CN117063945B - 一种促进沉香树结香的生物化学诱导剂 - Google Patents
一种促进沉香树结香的生物化学诱导剂 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
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Abstract
本发明属于生物工程技术领域，涉及一种促进沉香树结香的生物化学诱导剂。所述生物化学诱导剂包括化学组分和生物活性组分，其中，化学组分按质量百分比计包括：无机盐0.1％～2％、植物激素0.02％～0.05％、氧化剂0.1％～0.3％、有机酸0.05％～0.1％、增稠剂0.5％～1％和水余量；生物活性组分的添加量为化学组分体积的10％～50％。通过化学组分与生物活性组分相结合，能够有效提升醇溶性浸出物及沉香四醇的含量，在提高结香产量和品质的基础上，够缩短结香时间，为沉香的生产提供了技术支持，具有优异的经济效益。
Description
技术领域
本发明属于生物工程技术领域，更具体的说是涉及一种促进沉香树结香的生物化学诱导剂。
背景技术
沉香是由瑞香科沉香属树木木质部受损伤或受外界刺激后，分泌的一种带有香气的树脂，是一种珍贵的药材和天然香料，具有抗菌，止咳化痰，暖胃温脾，平喘等药用，有“药中黄金”和“香中之王”的美称。同时可用作香料，工艺品原料，有较大的现代经济价值。
由沉香形成的环境条件可分为自然结香和人工结香。自然沉香是由野生的健康的白木香在受到电击、虫咬、狂风等外界刺激下，在所产生的损伤部位分泌树脂，形成沉香。而人工沉香采用火烧、刀砍斧劈、凿洞等物理方式，或者采用化学诱导、真菌诱导的方式，对白香木施加刺激，从而刺激白香木结香。化学诱导为目前应用最广泛的促进沉香树结香的方法。目前的化学结香剂的成分主要包括植物激素、无机盐、有机酸，及其他刺激物，如过氧化氢等。化学结香剂主要利用化学试剂对沉香树的刺激作用，促使沉香树结香。真菌诱导促进结香的方式没有化学诱导应用广，通常采用在凿洞法的基础上接种真菌，通过液体菌剂滴注的方式，将菌液输入白香木木质组织，通过真菌侵染沉香树，促使其产生植物抗毒素，产生树脂形成结香。沉香树结香是感应刺激、发生防御反应并产生防御物质的动态过程，如何提供一种能更好的促进沉香树结香的诱导剂，缩短结香时间，提高沉香的质量和产量，实现高效结香，是本领域技术人员亟需解决的技术问题。
发明内容
为解决上述技术问题，本发明提供了一种促进沉香树结香的生物化学诱导剂。通过化学组分与生物活性组分相结合，能够有效提升醇溶性浸出物及沉香四醇的含量，在提高结香产量和品质的基础上，够缩短结香时间，为沉香的生产提供了技术支持，具有优异的经济效益。
为实现上述目的，本发明提供了如下技术方案：
本发明技术方案之一：提供一种促进沉香树结香的生物活性组分，包括烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌，以及所述烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌的代谢产物。
本发明技术方案之二：提供一种上述促进沉香树结香的生物活性组分的制备方法，包括：
将烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌按等质量比混合混匀得到真菌混合菌；
使用沉香树液体培养基对于所述真菌混合菌在28℃条件下扩大培养14～21天，得到的菌体及发酵代谢产物的混合物，即为所述生物活性组分。
进一步的，所述沉香树液体培养基的制备方法包括：将未结香白木香碎木块100g加入1000mL水中煮沸30min或在121℃条件下高压灭菌10min后，经过滤、调节pH值至7、定容至1000mL后，于121℃高压灭菌16min，冷却至室温后加入硫酸链霉素与氯霉素即得所述沉香树液体培养基。
进一步的，所述真菌混合菌的均含量不低于5×108cfu/g。
本发明所选的菌种能够在化学组分与苄基丙酮条件下正常生长。
本发明技术方案之三：提供一种促进沉香树结香的生物化学诱导剂，包括化学组分和上述生物活性组分；
所述化学组分按质量百分比计包括：无机盐0.1％～2％、植物激素0.02％～0.05％、氧化剂0.1％～0.3％、有机酸0.05％～0.1％、增稠剂0.5％～1％和水余量；
所述生物活性组分的添加量为所述化学组分体积的10％～50％。
进一步的，所述无机盐包括CaCl2、NaHSO3、NaCl和FeSO4中的至少一种。
进一步的，所述植物激素包括茉莉酸甲酯、脱落酸、6-苄基嘌呤和乙烯利中的至少一种。
进一步的，所述氧化剂包括氧化剂可以是FeCl3、过氧化氢和过氧乙酸中的至少一种。
进一步的，所述有机酸包括水杨酸、甲酸和乙酸中的至少一种。
进一步的，所述增稠剂包括明胶和/或琼脂。
本发明技术方案之四：提供一种促进沉香树结香的生物化学诱导剂的制备方法，包括：
将无机盐和水混合制备成盐溶液后加入氧化剂和有机酸制备得到溶液A；
将生物活性组分浓缩后与所述溶液A混合均匀，再加入增稠剂，搅拌均匀，得到溶液C；
在所述溶液C中加入植物激素，混合均匀后即得促进沉香树结香的生物化学诱导剂。
进一步的，所述浓缩的体积倍数为5～10倍。
本发明技术方案之五：提供一种上述生物化学诱导剂在人工结香中的应用。
进一步的，所述应用的方法为：选取离地30cm处的树干中部钻孔，用注射法将上述促进沉香树结香的生物化学诱导剂注射到沉香树树干的孔洞中，并封住孔洞，每3个月补注一次，首次注射1年后，收集沉香树分泌的树脂，结为沉香。
进一步的，所述注射的量为500～1000mL/次。
沉香形成机理是健康的白木香树干在意外受到损伤或者外界刺激情况下，细胞木质部会分泌一些具有黄褐色芳香气味的防御性树脂，进而在特定部位形成了黑色树脂，也就是沉香。白木香在自然状态下也能结香，在自然状态下树体受到意外伤害，可能会引起树脂的分泌，真菌的感染会加速和促进这个过程，但是自然状态下生成沉香的过程最少也要10年以上。
沉香实际上树体病变的产物，有观点认为真菌菌丝侵入木质部，可诱发植物体的免疫防御机制，导管及射线细胞营养输送渠道被堵塞，营养的减少使得菌丝侵入新细胞的速度放缓，最终形成沉香物质。植物激素等化学物质能够诱导白木香结香，通过植物激素对创口附近周边细胞的刺激作用，激活细胞的生长和分裂，进而增加含树脂在内的次生代谢产物的分泌增加，促进结香。
本发明制备的生物化学诱导剂，结合了化学诱导结香和真菌诱导结香的原理，通过对沉香树结香部位施用化学诱导成分与能够在苄基丙酮和化学诱导成分条件下正常生长的真菌，以及与真菌的发酵代谢产物的协同作用下，促进了沉香树结香，提升了结香速度与品质。
经由上述的技术方案可知，与现有技术相比，具有如下有益效果：
本发明通过将化学组分与生物活性组分复配得到生物化学诱导剂应用于人工结香能够有效提升沉香的产量和品质。
本发明增稠剂的添加，可以延长诱导剂中有效成分在结香部位的滞留时间，达到长期刺激木质部薄壁细胞产生免疫反应的目的，从而提高树脂的分泌量。
本发明的促进沉香结香的生物化学诱导剂可满足实际生产和市场需求，可用于大规模、标准化、商品化的生产，可提高沉香产量和结香品质，具有重要的经济效益。
附图说明
构成本申请的一部分的附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1为实施例1制备的结香诱导剂注射1个月后白木香树干部位结香图。
图2为对比例3中pH为7的缓冲液注射1个月后白木香树干部位结香图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例和对比例中所用菌种均通过购买途径得到，其中烟草镰刀菌购自中国林业微生物菌种保藏管理中心，保藏编号为cfcc 89013，菌种量109cfu/g；梅林青霉购自中国普通微生物菌种保藏管理中心，保藏编号为CGMCCNo.3.3262，菌种量6×108cfu/g；平滑正青霉菌购自上海生物保藏生物技术中心，保藏编号为SHBCC D23435，菌种量109cfu/g；淡紫拟青霉菌购自上海生物保藏生物技术中心，保藏编号为SHBCC D82309，菌种量5×108cfu/g；哈茨木霉菌购自上海生物保藏生物技术中心，保藏编号为SHBCC D65214，菌种量6×108cfu/g；尖孢镰孢菌购自上海生物保藏生物技术中心，保藏编号为SHBCC D68671，菌种量5×108cfu/g。
实施例和对比例中使用的生物活性组分的制备方法为：将烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌按照等质量比混合后，使用沉香树液体培养基在28℃条件下扩大培养14天，得到的菌体及发酵代谢产物的混合物，即为生物活性组分。
沉香树液体培养基的制备方法包括：将未结香白木香碎木块100g加入1000mL水中煮沸30min，经过滤、调节pH值至7、定容至1000mL后，于121℃高压灭菌16min，冷却至室温后加入硫酸链霉素与氯霉素即得所述沉香树液体培养基。
实施例1
促进沉香树结香的生物化学诱导剂的制备：
按质量百分比称取CaCl2 1％、茉莉酸甲酯0.02％、FeCl3 0.05％、水杨酸0.1％、琼脂1％和水余量。
称取水体积50％的生物活性组分。
将CaCl2和水混合制备成CaCl2水溶液，然后加入FeCl3和水杨酸制备得到溶液A；
将生物活性组分浓缩10倍后与溶液A混合均匀，再加入琼脂，搅拌均匀，得到溶液C；
在溶液C中加入茉莉酸甲酯，混合均匀后即得促进沉香树结香的生物化学诱导剂。
实施例2
促进沉香树结香的生物化学诱导剂的制备：
按质量百分比称取FeSO4 2％、6-苄基嘌呤0.03％、过氧乙酸0.3％、乙酸0.08％、琼脂1％和水余量。
称取水体积10％的生物活性组分。
将CaCl2和水混合制备成CaCl2水溶液，然后加入FeCl3和水杨酸制备得到溶液A；
将生物活性组分浓缩7倍后与溶液A混合均匀，再加入琼脂，搅拌均匀，得到溶液C；
在溶液C中加入茉莉酸甲酯，混合均匀后即得促进沉香树结香的生物化学诱导剂。
实施例3
促进沉香树结香的生物化学诱导剂的制备：
按质量百分比称取NaHSO3 0.1％、脱落酸0.05％、过氧化氢0.1％、甲酸0.05％、明胶0.5％和水余量。
称取水体积30％的生物活性组分。
将CaCl2和水混合制备成CaCl2水溶液，然后加入FeCl3和水杨酸制备得到溶液A；
将生物活性组分浓缩5倍后与溶液A混合均匀，再加入明胶，搅拌均匀，得到溶液C；
在溶液C中加入茉莉酸甲酯，混合均匀后即得促进沉香树结香的生物化学诱导剂。
对比例1
与实施例1相比，区别仅在于，未加入生物活性组分。
对比例2
与实施例1相比，区别仅在于，未加入琼脂。
对比例3
使用pH为7的缓冲液代替生物化学诱导剂。
对比例4
与实施例1相比，区别仅在于，将生物活性组分替换为未经扩大培养的六种生物菌的混合菌与溶液A混合。
试验例
选取七棵3年树龄的白木香树，从距离地面30cm处，用电钻进行钻孔，钻孔直径0.5cm，钻孔深度3cm，以螺旋状排列的方式钻6个孔。将实施例1～3与对比例1～4制备的诱导剂利用树木营养针向钻孔中注射1000mL，每3个月重复注射一次，首次注射12个月后，割取树干中棕褐色及黄褐色的变色木质部阴干后得到沉香。
对注射实施例1～3、对比例1～4得到的沉香进行醇溶性浸出物含量和沉香四醇相对含量进行检测：
醇溶性浸出物含量的检测：
参照2015年《中华人民共和国药典》所载醇溶性浸出物测定法制取沉香油，并对所得沉香样品的醇溶性浸出物含量进行了测定。取沉香磨成粉末状，称取沉香粉末2g于250mL的圆底烧瓶中，加入95％乙醇50mL，塞紧，称定重量，静置1小时萃取醇溶性物质。连接好蒸馏装置，加热至沸腾，并保持微沸1小时。待冷却后取下烧瓶，塞紧，称定重量，用95％乙醇补足减失的重量，摇匀，用干燥滤器滤过，即得沉香油。量取滤液25mL，置于蒸发皿中，水浴蒸干。105℃干燥3小时，移至干燥器中，冷却30分钟，记录精油重量，计算醇溶性浸出物的含量(质量分数)。
沉香四醇相对含量的检测：
方法参照2015年《中华人民共和国药典》所载HPLC方法测定。称取沉香粉末0.2g于离心管中，加入乙醇10mL，溶解1小时。超声处理(功率250W，频率40kHZ)破碎细胞，用乙醇补齐损失的质量。取样品10uL注入液相色谱仪，采用Agilent-C18柱进行分离检测，流动相A设置为乙腈，流动相B设置为0.1％的甲酸溶液，按下表中程序设置进行梯度洗脱；流速为每分钟0.7mL；柱温为30℃，检测波长为252nm。理论板数按沉香四醇峰计算应不低于6000。
检测结果如表1所示。
表1
由以上表数据可知，本发明实施例1～3中沉香醇溶性浸出物含量均高于2015版《中国药典》要求水平(醇溶性浸出物含量不得低于10％)。实施例1～3与对比例1～4相比，实施例1～3的醇溶性浸出物和沉香主要成分沉香四醇含量均高于对比例1～4。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (1)
1.一种促进沉香树结香的生物化学诱导剂，其特征在于，所述生物化学诱导剂由化学组分和生物活性组分组成；
所述化学组分按质量百分比计为：CaCl2 1%、茉莉酸甲酯 0.02%、FeCl3 0.05%、水杨酸0.1%、琼脂1%和水余量；
所述生物活性组分由烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌，以及所述烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌的代谢产物组成；
所述生物活性组分的制备方法为：将烟草镰刀菌、梅林青霉、平滑正青霉菌、淡紫拟青霉菌、哈茨木霉菌和尖孢镰孢菌按等质量比混合混匀得到真菌混合菌；使用沉香树液体培养基在28℃条件下对于所述真菌混合菌扩大培养14天，得到混合菌体及发酵代谢产物的混合物，即为所述生物活性组分；
所述沉香树液体培养基的制备步骤为：将未结香白木香碎木块100g加入1000mL水中煮沸30min，经过滤、调节pH值至7、定容至1000mL后，于121℃高压灭菌16min，冷却至室温后加入硫酸链霉素与氯霉素即得所述沉香树液体培养基；
所述生物活性组分的添加量为所述化学组分体积的50%；
所述的促进沉香树结香的生物化学诱导剂的制备步骤为：
将CaCl2和水混合制备成盐溶液后加入FeCl3和水杨酸制备得到溶液A；
将生物活性组分浓缩10倍后与所述溶液A混合均匀，再加入琼脂，搅拌均匀，得到溶液C；
在所述溶液C中加入茉莉酸甲酯，混合均匀后即得所述促进沉香树结香的生物化学诱导剂；
所述促进沉香树结香的生物化学诱导剂在人工结香中的应用方法为：选取离地30cm处的树干中部钻孔，用注射法将所述促进沉香树结香的生物化学诱导剂注射到沉香树树干的孔洞中，并封住孔洞，每3个月补注一次，首次注射1年后，收集沉香树分泌的树脂，即为沉香；
所述注射的量为500~1000mL/次。
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