【作者】 葛含静；

【导师】 李志西；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2012， 博士

【摘要】 细菌纤维素具有高结晶度、高纯度、机械性能良好、持水透水性强、生物可降解性和合成可调控性等优良特性，纤维丝粗细度可达纳米级（纤维直径0.01⁰.1um），广泛应用于食品、医学和器官再造、高级造纸、高档声学器材等领域。未来细菌纤维素在各行各业的应用将不断扩大，需求量将迅速增长而供不应求，因此，如何提高细菌纤维素产量是科学领域亟待解决的问题。优化培养基、改进发酵条件和培育高产菌株是提高纤维素产量的常用手段。其中，选育高产菌株是解决高产问题的根本，而高压诱变是其最为有效的途径之一。在细菌纤维素高产菌株的诱变育种中，高静水压诱变克服了传统物理诱变（如UV或X-射线等）的辐射性和化学诱变（如DES、亚硝基胍、氯化锂等）的毒性，成为重要的诱变育种方法之一。在超高压（P≥100MPa）环境中，微生物细胞形态、细胞膜和细胞壁都可能发生变化，它也会引起细胞内生化反应，还可改变微生物的基因表达、核酸结构及其生物学功能，这在微生物菌种诱变方面具有很大的应用潜力。本研究首先从本实验室自制荞麦醋中筛选出一株性能优良的纤维素产生菌野生菌株J2，然后对其进行高静水压诱变，从突变菌株中成功筛选出一株纤维素产量更高且传代稳定的突变菌株M₄₃₈。进而优化了两株菌生产纤维素的发酵培养基及培养条件，并测定和对比了所产纤维素的各项指标及性能，继而对两株菌的形态特征和生理生化特征进行了研究比较，结合系统发育分析对两株菌做了菌种鉴定，最后通过AFLP分子标记技术在DNA水平对细菌纤维素产生菌的高压诱变机理进行初步探讨，得到了以下主要研究结果：（1）从自制荞麦醋中分离出了性质稳定的纤维素产生菌野生菌株J2，并对其种子培养基及培养条件、发酵培养基及发酵条件进行优化。菌株J2最优种子培养基配方为：葡萄糖7%，酵母膏1%，K₂HPO₄0.5%，MgSO₄7H2O1.5%，无水乙醇2%（v/v），最佳培养条件为：温度30℃，种龄24h、接种量7%、摇床转速150r/min。优化后的发酵培养基为：碳源3%（葡萄糖:蔗糖＝1:2），酵母膏0.33%，FeSO₄0.4%，ZnSO₄0.09%，K₂HPO₄0.1%，MgSO₄7H₂O1%，苹果酸0.3%，无水乙醇0.7%（v/v）。细菌纤维素的产量为10.41g/100mL，是优化前（8.52g/100mL）的1.22倍。最佳发酵周期为7d，此时纤维素产量约为12g/100mL。（2）对野生菌株J2进行高静水压诱变处理，从突变株中筛选出纤维素产量高且传代稳定的突变菌株M₄₃₈。高静水压诱变的最适条件为压力250MPa，时间15min，温度25℃。突变菌株M₄₃₈的种子培养基和种子培养时摇床转速仍用其出发菌株优化的种子培养基配方及培养条件，优化后的种龄为24h，接种量为9%。优化后的发酵培养基为：碳源5%（葡萄糖/蔗糖=4:1），酵母浸出汁1.25%，CaCl₂0.15%，ZnSO₄0.2%，K₂HPO₄0.2%，MgSO₄7H₂O0.93%，富马酸0.3%，无水乙醇浓度为0.5%（v/v）。细菌纤维素的产量为28.99g/100mL，是优化前（15.75g/100mL）的1.84倍，是出发菌株J2产量（10.41g/100mL）的2.78倍。最佳发酵周期为7d，此时纤维素产量约为34g/100mL。（3）确定了菌株J2和菌株M₄₃₈所产凝胶状膜的主要成分是纤维素，纤维素含量分别为89.32%和89.35%。菌株J2所产细菌纤维素的各项理化指标分别为：纤维素湿膜含水量为98.68%，干膜复水率为81.76%，蛋白质含量为7.69%，脂肪含量为1.65%，持水力是其干膜的94倍，释水率为56h。菌株M₄₃₈所产细菌纤维素的各项理化指标分别为：纤维素湿膜含水量为98.73%，干膜复水率为80.63%，蛋白质含量为7.83%，脂肪含量为1.62%，持水力是其干膜的105倍，释水率为80h。菌株J2和菌株M₄₃₈所产细菌纤维素为致密的网状结构，结晶度分别为78%和82%，Iα型纤维素含量分别为52%和62%，最大抗拉强度分别为55.8MPa和78.4MPa。以上数据说明，菌株M₄₃₈所产的细菌纤维素比菌株J2所产细菌纤维素更具有优越性；高静水压不仅使细菌纤维素产生菌突变菌株的纤维素产量提高了，也使纤维素性能增强了。（4）通过表型测定和遗传学分类鉴定，菌株J2为典型Gluconacetobacter hansenii的变种，菌株M₄₃₈为Gluconacetobacter hansenii的亚种。虽然野生菌株J₂和高静水压诱变菌株M₄₃₈的最优发酵培养基配方以及纤维素产量均不同，但两者表型特征和系统发育关系完全相同。说明高静水压使纤维素产生菌代谢途径中的某些生化过程发生了改变，但并没有改变菌体的个体形态、群体形态、发酵状态、生理生化特征以及遗传关系。（5）建立了适合高静水压诱变前后葡糖醋杆菌菌株的AFLP多态性分析的反应体系，即双酶切反应模板DNA用量为600ng、反应时间为8h，连接反应时间为8h或过夜，预扩增反应的产物稀释500倍用于选择性扩增最为理想，筛选出了E+T/M+G为适合葡糖醋杆菌菌株高静水压诱变前后菌株多态性分析的引物组合。通过AFLP多态性分析，高静水压处理得到的纤维素高产突变菌株M₄₃₈是缺失突变株，这唯一的缺失片段的补码序列编码的小多重抗药蛋白cl00910能一定程度地抑制细菌纤维素的分泌，因而经高静水压诱变的缺失突变菌株M₄₃₈的细菌纤维素产量比它的野生菌株J2的纤维素产量显著提高。更多还原

【Abstract】 Due to high crystallinity, high purity, good mechanical property, strong waterpermeability and water holding capacity, well biodegradability and synthesis of controlled,and nanometer size of thin fibrils （diameter0.01⁰.1um）, bacterial cellulose （BC） was widelyused in the areas of food, medicine, organ reengineering, senior paper making and high-gradeacoustics etc. Because of the expanding application and the rapidly increased demand of BCin all walks of life in the future, how to increase the yield of BC comes to be a problemneeded to solve exigently. Optimizing culture medium, improving the conditions offermentation and cultivating high yield strains are the commonly used method of increasingBC yield. Cultivating high yield strains is solving the root of the high yield problem. Highpressure mutagenesis is one of the most effective ways. Among the methods of high-yield BCproducing strain mutation breeding, high hydrostatic pressure （HHP） technology bacomesone of the important ways of mutation breeding because of overcoming the shortages ofradioactive of traditional physical mutation （such as UV or X-ray, etc） and toxic of chemicalmutation （such as DES, nitrosoguanidine, lithium chloride and so on）. Ultra high pressure （P≥100MPa）, which can make changes in microbial cells form, cell membrane and cell wall,and cause intracellular biochemical reactions, even vary the microbial gene expression andthe nucleic acid structure and biological function, has great application potency in microbialmutation breeding.In the paper, a wild BC producing strain J2with good property was screened fromhomemade buckwheat vinegar in our laboratory firstly. Wild strain J2was treated by highhydrostatic pressure, then a mutant strain M₄₃₈with higher yield BC production and wellstablity in subculture was isolated successfully from all mutant strains. Both the fermentationmedium and the culture conditions of strain J2and strain M₄₃₈were optimized. Sequently, allindex parameters and properties of BC produced by strain J₂and strain M₄₃₈were measuredand compared. Next, the characterizations of morphology and physiology-biochemistry ofstrain J₂and strain M₄₃₈were researched and compared, and then the two strains wereidentified combining the phylogenetic analysis. At last, high pressure mutagenesismechanism of HHP technology on BC producing strain was primarily discussed in the level of DNA based on amplified fragment length polymorphism （AFLP）. The main researchresults are as follows:（1） The wild BC producing strain J2with stable property was screened from homemadebuckwheat vinegar. Its seed culture medium, fermentation medium and culture conditionswere optimized. The optimum seed culture medium contained D-glucose7%, yeast extract1%, K₂HPO₄0.5%, MgSO₄·7H₂O1.5%, ethanol2%（v/v）. The optimum cultivation time was24h at30℃with the shaking speed of150r/min in constant temperature shaking incubator.The optimized inoculation amount was7%. The optimum fermentation medium containedcarbon source3%（glucose:sucrose=1:2）, yeast extract0.33%, FeSO₄0.4%, ZnSO₄0.09%,K2HPO₄0.1%，MgSO₄7H₂O1%, malic acid0.3%and anhydrous alcohol0.7%（v/v）. Underthese conditions, BC yield could reach to10.41g/100mL, whch is1.22times as that under theconditions with no optimization （8.52g/100mL）. The optimum fermentation time was7d, andthe yield of BC was about12g/100mL at this time.（2） After treating the wild BC producing strain J2by HHP, a mutated strain M₄₃₈withhigher yield of BC and stable property during serial passage was isolated from the mutants.The optimal conditions of strain J2mutated by HHP were under250MPa with15min at25℃.Srtain M₄₃₈cultured in the same seed culture medium and the same culture conditions withstrain J2’s. The optimum cultivation time was24h at30℃with the shaking speed of150r/min in constant temperature shaking incubator. The optimized inoculation amount was9%.The optimum fermentation medium contained carbon source5%（glucose:sucrose=4:1）,yeast leaching juice1.25%, CaCl20.15%, ZnSO₄0.2%, K2HPO₄0.2%，MgSO₄7H₂O0.93%,fumaric acid0.3%and anhydrous alcohol0.5%（v/v）. Under these conditions, BC yield couldreach to28.99g/100mL, whch is1.84times as that under the conditions with no optimization（15.75g/100mL）. The optimum fermentation time was7d, and the yield of BC was about34g/100mL at this time.（3） The main component of the gelatinous membrane produced by strain J2and strainM₄₃₈was confirmed as cellulose, and the content of it produced by the two strains were89.32%and89.35%, respectively. The other physical and chemical characters of BCproduced by strain J2were as follows: water content of wet BC was98.68%, rehydration rateof dry BC after absorbing water was81.76%, protein content of dry BC was7.69%, fatcontent of dry BC was1.65%, water holding capacity of wet BC was as94times as that ofdry BC, and water release rate of wet BC was56h. All above physical and chemicalcharacters of BC produced by strain M₄₃₈were98.73%,80.63%,7.83%,1.62%,105timesand80h, respectively. BC produced by strain J2and strain M₄₃₈was observed as dense mesh structure.The crystallinity indexes were78%and82%, respectively. The cellulose Iα contents were52% and62%, repectively. And the maximum tensile strength were55.8MPa and78.4MPa,repectively.The data above indicated that BC produced by strain M₄₃₈has more advantages thanthat produced by strain J₂. Not only the yield of BC of the mutated strain treated by HHP hasincreased, but the properties were enhanced.（4） Based on identification through phenotype and hereditism, strain J₂was varietas oftypical Gluconacetobacter hansenii strain, while strain M₄₃₈was subspecies of Gluconaceto-bacter hansenii. The components of optimum fermentation medium, culture conditions andthe BC yield of strain J₂and strain M₄₃₈were not the same, but both phenotypiccharacterization and phylogenetic relationships were completely the same. It confirmed thatHHP treatment could make changes in some biochemical processes in the bacteria metabolicpathway of BC producing strain, but it couldnot change the bacteria individual form, thegroup form, the fermentation state, the physiological and biochemical characteristics andthe genetic relationship.（5） Amplified fragment length polymorphism （AFLP） reaction systerm was constructed.It was feasible to conduct the AFLP analysis of Gluconacetobacter sp. and its mutated straintreated by HHP. The optimum technical parameters of the AFLP reaction systerm were asfollows:600ng genomic DNA served as template should be used in the digestion system withthe reaction time8hours, the time of ligation should be8h or overnight （more than10h）,500times of dilution for the products of pre-amplification for selective amplification, and1pairsof primers （M+G/E+T） were selected which adapt the AFLP reaction system of Glucona-cetobacter sp. and its mutated strain treated by HHP. On the basis of AFLP analysis, strainM₄₃₈with higher BC yield was a deletion mutant induced by HHP, and the complement genesof the only deleted sequence code small multidrug resistance protein c100910, which inhibitBC producing in a certain extent. Therefore, the deletion mutant M₄₃₈muteted by HHP hasmuch higher BC yield than its initial strain J₂.更多还原