蒙古口蘑菌丝体高密度发酵及其多糖的研究
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摘要
蒙古口蘑为享誉全球的著名珍贵食、药用担子菌。由于蒙古口蘑人工栽培难度较大,长期以来其驯化栽培都未能获得实质性突破,加之目前野生口蘑的滋生环境受到破坏,蒙古口蘑资源锐减。从保护开发这一珍稀濒危资源出发,通过液体深层发酵,大规模工业化生产蒙古口蘑菌丝体与口蘑多糖,开发利用这一珍贵资源潜力巨大。
本论文系统地研究了碳源、氮源、无机盐等营养成分以及种龄、接种量、温度、转速、溶氧、pH、通气量等因素对蒙古口蘑液体深层发酵生物量与胞外多糖产量的影响,并通过摇瓶发酵进行培养基筛选、发酵罐发酵工艺优化以及蒙古口蘑菌丝体及胞外多糖的提取、纯化和分析,确定了深层发酵、多糖提取和分离的工艺条件,首次利用发酵罐实现了蒙古口蘑菌丝体高密度发酵培养生产菌丝体及口蘑多糖,为下一步工业化大生产提供了可能。
本论文主要试验结果和研究结论摘要如下:
1、采用RAPD指纹技术原理,通过琼脂糖凝胶电泳的方法对随机引物进行筛选,利用筛选出的有效随机引物对样品DNA进行扩增后,经微流控芯片电泳仪电泳,对获得的图谱利用分析软件进行统计分析。聚类结果表明本试验所用三株蒙古口蘑菌株是其子实体分离物的纯培养,并且证明锡林郭勒草原与呼伦贝尔盟草原的蒙古口蘑无地域遗传差异性。
2、采用PDA培养基进行平板培养,比较了三株蒙古口蘑菌株的生长情况,并确定其中的一株蒙古口蘑菌株Q97为实验菌株。比较了不同培养温度下菌丝体的生长速度,确定蒙古口蘑菌丝体最佳生长温度为25℃。
3、通过单因子试验和正交试验对蒙古口蘑菌株Q97的适宜C、N源、培养基组成及发酵工艺进行了研究,证明菌株Q97可以较好的利用大多数碳源与有机氮源,得到摇瓶液体发酵最佳培养基组成为:豆饼粉30 g/L,蔗糖50 g/L,K2HPO4 1 g/L,CaCO3 1 g/L,pH自然。
4、摇瓶发酵最佳周期为13天,最大生物量达到30 g/L,胞外多糖达到100 mg/L以上,这为工业化高密度规模化生产提供了可能性。
5、通过5 L发酵罐发酵工艺的优化,确定了发酵罐最佳培养基配方和合适的发酵工艺,缩短了发酵周期,提高了发酵产量。
6、利用20 L发酵罐进行了蒙古口蘑菌丝体生长与胞外多糖合成的动力学研究,包括菌体生长、产物形成和底物消耗在内的动力学数学模型,通过模型计算值与实验值的比较,模型计算值与实验值均能较好吻合,说明建立的动力学模型能够较好的反映蒙古口蘑在发酵罐发酵的过程。
7、确定250 L发酵罐发酵培养基配方为:豆饼粉30 g/L,蔗糖80 g/L,K2HPO4 1 g/L,CaCO3 1 g/L。发酵周期为7-8 d,生物量最大值可以稳定到45 g/L,胞外多糖最大产量可以稳定到300 mg/L。这为工业化高密度规模化生产打下坚实的基础。
8、对蒙古口蘑发酵液进行了多糖的提取、分离与纯化,获得了较纯的口蘑多糖,并应用高效凝胶渗透色谱法(HPGPC)测定蒙古口蘑多糖的分子量,示差检测器检测蒙古口蘑多糖洗脱结果共得到5个峰,证明蒙古口蘑多糖至少含有5种多糖组分,其主要组分重均分子量Mw=3919.
As one of notable edible-medicinal basidiomycete, Tricholoma mongolicum Imai enjoys a worldwide reputation. Mycelia and polysaccharide of Tricholoma mongolicum are produced on a large scale by liquid fermentation to be potential for protecting and exploring this precious resource in severe danger, because its domestication and manned planting is difficult and lacks of substantive breakthrough, as well as its natural habitats are destroyed to result in its wild resource reducing sharply.
It was investigated intensively in this study that effects of nutrients including carbon source, nitrogen source and inorganic salts, as well as seed age, inoculation volume, temperature, rotate rate, dissolved oxygen, pH value and aeration on yields of mycelia and polysaccharide during the submerged fermentation. Also, fermentation process, extraction and separation process of polysaccharide were determined and optimized. Mycelia and polysaccharide of Tricholoma mongolicum were produced by high-density fermentation using the fermentor, which establishes a base for the large scale production. The main results and conclusions are as follows.
1. Based on RAPD fingerprint technique, DNAs of the strains were amplified using the effective arbitrary primer screened by agarose gel electrophoresis. Maps of amplified DNAs obtained by microchip capillary electrophoresis were analyzed by statistical software. The results of cluster analysis show that three strains are all pure cultures of isolates from fruiting body of Tricholoma mongolicum, and Tricholoma mongolicum from Xilingol grassland has no any regionally genetic diversity with that from Hulunbeier grassland.
2. A comparison in growth characteristics of three strains was carried out using PDA medium. The strain Q97 was select and used in this study, and its optimal incubation temperature was determined to be 25℃based on growth rate under various temperatures.
3. Carbon and nitrogen sources, media ingredients and fermentation technics of the strain Q97 were studied with single factor test and orthogonal test. The experimental results show that Q97 can utilize most of carbon sources and organic nitrogen sources, and the optimum medium formula for shake-flask fermentation was determined as follows: bean cake powder 30g/L, sucrose 50g/L, K2HPO4 1g/L, CaCO3 1g/L, and pH non-adjusted.
4. The optimum fermentation period for shake-flask fermentation was 13 days, and maximum biomass was up to 30g/L with 100mg/L of exopolysaccharide.
5. The optimum medium formula for the fermentor fermentation was determined based on the experimental results using 5L of fermentor. Fermentation period was shortened and yield was increased by the optimization of fermentation process.
6. Fermentation kinetics including strain growth, product formation and substrate consumption kinetics was studied using 20L of fermentor, and the appropriate kinetic models were proposed. Experimental data were in agreement with calculated data according to kinetic models established, which suggests that models appear to provide a reasonable description for the fermentation process of Tricholoma mongolicum in the fermentor.
7. Fermentation medium formula for 250L of fermentor was as follows: bean cake powder 30g/L, sucrose 80g/L, K2HPO4 1g/L, and CaCO3 1g/L. The maximum biomass reached 45g/L with 300mg/L of maximum polysaccharide yield after 7~8 days’fermentation.
8. The polysaccharide was extracted, separated and purified from fermentation liquid, and its molecular weight was measured with high-performance gel-permeation chromatography (HPGPC) equipped with refractive index detector. The results show that polysaccharide of Tricholoma mongolicum includes at least five varieties of components, and molecular weight average of its main component is 3919.
本论文系统地研究了碳源、氮源、无机盐等营养成分以及种龄、接种量、温度、转速、溶氧、pH、通气量等因素对蒙古口蘑液体深层发酵生物量与胞外多糖产量的影响,并通过摇瓶发酵进行培养基筛选、发酵罐发酵工艺优化以及蒙古口蘑菌丝体及胞外多糖的提取、纯化和分析,确定了深层发酵、多糖提取和分离的工艺条件,首次利用发酵罐实现了蒙古口蘑菌丝体高密度发酵培养生产菌丝体及口蘑多糖,为下一步工业化大生产提供了可能。
本论文主要试验结果和研究结论摘要如下:
1、采用RAPD指纹技术原理,通过琼脂糖凝胶电泳的方法对随机引物进行筛选,利用筛选出的有效随机引物对样品DNA进行扩增后,经微流控芯片电泳仪电泳,对获得的图谱利用分析软件进行统计分析。聚类结果表明本试验所用三株蒙古口蘑菌株是其子实体分离物的纯培养,并且证明锡林郭勒草原与呼伦贝尔盟草原的蒙古口蘑无地域遗传差异性。
2、采用PDA培养基进行平板培养,比较了三株蒙古口蘑菌株的生长情况,并确定其中的一株蒙古口蘑菌株Q97为实验菌株。比较了不同培养温度下菌丝体的生长速度,确定蒙古口蘑菌丝体最佳生长温度为25℃。
3、通过单因子试验和正交试验对蒙古口蘑菌株Q97的适宜C、N源、培养基组成及发酵工艺进行了研究,证明菌株Q97可以较好的利用大多数碳源与有机氮源,得到摇瓶液体发酵最佳培养基组成为:豆饼粉30 g/L,蔗糖50 g/L,K2HPO4 1 g/L,CaCO3 1 g/L,pH自然。
4、摇瓶发酵最佳周期为13天,最大生物量达到30 g/L,胞外多糖达到100 mg/L以上,这为工业化高密度规模化生产提供了可能性。
5、通过5 L发酵罐发酵工艺的优化,确定了发酵罐最佳培养基配方和合适的发酵工艺,缩短了发酵周期,提高了发酵产量。
6、利用20 L发酵罐进行了蒙古口蘑菌丝体生长与胞外多糖合成的动力学研究,包括菌体生长、产物形成和底物消耗在内的动力学数学模型,通过模型计算值与实验值的比较,模型计算值与实验值均能较好吻合,说明建立的动力学模型能够较好的反映蒙古口蘑在发酵罐发酵的过程。
7、确定250 L发酵罐发酵培养基配方为:豆饼粉30 g/L,蔗糖80 g/L,K2HPO4 1 g/L,CaCO3 1 g/L。发酵周期为7-8 d,生物量最大值可以稳定到45 g/L,胞外多糖最大产量可以稳定到300 mg/L。这为工业化高密度规模化生产打下坚实的基础。
8、对蒙古口蘑发酵液进行了多糖的提取、分离与纯化,获得了较纯的口蘑多糖,并应用高效凝胶渗透色谱法(HPGPC)测定蒙古口蘑多糖的分子量,示差检测器检测蒙古口蘑多糖洗脱结果共得到5个峰,证明蒙古口蘑多糖至少含有5种多糖组分,其主要组分重均分子量Mw=3919.
As one of notable edible-medicinal basidiomycete, Tricholoma mongolicum Imai enjoys a worldwide reputation. Mycelia and polysaccharide of Tricholoma mongolicum are produced on a large scale by liquid fermentation to be potential for protecting and exploring this precious resource in severe danger, because its domestication and manned planting is difficult and lacks of substantive breakthrough, as well as its natural habitats are destroyed to result in its wild resource reducing sharply.
It was investigated intensively in this study that effects of nutrients including carbon source, nitrogen source and inorganic salts, as well as seed age, inoculation volume, temperature, rotate rate, dissolved oxygen, pH value and aeration on yields of mycelia and polysaccharide during the submerged fermentation. Also, fermentation process, extraction and separation process of polysaccharide were determined and optimized. Mycelia and polysaccharide of Tricholoma mongolicum were produced by high-density fermentation using the fermentor, which establishes a base for the large scale production. The main results and conclusions are as follows.
1. Based on RAPD fingerprint technique, DNAs of the strains were amplified using the effective arbitrary primer screened by agarose gel electrophoresis. Maps of amplified DNAs obtained by microchip capillary electrophoresis were analyzed by statistical software. The results of cluster analysis show that three strains are all pure cultures of isolates from fruiting body of Tricholoma mongolicum, and Tricholoma mongolicum from Xilingol grassland has no any regionally genetic diversity with that from Hulunbeier grassland.
2. A comparison in growth characteristics of three strains was carried out using PDA medium. The strain Q97 was select and used in this study, and its optimal incubation temperature was determined to be 25℃based on growth rate under various temperatures.
3. Carbon and nitrogen sources, media ingredients and fermentation technics of the strain Q97 were studied with single factor test and orthogonal test. The experimental results show that Q97 can utilize most of carbon sources and organic nitrogen sources, and the optimum medium formula for shake-flask fermentation was determined as follows: bean cake powder 30g/L, sucrose 50g/L, K2HPO4 1g/L, CaCO3 1g/L, and pH non-adjusted.
4. The optimum fermentation period for shake-flask fermentation was 13 days, and maximum biomass was up to 30g/L with 100mg/L of exopolysaccharide.
5. The optimum medium formula for the fermentor fermentation was determined based on the experimental results using 5L of fermentor. Fermentation period was shortened and yield was increased by the optimization of fermentation process.
6. Fermentation kinetics including strain growth, product formation and substrate consumption kinetics was studied using 20L of fermentor, and the appropriate kinetic models were proposed. Experimental data were in agreement with calculated data according to kinetic models established, which suggests that models appear to provide a reasonable description for the fermentation process of Tricholoma mongolicum in the fermentor.
7. Fermentation medium formula for 250L of fermentor was as follows: bean cake powder 30g/L, sucrose 80g/L, K2HPO4 1g/L, and CaCO3 1g/L. The maximum biomass reached 45g/L with 300mg/L of maximum polysaccharide yield after 7~8 days’fermentation.
8. The polysaccharide was extracted, separated and purified from fermentation liquid, and its molecular weight was measured with high-performance gel-permeation chromatography (HPGPC) equipped with refractive index detector. The results show that polysaccharide of Tricholoma mongolicum includes at least five varieties of components, and molecular weight average of its main component is 3919.
引文
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