技术领域technical field
本发明属于烟叶产品技术领域，具体涉及一种以重庆奉节云烟87花蕾为原料提取精油的工艺及其作为抗氧化剂和抑菌剂的应用The invention belongs to the technical field of tobacco leaf products, in particular to a process for extracting essential oil from Chongqing Fengjie Yunyan 87 flower buds and its application as an antioxidant and antibacterial agent
背景技术Background technique
对于植物精油，国内外的研究不胜枚举。凌育赵研究了水蒸气蒸馏法提取柑皮中的香精油，发现该方法不但减少了能源消耗，而且具有安装操作简单、节约试剂、减少污染等优点。王虹等利用CO2作为萃取剂提取苍耳子油，并与水蒸汽蒸馏法提取的物质相对比发现该方法并不能完全取代水蒸汽蒸馏法。Vian等利用微波辅助水扩散重力法提取薄荷精油。该方法具有快速、高效、安全、节能的优点。陈丽艳等研究植物精油抗菌活性时发现多种植物精油都具有抗菌、杀菌作用，在临床医药中都有广泛的应用。For plant essential oils, there are too many studies at home and abroad. Ling Yuzhao studied the steam distillation method to extract essential oils from citrus peels, and found that this method not only reduces energy consumption, but also has the advantages of simple installation and operation, saving reagents, and reducing pollution. Wang Hong et al. used CO2 as the extraction agent to extract cocklebur seed oil, and compared it with the material extracted by steam distillation and found that this method could not completely replace the steam distillation. Vian et al. used microwave-assisted water diffusion gravity method to extract peppermint essential oil. The method has the advantages of rapidity, high efficiency, safety and energy saving. When Chen Liyan and others studied the antibacterial activity of plant essential oils, they found that many plant essential oils have antibacterial and bactericidal effects, and are widely used in clinical medicine.
烟草是一种有争议，但有巨大经济效益的农作物，也是世界上种植最广泛的非粮食作物，烟草种植与产品加工在国民经济发展中发挥着重要的作用。我国引种烟草很早，产量颇大。烟草废弃物是指烟草原叶在采收以及烟草制品加工过程中，未能应用于卷烟中的烟草组成部分(卷烟中包括膨胀烟丝和烟草薄片)，包括农业生产中废弃的烟杆、烟草植株上部烟叶、烟花、烟种、叶脉、腋芽，复烤中废弃的烟梗、以及无法进行卷烟加工的低等级或等级外的烟叶，卷叶企业积存的烟末、烟根、根出条等。到目前为止我国烟草的种植面积和产量均居世界首位，也意味着我国每年能够产生数十万吨的烟草废弃物，这些废弃物不但污染环境，更是对资源的巨大浪费。Tobacco is a controversial crop with huge economic benefits, and it is also the most widely planted non-food crop in the world. Tobacco cultivation and product processing play an important role in the development of the national economy. Tobacco was introduced very early in my country, and the output is quite large. Tobacco waste refers to the tobacco components that cannot be used in cigarettes during the harvesting of tobacco leaves and the processing of tobacco products (cigarettes include expanded tobacco and tobacco sheets), including discarded tobacco rods and tobacco plants in agricultural production. Upper tobacco leaves, fireworks, tobacco seeds, leaf veins, axillary buds, discarded tobacco stems during re-curing, and low-grade or non-grade tobacco leaves that cannot be processed into cigarettes, tobacco dust, tobacco roots, root strips, etc. accumulated by leaf rolling enterprises. So far, my country's tobacco planting area and output rank first in the world, which also means that my country can produce hundreds of thousands of tons of tobacco waste every year. These wastes not only pollute the environment, but are also a huge waste of resources.
国外对烟草花蕾的基因工程、生理活性、生物活性等领域有大量的研究，LiuBF等研究毛细管电泳色谱对烟草花植物激素的分离条件，结果表明，在最佳条件下，完全分离赤霉素、脱落酸、吲哚乙酸等七种植物激素只需要30min。国内方面，康武成等将烟草花蕾经酒精、蒸馏水萃取用来提取香膏，目前已形成白肋烟花蕾香膏、烤烟花蕾香膏和白肋烟浸膏等“三膏”产品，并已在卷烟加香中应用。许春平等采用超临界CO2萃取烤烟花蕾中的挥发性成分，并对其进行成分研究，结果表明，其中含有11种醇类，7种酯类，8种酸类等，另外，还研究了白肋烟花蕾多糖的抗氧化活性。There have been a lot of research abroad on the genetic engineering, physiological activity, and biological activity of tobacco flower buds. LiuBF and others studied the separation conditions of tobacco flower phytohormones by capillary electrophoresis chromatography. The results showed that under the optimal conditions, gibberellin, Seven kinds of plant hormones such as abscisic acid and indole acetic acid only need 30 minutes. Domestically, Kang Wucheng and others extracted tobacco buds with alcohol and distilled water to extract balm. At present, they have formed "three pastes" products such as Burley smoke bud balm, cured tobacco bud balm and Burley tobacco extract, and have been used in cigarettes. Used in perfuming. Xu Chunping used supercriticalCO2 to extract the volatile components in flue-cured tobacco flower buds, and carried out component research on it. The results showed that it contained 11 kinds of alcohols, 7 kinds of esters, 8 kinds of acids, etc. Antioxidant activity of polysaccharides from burley flower buds.
近年来，较我国目前对于烟叶的研究，烟草废弃物的研究主要集中在烟草废弃物中有效物质的提取，制造烟草薄片、膨胀梗丝以及造纸等方面，比如从废次的烟叶和烟梗中提取烟碱、茄尼醇，从烟杆和烟梗中提取果胶，从新鲜的烟杆中提取蛋白质，采用造纸法将整个卷烟生产过程中废弃的物质再次处理，制成薄片，并应于用卷烟生产中等等。近年来随着生产成本的增加，环境压力的增大，以及生物化学、分析化学、植物学、医学等学科的飞速发展和交叉发展，人们对精油的提取分离、组成以及生物活性等有了越来越深入的认识，人们逐渐认识到精油作为一种天然的植物产品，其具有抗氧化、抗衰老、抑菌等一系列的生理功能。In recent years, compared with the current research on tobacco leaves in my country, the research on tobacco waste mainly focuses on the extraction of effective substances in tobacco waste, the manufacture of tobacco sheets, expanded stems, and papermaking, such as from waste tobacco leaves and stems. Extract nicotine and solanesol, extract pectin from tobacco stems and tobacco stems, extract protein from fresh tobacco stems, use papermaking method to reprocess waste materials in the whole cigarette production process, make thin slices, and should be In the production of cigarettes and so on. In recent years, with the increase of production cost, the increase of environmental pressure, and the rapid development and cross-development of biochemistry, analytical chemistry, botany, medicine and other disciplines, people have made more and more progress in the extraction, separation, composition and biological activity of essential oils. With more and more in-depth understanding, people gradually realize that essential oil, as a natural plant product, has a series of physiological functions such as anti-oxidation, anti-aging, and antibacterial.
烟草花蕾精油作为一种从烟草废弃物中提取出来的的一种天然的生物活性物质，极具开发潜质，其应用和开发前景非常广阔。加强对烟草废弃物中有利人类方面的综合开发利用研究，同时重视对环境的影响，扬长避短，让烟草充分发挥对人类的贡献。对烟草花蕾精油的生物活性的研究，开发出一种新型的烟草终端产品，为烟草的综合利用提供了一条新的途径，并拓展烟草的应用范围。目前，我国尚未有对烟草花蕾精油的研究和相关报道。As a natural biologically active substance extracted from tobacco waste, tobacco bud essential oil has great potential for development, and its application and development prospects are very broad. Strengthen the comprehensive development and utilization of tobacco waste that is beneficial to human beings. At the same time, pay attention to the impact on the environment, maximize the strengths and avoid weaknesses, and let tobacco give full play to its contribution to mankind. The research on the biological activity of tobacco flower bud essential oil has developed a new type of tobacco terminal product, which provides a new way for the comprehensive utilization of tobacco and expands the application range of tobacco. At present, there is no research and related reports on tobacco bud essential oil in my country.
发明内容Contents of the invention
本发明的目的在于提供一种以重庆奉节云烟87花蕾为原料提取精油的工艺及其作为抗氧化剂和抑菌剂的应用。The object of the present invention is to provide a process for extracting essential oil from the flower buds of Yunyan 87 in Fengjie, Chongqing, and its application as an antioxidant and antibacterial agent.
为实现上述目的，本发明采用如下的技术方案To achieve the above object, the present invention adopts the following technical solutions
一种以重庆奉节云烟87花蕾为原料提取精油的工艺，其由下述步骤获得：A kind of technology that takes Chongqing Fengjie Yunyan 87 flower bud as raw material to extract essential oil, and it is obtained by following steps:
将粉碎后的重庆奉节云烟87花蕾按料液比1：10～1：30加入蒸馏水后，按烟草花蕾质量的2％～10％加入纤维素酶，按总质量的5％～25％加入氯化钠后，超声处理10～40min，然后采用水蒸气蒸馏2-6h制备花蕾精油。水蒸汽蒸馏法制备精油采用本领域常规技术即可。Add distilled water to crushed Chongqing Fengjie Yunyan 87 flower buds at a material-to-liquid ratio of 1:10 to 1:30, add cellulase at 2% to 10% of the mass of tobacco buds, and add chlorine at 5% to 25% of the total mass. After dissolving sodium, sonicate for 10-40 minutes, and then use steam distillation for 2-6 hours to prepare flower bud essential oil. The steam distillation method can be used to prepare the essential oil using conventional techniques in the art.
本发明提取工艺优选的技术方案为：料液比为1：15～1：25，5％～7％加入纤维素酶，10％～14％加入氯化钠，超声处理10～25min，水蒸气蒸馏4-5h。The preferred technical scheme of the extraction process of the present invention is: the ratio of solid to liquid is 1:15 to 1:25, 5% to 7% of cellulase is added, 10% to 14% of sodium chloride is added, ultrasonic treatment is performed for 10 to 25 minutes, and steam Distilled for 4-5h.
本发明提取工艺最优选的技术方案为：料液比为1：20，6％添加纤维素酶，10％添加氯化钠，超声处理15min，水蒸气蒸馏4h。冷却后开启分离器下端活塞，使水分缓慢流出。用移液枪收集油层，并用二氯甲烷冲洗提取器三次，将冲洗液与油层合并，加入无水硫酸钠过夜，除去水分后，低温旋转蒸发，除去二氯甲烷得到烟草花蕾精油。The most preferred technical scheme of the extraction process of the present invention is: the ratio of solid to liquid is 1:20, 6% of cellulase is added, 10% of sodium chloride is added, ultrasonic treatment is performed for 15 minutes, and steam distillation is carried out for 4 hours. After cooling, open the lower piston of the separator to allow the water to flow out slowly. Collect the oil layer with a pipette, rinse the extractor three times with dichloromethane, combine the washing liquid with the oil layer, add anhydrous sodium sulfate overnight, remove the water, and rotate it at low temperature to remove the dichloromethane to obtain tobacco bud essential oil.
本发明同时提供了一种以重庆奉节云烟87花蕾为原料提取精油的工艺制备得到的烟草花蕾精油。The invention also provides a tobacco flower bud essential oil prepared by extracting essential oil from the flower buds of Yunyan 87 in Fengjie, Chongqing.
本发明同时提供了所述的烟草花蕾精油作为抗氧化剂、抑菌剂的应用。The invention also provides the application of the tobacco bud essential oil as an antioxidant and antibacterial agent.
本申请采用超声酶解法提取重庆奉节云烟87烟草花蕾精油，并通过五水平的单因素试验，研究氯化钠浓度A、纤维素酶添加量B、料液比C、超声时间D四个因素对花蕾精油提取率的影响。然后采用正交法进行优化试验设计，得出超声酶解法提取烟草花蕾精油的最佳提取工艺条件，为氯化钠添加量为10％，纤维素酶添加量6％、料液比1：20、超声时间15min，并且在最佳工艺条件下，重庆奉节烟草花蕾精油的得率为0.566％(g/g)。此外，采用邻二氮菲法、清除超氧阴离子自由基的方法检测了重庆奉节云烟87花蕾精油的抗氧化活性，即其对OH自由基和O2-自由基的清除能力。采用滤纸片法和微量稀释法检测了重庆奉节云烟87花蕾精油的抑菌性能。This application uses ultrasonic enzymolysis to extract the essential oil of Chongqing Fengjie Yunyan 87 tobacco flower buds, and through a five-level single factor test, studies the effects of four factors: sodium chloride concentration A, cellulase addition B, solid-liquid ratio C, and ultrasonic time D. Effects on the extraction rate of essential oils from flower buds. Then, the orthogonal method was used to optimize the experimental design, and the optimal extraction process conditions for the extraction of essential oil from tobacco flower buds by ultrasonic enzymolysis were obtained. The addition of sodium chloride was 10%, the addition of cellulase was 6%, and the ratio of solid to liquid was 1:20. , Ultrasonic time 15min, and under the optimal process conditions, the yield of Chongqing Fengjie Tobacco flower bud essential oil is 0.566% (g/g). In addition, the antioxidant activity of Yunyan 87 flower bud essential oil in Fengjie, Chongqing, ie its ability to scavenge OH free radicals and O2 - free radicals was tested by the method of o-phenanthroline and superoxide anion free radical scavenging. The antibacterial activity of Yunyan 87 flower bud essential oil in Fengjie, Chongqing was tested by filter paper method and microdilution method.
和现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
1)经济效益分析：近年来，由于生物化学、分析化学、植物学、医学等学科的飞速发展和交叉发展，人们对精油的提取分离、组成以及生物活性等有了越来越深入的认识，烟草花蕾精油作为烟草废弃物中提取出来的一种生物活性物质，具有一定的生理作用，并且由于其制备的原料为完全废弃的烟草花蕾，资源较丰富且价格低廉，其应用和开发前景非常广阔。高纯度花蕾精油的制备在实验室里已经可以实现，通过研究重庆奉节云南87花蕾精油的提取工艺优化，并分析花蕾精油的组成、抗氧化、抑菌性质，开发出一种新型的烟草相关产品，为烟草的综合利用提供了一条新的途径，提高烟草废弃物利用价值，并拓展烟草的应用范围。除此之外，由于烟草花蕾精油的制备工艺流程简单，成本低廉，可应用于大规模工业化生产中，因此，烟草花蕾精油终端产品的开发具有非常可观的经济效益。1) Economic benefit analysis: In recent years, due to the rapid development and cross-development of biochemistry, analytical chemistry, botany, medicine and other disciplines, people have more and more in-depth understanding of the extraction, separation, composition and biological activity of essential oils. As a biologically active substance extracted from tobacco waste, tobacco flower bud essential oil has certain physiological effects, and because the raw material for its preparation is completely discarded tobacco flower buds, the resource is relatively abundant and the price is low, its application and development prospects are very broad . The preparation of high-purity flower bud essential oil has been realized in the laboratory. By studying the optimization of the extraction process of Yunnan 87 flower bud essential oil in Fengjie, Chongqing, and analyzing the composition, antioxidant and antibacterial properties of flower bud essential oil, a new type of tobacco-related products has been developed. , providing a new way for the comprehensive utilization of tobacco, improving the utilization value of tobacco waste, and expanding the application range of tobacco. In addition, because the preparation process of tobacco bud essential oil is simple, the cost is low, and it can be applied in large-scale industrial production. Therefore, the development of tobacco bud essential oil terminal products has very considerable economic benefits.
2)社会效益分析：目前，世界各国对烟草综合利用，尤其是对于烟草废弃无的研究开发方兴未艾，而且正在不断的深入，与欧洲发达国家相比较，我国在该领域的研究相对较为落后。值得重视的是，我国既是烟草生产大国又是烟草的消费大国，每年产生数十万吨的烟草废弃物，不仅是对资源的浪费，还会产生很大的环境污染。因此，随着烟草废弃物综合利用研究的深入，以及一系列高附加值产品的开发，对于降低烟草生产成本、提高经济效益、减少环境污染，减轻环保压力等意义重大。烟草花蕾精油是从废弃的烟草花蕾中提取的一种天然的精油产品，其具有抗氧化、抗衰老、抑菌等生理功能。我们可以从对烟草废物的利用、烟草花蕾精油的提取、生物活性研究及应用研究着手，结合高新技术的发展，在烟草的综合开发与利用领域，开创出新的局面。2) Analysis of social benefits: At present, the comprehensive utilization of tobacco, especially the research and development of tobacco waste is in the ascendant in various countries in the world, and it is constantly deepening. Compared with European developed countries, my country's research in this field is relatively backward. It is worth noting that my country is both a big tobacco producer and a big consumer of tobacco, producing hundreds of thousands of tons of tobacco waste every year, which is not only a waste of resources, but also a great environmental pollution. Therefore, with the in-depth research on the comprehensive utilization of tobacco waste and the development of a series of high value-added products, it is of great significance to reduce the cost of tobacco production, improve economic benefits, reduce environmental pollution, and reduce the pressure on environmental protection. Tobacco bud essential oil is a natural essential oil product extracted from discarded tobacco buds, which has physiological functions such as anti-oxidation, anti-aging, and antibacterial. We can start from the utilization of tobacco waste, the extraction of essential oil from tobacco flower buds, the research on biological activity and application, combined with the development of high technology, we can create a new situation in the field of comprehensive development and utilization of tobacco.
附图说明Description of drawings
图1为重庆奉节云烟87烟草花蕾精油的超声酶解法提取工艺路线图；Figure 1 is a roadmap for the ultrasonic enzymolysis extraction process of Yunyan 87 tobacco flower bud essential oil in Fengjie, Chongqing;
图2为重庆奉节云烟87烟草花蕾精油对OH自由基的清除能力，烟草精油(●)BHT(○)；Figure 2 shows the scavenging ability of Chongqing Fengjie Yunyan 87 tobacco flower bud essential oil on OH free radicals, tobacco essential oil (●) BHT (○);
图3为重庆奉节云烟87烟草花蕾精油对O2-自由基的清除能力，烟草精油(●)BHT(○)；Figure 3 shows the scavenging ability of Chongqing Fengjie Yunyan 87 tobacco flower bud essential oil onO2- free radicals, tobacco essential oil (●) BHT (○);
具体实施方式Detailed ways
以下通过实施例是对本发明做进一步的说明，但本发明的保护范围不限于此。The present invention will be further described by the following examples, but the protection scope of the present invention is not limited thereto.
实施例1Example 1
一种以重庆奉节云烟87花蕾为原料提取精油的方法，其由下述步骤获得：将粉碎后的重庆奉节云烟87花蕾按料液比1：15～1：25加入蒸馏水后，按烟草花蕾质量的5％～7％加入纤维素酶，按总质量的10％～14％加入氯化钠后，超声处理10～25min，然后采用水蒸气蒸馏4h，冷却后开启分离器下端活塞，使水分缓慢流出。用移液枪收集油层，并用二氯甲烷冲洗提取器三次，将冲洗液与油层合并，加入无水硫酸钠过夜，除去水分后，低温旋转蒸发，除去二氯甲烷得到烟草花蕾精油。A method for extracting essential oil from Chongqing Fengjie Yunyan 87 flower buds as raw materials, which is obtained by the following steps: adding distilled water to the crushed Chongqing Fengjie Yunyan 87 flower buds at a material-to-liquid ratio of 1:15 to 1:25; Add cellulase at 5% to 7% of the total mass, add sodium chloride at 10% to 14% of the total mass, ultrasonically treat for 10 to 25 minutes, and then use steam distillation for 4 hours. After cooling, open the lower piston of the separator to make the water slowly flow out. Collect the oil layer with a pipette, rinse the extractor three times with dichloromethane, combine the washing liquid with the oil layer, add anhydrous sodium sulfate overnight, remove the water, and rotate it at low temperature to remove the dichloromethane to obtain tobacco bud essential oil.
通过五水平的单因素试验，研究氯化钠浓度A、纤维素酶添加量B、料液比C、超声时间D四个因素对花蕾精油提取率的影响。然后采用正交法进行优化试验设计。Through a five-level single factor test, the effects of four factors, namely sodium chloride concentration A, cellulase addition amount B, solid-liquid ratio C, and ultrasonic time D, on the extraction rate of flower bud essential oil were studied. Then the orthogonal method was used to optimize the experimental design.
氯化钠添加量：在料液比1：20，纤维素酶添加量4％(酶质量：花蕾质量)、超声时间15min，氯化钠添加量(氯化钠质量：蒸馏水体积)6％，8％，10％，12％，14％的条件下进行试验，得出最佳水平，选择8％，10％，12％三个水平进行正交试验。The amount of sodium chloride added: at a solid-liquid ratio of 1:20, the added amount of cellulase was 4% (enzyme mass: bud mass), the ultrasonic time was 15min, and the added amount of sodium chloride (mass of sodium chloride: volume of distilled water) was 6%, Under the conditions of 8%, 10%, 12%, and 14%, the test is carried out to obtain the optimal level, and three levels of 8%, 10%, and 12% are selected for orthogonal test.
纤维素酶添加量：在料液比1：20，氯化钠添加量10％(氯化钠质量：蒸馏水体积)、超声时间15min，纤维素酶添加量(酶质量：花蕾质量)分别为1％，2％，4％，6％，8％的条件下进行试验，得出最佳时间水平，选择4％、6％和8％三个水平设计正交试验。The amount of cellulase added: at a solid-to-liquid ratio of 1:20, an added amount of sodium chloride of 10% (mass of sodium chloride: volume of distilled water), and an ultrasonic time of 15 minutes, the amount of added cellulase (mass of enzyme: mass of flower buds) was 1 %, 2%, 4%, 6%, and 8% were tested to obtain the best time level, and three levels of 4%, 6%, and 8% were selected to design an orthogonal experiment.
料液比：在纤维素酶添加量4％(酶质量：花蕾质量)、氯化钠添加量10％(氯化钠质量：蒸馏水体积)、超声时间15min，料液比分别1:10，1:15，1:20，1:25，1:30W的条件下进行试验，得出最佳料液比。选择1：20，1：25，1：30三个水平进行正交试验设计。Ratio of solid to liquid: when adding 4% of cellulase (mass of enzyme: bud mass), adding 10% of sodium chloride (mass of sodium chloride: volume of distilled water), and ultrasonic time of 15 minutes, the ratio of solid to liquid is 1:10, 1 : 15, 1: 20, 1: 25, 1: 30W, the test is carried out to obtain the best material-liquid ratio. Choose three levels of 1:20, 1:25, and 1:30 for orthogonal experimental design.
超声时间：在料液比1：20，纤维素酶添加量4％(酶质量：花蕾质量)、氯化钠添加量10％(氯化钠质量：蒸馏水体积)，超声时间5，10，15，20，25min的条件下进行试验，得出最佳水平，选择10，15，20min三个水平进行正交试验。Ultrasonic time: at a solid-to-liquid ratio of 1:20, cellulase addition 4% (enzyme mass: flower bud mass), sodium chloride addition 10% (sodium chloride mass: distilled water volume), ultrasonic time 5, 10, 15 , 20, 25min under the conditions of the test, get the best level, choose 10, 15, 20min three levels for orthogonal test.
正交试验：在单因素实验分析的基础上，选择了1：20，1：25，1：30三个液料比水平，4％、6％和8％三个纤维素酶添加量(酶质量：花蕾质量)水平，8％，10％，12％三个氯化钠添加量(氯化钠质量：蒸馏水体积)水平，10，15，20min三个超声时间水平，进行正交试验设计，可得出的超声酶解法提取烟草花蕾精油的最佳提取工艺条件为：氯化钠添加量(氯化钠质量：蒸馏水体积)为10％，纤维素酶添加量6％、料液比1：20、超声时间15min，并且在最佳工艺条件下，重庆奉节烟草花蕾精油的得率为0.566％(g/g)。结果见表1。Orthogonal test: On the basis of single factor experimental analysis, three liquid-material ratio levels of 1:20, 1:25, and 1:30, and three cellulase additions of 4%, 6% and 8% (enzyme Quality: flower bud quality) level, 8%, 10%, 12% three sodium chloride addition levels (sodium chloride quality: distilled water volume) level, 10,15, 20min three ultrasonic time levels, carry out orthogonal experiment design, The optimal extraction process conditions that can be obtained by ultrasonic enzymatic hydrolysis to extract tobacco flower bud essential oil are: sodium chloride addition (sodium chloride quality: distilled water volume) is 10%, cellulase addition 6%, solid-liquid ratio 1: 20. The ultrasonic time was 15 minutes, and under the optimal process conditions, the yield of Chongqing Fengjie Tobacco flower bud essential oil was 0.566% (g/g). The results are shown in Table 1.
表1超声酶解法提取烟草花蕾精油正交试验结果表Table 1 Orthogonal test results of extraction of essential oil from tobacco buds by ultrasonic enzymatic hydrolysis
实施例2Example 2
一种以重庆奉节云烟87花蕾为原料提取精油的方法，其由下述步骤获得：A method for extracting essential oil from Chongqing Fengjie Yunyan 87 flower buds, which is obtained by the following steps:
将粉碎后的重庆奉节云烟87花蕾按料液比1：20加入蒸馏水后，按烟草花蕾质量的6％加入纤维素酶，按总质量的10％加入氯化钠后，超声处理15min，然后采用水蒸气蒸馏4h，冷却后开启分离器下端活塞，使水分缓慢流出。用移液枪收集油层，并用二氯甲烷冲洗提取器三次，将冲洗液与油层合并，加入无水硫酸钠过夜，除去水分后，低温旋转蒸发，除去二氯甲烷得到烟草花蕾精油。实施例3Add distilled water to the pulverized Chongqing Fengjie Yunyan 87 flower buds according to the material-to-liquid ratio of 1:20, add cellulase according to 6% of the mass of tobacco flower buds, add sodium chloride according to 10% of the total mass, ultrasonically treat for 15 minutes, and then use Steam distillation for 4 hours, after cooling, open the lower piston of the separator to let the water flow out slowly. Collect the oil layer with a pipette, rinse the extractor three times with dichloromethane, combine the washing liquid with the oil layer, add anhydrous sodium sulfate overnight, remove the water, and rotate it at low temperature to remove the dichloromethane to obtain tobacco bud essential oil. Example 3
应用试验：重庆奉节云烟87烟草花蕾的抗氧化研究Application test: Antioxidative study of Chongqing Fengjie Yunyan 87 tobacco flower buds
1)烟草花蕾精油对OH自由基清除率的测定：1) Determination of OH free radical scavenging rate of tobacco flower bud essential oil:
用50％的无水乙醇分别配置浓度为0.5、1.0、1.5、2.0mg/mL的烟草花蕾精油、BHT溶液作为样品溶液(BHT为阳性对照)。取样品溶液0.5mL，分别加入50μL吐温80，1mLPBS缓冲溶液(pH＝7.4)，1mL浓度为7.5mmol/L的邻二氮菲溶液，1mL浓度为3.25mmol/L的硫酸亚铁溶液，2mL浓度为1.5％的双氧水，充分混合后于37℃下放置1h，在536nm处测吸光度A。对照组用蒸馏水代替各个样品，重复上述操作，在536nm处测吸光度A1，空白组用蒸馏水代替邻二氮菲、硫酸亚铁溶液和双氧水，重复上述操作，在536nm处测吸光度A0。每组试验设三组平行试验。Tobacco flower bud essential oil and BHT solution with concentrations of 0.5, 1.0, 1.5, and 2.0 mg/mL were respectively prepared with 50% absolute ethanol as sample solutions (BHT was a positive control). Take 0.5mL of sample solution, add 50μL Tween 80, 1mL PBS buffer solution (pH=7.4), 1mL o-phenanthroline solution with a concentration of 7.5mmol/L, 1mL ferrous sulfate solution with a concentration of 3.25mmol/L, 2mL The hydrogen peroxide with a concentration of 1.5% was mixed thoroughly and placed at 37°C for 1 hour, and the absorbance A was measured at 536nm. The control group replaced each sample with distilled water, repeated the above operation, and measured the absorbance A1 at 536nm. The blank group used distilled water instead of phenanthroline, ferrous sulfate solution and hydrogen peroxide, repeated the above operation, and measured the absorbance A0 at 536nm. Three sets of parallel experiments were set up for each group of experiments.
烟草花蕾精油对OH自由基清除率的计算方法如下：The calculation method of the OH free radical scavenging rate of tobacco bud essential oil is as follows:
·OH清除率(％)＝[(A-A0)/(A1-A0)]*100％OH scavenging rate (%)=[(AA0 )/(A1 -A0 )]*100%
试验中还用BHT进行对照比较，结果如图2所示，在浓度0.5mg/mL～2mg/mL内，烟草花蕾精油的OH清除能力没有明显差异，均高于BHT的清除率。说明重庆奉节烟草花蕾精油对羟基自由基的清除活性比较显著，是一种良好的抗氧化物质，可以用作抗氧化剂。In the test, BHT was also used for comparison. The results are shown in Figure 2. Within the concentration of 0.5mg/mL-2mg/mL, there is no significant difference in the OH scavenging ability of tobacco flower bud essential oil, which is higher than the scavenging rate of BHT. It shows that Chongqing Fengjie Tobacco Bud Essential Oil has a significant scavenging activity on hydroxyl radicals, and it is a good antioxidant substance, which can be used as an antioxidant.
2)烟草花蕾精油对O2-自由基清除率的测定：2 ) Determination of O2- free radical scavenging rate of tobacco flower bud essential oil:
用50％的无水乙醇分别配置浓度为0.5、1.0、1.5、2.0mg/mL的烟草花蕾精油、BHT溶液作为样品溶液(BHT为阳性对照)。样品溶液取1mL，分别加入50μL吐温80，4.5mL浓度为0.05mol/L的Tris-HCL缓冲液(pH＝8.2),1mL浓度为1mmol/L的EDTA溶液，0.4mL浓度为25mmol/L的邻苯三酚溶液，置于25℃水浴锅中恒温4min后，迅速用1mL浓度为12mol/L的HCL终止反应。在320nm处测吸光度A。对照组用蒸馏水代替各个样品，重复上述操作，在320nm处测吸光度A1。空白组用蒸馏水代替EDTA、邻苯三酚和HCl溶液，重复上述操作，在320nm处测吸光度A0。每组试验设三组平行试验。Tobacco flower bud essential oil and BHT solution with concentrations of 0.5, 1.0, 1.5, and 2.0 mg/mL were respectively prepared with 50% absolute ethanol as sample solutions (BHT was a positive control). Take 1mL of the sample solution, add 50μL Tween 80, 4.5mL Tris-HCL buffer solution (pH=8.2) with a concentration of 0.05mol/L, 1mL EDTA solution with a concentration of 1mmol/L, and 0.4mL Tris-HCL buffer solution with a concentration of 25mmol/L. After the pyrogallol solution was placed in a water bath at 25°C for 4 minutes, the reaction was quickly terminated with 1 mL of HCL with a concentration of 12 mol/L. Absorbance A was measured at 320 nm. For the control group, each sample was replaced with distilled water, the above operation was repeated, and the absorbance A1 was measured at 320 nm. In the blank group, distilled water was used instead of EDTA, pyrogallol and HCl solutions, the above operations were repeated, and the absorbance A0 was measured at 320 nm. Three sets of parallel experiments were set up for each group of experiments.
烟草花蕾精油对O2-自由基清除率的计算方法如下：The calculation method of the scavenging rate of tobacco bud essential oil onO2- free radicals is as follows:
超氧阴离子清除率(％)＝[1-(A-A0)/A1]*100％Superoxide anion scavenging rate (%)=[1-(AA0 )/A1 ]*100%
试验中还用BHT进行对照比较，结果如图3所示，烟草花蕾精油的O2-清除率在0.5～1mg/mL范围内增加迅速，随后增加速率变缓。当浓度为0.5mg/mL时，BHT的O2-清除率为29.81±0.23％，而烟草花蕾精油的清除率为17.20±0.19％，当浓度为2mg/mL时，BHT的O2-清除率为43.66±1.54％，而烟草花蕾精油的清除率达到35.37±1.27％，两者差距逐渐变小。说明重庆奉节烟草花蕾精油对O2-的清除活性比较显著，是一种良好的抗氧化物质，可以用作抗氧化剂。实施例4：In the experiment, BHT was also used for comparison. As shown in Figure 3, the O2 - scavenging rate of tobacco bud essential oil increased rapidly in the range of 0.5-1 mg/mL, and then the increase rate slowed down. When the concentration is 0.5mg/mL, the O2- scavenging rate of BHT is 29.81±0.23%, while the scavenging rate of tobacco bud essential oil is 17.20±0.19%, and when the concentration is 2mg/mL, the O2- scavenging rate of BHT was 43.66±1.54%, while the clearance rate of tobacco bud essential oil reached 35.37±1.27%, and the gap between the two gradually narrowed. It shows that the essential oil of Chongqing Fengjie tobacco flower buds has a remarkable scavenging activity on O2 - and is a good antioxidant substance, which can be used as an antioxidant. Example 4:
烟草花蕾精油抑菌性能的测定：Determination of Antibacterial Properties of Tobacco Bud Essential Oil:
采用滤纸片法测定抑菌作用。用打孔器将滤纸制成直径为6mm的圆形纸片，置于洁净干燥的试管中，121℃高压灭菌30min。在无菌操作台上将滤纸片放入浓度为140mg/mL的精油-乙醇溶液中。用无菌移液枪吸取0.2mL含菌体浓度为106～107cfu/mL的菌悬液，滴加到新鲜无菌的固体培养基上，涂布均匀。用无菌镊子将浸有精油的滤纸片贴在固体培养基中心，每个菌种平行测定3次。恒温培养(细菌：37℃/24h)，测定抑菌圈直径，比较抑菌效果。采用3mg/mL的青霉素钠和10mg/mL的头孢噻肟钠作为阳性对照，100％的乙醇溶液为阴性对照。测定抑菌圈直径，比较抑菌效果。Bacteriostasis was determined by the filter paper method. Use a puncher to make filter paper into a circular disc with a diameter of 6mm, place it in a clean and dry test tube, and sterilize under high pressure at 121°C for 30 minutes. Put the filter paper piece into the essential oil-ethanol solution with a concentration of 140mg/mL on the sterile operating table. Use a sterile pipette gun to draw 0.2mL of the bacterial suspension containing bacteria with a concentration of 106-107cfu/mL, drop it onto a fresh sterile solid medium, and spread evenly. Use sterile tweezers to stick the filter paper soaked in essential oil on the center of the solid medium, and measure each strain 3 times in parallel. Cultivate at constant temperature (bacteria: 37°C/24h), measure the diameter of the inhibition zone, and compare the antibacterial effects. 3 mg/mL penicillin sodium and 10 mg/mL cefotaxime sodium were used as positive controls, and 100% ethanol solution was used as negative control. The diameter of the inhibition zone was measured to compare the antibacterial effect.
试验中采用青霉素钠、头孢噻肟钠和100％乙醇溶液作为对照样品，结果如表2所示。100％无水乙醇的抑菌圈为0，是由于乙醇具有挥发性，在细菌生长初期已经被挥发掉，因而不具有抑菌性，同时也说明了重庆奉节的抑菌圈均为精油所产生的。重庆奉节精油对大肠杆菌、枯草芽孢杆菌、多粘类芽孢杆菌的抑菌效果明显，抑菌圈直径达到青霉素钠抑菌圈直径的一半。说明重庆奉节烟草花蕾精油具有良好的抑制细菌生长的能力，可以用作抑菌剂。Penicillin sodium, cefotaxime sodium and 100% ethanol solution were used as control samples in the test, and the results are shown in Table 2. The inhibition zone of 100% absolute ethanol is 0, because ethanol is volatile and has been volatilized in the early stage of bacterial growth, so it has no antibacterial properties. It also shows that the inhibition zone in Fengjie, Chongqing is all produced by essential oils of. The antibacterial effect of Chongqing Fengjie essential oil on Escherichia coli, Bacillus subtilis and Paenibacillus polymyxa was obvious, and the diameter of the inhibition zone reached half of that of penicillin sodium. It shows that the essential oil of Chongqing Fengjie Tobacco Bud has a good ability to inhibit the growth of bacteria and can be used as a bacteriostatic agent.
表2不同菌种的抑菌圈直径(mm)Table 2 The diameter of the inhibition zone of different strains (mm)
烟草花蕾精油MIC和MBC的测定：Determination of MIC and MBC of tobacco bud essential oil:
利用微量稀释法在96孔板中测定最小抑菌浓度。首先第一列八个孔分别加入200μL液体培养基做空白对照，第二列加入180μL液体培养基和20μL浓度为140mg/mL的烟草花蕾精油，从第三列到第十一列均加入100μL的液体培养基。从第二列中分别吸取相应的100μL精油到对应的第三列各孔，依次稀释到第十一列，最后从第十一列中吸出100μL废弃。各列精油的浓度为(14、7、3.5、1.75、0.875、0.4375、0.2188、0.1094、0.0547、0.0273mg/mL)。最后向二到十一列各孔加入100μL菌悬液，其中第四行加100μL培养液做对照，第十二列各孔加200μL菌液做阴性对照，细菌的第四行以3mg/mL的青霉素钠的倍比稀释做阳性对照，于37℃下恒温培养24h。Minimal inhibitory concentrations were determined in 96-well plates using the microdilution method. First, add 200 μL of liquid medium to the eight wells in the first column as a blank control, add 180 μL of liquid medium and 20 μL of tobacco flower bud essential oil with a concentration of 140 mg/mL in the second column, and add 100 μL of tobacco bud essential oil from the third to eleventh columns. liquid medium. Pipette 100 μL of essential oil from the second column to the corresponding wells in the third column, dilute to the eleventh column in turn, and finally suck out 100 μL from the eleventh column to discard. The concentrations of essential oils in each column are (14, 7, 3.5, 1.75, 0.875, 0.4375, 0.2188, 0.1094, 0.0547, 0.0273 mg/mL). Finally, 100 μL of bacterial suspension was added to each well of columns 2 to 11, among which 100 μL of culture solution was added to the fourth row as a control, and 200 μL of bacterial solution was added to each well of the twelfth column as a negative control. The fourth row of bacteria was treated with 3 mg/mL Doubling dilution of penicillin sodium was used as positive control, and cultured at constant temperature at 37°C for 24h.
在MIC测定试验中，将无菌生长的液体培养基中取出20μL涂布平板，继续培养(37℃/24h)。观察有无菌落生长。无菌落生长的最小浓度即为MBC。In the MIC determination test, 20 μL of the coated plate was taken out of the aseptically grown liquid medium, and cultured (37° C./24 h) was continued. Observe for colony growth. The minimum concentration without colony growth is MBC.
试验中采用青霉素钠作为对照，结果如表3所示，青霉素钠和重庆奉节精油对细菌(大肠杆菌、枯草芽孢杆菌和多粘类芽孢杆菌)的抑菌效果均较显著。其中重庆奉节烟草花蕾精油对大肠杆菌和枯草芽孢杆菌抑菌能力较强，两者的MIC均为3.5mg/mL，大肠杆菌的MBC为7mg/mL，枯草芽孢杆菌的MBC为3.5mg/mL。说明重庆奉节烟草花蕾精油具有良好的抑制细菌生长的能力，可用作抑菌剂。Penicillin sodium was used as a control in the test, and the results are shown in Table 3. Penicillin sodium and Chongqing Fengjie essential oil had significant antibacterial effects on bacteria (Escherichia coli, Bacillus subtilis and Paenibacillus polymyxa). Among them, Chongqing Fengjie Tobacco Bud Essential Oil has strong antibacterial ability to Escherichia coli and Bacillus subtilis, the MIC of both is 3.5mg/mL, the MBC of Escherichia coli is 7mg/mL, and the MBC of Bacillus subtilis is 3.5mg/mL. It shows that the essential oil of Chongqing Fengjie Tobacco Bud has a good ability to inhibit the growth of bacteria and can be used as a bacteriostatic agent.
表3最小抑菌质量浓度(MIC)及最低杀菌质量浓度(MBC)Table 3 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
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| Date | Code | Title | Description |
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| C06 | Publication | ||
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20151230 |