阅读说明：本技术 在重组宿主中生产甜菊醇糖苷的方法 (Method for producing steviol glycosides in recombinant hosts ) 是由 陈宇杰 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种在重组宿主中生产甜菊醇糖苷的方法,包括以下步骤：选材、提取、分离、纯化和混合五到工序进行在重组宿主中生产甜菊醇糖苷,使得甜菊醇糖苷安全性高,经国内外药理实验证明,甜菊糖甙为非致癌性物质,无毒无副作用,食用安全。甜叶菊原产地南美巴拉圭、巴西等地的居民食用甜叶菊已有四百多年历史,至今未发现有任何毒害,低热值,可应用于低热量食品、饮料和医药。长期食用甜菊糖甙不会使人发胖,且有降血糖、防龋齿、健胃、解除疲劳之功能,并对健美减肥和糖尿病、心脏病、高血压、动脉硬化等患者食用有辅助疗效,是制作保健食品、保健饮料、保健化妆品和医药制品等最佳天然甜味剂。(The invention discloses a method for producing steviol glycosides in a recombinant host, comprising the steps of: the steviol glycoside is produced in a recombinant host through five working procedures of material selection, extraction, separation, purification and mixing, so that the steviol glycoside has high safety, and pharmacological experiments at home and abroad prove that the steviol glycoside is a non-carcinogenic substance, has no toxic or side effect and is safe to eat. The stevia rebaudiana is eaten by residents in places such as the original places of the stevia rebaudiana, paraguay, brazil and the like for more than four hundred years, has no toxic hazard and low calorific value so far, and can be applied to low-calorie foods, beverages and medicines. The stevioside can not cause people to be fat after long-term eating, has the functions of reducing blood sugar, preventing decayed teeth, invigorating stomach and relieving fatigue, has auxiliary curative effects on body building and weight losing and eating by patients with diabetes, heart disease, hypertension, arteriosclerosis and the like, and is the best natural sweetening agent for preparing health-care food, health-care beverage, health-care cosmetics, medical products and the like.)

1. A method of producing steviol glycosides in a recombinant host, comprising the steps of:

s1, selecting materials

Screening fresh stevia rebaudiana bertoni leaves of Compositae, cleaning the fresh stevia rebaudiana bertoni leaves of Compositae, drying or sun-drying the cleaned stevia rebaudiana bertoni leaves of Compositae in a dryer to obtain dry stevia rebaudiana bertoni leaves of Compositae, grinding the dry stevia rebaudiana bertoni leaves of Compositae into powder by a grinder, and finally sieving the powder by a sieve to obtain dry powder of the stevia rebaudiana bertoni leaves;

s2, extraction

Determining that the optimal extraction method is an enzyme-adding extraction method, wherein the optimal process conditions of the enzyme-adding method are as follows: adopting cellulase, wherein the optimal leaching temperature is 45 ℃, the optimal pH =5, the enzyme dosage is 0.3%, and the leaching time is 100-120 min;

s3, separation

Adding a flocculating agent into the feed liquid to achieve the aim of removing impurities, determining to adopt a novel efficient composite PAMCaOFeCl3+ + organic polymer flocculating agent, wherein the optimal flocculation conditions are that the optimal composite flocculating agent is prepared from PAM, CaO, FeCl3= 1: 2.5: 0.05, optimal flocculation pH =9, standing in a water bath at 70-90 ℃ for 10-30 min;

s4, purification

Adsorbing, decolorizing and refining with resin, decolorizing with CL type ion exchange fiber, adsorbing with macroporous resin, washing with acid and alkali, eluting with 70% -90% ethanol, adsorbing with macroporous resin at pH 8-10 and adsorption flow rate of 2BV/h to obtain stevioside with maximum adsorption amount, eluting with 70% ethanol at 1BV/h and 2BV/h

The optimal technological conditions for acid washing and refining are as follows: the concentration of acid (HCl) is 5%, the washing flow is 1BV/h, the acid consumption is 2BV, and the optimal process conditions for alkali washing and refining are as follows: the concentration of alkali (NaOH) is 3%, the flow rate is 1BV/h, and the amount of alkali liquor is 2 BV;

s5, mixing

Recombinant microorganisms and methods for the production of steviol glycosides using recombinant hosts.

2. The method for producing steviol glycosides in recombinant host according to claim 1, wherein stevia rebaudiana bertoni leaf of the family Compositae, which is a white powder, belongs to the glycosides of tetracyclic diterpenes

Terpenoids containing 20 carbon atoms, of the general formula (C5H 8) 4, isolated from stevia rebaudiana Bertoni at least 8 different sweetness levels of stevioside, with stevioside as the major component, 60% ~ 70%, followed by rebaudioside a in the range of 15% ~ 20%, along with rubusoside, steviol, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F and dulcoside a.

3. A method of producing steviol glycosides in a recombinant host according to claim 2, wherein the steviol glycosides are mainly classified into three main groups:

a. stevioside

Colorless crystals with sweetness 270 ~ 280 times that of sucrose, and the proportion of the crystals in the mixed crystal is 50% ~ 70%;

b. rebaudioside A

The god-delivering method can be further divided into four of RA, RC, RD, RE, etc., wherein RA is colorless JI crystal

The sweetness of sucrose is 450 times that of sucrose, RC is colorless and takes crystal, the sweetness is less than 50 times that of sucrose and the crystal is bitter

RD is colorless crystal, and sweetness is 150 times of that of sucrose

RE has sweetness 100, 150 times that of sucrose, RA accounts for about 15%, 20% of the sweet in dry leaves, and RC accounts for 10%

About 15%, the proportion of RD and RE is small;

c. bayer process for purifying

The glycoside can be divided into DA and DB, wherein DB and RC are the same substance, DA accounts for in dry leaves, and its sweetness is about 150 times that of sucrose.

4. The method for producing steviol glycosides in a recombinant host according to claim 2, wherein the mixing device in step S5 is mixed for 20-40 min.

5. A method of producing steviol glycosides in a recombinant host according to claim 2, characterized in that the steviol glycosides accumulate to a detectable concentration when cultured under the conditions.

6. A method of producing steviol glycosides in a recombinant host as claimed in claim 2, wherein the method produces steviol glycoside compositions wherein the composition has a steviol glycoside composition enriched in RebD or RebM relative to a steviol glycoside composition of a wild type stevia plant.

7. The method of claim 1, wherein the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.

8. The method of claim 7, wherein the bacterial cell comprises an Escherichia bacterial cell, a Lactobacillus bacterial cell, a lactococcus bacterial cell, a Corynebacterium bacterial cell, an Acetobacter bacterial cell, an Acinetobacter bacterial cell, or a Pseudomonas bacterial cell.

9. The method of claim 7, wherein the fungal cell comprises a yeast cell, and wherein the yeast cell is a yeast.

10. The method of claim 9, wherein the yeast cell is a cell from a species of saccharomyces cerevisiae, schizosaccharomyces pombe, yarrowia lipolytica, candida glabrata, ashbya gossypii, candida utilis, pichia pastoris, kluyveromyces lactis, hansenula polymorpha, candida boidinii, rhodobacter rhodochrous, or candida albicans.

Technical Field

The invention relates to the technical field of steviol glycosides, in particular to a method for producing steviol glycosides in a recombinant host.

Background

Sweeteners are well known as the most commonly used ingredient in the food, beverage or confectionery industries, either added to the final food product during manufacture or used alone, with appropriate dilution, as a table sweetener or as a household replacement for sugar in baking, including natural sweeteners (e.g., sucrose, high fructose corn syrup, molasses, maple syrup and honey) and artificial sweeteners (e.g., aspartame, saccharin and sucralose). Stevia extract is a natural sweetener that can be isolated and extracted from the evergreen shrub stevia. Stevia is widely grown in south america and asia for commercial production of stevia extracts. Stevia extracts of varying degrees of purification are used commercially as high intensity sweeteners in food products, as well as blended or individually as table sweeteners, and stevia plant extracts contain rebaudiosides and other steviol glycosides that contribute sweetness, but the amount of each glycoside often varies from product batch to product batch. The predominant commercial product available is rebaudioside a, and lesser amounts of other glycosides, such as rebaudioside C, D and F. Stevia extracts may also contain impurities such as plant-derived compounds that cause off-flavors. These off-tastes may be more or less problematic depending on the food system or application, and thus affect the rate of production in the steviol glycoside recombinant host, and thus the sweetness of the steviol glycoside.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for producing steviol glycoside in a recombinant host, which has the advantages of high sweetness and the like.

(II) technical scheme

In order to achieve the purpose of high sweetness, the invention provides the following technical scheme: a method of producing steviol glycosides in a recombinant host, comprising the steps of:

s1, selecting materials

Screening fresh stevia rebaudiana bertoni leaves of Compositae, cleaning the fresh stevia rebaudiana bertoni leaves of Compositae, drying or sun-drying the cleaned stevia rebaudiana bertoni leaves of Compositae in a dryer to obtain dry stevia rebaudiana bertoni leaves of Compositae, grinding the dry stevia rebaudiana bertoni leaves of Compositae into powder by a grinder, and finally sieving the powder by a sieve to obtain dry powder of the stevia rebaudiana bertoni leaves.

S2, extraction

Determining that the optimal extraction method is an enzyme-adding extraction method, wherein the optimal process conditions of the enzyme-adding method are as follows: adopting cellulase, wherein the optimal leaching temperature is 45 ℃, the optimal pH =5, the enzyme dosage is 0.3%, and the leaching time is 100-120 min.

S3, separation

Adding a flocculating agent into the feed liquid to achieve the aim of removing impurities, determining to adopt a novel efficient composite PAMCaOFeCl3+ + organic polymer flocculating agent, wherein the optimal flocculation conditions are that the optimal composite flocculating agent is prepared from PAM, CaO, FeCl3= 1: 2.5: 0.05, optimal flocculation pH =9, and standing in a water bath at 70-90 ℃ for 10-30 min.

S4, purification

The method comprises the steps of performing adsorption, decoloration and refining by using resin, decoloring by using CL type ion exchange fiber, adsorbing by using macroporous adsorption resin, washing by using acid and alkali, eluting by using 70-90% ethanol, performing dynamic adsorption by using the macroporous adsorption resin at the pH of 8-10 and at the adsorption flow rate of 2BV/h, wherein the adsorption amount of stevioside is the largest, and simultaneously, completely desorbing by using 2BV and using 70% ethanol as an eluent at the flow rate of 1 BV/h. The optimal technological conditions for acid washing and refining are as follows: the concentration of acid (HCl) is 5%, the washing flow is 1BV/h, the acid consumption is 2BV, and the optimal process conditions for alkali washing and refining are as follows: the concentration of alkali (NaOH) is 3%, the flow rate is 1BV/h, and the amount of alkali liquor is 2 BV.

S5, mixing

Recombinant microorganisms and methods for the production of steviol glycosides using recombinant hosts.

Preferably, the herb stevia rebaudiana Bertoni of Compositae belongs to glycosides of tetracyclic diterpenes, and is white powder, terpenoids containing 20 carbon atoms, which have a general formula of (C5H 8) 4, at least 8 kinds of stevioside with different sweetness are separated from stevia rebaudiana Bertoni, wherein stevioside (stevioside) is the main component accounting for 60% ~%, rebaudioside A (rebaudioside A) accounting for 15% ~%, and rubusoside, steviol, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F and dulcoside A.

Preferably, the stevioside is mainly classified into three main groups:

a. stevioside is colorless crystal, the sweetness of which is 270 ~ 280 times that of sucrose, is a main component of six stevia products, and the proportion of the stevioside in the mixed Xixi is 50% ~ 70%, the taste is good, and the aftertaste is long and slightly bitter;

b. rebaudioside A. The Shen-delivering method can be further divided into four types, namely RA, RC, RD, RE, etc., wherein RA is colorless crystals. The sweetness of sucrose is 450 times that of sucrose, the sweetness is pure and similar to that of sucrose, the most serious part of the sweetness of six wu tenu stevia products is that RC is colorless and crystal, and the sweetness is less than 50 times that of sucrose and has bitter taste. RD is colorless crystal, and the sweetness is 150 times of that of sucrose. RE has 100, 150 times of that of cane sugar, RA accounts for about 15%, 20% of in dry leaves, and RC accounts for 10%. About 15%, the proportion of RD and RE is small;

c. bayer process. The glycoside can be divided into DA and DB, wherein DB and RC are the same substance, DA accounts for in dry leaves, and its sweetness is about 150 times that of sucrose.

Preferably, the mixing time of the stirring device in the step S5 is 20-40 min.

Preferably, the steviol glycosides accumulate to a detectable concentration when cultured under such conditions.

Preferably, the method produces a steviol glycoside composition, wherein the composition has a steviol glycoside composition enriched in RebD or RebM relative to a steviol glycoside composition of a wild-type stevia plant.

Preferably, the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell or a bacterial cell.

Preferably, the bacterial cell includes an Escherichia bacterial cell, a Lactobacillus bacterial cell, a lactococcus bacterial cell, a Corynebacterium bacterial cell, an Acetobacter bacterial cell, an Acinetobacter bacterial cell or a Pseudomonas bacterial cell.

Preferably, the fungal cell comprises a yeast cell, which is a yeast.

Preferably, the yeast cell is a cell from the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Candida utilis, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Phaffia rhodozyma or Candida albicans.

5) Recombinant microorganisms and methods for the production of steviol glycosides using recombinant hosts.

(III) advantageous effects

Compared with the prior art, the invention provides a method for producing steviol glycoside in a recombinant host, which has the following beneficial effects:

according to the method for producing the steviol glycoside in the recombinant host, the steviol glycoside is produced in the recombinant host through five working procedures of material selection, extraction, separation, purification and mixing, so that the steviol glycoside is high in safety, and pharmacological experiments at home and abroad prove that the steviol glycoside is a non-carcinogenic substance, is non-toxic and free of side effect and is safe to eat. The stevia rebaudiana is eaten by residents in places such as the original places of the stevia rebaudiana, paraguay, brazil and the like for more than four hundred years, has no toxic hazard and low calorific value so far, and can be applied to low-calorie foods, beverages and medicines. The stevioside does not cause people to be fat after being eaten for a long time, has the functions of reducing blood sugar, preventing decayed teeth, invigorating stomach and relieving fatigue, has auxiliary curative effects on body building and weight losing and eating by patients with diabetes, heart disease, hypertension, arteriosclerosis and the like, is the best natural sweetener for manufacturing health-care food, health-care beverage, health-care cosmetics, pharmaceutical products and the like, has high sweetness, can reduce the cost by more than 60 percent by replacing cane sugar with the stevioside, reduces transportation amount, transportation cost and warehouse equipment, can be stored for a long time without deterioration, is easy to recombine microorganisms, has better taste and can improve sweetness.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.