CBD and CBDA Contents in Fermented Cannabis sativa L.: Study of Dynamic Changes

GUAN Xin, QI Kexiang, LI Wanru, ZHANG He, JIANG Shuo, WU Tong, ZHENG Chunying

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Chinese Agricultural Science Bulletin ›› 2023, Vol. 39 ›› Issue (21) : 144-150. DOI: 10.11924/j.issn.1000-6850.casb2022-0993

CBD and CBDA Contents in Fermented Cannabis sativa L.: Study of Dynamic Changes

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Abstract

The aim is to study the dynamic changes of CBD and CBDA contents of Cannabis sativa L. after fermentation, clarify the correlation between CBD and CBDA, and provide reference for efficient production of CBD raw materials. With CBD and CBDA as indexes, Cannabis sativa L. were fermented by Saccharomyces cerevisiae, Lactobacillus plantarum, Escherichia coli and endophytic bacterium WF17, respectively. The dynamic changes of CBD and CBDA contents of Cannabis sativa L. before and after fermentation were analyzed by HPLC method. The CBD content reached the maximum value (3.1161 mg/g) on the first day after the fermentation of Cannabis sativa L. by Saccharomyces cerevisiae. The CBD content reached the maximum value (3.7786 mg/g) on the 9th day after Lactobacillus plantarum fermentation of Cannabis sativa L.. The content of CBD reached the maximum value (3.5502 mg/g) at 3 d after fermentation of Cannabis sativa L. by Escherichia coli. After the fermentation of Cannabis sativa L. by endophytic bacteria WF17, the CBD content reached the maximum value (3.9182 mg/g) at the fermentation time of 7 days. The content of CBDA decreased the most in the first day of fermentation. All the four fermentation strains could significantly increase the CBD content and decrease the CBDA content in the fermented Cannabis sativa L. to a certain extent, and there might be a process of CBDA to CBD in the fermentation process of Cannabis sativa L.. Therefore, fermentation technology can be used for efficient production of CBD in Cannabis sativa L..

Key words

Cannabis sativa L. / cannabidiol / cannabidiolic / fermentation / HPLC

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GUAN Xin , QI Kexiang , LI Wanru , ZHANG He , JIANG Shuo , WU Tong , ZHENG Chunying. CBD and CBDA Contents in Fermented Cannabis sativa L.: Study of Dynamic Changes. Chinese Agricultural Science Bulletin. 2023, 39(21): 144-150 https://doi.org/10.11924/j.issn.1000-6850.casb2022-0993

0 引言

大麻(Cannabis satival L.)又称汉麻,属于大麻科(Cannabinaceae)大麻属(Cannabis)一年生草本植物[1],其植株含有精神活性成分Δ9-四氢大麻酚(Δ9-THC),是毒品原植物之一。为了便于管理,国际上将Δ9-THC含量低于0.3%的品种称为工业大麻[2]。大麻含有大麻酚类、黄酮类、生物碱等多种成分[3],作为大麻的专属性成分,大麻酚类化合物是目前的研究热点,其中,具有代表性的一种化合物为大麻二酚(CBD)[4]。该化合物是工业大麻中非成瘾性物质,具有抗炎[5]、抗菌[6]、抗惊厥[7]、抗氧化[8]、抗癌[9]等作用,并且对治疗神经系统疾病、心血管疾病等[10]具有显著疗效,可以有效地消除THC对人体产生的致幻作用,被称为“反毒品化合物”,是重要的食品、药品生产原料[11]
CBD最初是从工业大麻植株中提取分离得到的,研究其生物合成途径时发现,CBD前体化合物为CBDA,CBDA在CBD合酶作用下,通过脱去一分子CO2后生成CBD[12]。为了高效提取CBD,科研人员利用酶促法试图将CBDA转化为CBD,但酶促反应要求严格,不适合工业化生产[13]。此外,从工业大麻原料中提取CBD时,其提取分离工艺复杂,得率及纯度较低[14]。随着对CBD需求量的增加,研究学者开始致力于CBD的化学合成,其经典合成方法主要以BF3作为催化剂,以薄荷-2,8-二烯-1-醇与5-戊基-1,3-苯二酚作为原料,经过缩合反应后,进一步得到目标产物CBD[15]。化学合成虽然也能得到CBD原料,但在合成过程中,往往伴有副产物产生,从而使CBD纯度降低,因此其产量依然难以满足生产需求。
随着发酵技术在食品及中草药中的广泛应用,微生物发酵可以使底物中的活性成分发生增减变化,可以定向提高食品及中草药中某种主要活性成分含量,从而提高其活性成分的利用度[16]。郑春英等[17]以乳酸杆菌HD11对中药刺五加进行发酵后,其有效成分异嗪皮啶的含量明显提高,而且在其附近出现了新物质;王宇晴等[18]以甘草内生真菌RE4-RE11混合发酵甘草后,甘草次酸产量显著提高;许哲祥等[19]采用五味子内生真菌WJ1发酵五味子后,五味子中4种主要木脂素成分含量均较发酵前得到了显著提高。本研究采用微生物发酵技术对工业大麻进行发酵研究,观察工业大麻发酵后其CBD和CBDA的含量动态变化,确定微生物发酵对CBD和CBDA的影响,旨在采用发酵技术提高工业大麻中CBD的含量,为高效生产CBD原料提供参考。

1 材料与方法

1.1 材料与试剂

工业大麻(Cannabis sativa L.):‘龙麻5号'(采集地点:黑龙江省农科院绥化分院科技创新农场)。
发酵菌株:酿酒酵母(Saccharomyces cerevisiae)、植物乳杆菌(Lactobacillus plantarum)、大肠杆菌(Escherichia coli)(购自黑龙江省科学院微生物研究所)以及五味子内生细菌WF17(Bacillus subtilis)(菌种保藏号:CCTCC No:M 2011079)。
其他试剂均为色谱纯。

1.2 仪器与设备

主要实验仪器见表1
表1 实验主要仪器
仪器 型号规格 生产厂家
电子精密天平 PL303 梅特勒-托利多仪器有限公司
超净工作台 SZX 南通科学仪器有限公司
空气浴振荡器 HZQ-C 哈尔滨市东联电子技术开发有限公司
旋转蒸发仪 R-201 上海申胜生物技术有限公司
超声波清洗器 KQ-100DE 昆山市超声仪器有限公司
紫外可见波长检测器 FL2000 浙江温岭福立分析仪器有限公司
高效液相色谱仪 FL2000 浙江温岭福立分析仪器有限公司
400MH固体超导核磁共振波谱仪 AVANCE Ⅲ 美国Thermo Fisher公司
酵母蛋白胨葡萄糖培养基(YEPD):参阅文献[20]。配制方法如下:精密称取酵母膏10 g,蛋白胨20 g,葡萄糖20 g,琼脂粉20 g。分别将上述培养基中各组分放置于沸水中,待其完全溶解后,以蒸馏水将培养基总体积补至1 L,酸碱值调节至pH 7.1左右,分装,进行高压灭菌处理(121℃,30 min),取出,冷却,备用。
水杨素培养基(MRS):参阅文献[21]。配制方法如下:精密称取蛋白胨10 g,葡萄糖20 g,牛肉膏10 g,酵母膏5 g,磷酸氢二钾2 g,柠檬酸氢二铵2 g,乙酸钠5 g,MgSO4·7H2O 2 g,MnSO4·4H2O 0.05 g,琼脂粉20 g。分别将上述培养基中各组分放置于沸水中,待其完全溶解后,加入吐温-80 1 mL,以蒸馏水将培养基总体积补至1 L,调节pH 6.2~6.4,分装,进行高压灭菌处理(121℃,30 min),取出,冷却,备用。
牛肉膏蛋白胨固体培养基(NA):参阅文献[22]。配制方法如下:精密称取牛肉膏3 g,蛋白胨10 g,NaCl 5 g,琼脂粉20 g。分别将上述各培养基组分放置于沸水中,待其完全溶解后,以蒸馏水将培养基总体积补至1 L,将酸碱值调节至pH 7.1左右,分装,进行高压灭菌处理(121℃,30 min),取出,冷却,备用。

1.3 方法

1.3.1 菌悬液的制备

分别取上述的发酵菌株进行活化。无菌条件下,将酿酒酵母接种于YEPD斜面培养基,恒温培养(28℃,1 d);将植物乳杆菌接种于MRS斜面培养基,恒温培养(37℃,1 d);分别将大肠杆菌、五味子内生细菌WF17接种于NA斜面培养基,恒温培养(37℃,2 d)。
取上述活化后的各菌株(无菌条件下),分别加入5 mL无菌水,充分振荡摇匀后,于显微镜下观察计数(血球计数板计数法),将菌体浓度按比例稀释为1×107 CFU/mL,作为菌悬液,备用。

1.3.2 发酵样品的制备

取干燥的工业大麻叶,粉碎,过40目筛。精确称取10 g,置于100 mL的三角瓶内,加水(料液比为1:10),密封后,进行高压灭菌处理(121℃,30 min),取出,作为底物。
分别取“1.3.1”项下的各菌悬液5 mL,加入到工业大麻底物中(平行样品3份),置于空气浴摇床中培养(真菌:28℃,130 r/min;细菌:37℃,125 r/min),取出,冻干,作为工业大麻发酵样品,备用。

1.3.3 工业大麻发酵后CBD及CBDA含量的动态变化

参阅文献[23],采用HPLC法,以CBD的含量为指标,分别对不同菌株发酵工业大麻后CBD的含量变化进行分析检测。
(1)色谱条件
色谱条件见表2
表2 色谱条件
参数 设置条件
色谱柱 Venusil XBP-C18柱(4.6 mm×250 mm,5 μm,USA)
流动相及波长 ①号流动相:乙腈-0.1%甲酸水溶液(75:25),220 nm;
②号流动相:甲醇-乙腈-0.1%磷酸水溶液(40:30:30),254 nm
流速 1 mL/min
柱温 25℃
进样量 10 μL
(2)对照品溶液的制备
分别精密称取CBD、CBDA对照品,采用色谱甲醇配成每1 mL含0.1 mg的对照品溶液,备用。
(3)供试品溶液的制备
精密称取“1.3.2”项下的发酵样品1.0 g,加入95%乙醇(料液比1:50),以50℃超声提取40 min,过滤,减压浓缩至近干(50℃),适量蒸馏水溶解,以乙酸乙酯进行萃取(3×100 mL),合并萃取液,减压回收(50℃),残渣以1 mL色谱甲醇溶解,作为供试品溶液,备用。
(4)线性关系考察
分别精确吸取“1.3.3(2)”项中所述的CBD、CBDA对照品溶液,按一定梯度比例,制备得到系列浓度的各对照品溶液。在“1.3.3(1)”项的色谱条件下,各吸取10 μL进样检测。以各对照品溶液的系列质量浓度(mg/mL)为横坐标(X),峰面积为纵坐标(Y),绘制标准曲线。
(5)方法学考察
参阅文献[24]。分别进行日内稳定性实验、日间稳定性实验、重现性实验、精密度实验以及加样回收率实验。

1.3.4 样品测定

分别对“1.3.2”项中不同发酵菌株制备的发酵样品,在“1.3.3(1)”项的色谱条件下,分别精密吸取(2)项下对照品溶液和(3)项下供试品溶液各10 μL,进行HPLC分析(n=3)。

2 结果与分析

2.1 工业大麻发酵后CBD及CBDA含量的动态变化结果

2.1.1 色谱条件下CBD和CBDA的分离结果

表1①项色谱条件下,供试品中CBD得到较好的分离;在表1②项色谱条件下,供试品中CBDA得到较好的分离,其结果见图1图2
图1 色谱条件下CBD的分离
A为生药样品;B为CBD对照品

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图2 色谱条件下CBDA的分离
A为生药样品;B为CBDA对照品

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2.1.2 线性关系考察结果

CBD、CBDA的线性关系考察结果见表3
表3 线性关系考察结果
组分 回归方程 相关系数r 线性范围/(mg/mL)
CBD Y=6×106X+264479 0.9998 0.0625~1.00
CBDA Y=8.5198×105X+47651 0.9998 0.0625~1.00
实验结果表明,在0.0625~1.00 mg/mL的浓度范围内,CBD和CBDA这2种对照品的质量浓度与峰面积均呈现良好的线性关系。

2.1.3 方法学考察结果

方法学考察结果见表4
表4 方法学考察结果
日内稳定性 日间稳定性 精密度 重现性 平均加样回收率(101.28%)
CBD 1.47% 1.40% 1.25% 2.08% 1.79%(n=6)
CBDA 1.45% 1.41% 1.24% 2.09% 1.77%(n=6)
上述结果表明该方法稳定、重现性高,能够满足定量分析的要求。

2.1.4 样品测定结果

采用HPLC法测定工业大麻发酵后其CBD及CBDA的含量动态变化,结果见图3图4
图3 工业大麻发酵前后CBD的变化
A为工业大麻发酵样品;B为CBD对照品;C为工业大麻生药样品(发酵菌株为内生细菌WF17)

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图4 工业大麻发酵前后CBDA的变化
A为工业大麻发酵样品;B为CBDA对照品;C为工业大麻生药样品(发酵菌株为内生细菌WF17)

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图3图4可知,工业大麻发酵后其CBD含量升高,其CBDA含量降低。上述结果说明,在工业大麻在发酵过程中,存在着物质间的相互转化。

2.1.5 发酵菌株对工业大麻中CBD及CBDA的影响结果

工业大麻经酿酒酵母(Saccharomyces cerevisiae)发酵后,分别于不同发酵时间内取样测定其CBD及CBDA的动态变化,结果见图5
图5 Saccharomyces cerevisiae发酵工业大麻后CBD及CBDA动态变化

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酿酒酵母发酵工业大麻后CBD含量在1 d时达到最大值(3.1161 mg/g),此后,随着发酵时间的增加,其CBD含量不再增加,并出现降低趋势,这可能是CBD成分不稳定造成的。而酿酒酵母发酵工业大麻后CBDA含量急剧下降,尤其在发酵第1 d内降低幅度最大,这可能是发酵菌株将其降解所造成的。
工业大麻经植物乳杆菌(Lactobacillus plantarum)发酵后,分别于不同发酵时间内取样测定其CBD及CBDA含量的动态变化,结果见图6
图6 Lactobacillus plantarum发酵工业大麻后CBD及CBDA动态变化

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植物乳杆菌发酵工业大麻后,CBD含量出现先增高后降低的趋势,在第9天时达到最大值(3.7786 mg/g);而CBDA含量随着发酵时间的增加出现逐渐降低的趋势,尤其是在第1天内降低幅度最大。
工业大麻经大肠杆菌(Escherichia coli)发酵后,分别于不同发酵时间内取样测定其CBD及CBDA含量的动态变化,结果见图7
图7 Escherichia coli发酵工业大麻后CBD及CBDA动态变化

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大肠杆菌发酵工业大麻后,其CBD含量随着发酵时间的增加出现先增高后降低的趋势,当发酵3 d时,其CBD含量达到最大值(3.5502 mg/g);而CBDA含量随着发酵时间的增加出现逐渐下降的趋势,尤其在发酵第1天时降低幅度最大。
工业大麻经五味子内生细菌WF17(Bacillus subtilis)发酵后,分别于不同发酵时间内取样测定其CBD及CBDA的含量动态变化,结果见图8
图8 Bacillus subtilis (WF17)发酵工业大麻后CBD及CBDA动态变化

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内生细菌WF17发酵工业大麻后,其CBD含量随着发酵时间的增加出现先增加后降低的趋势,当发酵时间为7 d时,其含量达到最大值(3.9182 mg/g);而CBDA含量则表现下降,尤其在发酵第1天时降低幅度最大。
图5图8可以看出,随着发酵时间的增加,发酵工业大麻样品中CBD的含量先增加后降低,而CBDA的含量则出现骤降并趋于平稳的趋势。出现这样趋势的原因可能是上述发酵菌株将底物中CBDA转化为CBD所造成的,并且随着发酵时间的增加,由于CBD不稳定,出现含量降低的趋势。

2.1.6 4种不同菌株发酵工业大麻后CBD的含量结果

以工业大麻为底物,分别以上述4种不同菌株进行发酵,其CBD的含量结果见图9。上述4种发酵菌株均能使工业大麻中CBD含量存在不同程度的提高,经过对比分析,内生细菌WF17的发酵效果最好。因此,可以采用内生细菌WF17作为发酵菌株对工业大麻进行发酵,以期可以最大限度提高工业大麻中CBD的含量。
1:Saccharomyces cerevisiae,2:Lactobacillus plantarum,3:Escherichia coli,4:Bacillus subtilis

图9 4种不同菌株发酵工业大麻后CBD的含量结果

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3 讨论与结论

目前,CBD原料的生产主要包括从高CBD、低四氢大麻酚(THC)的工业大麻植株中提取分离CBD;或采用化学合成的方法生产CBD,但CBD产率依然是亟待解决的问题。本研究采用微生物发酵工业大麻,旨在提高CBD的含量以高效生产CBD原料,并最大限度利用工业大麻资源。关于采用微生物发酵法生产宿主植物次生代谢产物的研究有着悠久的历史,CHEN等[25]研究发现,利用任何4种不同乳杆菌和双歧杆菌发酵豆奶后,其大豆异黄酮的水解均有显著增加,苷元含量从起初的36%增加到90%以上;孟欣桐等[26]研究发现,使用香菇、灵芝、姬松茸和桑黄等不同的药(食)用真菌发酵黄芪,均可以将黄芪甲苷转化成异黄芪甲苷。上述研究说明,采用微生物发酵法可定向提高宿主植物中某种次生代谢产物含量,而关于采用微生物发酵法工业大麻的次生代谢产物研究还未见报道。
本研究采用4种目的菌株分别发酵工业大麻后,研究发现4种目的菌株均能在一定程度上使工业大麻中CBD的含量显著提高,而CBDA含量显著下降。工业大麻经酿酒酵母发酵后,其CBD含量在1 d时达到最大值(3.1161mg/g);工业大麻经植物乳杆菌发酵后,其CBD含量在第9天时达到最大值(3.7786 mg/g);工业大麻经大肠杆菌发酵后,其CBD含量在发酵3 d时达到最大值(3.5502 mg/g);工业大麻经内生细菌WF17发酵后,其CBD含量在发酵时间为7 d时达到最大值(3.9182 mg/g),而CBDA含量均在发酵第一天内大幅度降低。
上述说明,工业大麻在发酵过程中存在着次生代谢产物的增减变化,工业大麻在发酵过程中可能通过微生物转化的方式将CBDA及其他次生代谢产物转化为CBD,从而使发酵工业大麻中CBD含量显著增高。此外,通过发酵菌株的筛选,说明不同的发酵菌株,其转化底物的能力不同,在上述4种发酵菌株中,以内生细菌WF17的作用最为显著,在发酵7 d时其CBD含量可达到3.9182 mg/g。因此内生细菌WF17为CBD的最佳转化菌,后续可选取内生细菌WF17作为目的菌株,以该菌株发酵工业大麻,可高效生产CBD原料,最大限度利用工业大麻资源。此外,将工业大麻发酵药渣回收后可以进行微生物菌肥的制作[27],不仅有利于改善环境污染,还可以达到完善循环产业链的目的,从而降低工艺成本。

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