WO2019227532A1 - 一种制备低接枝数葡萄糖基甜菊糖苷的方法 - Google Patents

一种制备低接枝数葡萄糖基甜菊糖苷的方法 Download PDF

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WO2019227532A1
WO2019227532A1 PCT/CN2018/091271 CN2018091271W WO2019227532A1 WO 2019227532 A1 WO2019227532 A1 WO 2019227532A1 CN 2018091271 W CN2018091271 W CN 2018091271W WO 2019227532 A1 WO2019227532 A1 WO 2019227532A1
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glucosyl
low
glycosides
steviol glycoside
glucosyl steviol
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French (fr)
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夏咏梅
朱理平
张童童
杜国营
刘湘
方云
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东台市浩瑞生物科技有限公司
江南大学
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Priority to US16/525,706 priority Critical patent/US11180788B2/en
Publication of WO2019227532A1 publication Critical patent/WO2019227532A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose

Definitions

  • the invention relates to a method for preparing a low graft number glucosyl steviol glycoside, and belongs to the technical field of biosynthesis of sweeteners.
  • Steviol glycosides are a collective term for about 40 glycoside molecules in stevia extracts, all of which are composed of a hydrophilic sugar group with a sweet taste and a steviol aglycon that is hydrophobic.
  • Rebaudioside D and Rebaudioside M have better taste and texture
  • Rebaudioside A (RA) has a good taste but a little long-lasting sweetness
  • Stevioside (St) has a slight bitter taste or licorice aftertaste, this slightly bitter aftertaste reduces its quality as a natural sweetener.
  • the steviol aglycone molecular structure of steviol glycoside itself is also a factor that produces bitter aftertaste and after-sweetness (ie, long-lasting sweetness). From the perspective of taste biology, the difference in binding energy between different steviol glycosides and sweet taste receptors and bitter taste receptors is an important factor.
  • One of the methods to improve the taste of steviol glycosides is to introduce glycosyl groups by methods such as enzymatic glycosylation, and use the steric hindrance of access to the glycosyl groups to change the difference in the binding energy between steviol glycosides and sweet taste receptors and bitter taste receptors. Improve its taste and texture.
  • enzymatic glycosylation can result in a variety of random transglycosyl products, such as various glucosyl steviol glycosides with various glucosyl substitution numbers or grafting numbers. The number of grafts in the product ranges from 1 to 20.
  • the mass percentages of low-grafted products such as mono- and diglucosyl-grafted steviol glycosides generally total less than 50% of the total glycosides.
  • the sweetness of all glucoside steviol glycosides will be reduced to a different degree than the raw steviol glycosides, and the aftertaste and mouthfeel will be improved.
  • Products with a high number of glucosyl substitutions can be used as flavoring agents and table sugars, but the sweetness decreases too much.
  • Products with a low number of glucosyl substitutions such as the mono and diglucosyl substitutes of steviol glycosides, have good sweetness and taste correction functions; their structures are similar to those of RD and RM, which have the best taste in natural stevia. Constituency.
  • a method for improving the taste of steviol glycosides by introducing glycosyl groups through enzymatic glycosylation and other methods is to use a cyclodextrin glucosyltransferase from Bacillus alkalophilic Bacillus catarrhalis to catalyze the conversion of steviol glycosides St.
  • the product, structural analysis shows that 1 to 3 glucosyl groups are connected at the C-13 and C-19 positions through ⁇ -1,4 glycosidic bonds, and 1 or 2 glucosyl groups are connected at the C-13 position. It has been improved compared to St; when 3 glucose groups are connected at C-13, the sweetness is greatly reduced (Fukunaga, 1989).
  • Kochikyan uses six strains to produce different types of cyclodextrin glucosyltransferases, and uses liquefied starch as a glycosyl donor to catalyze stevioside transglycosides to obtain seven transglycosyl products (Kochikyan VT, 2006).
  • Another method to improve the taste of steviol glycosides is to use ⁇ -cyclodextrin glucosyltransferase Toruzyme 3.0L and ⁇ -moderate amylase to catalyze the hydrolysis of starch modified St by a double-enzyme method. After 4 hours of reaction, the St conversion rate reaches 77.11 %, To obtain fourteen steviol glycoside derivatives; the bitterness and astringency of the product are obviously improved, and the taste is clear and sweet. St-Glc1 and St-Glc2 with the highest sweetness had the highest yields. With cyclodextrin and steviol glycoside as substrate, and the addition amount of 10 U / g of stevia glycoside, the conversion rate of stevia glycoside can reach 87.8% after 5 hours of reaction (Li W, 2013).
  • US patents US4219571 and US7807206 use cyclodextrin glucosyltransferase produced by Bacillus stearothermophilus to obtain ⁇ -1,4 glucosyl derivatives with a degree of polymerization of up to 10.
  • Chinese patent CN105899670A Add a mixture of amylase and cyclodextrin glucosyltransferase to a starch suspension and incubate at about 75-80 ° C for about 0.5 to 2 hours. After the liquefied starch suspension is formed, the starch is processed by low pH heat treatment.
  • the enzyme is inactivated; then the steviol glycoside is added to the liquefied starch suspension to form a reaction mixture, and a second batch of cyclodextrin glucosyltransferase is added to the reaction mixture and incubated at about 5-125 ° C for about 1 to 168 hours; subsequently adding one or more saccharidases, and incubating the reaction mixture at about 5-125 ° C for about 0.001-1168 hours, the resulting glucosteviol steviol glycoside composition contains ⁇ -1 having twenty or less, Steviol glycoside derivatives of 4-glucosyl residues.
  • the resulting product is decolorized using an ion exchange resin or membrane; the decolorized reaction mixture is then contacted with a macroporous adsorption resin to remove non-steviol glycoside compounds and then the adsorbed steviol glycosides are eluted with an alcohol or aqueous alcohol; the eluate Desalting is performed through a column or membrane filled with an ion exchange resin; alcohol is removed from the eluate to obtain an aqueous eluate; the aqueous eluate is concentrated and dried to obtain a dried glucosteviolside.
  • the purpose of the present invention is to provide a simple and economical method to prepare a low graft number of glucosyl steviol glycosides, that is, the content of mono- and diglucosyl substitutes of steviol glycosides in the product is 60% of the total glycosides.
  • the mass percentage of glucosyl steviol glycosides with a glucosyl graft number of 3 or less is higher than 70% of the total glycosides; the total glycosides refer to glucosteviol and steviol glycosides.
  • amylase is used to catalyze the hydrolysis of high-grafted steviol glycosides to prepare low-grafted steviol glycosides.
  • the product is directly spray-dried or slightly concentrated and dried to obtain a crude glucosteviolside.
  • Reductive sugar can be removed by recrystallization, and reductive sugar can also be adsorbed on the macroporous adsorption resin and eluted with water.
  • the mass percentage of glucosyl steviol glycosides with a high graft number of 4 or more is greater than the total mass of total glycosides (i.e., all steviol glycosides, including glucosyl stevioside and steviol glycoside).
  • 40% (determined by HPLC method) or the mass percentage of monoglucosyl-grafted steviol glycoside and diglucosyl-grafted steviol glycoside in the glucosyl steviol glycoside molecule with a high number of grafts of the raw material is lower than the total glycosides in total 50% (determined by HPLC method) is a commercial product well known to those skilled in the art.
  • the glucosyl steviol glycoside with high graft number of the raw material is a product of an enzymatic transglycosyl method, and the enzyme is a cyclodextrin glucosyltransferase.
  • amylase is derived from Aspergillusniger sp., Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Rhizopus oryzae Aspergillus (oryzae), a commercial product that complies with GB2760-2014.
  • an ⁇ -amylase derived from Bacillus subtilis is used as a catalyst, and a glucosyl steviol glycoside having a high graft number of 60 to 300 g / L is used as a substrate at 55 to 60 ° C.
  • the reaction takes 3 to 5 hours.
  • the amount of the ⁇ -amylase may be 1000 to 4000 U / g.
  • a saccharifying enzyme derived from Aspergillus niger is used as a catalyst, and a glucosyl steviol glycoside with a high graft number of 60 to 240 g / L is used as a substrate, and the reaction is performed at a temperature of 55 to 60 ° C for 3 to 24h.
  • the substrate concentration is preferably 60 to 240 g / L.
  • the amount of the saccharifying enzyme is preferably 50 to 800 U / g.
  • an ⁇ -amylase derived from Bacillus amyloliquefaciens is used as a catalyst, and a glucosyl steviol glycoside with a high grafting number of 60 to 240 g / L is used as a substrate at 55 to 60 ° C. Incubate for 3 to 24 hours.
  • the substrate concentration is preferably 60 g / L.
  • the amount of the ⁇ -amylase is preferably 50 to 800 U / g.
  • a ⁇ -amylase derived from Bacillus subtilis is used as a catalyst, and a glucosyl steviol glycoside with a high grafting number of 60 to 240 g / L is used as a substrate at 55 to 60 ° C.
  • the reaction takes 3 to 24 hours.
  • the substrate concentration is preferably 60 g / L.
  • the amount of the ⁇ -amylase is preferably 50 to 800 U / g.
  • the invention discloses a method for preparing a low grafting number of glucosyl steviol glycosides, which adopts a single enzyme and uses a high grafting number of glucosyl steviol glycosides as a substrate.
  • the reaction time is short, the process is simple, and sweetness can be obtained in high throughput.
  • the composition which is excellent in taste, is relatively simple and has a low number of grafted glucostein.
  • RD and RM are mono- and di-substituted glucose-based products with RA at the C19 position, respectively.
  • the mono- and di-substituted glucosyl steviol glycosides obtained by the enzyme catalysis of the present invention are similar in structure and belong to a mixture of isomers with good sweetness and taste correction functions.
  • Figure 1 HPLC chart of Bacillus subtilis alpha-amylase (4000 U / g sugar) for catalyzed hydrolysis of glucosyl stevioside.
  • FIG. 2 HPLC chart of Aspergillus niger saccharifying enzyme (50U / g sugar) for catalyzing hydrolysis of glucosteviolin.
  • FIG. 3 HPLC chart of Aspergillus niger saccharifying enzyme (100 U / g sugar) for catalyzing the hydrolysis of glucostein.
  • Figure 4 HPLC chart of Aspergillus niger saccharifying enzyme (300U / g sugar) for catalyzing the hydrolysis of glucosteviolside.
  • FIG. 5 HPLC chart of Bacillus amyloliquefaciens alpha-amylase (800 U / g sugar) for catalyzed hydrolysis of glucostein.
  • Figure 6 HPLC chart of Bacillus licheniformis ⁇ -amylase (300 U / g sugar) for catalytic hydrolysis of glucosyl stevioside.
  • FIG. 7 HPLC chart of Bacillus licheniformis alpha-amylase (300 U / g sugar) for catalytic hydrolysis of glucosyl stevioside.
  • FIG. 8 HPLC chart of Aspergillus niger saccharifying enzyme (50 U / g sugar, 60 ° C.) for catalyzing hydrolysis of glucostein.
  • Fig. 11 Effect of the amount of enzyme added on the preparation of low-graft glucosyl stevioside.
  • the saccharifying enzyme activity and ⁇ -amylase enzyme activity are determined by the method for measuring enzyme activity in the national standard GB1886.174-2016.
  • a low-graft glucosteviol glycoside was prepared from glucosteviol glycoside derived from rebaudioside A.
  • the raw material is a commercial product obtained by using rebaudioside A as a raw material and dextrin transglycoside catalyzed by cyclodextrin glucose transferase.
  • the mass fraction of rebaudioside A in the raw material was 8.3%, and the mass fractions of mono- and di-substituted products (monoglucosyl-grafted stevioside and diglucosyl-grafted stevioside) totaled 23.3%.
  • the total glycoside content in the product is 98.7%, the mono- and di-substituted glucosyl steviol glycoside content is 36.8% and 34.4%, accounting for 71.2%; the mono-, di- and tri-substituted glucosteviol glycoside content accounts for 86.1%, sweetness And taste correction function is better.
  • the high-pressure liquid phase diagram of the hydrolyzed product is shown in Figure 1 of the specification.
  • a low-graft glucosteviol glycoside was prepared from glucosteviol glycoside derived from rebaudioside A.
  • the mass fraction of rebaudioside A in the raw material was 9.8%, and the mass fraction of mono- and di-substituted products accounted for 30.5% in total.
  • the mono-, di-, and tri-substituted glucosteviol glycosides accounted for 76.3% in total, with good sweetness and taste correction functions.
  • the high-pressure liquid phase diagram of the hydrolyzed product is shown in Figure 2 of the specification.
  • a low-graft glucosteviol glycoside was prepared from glucosteviol glycoside derived from rebaudioside A.
  • the mass fraction of rebaudioside A in the raw material was 9.8%, and the mass fraction of mono- and di-substituted products accounted for 30.5% in total.
  • a low-graft glucosteviol glycoside was prepared from glucosteviol glycoside derived from rebaudioside A.
  • the mass fraction of rebaudioside A in the raw materials was 6.8%, and the mass fractions of mono- and di-substituted products accounted for 21.2%.
  • a low-graft number of glucosyl steviol glycosides was prepared using glucoside steviol glycoside with a mass fraction of rebaudioside A of 8.3% and a mass fraction of mono- and di-substituents of 23.3%.
  • a glucosyl steviol glycoside with a content of 9.1% and mono- and di-substituents accounting for a total of 23.7% was prepared with low-graft glucosyl steviol glycosides.
  • a low-glucose steviol glycoside was prepared using glucoside steviol glycoside with a content of 8.3% and mono- and di-substituents accounting for 23.3%.
  • Example 8 Synthesis of a low-graft glucosyl steviol glycoside using a saccharifying enzyme derived from Aspergillus sp. As a catalyst
  • a low-glucose steviol glycoside was prepared by using glucoside steviol glycoside with a content of 9.8% and 30.5% of mono- and di-substituents.
  • the raw material glycosides were prepared at a concentration of 100 g / L, 150 g / L, and 200 g / L at 60 ° C., and an a-amylase aqueous solution derived from Bacillus subtilis was added to the above solution at 300 U / g of the raw material glycoside under stirring.
  • the contents of various steviol glycosides in the product were measured after reacting at 60 ° C for different times. The results are shown in Figure 9 of the specification.
  • the raw material glycosides were prepared at a concentration of 100 g / L by using sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solutions (10 mmol / L) of different pH at 60 ° C, and the source was added to the above solution at 300 U / g of raw material glycosides under stirring.
  • Alpha-amylase in Bacillus subtilis The contents of various steviol glycosides in the product were measured after reacting at 60 ° C for different times, and the results are shown in the attached figure 10 of the specification.
  • the raw material glycosides were respectively prepared to a concentration of 100 g / L with water, and an a-amylase aqueous solution derived from Bacillus subtilis was added to the above solution at 300 U / g of the raw material glycosides under stirring.
  • the contents of various steviol glycosides in the product were measured after reacting at 70 ° C for different times. The results are shown in Figure 11 of the specification.

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Abstract

一种制备低接枝数葡萄糖基甜菊糖苷的方法，是用淀粉酶催化水解高接枝数的葡萄糖基甜菊糖苷以制备低接枝数的葡萄糖基甜菊糖苷，所述低接枝数的葡萄糖基甜菊糖苷是甜菊糖苷的单和二葡萄糖基取代物含量为总苷的60％以上，葡萄糖基接枝数小于等于3的葡萄糖基甜菊糖苷的质量百分数总计高于总苷的70％；所述总苷包括葡萄糖基甜菊糖苷和甜菊糖苷。还涉及由该方法制得的低接枝数葡萄糖基甜菊糖苷产品，包含该产品的甜味剂或矫味剂以及该产品在制备甜味剂或矫味剂中的应用，包含该产品的的食品、饮料或调味料以及该产品在制备食品、饮料或调味料中的应用。

Description

一种制备低接枝数葡萄糖基甜菊糖苷的方法 技术领域
本发明涉及一种制备低接枝数葡萄糖基甜菊糖苷的方法，属于甜味剂的生物合成技术领域。
背景技术
甜菊糖苷是甜叶菊提取物中约40种糖苷分子的总称，均由具有甜味的亲水性糖基与疏水性的甜菊醇苷元组成。其中Rebaudioside D和Rebaudioside M的味质口感较好，莱鲍迪苷A(Rebaudioside A，RA)口感较好但甜味略嫌持久，而斯替夫苷(Stevioside，St)、莱鲍迪苷C和其它低分子量甜菊糖苷则带有一定的轻微苦味或甘草余味，这种微苦余味降低了它作为天然甜味剂的品质。除了具有苦味的杂质对甜菊糖苷产品口感味质有影响外，甜菊糖苷自身的甜菊醇苷元分子结构也是产生苦涩余味和后甜(即甜味持久)的因素。而从味觉角度的生物学来看，不同甜菊糖苷与甜味受体和苦味受体的结合能差异是一个重要因素。
对甜菊糖苷口感进行改善的方法之一是利用酶法糖基化等方法引入糖基，利用接入糖基的空间位阻，改变甜菊糖苷与甜味受体和苦味受体的结合能差异，对其口感味质进行改善。但酶法糖基化会得到很多种无规的转糖基产物，典型的如各种葡萄糖基取代数或称接枝数的葡萄糖基甜菊糖苷，产品中接枝数从1到20。葡萄糖基甜菊糖苷分子中，低接枝数产品如单和二葡萄糖基接枝的甜菊糖苷的质量百分数总计通常低于总苷的50％。所有葡萄糖基甜菊糖苷的甜度都会比原料甜菊糖苷的甜度有不同程度的降低，后味即口感也会有所改善。葡萄糖基取代数高的产品，可以用作矫味剂和餐桌糖，但甜度下降过多。葡萄糖基取代数低的产品，如甜菊糖苷的单和二葡萄糖基取代物，其甜度和矫味功能均较好；其结构同于天然甜菊糖中味质最好的RD和RM的同分异构体。
一种利用酶法糖基化等方法引入糖基对甜菊糖苷口感进行改善的方法，是采用来自嗜碱软性芽胞杆菌的环糊精葡萄糖基转移酶催化甜菊苷St的转苷，得到9种产物，结构分析显示在C-13位和C-19位通过α-1,4糖苷键分别连接了1到3个葡萄糖基，而C-13位连接1个或2个葡萄糖基时，味质与St相比都已改善；当C-13位连接3个葡萄糖基时，甜度则大幅下降(Fukunaga,1989)。Lobov等利用普鲁兰酶(Pullulanase)和真菌淀粉酶(Biozyme L)，用于甜菊糖苷的转苷反应，分别得到三种产物；但反应所需时间较长且产物收率小于10％(Lobov,1991)。Abelyan用环糊精作为糖基供体，得到九种转糖基产物，其中两种葡萄糖基甜菊糖苷的甜味特性较好，但产率较低，产率之和为11.6％(Abelyan V A,2004)。Kochikyan用六种菌 株生产出不同种类的环糊精葡萄糖基转移酶，以液化淀粉作为糖基供体催化甜菊糖苷转苷，得到七种转糖基产物(Kochikyan V T,2006)。
另一种对甜菊糖苷口感进行改善的方法，是用α-环糊精葡萄糖基转移酶Toruzyme 3.0L和α-中温淀粉酶双酶法催化水解淀粉改性St，反应4h，St转化率达到77.11％，得到十四种甜菊糖苷衍生物；产物的后苦涩味明显改善，口感清甜。甜度最高的St-Glc1和St-Glc2的产率最高。以环糊精和斯替夫苷为底物，以10U/g斯替夫苷的加酶量，反应5h后斯替夫苷的转化率最高可达到87.8％(Li W,2013)。
美国专利US4219571和US7807206利用嗜热脂肪芽孢杆菌产的环糊精葡萄糖基转移酶，得到具有高达10的聚合度的α-1,4葡糖基衍生物。
中国专利CN105899670 A将淀粉酶和环糊精葡萄糖基转移酶的混合物加入到淀粉悬浮液中并在约75-80℃下孵育约0.5至2小时，生成液化淀粉悬浮液后通过低pH热处理将淀粉酶灭活；然后将甜菊醇糖苷加入到液化淀粉悬浮液中，生成反应混合物，再将第二批环糊精葡萄糖基转移酶加入到反应混合物中并在约5-125℃下孵育约1至168小时；随后加入一种或几种糖酶，将反应混合物在约5-125℃下孵育约0.0001-168小时，所得葡萄糖基甜菊糖苷组合物包含具有二十个或更少的α-1，4-葡糖基残基的甜菊醇糖苷衍生物。所得到的产品使用离子交换树脂或膜进行脱色；再将脱色后的反应混合物与大孔吸附树脂接触来除去非甜菊糖苷化合物并随后用醇或含水醇洗脱吸附的甜菊糖苷；将洗脱液通过填充有离子交换树脂的柱或膜来进行脱盐；从洗脱液中除去醇，获得含水洗脱液；将含水洗脱液浓缩并干燥以获得干燥的葡萄糖基甜菊糖苷。
综上所述，需要提供一种简便的制备方法来获得以低接枝数为主的葡萄糖基甜菊糖苷，即单和二取代葡萄糖基甜菊糖苷的质量含量超过50％的产品，这样可以兼顾产品甜度和矫味功能。
发明内容
本发明的目的在于针对以上现状的不足，提供一种简单、经济的方法以制备低接枝数葡萄糖基甜菊糖苷，即产品中甜菊糖苷的单和二葡萄糖基取代物含量为总苷的60％以上，葡萄糖基接枝数小于等于3的葡萄糖基甜菊糖苷的质量百分数总计高于总苷的70％；所述总苷指葡萄糖基甜菊糖苷和甜菊糖苷。
具体地，用淀粉酶催化水解高接枝数的葡萄糖基甜菊糖苷以制备低接枝数葡萄糖基甜菊糖苷。产品经直接喷雾干燥或者稍经浓缩并干燥即得葡萄糖基甜菊糖苷粗产品，可以用重结晶脱去副产品还原糖，还可以将副产品还原糖等经大孔吸附树脂吸附后用水洗脱。
所述原料高接枝数的葡萄糖基甜菊糖苷分子中接枝数大于等于4的葡萄糖基甜菊糖苷的质量百分数总计高于总苷(即所有甜菊糖苷，包括葡萄糖基甜菊糖苷和甜菊糖苷)总质量的40％(HPLC法测定)，或者，所述原料高接枝数的葡萄糖基甜菊糖苷分子中单葡萄糖基接枝的甜菊糖苷和二葡萄糖基接枝的甜菊糖苷的质量百分数总计低于总苷的50％(HPLC法测定)，是本领域技术人员公知的商业化产品。
所述原料高接枝数的葡萄糖基甜菊糖苷是酶促转糖基法的产物，所述酶是环糊精葡萄糖基转移酶。
所述淀粉酶来源于黑曲霉(Aspergillusniger sp.)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、米根霉(Rhizopus oryzae)或米曲霉(Aspergillus oryzae)，符合GB2760-2014的商业化产品。
在本发明一种实施方式中，以来源于枯草芽孢杆菌的α-淀粉酶为催化剂，以60～300g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～5h。所述α-淀粉酶的用量可以是1000～4000U/g。
在本发明一种实施方式中，以来源于黑曲霉的糖化酶为催化剂，以60～240g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～24h。所述底物浓度优选60～240g/L。所述糖化酶的用量优选50～800U/g。
在本发明一种实施方式中，以来源于解淀粉芽孢杆菌的α-淀粉酶为催化剂，以60～240g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～24h。所述底物浓度优选60g/L。所述α-淀粉酶的用量优选50～800U/g。
在本发明一种实施方式中，以来源于枯草芽孢杆菌的β-淀粉酶为催化剂，以60～240g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～24h。所述底物浓度优选60g/L。所述β-淀粉酶的用量优选50～800U/g。
[有益效果]
本发明公开的一种制备低接枝数葡萄糖基甜菊糖苷的方法，采用单酶，以高接枝数的葡萄糖基甜菊糖苷为底物，反应时间短，工艺简洁，可以高通量得到甜味和口感俱佳的组成相对简单、低接枝数葡萄糖基甜菊糖苷。
目前市场上最欢迎的甜菊醇糖苷是RD和RM，即分别为RA在C19位的单和二取代葡萄糖基产品。本发明酶催化所得的单和二取代葡萄糖基甜菊糖苷在结构上与之类似，属于其同分异构体的混合物，甜度和矫味功能均较好。
附图说明
图1枯草芽孢杆菌α-淀粉酶(4000U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图2黑曲霉糖化酶(50U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图3黑曲霉糖化酶(100U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图4黑曲霉糖化酶(300U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图5解淀粉芽孢杆菌α-淀粉酶(800U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图6地衣芽孢杆菌β-淀粉酶(300U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。
图7地衣芽孢杆菌α-淀粉酶(300U/g糖)催化水解葡萄糖基甜菊糖苷的HPLC图。图8黑曲霉糖化酶(50U/g糖，60℃)催化水解葡萄糖基甜菊糖苷的HPLC图。
图9底物浓度对制备低接枝数葡萄糖基甜菊糖苷的影响。
图10pH对制备低接枝数葡萄糖基甜菊糖苷的影响。
图11加酶量对制备低接枝数葡萄糖基甜菊糖苷的影响。
具体实施方式
分析计算方法：
(1)葡萄糖基甜菊糖苷的定性分析：采用液相色谱串联四极杆飞行时间质谱仪定性转苷产物，检测条件如下：ACQUITYUPLC BEH HILIC氨基色谱柱，柱温为30℃，乙腈：水＝80：20,(2min)-50：50(30min)(v/v)下梯度洗脱，进样量1μL，进样浓度为5mg/mL，流速为0.3mL/min；质谱条件为碰撞电压为6eV；离子化方式电喷雾电离(ESI)，负离子检测模式，分子量范围：200-2000。
(2)葡萄糖基甜菊糖苷的定量分析依据GB2760-2014卫计委增补文件第8号文件中葡萄糖基甜菊糖苷的分析检测方法。
(3)糖化酶酶活、α-淀粉酶酶活用国标GB1886.174—2016中酶活测定方法测定。
实施例1以源于Bacillus subtilis的α-淀粉酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
用源于莱苞迪苷A的葡萄糖基甜菊糖苷为原料制备低接枝数葡萄糖基甜菊糖苷。该原料是以莱苞迪苷A为原料和糊精在环糊精葡萄糖转移酶的催化下转苷得到的商品。原料中莱苞迪苷A的质量分数为8.3％，单、二取代物(单葡萄糖基接枝的甜菊糖苷和二葡萄糖基接枝的甜菊糖苷)的质量分数共计23.3％。
在夹套反应器中加入20g水，加热到60℃后先后投入1.2g原料糖苷，搅拌溶解后，搅拌条件下向上述夹套反应器中按4000U/g原料加入来源于Bacillus subtilis的α-淀粉酶(无锡雪梅酶制剂厂出品)水溶液。60℃下反应5h，结束反应。产品中总苷含量为98.7％，单、二取代葡萄糖基甜菊糖苷的含量分别为36.8％和34.4％，共占71.2％；单、双、三取代葡萄糖 基甜菊糖苷含量共占86.1％，甜度和矫味功能均较好。水解后产品的高压液相图见说明书附图1。
实施例2以来源于Aspergillus sp.的糖化酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
用源于莱苞迪苷A的葡萄糖基甜菊糖苷为原料制备低接枝数葡萄糖基甜菊糖苷。原料中莱苞迪苷A的质量分数为9.8％，单、二取代物的质量分数共占30.5％。
在夹套反应器中加入20g水，加热到55℃后投入4.8g原料糖苷，搅拌溶解后，搅拌下向上述夹套反应器中按50U/g原料糖苷加入来源于Aspergillus sp.的糖化酶(山东西亚化学工业有限公司出品)水溶液。55℃下反应7.5h，结束反应。产品中总苷含量为99.4％，单、二取代葡萄糖基甜菊糖苷的含量分别为43.9％和25.9％，共占69.8％。单、双、三取代葡萄糖基甜菊糖苷含量共占76.3％，甜度和矫味功能均较好。水解后产品的高压液相图见说明书附图2。
实施例3以来源于Aspergillus sp.的糖化酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
用源于莱苞迪苷A的葡萄糖基甜菊糖苷为原料制备低接枝数葡萄糖基甜菊糖苷。原料中莱苞迪苷A的质量分数为9.8％，单、二取代物的质量分数共占30.5％。
在夹套反应器中加入20g水，加热到55℃后投入1.2g原料糖苷，搅拌溶解后，搅拌下向上述夹套反应器中按100U/g原料糖苷加入来源于Aspergillus sp.的糖化酶水溶液。55℃下反应2.5h，结束反应。产品中总苷含量98.0％，单、二取代葡萄糖基甜菊糖苷的含量分别为40.2％和25.3％，共占65.5％。单、双、三取代葡萄糖基甜菊糖苷含量占75.6％，甜度和矫味功能均较好。水解后产品的高压液相图见说明书附图3。
实施例4以源于Aspergillus sp.的糖化酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
用源于莱苞迪苷A的葡萄糖基甜菊糖苷为原料制备低接枝数葡萄糖基甜菊糖苷。原料中莱苞迪苷A的质量分数为6.8％，单、二取代物的质量分数共占21.2％。
在夹套反应器中加入20g水，加热到55℃后投入1.2g原料糖苷，搅拌溶解后，搅拌下向上述夹套反应器中按300U/g原料糖苷加入来源于Aspergillus sp.的糖化酶水溶液。55℃下反应0.5h，结束反应。产品中总苷含量为99.0％，单、二取代葡萄糖基甜菊糖苷的含量分别为36.5％和28.9％，共占65.4％。单、双、三取代葡萄糖基甜菊糖苷含量共占80.2％，甜度和矫味功能均较好。水解后产品的高压液相图见说明书附图4。
实施例5以来源于Bacillus amyloliquefaciens的α-淀粉酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
以莱苞迪苷A的质量分数为8.3％，单、二取代物的质量分数共占23.3％的葡萄糖基甜 菊糖苷为原料，制备低接枝数葡萄糖基甜菊糖苷。
在夹套反应器中加入20g水，加热到60℃后先后投入1.2g糖，搅拌溶解后，搅拌下向上述夹套反应器中按800U/g原料糖苷加入来源于Bacillus amyloliquefaciens的α-淀粉酶水溶液。60℃下反应24h，结束反应。产品中总苷含量为99.0％，单、二取代葡萄糖基甜菊糖苷的含量分别为33.7％和34.0％，共占67.7％。单、双、三取代葡萄糖基甜菊糖苷含量共占85.1％，甜度和矫味功能均较好。水解后产品的高压液相图见说明书附图5。
实施例6以源于Bacillus licheniformis的β-淀粉酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
以莱苞迪苷A的含量为9.1％，单、二取代物共占23.7％的葡萄糖基甜菊糖苷为原料，制备低接枝数葡萄糖基甜菊糖苷。
在夹套反应器中加入20g水，加热到60℃后先后投入4g糖，搅拌溶解后，搅拌下向上述夹套反应器中按300U/g原料糖苷加入Bacillus licheniformis β-淀粉酶水溶液。60℃下反应3h，结束反应。产品中总苷含量为92.4％，单、二取代葡萄糖基甜菊糖苷的含量分别为33.3％和27.2％，共占60.5％，甜度和矫味功能均较好。单、双、三取代葡萄糖基甜菊糖苷含量共占71.7％。水解后产品的高压液相图见说明书附图6。
实施例7以源于Bacillus licheniformis的α-淀粉酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
以莱苞迪苷A的含量为8.3％，单、二取代物共占23.3％的葡萄糖基甜菊糖苷为原料，制备低接枝数葡萄糖基甜菊糖苷。
在夹套反应器中加入20g水，加热到85℃后先后投入6g糖，搅拌溶解后，搅拌下向上述夹套反应器中按300U/g原料糖苷加入Bacillus licheniformis α-淀粉酶水溶液。85℃下反应3h，结束反应。产品中总苷含量为95.6％，单、二取代葡萄糖基甜菊糖苷的含量分别为37.7％和25.9％，共占63.6％。单、双、三取代葡萄糖基甜菊糖苷含量共占71.6％。水解后产品的高压液相图见说明书附图7。
实施例8以源于Aspergillus sp.的糖化酶为催化剂合成低接枝数葡萄糖基甜菊糖苷
以莱苞迪苷A的含量为9.8％，单、二取代物共占30.5％的葡萄糖基甜菊糖苷为原料，制备低接枝数葡萄糖基甜菊糖苷。
在夹套反应器中加入20g水，加热到60℃后先后投入5g糖，搅拌溶解后，搅拌下向上述夹套反应器中按50U/g原料糖苷加入Aspergillus sp.的糖化酶水溶液。60℃下反应7h，结束反应。产品中总苷含量为96.1％，单、二取代葡萄糖基甜菊糖苷的含量分别为47.6％和15.3％，共占62.9％。单、双、三取代葡萄糖基甜菊糖苷含量共占71.9％。水解后产品的高压液相图见说明书附图8。
实施例9原料糖苷浓度对制备低接枝数葡萄糖基甜菊糖苷的影响
60℃下分别将原料糖苷配制成100g/L、150g/L和200g/L的浓度反应，搅拌下向上述溶液中按300U/g原料糖苷加入来源于Bacillus subtilis的α-淀粉酶水溶液。60℃下反应不同时间后测定产品中各种甜菊糖苷的含量，结果见说明书附图9。
由图9可以看出，实验范围内底物浓度的升高对制备低接枝数葡萄糖基甜菊糖苷没有显著影响；当然，影响程度可能会因淀粉酶品种而异的。随着反应时间的延长，一定范围内，高接枝数的产物浓度迅速降低，而低接枝数物的浓度迅速升高。
实施例10pH对制备低接枝数葡萄糖基甜菊糖苷的影响
60℃下分别用不同pH的磷酸二氢钠—磷酸氢二钠缓冲液(10mmol/L)将原料糖苷配制成100g/L的浓度反应，搅拌下向上述溶液中按300U/g原料糖苷加入来源于Bacillus subtilis的α-淀粉酶水溶液。60℃下反应不同时间后测定产品中各种甜菊糖苷的含量，结果见说明书附图10。
实施例11加酶量对制备低接枝数葡萄糖基甜菊糖苷的影响
70℃下分别用水将原料糖苷配制成100g/L的浓度反应，搅拌下向上述溶液中按300U/g原料糖苷加入来源于Bacillus subtilis的α-淀粉酶水溶液。70℃下反应不同时间后测定产品中各种甜菊糖苷的含量，结果见说明书附图11。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，用淀粉酶催化水解高接枝数的葡萄糖基甜菊糖苷以制备低接枝数的葡萄糖基甜菊糖苷。
  2. 根据权利要求1所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述低接枝数的葡萄糖基甜菊糖苷是甜菊糖苷的单和二葡萄糖基取代物含量为总苷的60％以上，葡萄糖基接枝数小于等于3的葡萄糖基甜菊糖苷的质量百分数总计高于总苷的70％；所述总苷包括葡萄糖基甜菊糖苷和甜菊糖苷。
  3. 根据权利要求1或2所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述原料高接枝数的葡萄糖基甜菊糖苷分子中接枝数大于等于4的葡萄糖基甜菊糖苷的质量百分数总计高于总苷总质量的40％，或者，所述原料高接枝数的葡萄糖基甜菊糖苷分子中单葡萄糖基接枝的甜菊糖苷和二葡萄糖基接枝的甜菊糖苷的质量百分数总计低于总苷的50％。
  4. 根据权利要求1～3任一所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述淀粉酶来源于黑曲霉(Aspergillusniger sp.)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、米根霉(Rhizopus oryzae)或米曲霉(Aspergillus oryzae)。
  5. 根据权利要求1～4任一所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，酶催化产物经直接喷雾干燥或者稍经浓缩并干燥即得葡萄糖基甜菊糖苷粗产品；所得粗产品还可以用重结晶脱去副产品还原糖，或者将副产品还原糖等经大孔吸附树脂吸附后用水洗脱。
  6. 根据权利要求1～4任一所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，以来源于枯草芽孢杆菌的α-淀粉酶为催化剂，以60～300g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～5h。
  7. 根据权利要求6所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述α-淀粉酶的用量是1000～4000U/g。
  8. 根据权利要求1～4任一所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，以来源于黑曲霉的糖化酶为催化剂，以60～240g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～24h。
  9. 根据权利要求8所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述糖化酶的用量为50～800U/g。
  10. 根据权利要求1～4任一所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，以来源于解淀粉芽孢杆菌的α-淀粉酶为催化剂或者来源于枯草芽孢杆菌的β-淀粉酶为催化剂，以60～240g/L的高接枝数的葡萄糖基甜菊糖苷为底物，在55～60℃条件下反应3～24h。
  11. 根据权利要求8所述的一种制备低接枝数葡萄糖基甜菊糖苷的方法，其特征在于，所述β-淀粉酶的用量为50～800U/g。
  12. 根据权利要求1～11任一所述方法制备得到的低接枝数葡萄糖基甜菊糖苷。
  13. 权利要求12所述低接枝数葡萄糖基甜菊糖苷在制备甜味剂或矫味剂中的应用。
  14. 含有权利要求12所述低接枝数葡萄糖基甜菊糖苷的甜味剂或矫味剂。
  15. 权利要求12所述低接枝数葡萄糖基甜菊糖苷在制备食品、饮料或调味料中的应用。
  16. 含有权利要求12所述低接枝数葡萄糖基甜菊糖苷的食品、饮料或调味料。
PCT/CN2018/091271 2018-05-31 2018-06-14 一种制备低接枝数葡萄糖基甜菊糖苷的方法 WO2019227532A1 (zh)

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