阅读说明：本技术 一种提高赤藓糖醇生产转化效率的方法及其应用 (Method for improving production conversion efficiency of erythritol and application of method ) 是由 李丽 聂在建 程保华 李德春 于 2019-10-24 设计创作，主要内容包括：本发明涉及一种提高赤藓糖醇生产转化效率的方法及其应用,所述方法为：以葡萄糖为原料,以酵母为发酵转化菌株,在发酵转化过程中添加蔗糖合酶和环麦芽糊精-葡聚糖转移酶,从而提高从葡萄糖到赤藓糖醇的转化率。该方法操作简单,成本低,转化率的提高显著。(The invention relates to a method for improving the production conversion efficiency of erythritol and application thereof, wherein the method comprises the following steps: the method is characterized in that glucose is used as a raw material, yeast is used as a fermentation transformation strain, and sucrose synthase and cyclomaltodextrin-glucanotransferase are added in the fermentation transformation process, so that the conversion rate from glucose to erythritol is improved. The method has the advantages of simple operation, low cost and remarkable improvement of the conversion rate.)

1. A method for improving the conversion efficiency of erythritol production is characterized by comprising the following steps: the method is characterized in that glucose is used as a raw material, yeast is used as a fermentation transformation strain, and sucrose synthase and cyclomaltodextrin-glucanotransferase are added in the fermentation transformation process, so that the conversion rate from glucose to erythritol is improved.

2. The method for improving the conversion efficiency in erythritol production according to claim 1, wherein the yeast comprises candida lipolytica, moniliforme, or trichosporoid, preferably candida lipolytica.

3. The method for improving the conversion efficiency in erythritol production according to claim 1 or 2, wherein the sucrose synthase is added at a concentration of 100-500000U.

4. The method for improving the conversion efficiency of erythritol production according to any one of claims 1-3, wherein the concentration of the added cyclodextrin-glucanotransferase is 1000-500000U.

5. The method for increasing the conversion efficiency of erythritol production according to any one of claims 1-4, comprising the steps of:

(1) inoculating yeast into a fermentation culture medium for culture and fermentation;

(2) and transferring the erythritol into a production fermentation culture medium for conversion.

6. The method for improving the conversion efficiency in erythritol production according to claim 5, wherein the fermentation medium is treated at a temperature of 80-90 ℃ for 20-40min before inoculation in step (1), and is inoculated with yeast for fermentation culture after cooling;

preferably, the fermentation medium in step (1) comprises 400g/L of carbon source 100-;

preferably, the fermentation medium in the step (1) comprises starch hydrolysate, diammonium hydrogen phosphate, yeast powder, corn steep liquor dry powder, magnesium sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride.

7. The method for improving the conversion efficiency of erythritol production according to claim 5 or 6, wherein the initial temperature of the culture fermentation in step (1) is 25-35 ℃;

preferably, the initial pH value of the culture fermentation in the step (1) is 5.0-7.0, and the pH value is adjusted to 3.1-4.0 when the fermentation is carried out until the cell density OD600 value is 20 or more;

preferably, the culture fermentation in step (1) comprises primary culture and secondary expansion culture.

8. The method for improving the conversion efficiency in erythritol production according to any one of claims 5-7, wherein the production fermentation medium in step (2) comprises 400g/L of carbon source 100-;

preferably, the production fermentation medium in the step (2) comprises starch hydrolysate, diammonium hydrogen phosphate, yeast powder, corn steep liquor dry powder, magnesium sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride;

preferably, glucose, sucrose synthase and cyclomaltodextrin-glucanotransferase are added to the production fermentation medium during the conversion in step (2);

preferably, the concentration of the glucose is 500-600 g/L.

9. The method for improving the conversion efficiency of erythritol production according to any one of claims 5-8, wherein erythritol is purified after the conversion in step (2) is finished to obtain erythritol crystals;

preferably, the purification includes filter sterilization, concentration and crystallization.

10. The method for improving the production conversion efficiency of erythritol according to any one of claims 1-9, wherein erythritol is prepared by a biological method.

Technical Field

The invention belongs to the technical field of biological fermentation, particularly relates to a method for preparing erythritol and application thereof, and particularly relates to a method for improving the production conversion efficiency of erythritol and application thereof.

Background

Erythritol (also called erythritol) is a quaternary polyol with a molecular weight of 122.1Da, and is present in many fruits and vegetables and brewed foods (e.g., fruits such as pears, glucose, and wines), but in very small amounts. Due to the advantages of zero calorie, no dosage limitation and pure biological fermentation preparation, the erythritol has wider application in foods and is widely used in foods such as candies, baked foods, sugar-free beverages and the like. Such as: the invention patent with the application number of 200710014055.8 discloses a method for preparing a sandwich chocolate containing erythritol; the invention patent with the application number of 201110420379.8 discloses a composite sugar-free beverage containing erythritol and a preparation method thereof; the invention patent with application number 201180012533.6 discloses a candy product containing erythritol and a preparation method thereof; the invention patent with application number 201010569981.3 discloses a liver-protecting and stomach-invigorating health-care beverage containing erythritol and a preparation method thereof.

In addition, the erythritol can be matched with other functional sugar or functional sugar alcohol to be applied to food. Such as: the invention patent with application number 200610168838.7 discloses a sugar-free dessert containing xylitol and erythritol and having no cooling effect; US invention patent US7754268B2 discloses a method for making a baked food product comprising xylitol and/or erythritol; both the invention patents with application numbers 201010289677.3 and 201010289668.4 and the US invention patent US7579032B2 disclose a zero-or low-calorie beverage containing erythritol and tagatose and its use in food products; european patent application EP1057414B1 discloses a method for preparing sugar-free candy containing erythritol and sorbitol.

In addition, erythritol is used in the field of toothpastes, mouthwashes and cosmetics (see for details US8287842B2, WO2001074323a1, US20060067902a1 and US20060062752a 1.

Erythritol is currently approved for use in food products in many countries. Erythritol is listed as a new food additive variety in 2008 of China, and the national standard of erythritol is established in 2011 (GB26404-2011), and a yeast strain Candidalipolytica (Candida lipolytica) for synthesizing erythritol is added, and the yeast is internationally named as Yarrowia lipolytica (Yarrowia lipolytica or Yarrowia lipolytica). The three erythritol-producing yeasts specified in this national standard are Candida lipolytica (Candida lipolytica), Monilia pollenizalis (Monilliella pollenis) and Trichosporoides (Trichosporoides megachilies), respectively.

Chinese invention patent 200610163644.8 discloses a yeast strain producing erythritol, which is closest to Moniliella acetobacter, and the efficiency of converting glucose into erythritol is more than 30%. The strain does not meet the national standard established in China and cannot be used in China. Chinese patent ZL200510102929 (grant publication No. CN100506972C) discloses a Candida lipolytica and a method for producing erythritol, the used yeast is a strain (Candida lipolytica) specified by national standard in China and has been used and popularized in related enterprises in China, but the conversion rate of synthesizing erythritol from glucose is low (47%). European patent application EP0770683A1 discloses a process for synthesizing erythritol from glucose using Yarrowia lipolytica, but the highest conversion obtained is only 32.9%, and the conversion is low. The invention patent US6110715 discloses that the yeast Trichosporoides megachilies can ferment glucose to synthesize erythritol, although the strain meets the national standard of China, the highest conversion rate reported is only 35.1%, and the conversion rate is low and has no practical use value.

Therefore, it is very interesting to develop a method for preparing erythritol with high conversion rate and simple and easy operation.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for preparing erythritol and application thereof, and particularly provides a method for improving the production conversion efficiency of erythritol and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a method for improving the conversion efficiency of erythritol production, wherein the method comprises the following steps: the method is characterized in that glucose is used as a raw material, yeast is used as a fermentation transformation strain, and sucrose synthase and cyclomaltodextrin-glucanotransferase are added in the fermentation transformation process, so that the conversion rate from glucose to erythritol is improved.

The inventors surprisingly found that the addition of sucrose synthase (EC2.4.1.13) and cyclomaltodextrin-glucanotransferase (EC2.4.1.19) to the fermentation system significantly improved the conversion of erythritol to 51% or more. And sucrose synthase (EC2.4.1.13) and cyclomaltodextrin-glucanotransferase (EC2.4.1.19) need to be added simultaneously.

Preferably, the yeast comprises candida lipolytica, moniliforme, or trichosporoid, preferably candida lipolytica.

Preferably, the sucrose synthase is added at a concentration of 100-500000U, such as 100U, 200U, 500U, 800U, 1000U, 5000U, 8000U, 10000U, 50000U, 100000U, 200000U, 300000U or 500000U, etc.

Preferably, the cyclomaltodextrin glucanotransferase is added at a concentration of 1000-500000U, such as 1000U, 5000U, 8000U, 10000U, 50000U, 100000U, 200000U, 300000U or 500000U and the like.

The concentrations of the sucrose synthase and the cyclomaltodextrin-glucanotransferase added in the present invention are not particularly limited, but from the viewpoint of the production cost, the sucrose synthase is preferably added at a concentration of 500000U-100 and the cyclomaltodextrin-glucanotransferase is preferably added at a concentration of 500000U-1000.

Preferably, the method for improving the conversion efficiency of erythritol production comprises the following steps:

(1) inoculating yeast into a fermentation culture medium for culture and fermentation;

(2) and transferring the erythritol into a production fermentation culture medium for conversion.

Preferably, the fermentation medium is treated at 80-90 deg.C (e.g. 80 deg.C, 82 deg.C, 83 deg.C, 84 deg.C, 86 deg.C, 88 deg.C or 90 deg.C) for 20-40min (e.g. 20min, 22min, 25min, 30min, 35min or 40 min) before inoculation in step (1), and then inoculated with yeast for fermentation culture after cooling.

Preferably, the fermentation medium in step (1) comprises 400g/L of carbon source 100-.

The concentration of the carbon source may be 100g/L, 150g/L, 200g/L, 250g/L, 300g/L, 350g/L, 400g/L, or the like.

The nitrogen source may be at a concentration of 2g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, or the like.

The concentration of the inorganic salt may be 0g/L, 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, or the like.

Preferably, the fermentation medium in the step (1) comprises starch hydrolysate, diammonium hydrogen phosphate, yeast powder, corn steep liquor dry powder, magnesium sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride. Wherein the starch hydrolysate is a carbon source, diammonium hydrogen phosphate, yeast powder and corn steep liquor dry powder are nitrogen sources, and magnesium sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride are inorganic salt components.

Preferably, the initial temperature of the culture fermentation in step (1) is 25-35 deg.C, such as 25 deg.C, 27 deg.C, 29 deg.C, 30 deg.C, 32 deg.C, 34 deg.C or 35 deg.C.

Preferably, the initial pH of the culture fermentation in step (1) is 5.0 to 7.0 (e.g., pH 5.0, pH 5.5, pH 6.0, pH 7.0, etc.), and the pH is adjusted to 3.1 to 4.0 (e.g., pH 3.1, pH 3.5, pH 3.8, pH 4.0, etc.) when the fermentation is conducted until the cell density OD600 value is 20 or more.

Preferably, the culture fermentation in step (1) comprises primary culture and secondary expansion culture.

The first stage culture may be carried out in a shake flask and the second stage culture in a jar fermenter (volume 250L or less).

Preferably, the production fermentation medium in step (2) comprises 400g/L of carbon source 100-.

The concentration of the carbon source may be 100g/L, 150g/L, 200g/L, 250g/L, 300g/L, 350g/L, 400g/L, or the like.

The nitrogen source may be at a concentration of 2g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, or the like.

The concentration of the inorganic salt may be 0g/L, 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, or the like.

Preferably, the production fermentation medium in the step (2) comprises starch hydrolysate, diammonium hydrogen phosphate, yeast powder, corn steep liquor dry powder, magnesium sulfate, manganese chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride.

Preferably, glucose, sucrose synthase and cyclomaltodextrin-glucanotransferase are added to the production fermentation medium during the transformation in step (2).

Preferably, the concentration of the glucose is 500-600 g/L.

The main components of the fermentation medium and the production fermentation medium are the same, and the difference is only that the production fermentation medium is supplemented with a high-sugar medium component, namely glucose, and a component for improving the conversion rate, namely sucrose synthase and cyclomaltodextrin-glucanotransferase.

More preferably, erythritol is purified after the conversion in step (2) is finished, so that erythritol crystals are obtained.

Preferably, the purification includes filter sterilization, concentration and crystallization.

The filtration sterilization specifically comprises the following steps: after fermentation, separating yeast cells from fermentation liquor by a centrifugal or ceramic membrane filtration method, and separating and removing macromolecular viscous substances with the molecular weight of more than 1000Da in the fermentation liquor by a nanofiltration membrane to obtain clear and transparent fermentation liquor.

The concentration is specifically as follows: and (3) concentrating the clear fermentation liquor in an evaporator to a solid content of 50-80% (mass volume percentage) to obtain the erythritol-rich syrup.

The crystallization is specifically as follows: and cooling the syrup rich in erythritol to below 10 ℃ at the speed of 1-5 ℃/h, starting to crystallize erythritol, and carrying out centrifugal separation to obtain the coarse erythritol crystals.

As a preferred technical scheme, the method for improving the conversion efficiency of erythritol production comprises the following steps:

(1) treating the fermentation culture medium at 80-90 deg.C for 20-40min, cooling, inoculating yeast into the fermentation culture medium, culturing at initial temperature of 25-35 deg.C and initial pH of 5.0-7.0, and adjusting pH to 3.1-4.0 when the cell density OD600 value is 20 or above;

(2) transferring into production fermentation culture medium for converting erythritol, and supplementing glucose, sucrose synthase and cyclomaltodextrin-glucanotransferase.

(3) And after the fermentation is finished, filtering, sterilizing, concentrating and crystallizing to obtain the erythritol crystals.

In another aspect, the invention provides an application of the method for improving the production conversion efficiency of erythritol in preparing erythritol by a biological method.

Compared with the prior art, the invention has the following beneficial effects:

the inventor unexpectedly finds that in the process of preparing the erythritol by a biological method, the addition of the sucrose synthase (EC2.4.1.13) and the cyclomaltodextrin-glucanotransferase (EC2.4.1.19) into a fermentation system can obviously improve the conversion rate of the erythritol, and the conversion rate can be improved to 51 percent or more. And sucrose synthase (EC2.4.1.13) and cyclomaltodextrin-glucanotransferase (EC2.4.1.19) need to be added simultaneously. The method has the advantages of simple operation, low cost and remarkable improvement of the conversion rate.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

The erythritol conversion referred to in the examples below is the ratio of the mass of the crude crystals of erythritol to the mass of glucose.