验确定培养基最佳配方为：降解油茶粕13g,可溶性淀粉1g,尿素1.5g,磷酸氢二钠0.05g,七水硫酸镁0.015g,牛肉膏0.2g,七水硫酸亚铁0.03g,酵母膏0.5g,蒸馏水100mL,自然pH,121℃灭菌30min后接种5mL菌悬液,37℃,150r/min摇床培养24h,得到含108个/mL的枯草芽孢杆菌液态发酵液。
运用单因素实验及Box-Behnken实验设计,以活菌数为指标研究液态发酵生产枯草芽孢杆菌益生菌的工艺条件,确定最佳发酵条件为：500mL摇瓶中装液120mL培养基,调节pH6.8,灭菌30min后,接种9mL菌悬液摇匀后,在转速179r/min、35.2℃摇床中培养36.8h后计数,得到活菌数为2.04×1010个/mL的发酵液,与培养基优化时相比,活菌倍增提高了近5倍。
Camellia Oleifera is rich in the south foothills of our country and in parts of Southeast Asia, it has high comprehensive utilization value in the forestry and subsidiary agricultural products. A large number of Oil-tea-cake is formed as tea oil refining from oleifera seed, it contains many types of organic compounds and nutrients, and has high value in chemical industry, light industry, food and feed industry. Tea saponin as three glucoside terpenoid, has much of physical, chemical and biological physiological activity, and always stimulates the development of the chemical industry, agriculture, animal husbandry and fishery. In the application of feedstuff, a large amount of tea saponin has hemolysis and toxicity of fish, stimulates the nasal mucosa. Few of peroral tea saponin has lots of functions like impervious, antiphlogosis, spleen-tonifying, liquid-lowering, antibiosis, antianaphylaxis, intestines and stomach-protection, and improvement animal growth performance.
The production of probiotics feed additive completes the fermentation feed technology. As functional feed, probiotics feed can adjust the bacterial colony balance in animal gut flora balance, so never produce resistance to drugs. It also has great palatability, high animal digestion rate and stable performance. Bacillus subtilis has typical Bacillus characteristics, as one of40kinds of probiotics approved by the U.S. food and drug administration (FAD) and the United States official feed association (AAFCO) published.
Therefore, the study on the technique in degrading tea saponin and the bacillus subtilis production using oil-tea-cake application in feed additives as feed raw materials expands the forage sources and breaks through the bottleneck of agricultural development bottleneck. There is a lot of positive significance.
This paper was study on camellia meal, analysed the nutritional compositions. Combined with microbial growth characteristics, finded out the microbial used of degrading tea saponin, and used degradation camellia meal producted bacillus subtilis. The main results are as follows:
The content of nutrient in camellia meal was measured with various methods. Combined with bacillus subtilis, white-rot fungus and aspergillus, aspergillus was screened to degrade tea saponin.
Combined with high performance liquid chromatography(HPLC),single factor experiment and Box-Behnken experimental design, the solid fermentation process conditions of degradeting tea saponin was eight layer of gauze for regulating the mass and oxygen transfer, initial moisture content70%, pH4.35, inoculated5mL liquid of2.18×107single spore per milliliter, fermentation temperature34.5℃, fermentation time84h. Tea saponin content after fermentation is2.04%and the degradation rate reached89.99%.
For studying product bacillus subtilis probiotics by liquid fermentation, I used degradation camellia meal as raw material, the living bacterium number as index, and then via single factor experiment, Plackett-Burman experiment and the steepest climbing experiment to select factors, used the orthogonal experiment to determine the best formula for medium was degradation camellia meal13g, soluble starch1g, urea1.5g, disodium phosphate0.05g, seven water magnesium sulfate0.015g, beef cream0.2g, seven water ferrous sulfate0.03g, yeast anointed0.5g, distilled water100mL, natural pH,121℃sterilization30min,5mL bacteria liquid,37℃,150r/min table concentrator,24h. I got fermented liquid with108per mL of bacillus subtilis.
And then, I researched the fermentation condation of producing bacillus subtilis by single factor experiment and Box-Behnken experimental design. The optimal fermentation conditions was500mL culture bottle containing120mLculture medium, pH6.8, sterilization30min, and5mL bacteria liquid, in speed179r/min,35.2℃table concentrator, for36.8h. The living bacterium number was2.04×1010per ml in the fermentation liquid. By comparing the culture medium optimization, the living bacterium multiply increased nearly five times.
益生菌饲料添加剂的生产是对发酵饲料技术的完善。作为功能性饲料,益生菌饲料调节动物肠道菌落平衡,不产生抗药性,适口性好,动物消化率高且功能稳定。枯草芽孢杆菌(Bacillus subtilis)具有典型芽孢杆菌特征,是经美国食品药物管理局(FAD)和美国饲料公定协会(AAFCO)公布的40余种益生菌之一
因此,研究油茶粕中的茶皂素的实用降解技术及利用降解油茶粕发酵生产枯草芽孢杆菌,可为油茶粕在饲料中的应用提供有力的实验依据。为饼粕作为饲料原料在饲料添加剂中的应用,扩大了饲料来源,为饲料业的发展提供的新方向及新思路对突破农副业发展瓶颈具有积极的现实意义。
本文以油茶粕作为研究对象,对其进行营养分析；结合微生物生长特性,研究微生物降解茶皂素的发酵工艺,利用降解油茶粕发酵生产枯草芽孢杆菌,主要结果如下：
利用各种测定方法,确定油茶粕的营养成分含量,结合枯草芽孢杆菌、白腐菌及黑曲霉对油茶粕的利用特性,筛选确定可降解茶皂素的微生物为黑曲霉。
对黑曲霉固态发酵降解油茶粕中的茶皂素进行了研究,运用单因素实验及Box-Behnken实验设计,结合高效液相色谱法(HPLC)检测技术,优化其固态发酵工艺,确定其最佳降解条件为：利用8层纱布控制传质传氧,初始含水量70%,pH4.35,接种2.18×107个单孢子/mL的悬液5mL,发酵温度34.5℃,发酵时间84h,发酵结束油茶粕中茶皂素降为2.04%,降解率达到89.99%。
利用降解油茶粕为原料,以活菌数为指标研究液态发酵生产饲料添加剂枯草芽孢杆菌益生菌。运用单因素实验、Plackett-Burman实验、最陡爬坡实验筛选液态发酵培养基因素,利用正交优化实验确定培养基最佳配方为：降解油茶粕13g,可溶性淀粉1g,尿素1.5g,磷酸氢二钠0.05g,七水硫酸镁0.015g,牛肉膏0.2g,七水硫酸亚铁0.03g,酵母膏0.5g,蒸馏水100mL,自然pH,121℃灭菌30min后接种5mL菌悬液,37℃,150r/min摇床培养24h,得到含108个/mL的枯草芽孢杆菌液态发酵液。
运用单因素实验及Box-Behnken实验设计,以活菌数为指标研究液态发酵生产枯草芽孢杆菌益生菌的工艺条件,确定最佳发酵条件为：500mL摇瓶中装液120mL培养基,调节pH6.8,灭菌30min后,接种9mL菌悬液摇匀后,在转速179r/min、35.2℃摇床中培养36.8h后计数,得到活菌数为2.04×1010个/mL的发酵液,与培养基优化时相比,活菌倍增提高了近5倍。
Camellia Oleifera is rich in the south foothills of our country and in parts of Southeast Asia, it has high comprehensive utilization value in the forestry and subsidiary agricultural products. A large number of Oil-tea-cake is formed as tea oil refining from oleifera seed, it contains many types of organic compounds and nutrients, and has high value in chemical industry, light industry, food and feed industry. Tea saponin as three glucoside terpenoid, has much of physical, chemical and biological physiological activity, and always stimulates the development of the chemical industry, agriculture, animal husbandry and fishery. In the application of feedstuff, a large amount of tea saponin has hemolysis and toxicity of fish, stimulates the nasal mucosa. Few of peroral tea saponin has lots of functions like impervious, antiphlogosis, spleen-tonifying, liquid-lowering, antibiosis, antianaphylaxis, intestines and stomach-protection, and improvement animal growth performance.
The production of probiotics feed additive completes the fermentation feed technology. As functional feed, probiotics feed can adjust the bacterial colony balance in animal gut flora balance, so never produce resistance to drugs. It also has great palatability, high animal digestion rate and stable performance. Bacillus subtilis has typical Bacillus characteristics, as one of40kinds of probiotics approved by the U.S. food and drug administration (FAD) and the United States official feed association (AAFCO) published.
Therefore, the study on the technique in degrading tea saponin and the bacillus subtilis production using oil-tea-cake application in feed additives as feed raw materials expands the forage sources and breaks through the bottleneck of agricultural development bottleneck. There is a lot of positive significance.
This paper was study on camellia meal, analysed the nutritional compositions. Combined with microbial growth characteristics, finded out the microbial used of degrading tea saponin, and used degradation camellia meal producted bacillus subtilis. The main results are as follows:
The content of nutrient in camellia meal was measured with various methods. Combined with bacillus subtilis, white-rot fungus and aspergillus, aspergillus was screened to degrade tea saponin.
Combined with high performance liquid chromatography(HPLC),single factor experiment and Box-Behnken experimental design, the solid fermentation process conditions of degradeting tea saponin was eight layer of gauze for regulating the mass and oxygen transfer, initial moisture content70%, pH4.35, inoculated5mL liquid of2.18×107single spore per milliliter, fermentation temperature34.5℃, fermentation time84h. Tea saponin content after fermentation is2.04%and the degradation rate reached89.99%.
For studying product bacillus subtilis probiotics by liquid fermentation, I used degradation camellia meal as raw material, the living bacterium number as index, and then via single factor experiment, Plackett-Burman experiment and the steepest climbing experiment to select factors, used the orthogonal experiment to determine the best formula for medium was degradation camellia meal13g, soluble starch1g, urea1.5g, disodium phosphate0.05g, seven water magnesium sulfate0.015g, beef cream0.2g, seven water ferrous sulfate0.03g, yeast anointed0.5g, distilled water100mL, natural pH,121℃sterilization30min,5mL bacteria liquid,37℃,150r/min table concentrator,24h. I got fermented liquid with108per mL of bacillus subtilis.
And then, I researched the fermentation condation of producing bacillus subtilis by single factor experiment and Box-Behnken experimental design. The optimal fermentation conditions was500mL culture bottle containing120mLculture medium, pH6.8, sterilization30min, and5mL bacteria liquid, in speed179r/min,35.2℃table concentrator, for36.8h. The living bacterium number was2.04×1010per ml in the fermentation liquid. By comparing the culture medium optimization, the living bacterium multiply increased nearly five times.
引文
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