CN116716215A - 一株产细菌纤维素的汉逊氏醋杆菌及应用 - Google Patents
一株产细菌纤维素的汉逊氏醋杆菌及应用 Download PDFInfo
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Abstract
本发明公开了一株产细菌纤维素的汉逊氏醋杆菌及应用。该菌株命名为汉逊氏醋杆菌Novacetimonas hansenii YZHY21.A11,所述菌株已于2022年6月13日保藏在“广东省微生物菌种保藏中心”,保藏号为GDMCC NO.62534。本发明提供的菌株可发酵产生细菌纤维素。发酵7天干膜产量达到6.38g/L,该菌在生产细菌纤维素的时候同时产酸。该菌株是一株极具研究开发价值的菌株。
Description
技术领域
本发明属于生物技术领域,涉及一株产细菌纤维素的汉逊氏醋杆菌及应用。
背景技术
利用微生物发酵以生产的纤维素称为细菌纤维素(BC)。BC是由农杆菌、无色杆菌、固氮杆菌、根瘤菌和沙门菌等细菌合成的天然纤维素聚合物,具有高纯度、高结晶度、高吸水能力等诸多优于棉或木纤维素的理化性能。同时BC具有优良的生物相容性、抗菌活性和生物可降解性。BC的产生始于6-磷酸-葡萄糖,磷酸葡萄糖异构酶促进6-磷酸-葡萄糖异构化为1-磷酸-葡萄糖,接着1-磷酸-葡萄糖在酶的催化下得到尿苷-5'-二磷酸-α-D-葡萄糖(UDPG),最后再通过纤维素合成酶将葡萄糖残基转移到新生的β-D-1,4-葡聚糖链,将大量的UDP-葡萄糖引导到纤维素生物合成中,导致细胞代谢的重编程,有利于糖异生。木醋杆菌细胞在极短时间就可以聚合大量的葡萄糖分子,BC在生物合成过程中,将细胞内形成的葡萄糖链通过细菌细胞膜上的微小孔挤出来,从而葡萄糖链形成微纤维,微纤维再进行聚合产生纤维素带,这些纤维素带会产生蛛网状的网络结构,纤维中间有大量的空洞,BC的纳米微纤维分离良好,形成了扩展的表面积和高孔基质。BC微纤维大约比植物纤维素纤维小100倍。
BC的生产分为静态发酵和动态发酵,不同的发酵方式导致BC的形态不同。BC膜是通过在各种静态反应器中静态培养的,在静态培养中产生的BC通常是均匀光滑的凝胶状薄膜。静态条件下纤维素的形成受来自介质表面的空气供应的调节,而产量取决于碳源的浓度。生长时间越长,通过C-H键形成的BC就越多。然而,当膜向下生长并捕获细菌时,随着膜厚度的增加,BC产量达到极限,然后由于氧气不足而变得不活跃降低合成速率。由于静态发酵所生产的纤维素纤维结构致密、纯度高、生物相容性好,目前采用最多的培养方法还是静态培养法。动态培养通常采用搅动或摇动培养,是在高速旋转条件下生产颗粒状或糊状BC的另一种方法。在动态培养中,细胞与循环空气有更好的接触,导致更好的生长速度,最终提高BC的生产力和产量。动态发酵所得到的纤维素通常为球形或团状,其具有更大的比表面积,广泛应用在药物缓释、污水处理等领域。但是同时在液体培养基中,BC的生产会导致发酵液黏度的增大,从而培养基中的空气含量会减少,导致葡萄糖醛酸、醋酸或乳酸的堆积,这将严重影响pH,对微生物生长和BC的生产造成不良影响。很多研究者用不同菌种采用静态或动态方法培养BC。中国科学院微生物研究所毛贤君等人公开了木驹形杆菌(komagataeibacter xylinus BJ11,CGMCC No.18422)通过动态发酵罐生产BC,具有稳定、发酵时间短、生产量大等特点(专利号:CN 201911147868.3)。北京爱发科技公司刁刘洋等人筛选了一株产细菌纤维素的驹形杆菌(Komagataeibacter sp.ATC301,CGMCCNo.17875),该菌株能够在在酸性条件下生长并高效生产细菌纤维素(专利号:CN201910801923.X)。华东师范大学易正芳等人筛选了一种驹形杆菌(Komagataeibactersp.171129Z2-3,CGMCC No.17276)够在静置培养和振荡培养时产生大量细菌纤维素,在发酵4天时产量分别可达5.12g/L和2.571g/L(专利号:CN 201911275504.3)。该单位高红亮等人从柿子中分离筛选获得适合动态发酵的木葡糖酸醋杆菌(Gluconacetobacterxylinus171027-PER1,CGMCC No.15234),其动态发酵纤维素产量可达6g/L,纤维直径低,有更强的持水能力(专利号:CN 202110154593.7)。
发明内容
本发明提供的汉逊氏醋杆菌Novacetimonas hansenii YZHY21.A11菌株能产细菌纤维素,发酵7天干膜产量达到6.38g/L。本发明得到的汉逊氏醋杆菌和细菌纤维素均具有研究和应用价值。
本发明技术方案如下。
本发明所述一株产细菌纤维素的细菌,其特征在于,该菌株命名为汉逊氏醋杆菌Novacetimonas hansenii YZHY21.A11,是从腐烂的杨桃中分得,所述菌株已于2022年6月13日保藏在“广东省微生物菌种保藏中心”,保藏地址为:广州市先烈中路100号大院59号楼5楼,保藏号为GDMCC NO.62534。
优选地,所述菌株分离自腐烂的杨桃,采用以下方法分离。
一株产细菌纤维素的细菌的分离方法,取5g自然腐烂的杨桃放入装有50mL富集培养基的并带有玻璃珠的300mL三角瓶中,30℃,150r/min震荡12h,取0.1mL菌悬液于10mL灭菌离心管中,进行梯度稀释并涂布到分离培养基上,30℃倒置恒温培养,待长出黄色且有透明圈的菌落后再次稀释涂布纯化,之后将单一菌落接种到液体种子培养基中,30℃、150r/min过夜培养后以5%(v/v)接种量接种于发酵培养基中,30℃静置培养确定该菌株是否产膜。纯化复筛后,挑选生长良好,产细菌纤维素膜的目标菌落转接至试管斜面中,30℃培养3天后于4℃下保存并进行测序。
该菌株得序列测序结果附表1。
优选地,所述培养基组成为:
富集培养基:蛋白胨5g/L,酵母浸粉5g/L,葡萄糖5g/L,甘露醇5g/L,无水硫酸镁1g/L,pH 6.0±0.2。
优选地,分离培养基:酵母浸粉10g/L,葡萄糖10g/L,碳酸钙20g/L,溴甲酚紫0.015g/L,琼脂20g/L,pH 6.0±0.2。
优选地,试管斜面培养基:富集培养基加入20g/L琼脂,pH 6.0±0.2。
优选地,液体种子培养基:同富集培养基。
优选地,液体发酵培养基(免灭菌):950mL沸水中加入50g蔗糖,蔗糖溶解后,加入约5g的红茶包继续煮沸10min,取出茶包后,加入50mL白醋,冷却至室温。
细菌纤维素采用以下方法发酵生产:
将所筛选菌种接种到液体种子培养基中,在摇床中30℃,150rpm震荡培养24h,以5%(v/v)接种量接种到发酵培养基中,在恒温培养箱中30℃静置培养7天,待液体表面长出纤维素膜,将膜取出,在0.3mol/L NaOH溶液中加热煮沸30min,以去除残留的菌体和杂质,然后用清水漂洗直至膜呈中性。
与现有技术相比,本发明的优势在于:
本发明的菌株可利用廉价的蔗糖和茶叶提取物发酵生产细菌纤维素,并可同时产酸,有较高的生产能力,可发酵生产出具有高度的网络多孔结构、高纯度和高结晶度的细菌纤维素。
附图说明
图1为Novacetimonas hansenii YZHY21.A11在分离培养基上的菌落形态和革兰氏染色显微镜图;
图2为Novacetimonas hansenii YZHY21.A11的进化树图;
图3为Novacetimonas hansenii YZHY21.A11发酵生产的细菌纤维素图;
图4为细菌纤维素产量和发酵液pH随发酵天数变化图;
图5为细菌纤维素扫描电镜图;
图6为细菌纤维素红外谱图;
图7为细菌纤维素X射线衍射图。
具体实施方式
下面结合具体实施例对本发明作进一步地具体详细描述,但本发明地实施方式不限于此,对于未特别注明地工艺参数,可参照常规技术进行。
实施例1汉逊氏醋杆菌菌株筛选
取5g自然腐烂的杨桃放入装有50mL富集培养基的并带有玻璃珠的300mL三角瓶中,30℃,150r/min震荡12h,取0.1mL菌悬液于10mL灭菌离心管中,进行梯度稀释并涂布到分离培养基上,30℃倒置恒温培养,待长出黄色且有透明圈的菌落后再次稀释涂布纯化,之后将单一菌落接种到液体种子培养基中,30℃、150r/min过夜培养后以5%(v/v)接种量接种于发酵培养基中,30℃静置培养确定该菌株是否产膜。纯化复筛后,挑选生长良好,产细菌纤维素膜的目标菌落转接至试管斜面中,30℃培养3天后于4℃下保存并送至基因公司进行测序。最终获得一株汉逊氏醋杆菌Novacetimonas hansenii YZHY21.A11,所述菌株已于2022年6月17日保藏在“广东省微生物菌种保藏中心”,保藏号为GDMCC NO.62534。该菌株的菌落表面光滑,圆形,略凸起,颜色为米黄色。该菌株能产酸使溴甲酚紫变黄色,并溶解碳酸钙产生透明圈。用200×显微镜观察,该菌呈现中间杆状两端圆的形态,革兰氏染色为阴性(见图1)。
其中所述培养基组成为:
富集培养基:蛋白胨5g/L,酵母浸粉5g/L,葡萄糖5g/L,甘露醇5g/L,无水硫酸镁1g/L,pH 6.0±0.2。
分离培养基:酵母浸粉10g/L,葡萄糖10g/L,碳酸钙20g/L,溴甲酚紫0.015g/L,琼脂20g/L,pH 6.0±0.2。
试管斜面培养基:富集培养基加入20g/L琼脂,pH 6.0±0.2。
液体种子培养基:同富集培养基。
液体发酵培养基(免灭菌):950mL沸水中加入50g蔗糖,蔗糖溶解后,加入约5g的红茶包继续煮沸10min,取出茶包后,加入50mL白醋,冷却至室温。
实施例2菌株***发育树的建立
将该菌株基因序列在NCBI网站(https://www.ncbi.nlm.nih.gov/)经过与标准数据库快速的局域序列对位排列(BLAST)比对,检索出相似性、覆盖率最高、E值最小的相关菌株的基因序列,BLAST分析表明,该分离菌株与汉逊氏醋杆菌Novacetimonas hansenii(GenBank登录号NR 112227.1)具有最高的同源性。利用分子进化遗传学分析软件MEGA11,采用邻接法对高度同源的DNA序列构建***发育树(见图2)。
实施例3GDMCC NO.62534菌株静置发酵生产细菌纤维素
将所筛选菌种接种到液体种子培养基中,在摇床中30℃,150rpm震荡培养24h,以5%(v/v)接种量接种到发酵培养基中,在恒温培养箱中30℃静置培养7天,待液体表面长出纤维素膜,将膜取出,在0.3mol/L NaOH溶液中加热煮沸30min,以去除残留的菌体和杂质,然后用清水漂洗直至膜呈中性(见图3)。在培养期间每天测定干膜重量及培养基pH,产量逐渐上升,在发酵第二天时产量增加最大,到第7天时干膜产量达到6.38g/L。培养基pH呈下降趋势,可能是由于该菌在生产细菌纤维素的时候同时产酸。Novacetimonas hanseniiYZHY21.A11的转化率和产率分别为0.13g BC/g蔗糖和0.91g/(L·d)(见图4)。利用扫描电镜(SEM)对细菌纤维素干膜的表面形貌和微观结构进行了检测。细菌纤维素的显微照片显示了一个密集排列的网状结构,直径从40到100nm不等。当纤维在静态生产被高度拉伸时,纤维缠结和弯曲形成网状致密结构。这些原纤维聚集在一起形成具有高纵横比的多孔结构(见图5)。细菌纤维素的红外光谱(FT-IR)峰的位置和强度,显示了细菌纤维素的组成,具有纤维素在FT-IR光谱中的典型波段,在2900cm-1附近的特征带归因于纤维素纤维组分中常见的CH2和CH3基团的C-H拉伸振动,C-O键在1055cm-1左右伸缩,由于O-H伸缩,在3340cm-1处和1500-1235cm-1处有典型的特征峰。约1650cm-1处的条带是由于被吸收的水分子的变形振动造成的。在约1424cm-1、1360cm-1和1060cm-1处均有较强的条带,分别对应碳水化合物的CH2剪切键、C-H弯曲键和C-O-H键的振动。896cm-1处的峰对应于C-O-C拉伸和1,4-β键的弯曲振动。在该谱图中没有发现在1735cm-1附近的波段,这部分波段对应于木质素、半纤维素中乙酰键和酯键的C-O拉伸振动,这也证实了BC的纯度(不含木质素,半纤维素)(见图6)。细菌纤维素膜的X射线衍射谱图(XRD)在14.5°和22.5°表现出非常好分辨的窄峰,经计算,所得细菌纤维素的结晶度为68.05%(见图7)。
其中所述液体种子培养基和发酵培养基组成为:
种子培养基:蛋白胨5g/L,酵母浸粉5g/L,葡萄糖5g/L,甘露醇5g/L,无水硫酸镁1g/L,pH 6.0±0.2。
发酵培养基(免灭菌):950mL沸水中加入50g蔗糖,蔗糖溶解后,加入约5g的红茶包继续煮沸10min,取出茶包后,加入50mL白醋,冷却至室温。
本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一株产细菌纤维素的细菌,其特征在于,该菌株命名为汉逊氏醋杆菌Novacetimonashansenii YZHY21.A11,所述菌株已于2022年6月13日保藏在“广东省微生物菌种保藏中心”,保藏地址为:广州市先烈中路100号大院59号楼5楼,保藏号为GDMCC NO.62534。
2.根据权利要求1所述产细菌纤维素的细菌,其特征在于,所述菌种分离自腐烂的杨桃。
3.根据权利要求1或2所述产细菌纤维素的细菌,其特征在于,所述菌株采用以下方法分离:
取自然腐烂的杨桃放入装有富集培养基的并带有玻璃珠的三角瓶中,震荡,取菌悬液于灭菌离心管中,进行梯度稀释并涂布到分离培养基上,倒置恒温培养,待长出单菌落后挑取黄色且有透明圈菌落进行再次稀释涂布,恒温培养,之后将单一菌落接种到液体种子培养基中,过夜培养后接种于发酵培养基中,静置培养确定该菌株是否产膜,挑选生长良好且产膜的目标菌落转接至试管斜面,培养完成后放冰箱保存。
4.根据权利要求3所述产细菌纤维素的细菌,其特征在于,具体方法为:
取5g自然腐烂的杨桃放入装有50mL富集培养基的并带有玻璃珠的300mL三角瓶中,30℃,150r/min震荡12h,取0.1mL菌悬液于10mL灭菌离心管中,进行梯度稀释并涂布到分离培养基上,30℃倒置恒温培养,待长出黄色且有透明圈的菌落后再次稀释涂布纯化,之后将单一菌落接种到液体种子培养基中,30℃、150r/min过夜培养后以5%(v/v)接种量接种于发酵培养基中,30℃静置培养确定该菌株是否产膜,纯化复筛后,挑选生长良好,产细菌纤维素膜的目标菌落转接至试管斜面中,30℃培养3天后于4℃下保存。
5.根据权利要求3或4所述产细菌纤维素的细菌,其特征在于,所述富集培养基的组成为:蛋白胨5g/L,酵母浸粉5g/L,葡萄糖5g/L,甘露醇5g/L,无水硫酸镁1g/L,pH 6.0±0.2;
所述分离培养基的组成为:酵母浸粉10g/L,葡萄糖10g/L,碳酸钙20g/L,溴甲酚紫0.015g/L,琼脂20g/L,pH 6.0±0.2。
6.根据权利要求3或4所述产细菌纤维素的细菌,其特征在于,所述试管斜面培养基的组成为:富集培养基加入20g/L琼脂,pH 6.0±0.2。
7.根据权利要求3或4所述产细菌纤维素的细菌,其特征在于,所述液体种子培养基的组成为:蛋白胨5g/L,酵母浸粉5g/L,葡萄糖5g/L,甘露醇5g/L,无水硫酸镁1g/L,pH 6.0±0.2。
8.根据权利要求3或4所述产细菌纤维素的细菌,其特征在于,所述发酵培养基的制备方法为:950mL沸水中加入50g蔗糖,蔗糖溶解后,加入约5g的红茶包继续煮沸10min,取出茶包后,加入50mL白醋,冷却至室温。
9.权利要求1所述产细菌纤维素的细菌应用于发酵细菌纤维素。
10.根据权利要求9所述应用,其特征在于,所述细菌纤维素采用以下方法发酵生产:
将所筛选菌种接种到液体种子培养基中,在摇床中30℃,150rpm震荡培养24h,以5%(v/v)接种量接种到发酵培养基中,在恒温培养箱中30℃静置培养7天,待液体表面长出纤维素膜,将膜取出,在0.3mol/L NaOH溶液中加热煮沸30min,以去除残留的菌体和杂质,然后用清水漂洗直至膜呈中性。
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