WO2020063892A1 - 一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法 - Google Patents

一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法 Download PDF

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WO2020063892A1
WO2020063892A1 PCT/CN2019/108615 CN2019108615W WO2020063892A1 WO 2020063892 A1 WO2020063892 A1 WO 2020063892A1 CN 2019108615 W CN2019108615 W CN 2019108615W WO 2020063892 A1 WO2020063892 A1 WO 2020063892A1
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chlorogenic acid
acid
stevia
extraction
stevioside
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PCT/CN2019/108615
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English (en)
French (fr)
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连运河
徐美利
高伟
牛志平
王艳芳
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晨光生物科技集团股份有限公司
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Priority to MYPI2021001741A priority Critical patent/MY197531A/en
Priority to KR1020217013032A priority patent/KR102580703B1/ko
Publication of WO2020063892A1 publication Critical patent/WO2020063892A1/zh
Priority to US17/215,910 priority patent/US20210230200A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids

Definitions

  • the invention belongs to the technical field of plant extracts and relates to an industrialized method for simultaneously and efficiently preparing stevia chlorogenic acid and steviol glycosides.
  • Stevia rebaudiana is a perennial herb of the Asteraceae family. It is native to Paraguay and Brazil in South America. It is one of the currently known sweeter sugar plants and has become the third natural sugar after sucrose and beet sugar. source. At present, China is the world's largest producer and supplier of stevioside, accounting for more than 80% of the global total. In addition to steviol glycosides, stevia contains relatively high levels of phenols, and these phenols have important biological activities. They have been used as sweet tea and medicinal tea for more than a century.
  • Stevioside is a sweet component in stevia. It is a kauriene diterpene glycoside. It contains multiple glycosyl fragments in the molecule and is easily soluble in water. It is a zero-calorie sweetener (its calories are sucrose 300-500 times).
  • the index components specified in GB 8270-2014 mainly include: stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, ducoside A, 9 components including steviol glycosides and stevioside.
  • the phenolic substances in stevia are mainly chlorogenic acid (raw material content: 4-6%, HPLC), in which the dicaffeoyl-substituted isochlorogenic acid accounts for 80% of the total acid.
  • isochlorogenic acid is antioxidant (J. Agric. Food Chem., 2004, 52 (15), 4893), anti-inflammatory (J. Nat. Prod., 1995, 58 (5), 639), antibacterial Anti-virus (JASHS, 2008, 133 (4), 492; Fitorick, 2012, 83, 1281; PLoS One, 2011, 6 (4), 18127; J. Ethnopharmacol, 2006, 106 (2), 187) and many more An important biological effect.
  • the traditional stevia industry uses water extraction, but the isochlorogenic acid is easily hydrolyzed during the extraction process. As shown in Figure 1-3, the proportion of isochlorogenic acid (dicaffeoylquinic acid) in the water extraction solution is greatly reduced. The ratio of caffeoquinic acid and caffeic acid is greatly increased, and the proportion of isochlorogenic acid contained in the product obtained by this patented technology is close to that of the raw materials.
  • isochlorogenic acid Compared with monocaffeoyl chlorogenic acid, isochlorogenic acid has less polarity and enhanced fat solubility (monocaffeoyl chlorogenic acid is easily soluble in water, the solubility in water at 4 ° C is 4%, and it is slightly soluble in ethyl acetate. Insoluble in lipophilic organic solvents).
  • This patented technology utilizes the dissolution characteristics of stevia chlorogenic acid and separates it from stevioside by extraction.
  • Patent CN 102617667 B uses organic solvent extraction to separate the chlorogenic acid component in stevia.
  • chlorogenic acid still has a certain solubility in water, such as direct extraction, the effect is not ideal, only partial chlorogenic acid products can be obtained, such as in the comparative patent CN102617667B, the product spectrum obtained from the patent CN102617667B Figure 1 It seems that the peak corresponding to 17.808min is the main component (accounting for about 40%), but this component is not obtained in the product spectrum ( Figures 2 and 3).
  • stevia chlorogenic acid has important biological effects. As of now, there are still no related products on the market. The main reasons are:
  • the present invention provides an industrialized method for simultaneously and efficiently preparing stevia chlorogenic acid and steviol glycosides, which overcomes the above technical difficulties, and on the premise of ensuring that the quality of steviol glycosides and the functional components of stevia chlorogenic acid are not destroyed It can realize the industrial production of stevia chlorogenic acid and steviol glycosides at the same time, laying a foundation for promoting the comprehensive utilization of stevia resources.
  • this process also effectively reduces the production water consumption, reduces the discharge of sewage and flocculent slag, is a high-efficiency green production process, and can greatly promote the progress of the industry.
  • the present invention provides an industrialized method for simultaneously preparing stevia chlorogenic acid and steviol glycosides.
  • An industrialized method for the simultaneous preparation of stevia chlorogenic acid and steviol glycosides includes the following steps: using stevia as a raw material, after alcohol extraction, adjusting the state of the feed liquid to make chlorogenic acid in a free molecular state, and then using it with water Mutually soluble and polar organic solvents are extracted and separated to obtain an organic layer enriched with chlorogenic acid and an aqueous layer enriched with stevioside.
  • the reagent with pKa ⁇ 4.7 is selected from NaH 2 PO 4 , H 3 PO 4 , HCl, NaHSO 4 , H 2 SO 4 , H 2 CO 3 , HNO 3 , citric acid, formic acid, oxalic acid, amber One or a combination of acids and benzoic acid.
  • the alcohol extraction is extraction with a short-chain alcohol aqueous solution containing 1-3 carbon atoms; preferably, the concentration of the short-chain alcohol aqueous solution is at least 70%; more preferably, the extraction is performed at 40-60 ° C.
  • the polarity of the water-immiscible medium-polar organic solvent is 2.0-4.5, more preferably one or more selected from the group consisting of ethyl acetate, dichloromethane, chloroform, ether, and propyl ether; Further preferably, the volume usage of the organic solvent is 0.8-1.5 times that of the solution to be extracted.
  • the stevia is leached with the short-chain alcohol before extraction, and the ratio of material to liquid is 1: (3-7); preferably, the step of alcohol extraction is repeated 1-3 times.
  • the alcohol extraction liquid is concentrated under the conditions of a temperature of 50-60 ° C. and a degree of vacuum of -0.08 MPa; the concentration is preferably 5-10 times.
  • the proportion of the active ingredient is similar to that of the stevia raw material, and it can be seen that there is almost no waste of the active substance.
  • the total isochlorogenic acid of stevia is greater than 60%.
  • the water layer obtained by the extraction and separation is separated by resin to obtain stevioside;
  • the resin is a low-polar divinylbenzene type adsorption resin, and the resin includes T28, ADS-750, 69M, DM30, 201-H, etc. ;
  • the amount of the resin is 0.5-1 times the weight of the water layer obtained from the separation.
  • the solid content of the separated water layer is adjusted to 8-12%. Adjusting the solid content can save water and reduce sewage.
  • the resin adsorption and separation includes: steviol sugar adsorption resin, desalting, decoloring, and fine resin removal and purification;
  • the adsorption flow rate during the adsorption is 0.1-0.4 BV / h.
  • a short-chain alcohol solution is used for the analysis, wherein the concentration of the short-chain alcohol solution for analysis is lower than the concentration of the short-chain alcohol solution for extraction; further, the concentration of the short-chain alcohol solution for analysis is controlled to 70-75 %; Dosage is 1-2BV, flow rate during analysis is 1-2BV / h.
  • the method for simultaneously preparing stevia chlorogenic acid and stevioside according to the present invention includes the following steps:
  • the invention overcomes many technical difficulties (including the current situation that the stevia chlorogenic acid is easily hydrolyzed during the extraction process and the stevia glycoside and stevia chlorogenic acid cannot be separated), and provides an industrialized method for efficiently preparing stevia chlorogenic acid and steviol glycosides.
  • the industrial production of stevia chlorogenic acid and steviol glycosides can be achieved at the same time, laying a foundation for the comprehensive utilization of stevia resources.
  • this process also effectively reduces the production water consumption, reduces the discharge of sewage and flocculent slag, is a high-efficiency green production process, and can greatly promote the progress of the industry.
  • the invention also provides a stevia chlorogenic acid extract and a steviol glycoside extract prepared according to any one of the above technical solutions.
  • the purity (content) of isochlorogenic acid is> 60%.
  • the patented extraction technology can prevent the hydrolysis of stevia isochlorogenic acid components, so as to ensure the content of the effective ingredients and the efficacy of stevia chlorogenic acid products.
  • this process significantly reduces the production water consumption and reduces the discharge of sewage and flocculent slag. It is a high-efficiency green production process and can greatly promote the progress of the industry.
  • Figure 1 is a chlorogenic acid spectrum in stevia raw materials
  • FIG. 2 is a chlorogenic acid spectrum in a water stevia extract
  • FIG. 3 is a chlorogenic acid spectrum in the product obtained in Example 2.
  • the present invention provides an industrialized method for preparing stevia chlorogenic acid and the obtained product.
  • the specific steps are as follows:
  • the present invention provides an industrialized method for preparing stevia chlorogenic acid and the obtained product.
  • the specific steps are as follows:
  • the present invention provides an industrialized method for preparing stevia chlorogenic acid and the obtained product.
  • the specific steps are as follows:
  • the present invention provides an industrialized method for preparing stevia chlorogenic acid and the obtained product.
  • the specific steps are as follows:
  • the present invention provides an industrialized method for preparing stevia chlorogenic acid and the obtained product.
  • the specific steps are as follows:
  • This embodiment provides an industrialized method for preparing stevioside and the resulting product.
  • the specific steps are as follows:
  • T28 resin adsorption was performed after the solid content of the aqueous layer was extracted 3 times in the step (4) in Example 1 and the solid content of the water layer was 10%.
  • the resin amount was 1.5L
  • the adsorption flow rate was 0.2BV / h
  • 2BV was washed after the adsorption was completed.
  • the first BV flow rate is 0.2BV / h
  • the second BV flow rate is 1BV / h.
  • a 2BV 70% ethanol aqueous solution was used for analysis, and the analysis flow rate was 1BV / h.
  • the analytical solution was concentrated, and then desalted, decolorized, refined, and dried to obtain 99 g of stevioside product.
  • the product was a white powder with a TSG of 94.3%, a light transmittance of 90.8% at 420 nm, and a light absorption of 0.012 at a concentration of 370 nm.
  • This embodiment provides an industrialized method for preparing stevioside and the resulting product.
  • the specific steps are as follows:
  • the solid content of the water layer was adjusted to 8%, and then the 201-H resin was adsorbed.
  • the amount of resin was 1.5L, and the adsorption flow rate was 0.25 BV / h.
  • the first BV flow rate is 0.25BV / h, and the second BV flow rate is 1BV / h.
  • a 2BV 70% ethanol aqueous solution was used for analysis, and the analysis flow rate was 1BV / h.
  • the analysis solution was concentrated, desalting, decoloring, fine dehydration, and drying were performed to obtain 100.5 g of a stevioside product.
  • the product was a white powder with a TSG of 92.9%, a light transmittance of 90.3% at 420 nm, and a light absorption of 0.015 at 370 nm.
  • This embodiment provides an industrialized method for preparing stevioside and the resulting product.
  • the specific steps are as follows:
  • the solid content of the water layer was adjusted to 6% and then DM30 resin was adsorbed.
  • the amount of resin was 1.5L, the adsorption flow rate was 0.3BV / h, and 2BV was washed after the adsorption was completed The first BV flow rate is 0.3BV / h, and the second BV flow rate is 1BV / h.
  • a 2BV 70% ethanol aqueous solution was used for analysis, and the analysis flow rate was 1BV / h.
  • the analytical solution was concentrated, and then desalted, decolorized, refined, and dried to obtain 99.7 g of a stevioside product.
  • the product was a white powder with a TSG of 93.6%, a transmittance of 420 nm of 90.6%, and a concentration of 1% at 370 nm.
  • the specific absorption was 0.010.
  • This comparative example provides a method for preparing stevioside and chlorogenic acid, which is operated by the method provided by Chinese Patent Publication No. CN105001281.
  • This comparative example provides a method for separating steviol phenols and steviol glycosides, which is operated by the method provided by Chinese Patent Publication No. CN106236808B.
  • This test example provides a comparison of the contents of some components of stevia chlorogenic acid extracted in Example 2 and Comparative Example 1 (traditional water extraction method), as shown in Table 1.
  • This test example provides a comparison of the technical effects of separation / preparation of stevioside provided in Examples 4-6 and Comparative Examples 1-3, as shown in Table 2.
  • Example 1 Comparative Example 2
  • Example 4 Example 5
  • Example 6 TSG% 90 91 94.3 92.9 93.6 Transmittance (420nm) 81 83 90.8 90.3 90.6 Specific absorption (370nm) 0.031 0.035 0.012 0.015 0.01
  • the content of total glycosides in this application is determined by the GB 8270-2014 method.
  • the transmittance is the transmittance at 14% solids concentration at 420nm detected by UV.
  • the steviol glycoside at 1% concentration at 370nm adopts GB8270-1999 method.
  • the measurement was performed.
  • the content of total chlorogenic acid and the proportion of each component were measured by the T / CCCMHPIE 1.17-2016 method.
  • the invention provides an industrialized method for simultaneously preparing stevia chlorogenic acid and stevioside.
  • the method of the present invention uses stevia as a raw material, and after alcohol extraction, adjusts the state of the feed liquid so that chlorogenic acid is in a free molecular state, and is extracted and separated with a medium-polar organic solvent that is immiscible with water. Acid, water layer is enriched with stevioside.
  • the present invention can prevent the chlorogenic acid component in stevia from being hydrolyzed, so as to ensure the content and efficacy of the effective components of the stevia chlorogenic acid product.

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Abstract

一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法，以甜叶菊作为原料，经醇提后，调节料液状态，使绿原酸呈自由分子状态，用与水不互溶的中极性有机溶剂萃取分离，有机层富集甜叶菊绿原酸，水层富集甜菊糖苷。较传统水提取工艺，所述方法可防止甜叶菊中绿原酸成分水解，以保证甜叶菊绿原酸产品有效成分含量及功效。在不影响甜菊糖苷产品质量和生产效率前提下，实现有效分离，生产效率提高，所得产品异绿原酸比例和原料接近。降低了生产水耗，减少污水及絮凝渣的排放，为一种高效益的绿色生产工艺。

Description

一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法
交叉引用
本申请要求2018年9月30日提交的专利名称为“一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法”的第201811159815.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于植物提取物技术领域，涉及一种同步高效制备甜叶菊绿原酸和甜菊糖苷的工业化方法。
背景技术
甜叶菊(Stevia rebaudiana)属菊科多年生草本植物，原产于南美巴拉圭和巴西，是目前已知甜度较高的糖料植物之一，已成为继蔗糖、甜菜糖之后的第三种天然糖源。目前，中国是世界最大的甜菊糖苷生产及供应国，占全球总量的80％以上。甜叶菊中除甜菊糖苷外还含有较高含量的酚类，且这些酚类物质具有重要的生物活性，在其发源地作为甜茶、药茶饮用已有一百多年的历史。
甜菊糖苷为甜叶菊中的甜味成分，为贝壳杉烯二萜苷类物质，分子中含有多个糖基片段，易溶于水，是一种零热量的高倍甜味剂(其热量为蔗糖的300-500倍)。GB 8270-2014中规定的指标成分主要包括：甜菊苷、瑞鲍迪苷A、瑞鲍迪苷B、瑞鲍迪苷C、瑞鲍迪苷D、瑞鲍迪苷F、杜克苷A、甜茶苷、甜菊双糖苷等9种成分。
Figure PCTCN2019108615-appb-000001
Figure PCTCN2019108615-appb-000002
Figure PCTCN2019108615-appb-000003
甜叶菊中的酚类物质的主要为绿原酸(原料含量4-6％，HPLC)，其中二咖啡酰基取代的异绿原酸占总酸比例达80％。研究表明，异绿原酸具有抗氧化(J.Agric.Food Chem.,2004,52(15),4893)，抗炎(J.Nat.Prod.,1995,58(5),639)，抗菌、抗病毒(JASHS,2008,133(4),492；Fitoterapia,2012,83,1281；PLoS One,2011,6(4),18127；J.Ethnopharmacol,2006,106(2),187)等多种重要的生物功效。
传统甜菊行业均采用水提取，但提取过程中异绿原酸易发生水解，如图1-3所示，水提取液中异绿原酸(二咖啡酰奎宁酸)占比大幅降低、单咖啡酰基奎宁酸及咖啡酸比例大幅升高，该专利技术所得产品所含异绿原酸比例和原料接近。
对于甜叶菊中的酚类物质和甜菊糖苷的分离多通过树脂分离实现，如专利200710111313.4和201610745221，均是利用甜叶菊中酚类物质和糖苷极性差异，通过树脂环节实现了两类成分的分离，采用该方法，甜叶菊酚类物质和糖苷之间存在竞争吸附，吸附过程甜叶菊绿原酸占用部分甜菊糖苷吸附位点，使甜菊糖苷的树脂吸附量降低，导致生产效率降低、生产成本升高。而该申请专利技术，在吸附树脂前实现了甜叶菊绿原酸和甜菊 糖苷的分离，避免了该类问题的发生。
相较于单咖啡酰基绿原酸，异绿原酸极性较小，脂溶性增强(单咖啡酰基绿原酸易溶于水，25℃水中溶解度为4％，极微溶于醋酸乙酯，难溶于亲脂性有机溶剂)。该专利技术利用甜叶菊绿原酸的溶解特性，通过萃取将其和甜菊糖苷分离。专利CN 102617667 B中采用了有机溶剂萃取对甜叶菊中绿原酸成分进行了分离。但绿原酸在水中仍具有一定的溶解性，如直接萃取，效果不理想，只能得到部分绿原酸产品，如对比专利CN 102617667 B中，从专利CN 102617667 B所得产品谱图附图1看，17.808min对应的峰为主要成分(占比约40％)，但产品谱图(附图2和3)中并未得到该成分。如前所述，甜叶菊绿原酸具有重要的生物功效，截止目前为止，市场上仍然没有相关的产品推出，主要原因：
(1)传统水提取过程，甜叶菊中异绿原酸发生水解，且该过程受酶及温度等多种因素影响，过程不易控制，甜叶菊异绿原酸含量降低，产品质量稳定性差。
(2)甜菊糖苷和甜叶菊绿原酸极性接近分离困难。
发明内容
由于上述缺陷，本发明提供了一种同步高效制备甜叶菊绿原酸和甜菊糖苷的工业化方法，克服了上述技术困难，在保证甜菊糖苷质量和甜叶菊绿原酸功效成分不被破坏的前提下，可同时实现甜叶菊绿原酸和甜菊糖苷的工业化生产，对推动甜菊资源的综合利用奠定基础。此外，和传统的水提取工艺相比，该工艺还有效的降低了生产水耗，减少污水及絮凝渣的排放，为一种高效益的绿色生产工艺，可大幅度推进行业进步。
发明人于研究中发现，传统甜菊行业均采用水提取，但提取过程中异绿原酸易发生水解，水提取液中绿原酸占比大幅降低、单咖啡酰基奎宁酸及咖啡酸比例大幅升高，且该过程受酶及温度等多种因素影响，过程不易控制，使甜叶菊绿原酸含量降低，产品质量稳定性差。
为提高甜叶菊综合利用价值、加快其综合利用进程，本发明提供了一 种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法。
为实现上述发明目的，本发明采用以下技术方案予以实现：
一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法，包括如下步骤：以甜叶菊作为原料，经醇提后，调节料液状态，使绿原酸呈自由分子状态，再用与水不互溶的中极性的有机溶剂萃取分离，得到富集绿原酸的有机层以及富集甜菊糖苷的水层。
所述调节料液状态使绿原酸呈自由分子状态，是以pKa＜4.7的试剂对料液进行调整，至绿原酸呈自由的分子状态；
优选地，所述pKa＜4.7的试剂选自NaH 2PO 4、H 3PO 4、HCl、NaHSO 4、H 2SO 4、H 2CO 3、HNO 3、枸橼酸、蚁酸、草酸、琥珀酸、苯甲酸中的一种或几种的组合。
所述醇提是以含1-3个碳原子的短链醇水溶液进行提取；优选地，所述短链醇水溶液的浓度至少70％；更优选地，在40-60℃进行提取。
优选地，所述与水不互溶的中极性的有机溶剂的极性为2.0-4.5，更优选选自醋酸乙酯、二氯甲烷、氯仿、乙醚、丙醚中的一种或几种；进一步优选地，所述有机溶剂的体积用量是待萃取溶液的0.8-1.5倍。
本发明所述的方法，在萃取前，以所述短链醇浸提甜叶菊，料液比为1：(3-7)；优选地，所述醇提的步骤重复1-3次。
进一步地，在醇提后、调整料液状态前，在温度50-60℃，真空度-0.08MPa条件下对醇提液进行浓缩；优选浓缩5-10倍。
通过上述方法提取得到的富集甜叶菊绿原酸的提取物中，活性成分的比例和甜叶菊原料相接近，可知对活性物质几乎没有浪费。利用本发明所述的工业化方法，所得的富集甜叶菊绿原酸的提取物中，甜叶菊总异绿原酸纯度＞60％。
所述萃取分离得到的水层经树脂吸附分离后即得甜菊糖苷；所述树脂为低极性二乙烯苯型吸附树脂，所述树脂包括T28、ADS-750、69M、DM30、201-H等；优选地，所述树脂的用量为分离所得水层重量的0.5-1 倍。
优选地，在树脂吸附前，对分离得到的水层调固含量至8-12％。调节固含量能够节省用水，减少污水。
本发明所述的甜菊糖苷制备方法中，所述树脂吸附分离包括：甜菊糖吸附树脂、脱盐、脱色、精脱树脂分离纯化；
优选地，所述吸附时的吸附流速为0.1-0.4BV/h。
吸附完成后，进行后续水洗、解析，解析液经甜菊糖脱盐、脱色、精脱，干燥得甜菊糖苷产品。
优选地，所述解析时选用短链醇溶液，其中，解析用短链醇溶液的浓度低于提取用短链醇溶液的浓度；进一步地，解析用短链醇溶液的浓度控制在70-75％；用量为1-2BV，解析时的流速为1-2BV/h。
作为优选，本发明所述的同步制备甜叶菊绿原酸和甜菊糖苷方法包括如下步骤：
(1)将甜叶菊粉末以短链醇提取，得提取液；
(2)将提取液浓缩回收短链醇溶液，得浓缩液；
(3)调节料液状态使绿原酸呈自由分子状态；
(4)对(3)所得溶液萃取分离，取有机层得到甜叶菊绿原酸提取物；
(5)对(4)所得水层进行树脂吸附，得到甜菊糖苷提取物。
本发明克服了诸多技术困难(包括甜叶菊绿原酸提取过程易水解，以及甜菊糖苷和甜叶菊绿原酸无法分离的现状)，提供了高效制备甜叶菊绿原酸和甜菊糖苷的工业化方法，在保证甜菊糖苷质量和甜叶菊绿原酸功效成分不被破坏的前提下，可同时实现甜叶菊绿原酸和甜菊糖苷的工业化生产，对推动甜菊资源的综合利用奠定基础。此外，和传统的水提取工艺相比，该工艺还有效的降低了生产水耗，减少污水及絮凝渣的排放，为一种高效益的绿色生产工艺，可大幅度推进行业进步。
本发明同时提供依据上述任一项技术方案所制备得到的甜叶菊绿原酸提取物和甜菊糖苷提取物。
优选地，所述甜叶菊绿原酸提取物中，异绿原酸纯度(含量)＞60％。
采用上述技术方案所产生的有益效果是：
(1)较传统水提取工艺，该专利公布的提取技术可防止甜叶菊异绿原酸成分的水解，以保证甜叶菊绿原酸产品的有效成分含量及功效。
(2)该工艺在不影响甜菊糖苷产品质量和生产效率的前期下，实现了甜叶菊绿原酸的有效分离，生产效率大幅提高，所得产品所含异绿原酸比例和原料接近。
(3)和传统的水提取工艺相比，该工艺大幅降低了生产水耗，减少污水及絮凝渣的排放，为一种高效益的绿色生产工艺，可大幅度推进行业进步。
附图说明
图1为甜叶菊原料中绿原酸谱图；
图2为甜叶菊水提取液中绿原酸谱图；
图3为实施例2所得产品中绿原酸谱图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
本发明提供一种制备甜叶菊绿原酸的工业化方法及所得产品，具体步骤如下：
(1)称取甜叶菊粉末1kg，85％的乙醇水溶液为提取液，料液比分别为1:5/3.5/3.5，50℃提取三次，第1遍提取时间为1.5h，2、3遍提取时间均为1h，合并滤液作为提取液。
(2)提取液水浴60℃、真空-0.08MPa条件下10倍浓缩回收乙醇。
(3)使用电位滴定仪进行在线监测，使用H 3PO 4水溶液调整料液状态，且过程中不断搅拌，至电极电位发生突跃时停止加液。
(4)使用等体积氯仿对上一工艺步骤所得料液萃取3次，有机相浓缩、树脂纯化后得到78.5g甜叶菊绿原酸产品，甜叶菊总绿原酸82％，异 绿原酸含量为65％。
实施例2
本发明提供一种制备甜叶菊绿原酸的工业化方法及所得产品，具体步骤如下：
(1)称取甜叶菊粉末1kg，95％的甲醇水溶液为提取液，料液比分别为1:6/4.5，50℃提取两次，第1遍提取时间为1.5h，第2遍提取时间均为1h，合并滤液作为提取液。
(2)提取液水浴60℃、真空-0.08MPa条件下10倍浓缩回收乙醇。
(3)使用PHS-3C pH计进行在线监测，使用H 2SO 4水溶液调整料液状态，且过程中不断搅拌，至电极电位为180mV停止加液。
(4)使用等体积醋酸乙酯对上一工艺步骤所得料液萃取3次，有机相浓缩、树脂纯化后得到75.2g甜叶菊异绿原酸产品，甜叶菊总绿原酸含量为84％，异绿原酸含量为66％。
实施例3
本发明提供一种制备甜叶菊绿原酸的工业化方法及所得产品，具体步骤如下：
(1)称取甜叶菊粉末1kg，75％的丙醇水溶液为提取液，料液比分别为1:6/4.5，50℃提取两次，第1遍提取时间为1.5h，第2遍提取时间均为1h，合并滤液作为提取液。
(2)提取液水浴60℃、真空-0.08MPa条件下10倍浓缩回收乙醇。
(3)使用PHS-3C pH计进行在线监测，使用蚁酸水溶液，且过程中不断搅拌，至pH 3.0停止加液。
(4)使用等体积乙醚对上一工艺步骤所得料液萃取3次，有机相浓缩后、树脂纯化得到72.8g甜叶菊绿原酸产品，甜叶菊总绿原酸含量为90％，异绿原酸含量为73％。
实施例4
本发明提供一种制备甜叶菊绿原酸的工业化方法及所得产品，具体步 骤如下：
(1)称取甜叶菊粉末1kg，70％的乙醇水溶液为提取液，料液比分别为1:5/3.5/3.5，50℃提取三次，第1遍提取时间为1.5h，2、3遍提取时间均为1h，合并滤液作为提取液。
(2)提取液水浴60℃、真空-0.08MPa条件下10倍浓缩回收乙醇。
(3)使用电位滴定仪进行在线监测，使用HNO 3水溶液调整料液状态，且过程中不断搅拌，至电极电位发生突跃时停止加液。
(4)使用等体积二氯甲烷对上一工艺步骤所得料液萃取3次，有机相浓缩、树脂纯化后得到79.8g甜叶菊绿原酸产品，甜叶菊总绿原酸82％，异绿原酸含量为64％。
实施例5
本发明提供一种制备甜叶菊绿原酸的工业化方法及所得产品，具体步骤如下：
(1)称取甜叶菊粉末1kg，80％的甲醇水溶液为提取液，料液比分别为1:5/4/3.5，50℃提取两次，第1遍提取时间为1.5h，第2遍提取时间均为1h，合并滤液作为提取液。
(2)提取液水浴60℃、真空-0.08MPa条件下10倍浓缩回收乙醇。
(3)使用电位滴定仪进行在线监测，使用HCl水溶液调整料液状态，且过程中不断搅拌，至电极电位发生突跃时停止加液。
(4)使用等体积丙醚对上一工艺步骤所得料液萃取3次，有机相浓缩、树脂纯化后得到76.3g甜叶菊绿原酸产品，甜叶菊总绿原酸85％，异绿原酸含量为65％。
实施例6
本实施例提供一种制备甜菊糖苷的工业化方法及所得产品，具体步骤如下：
将实施例1中的步骤(4)中所得料液萃取3次后的水层调固含量10％后进行T28树脂吸附，树脂用量为1.5L，吸附流速0.2BV/h，吸附完成后 2BV水洗，第1BV流速0.2BV/h，第2BV流速1BV/h。水洗完成后使用2BV 70％的乙醇水溶液进行解析，解析流速1BV/h。解析液浓缩后进行脱盐、脱色、精脱、干燥，得99g甜菊糖苷产品，产品为白色粉末，TSG为94.3％，420nm透光度90.8％，1％浓度370nm比吸光0.012。
实施例7
本实施例提供一种制备甜菊糖苷的工业化方法及所得产品，具体步骤如下：
将实施例2中的步骤(4)中所得料液萃取3次后的水层调固含量8％后进行201-H树脂吸附，树脂用量为1.5L，吸附流速0.25BV/h，吸附完成后2BV水洗，第1BV流速0.25BV/h，第2BV流速1BV/h。水洗完成后使用2BV 70％的乙醇水溶液进行解析，解析流速1BV/h。解析液浓缩后进行脱盐、脱色、精脱、干燥，得100.5g甜菊糖苷产品，产品为白色粉末，TSG为92.9％，420nm透光度90.3％，1％浓度370nm比吸光0.015。
实施例8
本实施例提供一种制备甜菊糖苷的工业化方法及所得产品，具体步骤如下：
将实施例3中的步骤(4)中所得料液萃取3次后的水层调固含量6％后进行DM30树脂吸附，树脂用量为1.5L，吸附流速0.3BV/h，吸附完成后2BV水洗，第1BV流速0.3BV/h，第2BV流速1BV/h。水洗完成后使用2BV 70％的乙醇水溶液进行解析，解析流速1BV/h。解析液浓缩后进行脱盐、脱色、精脱、干燥，得99.7g甜菊糖苷产品，产品为白色粉末，TSG为93.6％，420nm透光度90.6％，1％浓度370nm比吸光0.010。
对比例1
本对比例提供一种制备甜菊糖苷和绿原酸的方法，采用中国专利公开号CN 105001281 B所提供的方法进行操作。
所得提取物的绿原酸谱图见附图2。
对比例2
本对比例提供一种甜叶菊酚类和甜菊糖苷的分离方法，采用中国专利公开号CN106236808B所提供的方法进行操作。
试验例1
本试验例提供实施例2和对比例1(传统水提法)所提取得到的甜叶菊绿原酸部分成分的含量对比，如表1所示。
其中甜叶菊原料的绿原酸谱图如附图1所示；
甜叶菊水提物(对比例1)中绿原酸谱图如附图2所示；
实施例2所得到的提取物中绿原酸谱图如附图3所示。
表1
Figure PCTCN2019108615-appb-000004
试验例2
本试验例提供实施例4-6和对比例1-3所提供的分离/制备甜菊糖苷的技术效果对比，如表2所示。
表2
检测指标 对比例1 对比例2 实施例4 实施例5 实施例6
TSG％ 90 91 94.3 92.9 93.6
透光度(420nm) 81 83 90.8 90.3 90.6
比吸光(370nm) 0.031 0.035 0.012 0.015 0.01
本申请中总苷含量采用GB 8270-2014方法进行测定，透光度为UV检测的14％固形物浓度420nm下的透光度，1％浓度的甜菊糖苷在370nm比吸光采用GB 8270-1999方法进行测定，总绿原酸的含量及各成分比例采用T/CCCMHPIE 1.17-2016方法进行测定。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法。本发明方法以甜叶菊作为原料，经醇提后，调节料液状态，使绿原酸呈自由分子状态，用与水不互溶的中极性有机溶剂萃取分离，有机层富集甜叶菊绿原酸，水层富集甜菊糖苷。较传统水提取工艺，本发明可防止甜叶菊中绿原酸成分水解，以保证甜叶菊绿原酸产品有效成分含量及功效。在不影响甜菊糖苷产品质量和生产效率前提下，实现有效分离，生产效率大幅提高，所得产品异绿原酸比例和原料接近。大幅降低了生产水耗，减少污水及絮凝渣的排放，为一种高效益的绿色生产工艺，可大幅度推进行业进步，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种同步制备甜叶菊绿原酸和甜菊糖苷的工业化方法，其特征在于，包括如下步骤：
    以甜叶菊作为原料，经醇提后，调节料液状态，使绿原酸呈自由分子状态，再用与水不互溶的中极性的有机溶剂萃取分离，得到富集绿原酸的有机层以及富集甜菊糖苷的水层。
  2. 根据权利要求1所述的方法，其特征在于，所述调节料液状态，是以pKa＜4.7的试剂对料液进行调节，至绿原酸呈自由的分子状态；
    优选地，所述试剂选自NaH 2PO 4、H 3PO 4、HCl、NaHSO 4、H 2SO 4、H 2CO 3、HNO 3、枸橼酸、蚁酸、草酸、琥珀酸、苯甲酸中的一种或几种的组合。
  3. 根据权利要求1或2所述的方法，其特征在于，所述醇提是以含1-3个碳原子的短链醇水溶液进行提取；
    优选地，所述短链醇水溶液的浓度至少70％；
    更优选地，在40-60℃进行提取。
  4. 根据权利要求1-3任一项所述的方法，其特征在于，所述与水不互溶的中极性的有机溶剂的极性为2.0-4.5；
    优选地，选自醋酸乙酯、二氯甲烷、氯仿、乙醚、丙醚中的一种或几种；
    更优选地，所述有机溶剂的体积用量是待萃取溶液的0.8-1.5倍。
  5. 根据权利要求1-4任一项所述的方法，其特征在于，萃取前，以所述短链醇溶解甜叶菊，料液比为1：(3-7)；
    优选地，所述醇提的步骤重复1-3次。
  6. 根据权利要求1-5任一项所述的方法，其特征在于，在醇提后、调整料液状态前，在温度50-60℃，真空度-0.08MPa条件下对醇提液进行浓缩；
    优选浓缩5-10倍。
  7. 根据权利要求1-6任一项所述的方法，其特征在于，所述萃取分离得到的水层经树脂吸附分离后即得甜菊糖苷；
    优选地，所述树脂为低极性二乙烯苯型吸附树脂；
    更优选所述树脂包括T28、ADS-750、69M、DM30、201-H；
    和/或，所述树脂的用量为原料的0.5-1倍。
  8. 根据权利要求7所述的方法，其特征在于，在所述树脂吸附前，对分离得到的水层调固含量至8-12％；所述树脂吸附分离时的吸附流速为0.1-0.4BV/h；
    和/或，吸附完成后，进行水洗、解析，得甜菊糖苷产品；所述解析时选用短链醇溶液，其中，解析用短链醇溶液的浓度低于提取用短链醇溶液的浓度；
    优选地，解析用短链醇溶液的浓度控制在70-75％；用量为1-2BV，解析时的流速为1-2BV/h。
  9. 一种富含绿原酸的提取物，其特征在于，由权利要求1-8任一项所述的方法得到；
    优选地，所述富含绿原酸的提取物中，异绿原酸含量＞60％。
  10. 一种富含甜菊糖苷的提取物，其特征在于，由权利要求1-8任一项所述的方法得到。
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