阅读说明：本技术 一种麦芽糖浆的制备方法及其应用 (Preparation method and application of maltose syrup ) 是由 张锦雯 单逸蓝 赵兰兰 沈微 陈献忠 杨海泉 夏媛媛 陈磊 于 2021-11-02 设计创作，主要内容包括：本发明涉及一种麦芽糖浆的制备方法及其应用,属于酶工程和发酵工程技术领域。其以一种酵母来源的淀粉酶SfA在特定条件下水解玉米淀粉获得的糖浆用于YCC166的发酵；发酵液中酪醇的水平与采用麦芽糖为原料得到的产量相当而发酵时间则进一步缩短。本发明提供的大肠杆菌YC166发酵产酪醇的原料主要是玉米淀粉,价格相对低廉,获得的酪醇发酵水平较高,发酵周期短于已见报道,具有良好的工业化应用前景,具备产业化价值。(The invention relates to a preparation method and application of maltose syrup, belonging to the technical field of enzyme engineering and fermentation engineering. It uses a syrup obtained by hydrolyzing corn starch with yeast-derived amylase SfA under specific conditions for fermentation of YCC 166; the level of tyrosol in the fermentation broth is comparable to the yield obtained with maltose as the starting material and the fermentation time is further shortened. The raw material for producing tyrosol by fermenting escherichia coli YC166 provided by the invention is mainly corn starch, the price is relatively low, the obtained tyrosol has higher fermentation level, the fermentation period is shorter than that of the obtained tyrosol, and the tyrosol has good industrial application prospect and industrial value.)

1. A preparation method of maltose syrup is characterized by comprising the following steps: the corn starch is hydrolyzed by yeast amylase SfA to obtain maltose syrup containing maltose and maltotriose as main components.

2. The method for producing maltose syrup as set forth in claim 1, characterized by comprising the steps of: taking corn starch as a raw material, adding water to prepare a corn starch suspension with the mass concentration of 8-9%, heating to 78-82 ℃ to fully gelatinize the corn starch, and naturally cooling to below 40 ℃ after gelatinization; adding yeast amylase SfA into the gelatinized starch solution according to the enzyme activity unit amount of 0.055-0.065U per gram of starch, and reacting at 39-41 deg.C for 11-12h to obtain the final product.

3. The method for producing maltose syrup as set forth in claim 1, wherein: the yeast amylase SfA is a recombinant amylase prepared by fermenting recombinant kluyveromyces lactis YW 07.

4. Use of a maltose syrup prepared by the process of claim 1 wherein: it is applied to recombinant Escherichia coli fermentation.

5. Use of maltose syrup according to claim 4 characterized in that: the recombinant Escherichia coli is specifically recombinant Escherichia coli YC 166.

6. Use of maltose syrup according to claim 5 characterized by: firstly, preparing a fermentation medium of a fermentation tank by adopting maltose syrup; preparing a corn starch suspension, adding medium-temperature alpha-amylase and fungal amylase into the starch suspension to prepare a fungal amylase hydrolysate, and then adding yeast powder to prepare a supplemented medium; and fermenting the recombinant Escherichia coli YC166 by adopting a fermentation tank culture medium, and supplementing materials by adopting a supplementing culture medium to finally prepare the tyrosol.

7. Use of maltose syrup according to claim 6 characterized in that: the preparation process of the fermentation medium of the fermentation tank comprises the following steps: adding maltose syrup according to 40-60% of the total volume of the initial fermentation liquid, sequentially adding 16-18g/L of disodium hydrogen phosphate dodecahydrate, 1-2g/L of monopotassium phosphate, 3-5g/L of ammonium sulfate, 5-8g/L of yeast powder and 1-2g/L of magnesium sulfate heptahydrate, and stirring for dissolving; adding water until the amount of starch is 35-45 g/L; sterilizing at 110-.

8. Use of maltose syrup according to claim 6 characterized in that: the preparation process of the fungal amylase hydrolysate comprises the following steps: firstly, preparing starch suspension with the mass concentration of 40-60%, adding medium-temperature alpha-amylase according to the amount of 0.4-0.6U per gram of starch, and heating in a water bath kettle at 74-76 ℃ until the starch is completely liquefied; cooling to 50 deg.C, accurately adding fungal amylase at an amount of 0.12-0.14U per gram of starch, strictly controlling reaction time at 3.3-3.4h, and controlling reaction temperature at 48-52 deg.C to obtain fungal amylase hydrolysate.

9. Use of maltose syrup according to claim 8 characterized in that: adding yeast powder with the concentration of 180-220g/L into the fungal amylase hydrolysate to obtain the supplemented medium.

10. Use of maltose syrup according to claim 6 characterized in that: the fermentation process specifically comprises the following steps: adding a fermentation medium of a fermentation tank into the fermentation tank, carrying out shake-flask culture on recombinant Escherichia coli YC166 by using an LB liquid medium, carrying out culture for 24h at 30 ℃, and then inoculating a new LB liquid medium according to the inoculation amount of 1-2% to continue the culture for 2-3h to obtain a seed culture solution; inoculating the seed culture solution into an initial culture medium of a fermentation tank according to the inoculation amount of 10% for fermentation; in the fermentation process, ammonia water is used for controlling the pH value to be 6.5 +/-0.5, the residual sugar of the culture medium is detected at intervals of 4 hours, when the residual sugar content of the culture medium is lower than 20g/L, the supplemented culture medium is supplemented until the total sugar concentration is 30-40g/L, and the tyrosol is prepared by fermentation.

Technical Field

The invention relates to a preparation method and application of maltose syrup, belonging to the technical field of enzyme engineering and fermentation engineering.

Background

Tyrosol is a phenolic organic compound with specific pharmacological actions. The method for producing the tyrosol by adopting the microbial fermentation method mainly takes the glucose as the raw material, has low cost and can be regenerated, thereby being a tyrosol preparation method with development prospect. Xu et al constructed a recombinant Escherichia coli YMG5A (Xu W, Yang C, Xiao Y, et al, high-level production of type with non-fermented recombinant Escherichia coli by means of microbial engineering, agricultural and Food Chemistry,2020,68: 4616-containing 4623) which can produce tyrosol with high yield, wherein the highest yield of tyrosol produced by the fermentation of the strain in the literature is 3.9g/L, and the fermentation time is 48 h. Optimization of fermentation conditions was performed later on the morning and so on based on the above-mentioned YMG5A strain (

Longmorning, zhanghaibing, zhanglihua, et al high density fermentation of escherichia coli produces tyrosol [ EB/OL ]. beijing: the Chinese scientific and technological article on line, [2021-03-26]. http:// www.paper.edu.cn/releaseppaper/content/202103-. The fermentation level of tyrosol reported above is higher, but the fermentation time is longer, and is 48 hours. The invention discloses a tyrosol-producing recombinant escherichia coli with a shortened fermentation period and application thereof, wherein the tyrosol fermentation time is greatly shortened by combining natural screening with genetic engineering modification in the morning and the like (the recombinant escherichia coli YC166 is obtained by combining natural screening with genetic engineering modification in the morning and the like (the recombinant escherichia coli is a tyrosol-producing recombinant escherichia coli with a shortened fermentation period and application thereof, Chinese patent application No. 202110598376.7).

The host bacterium YEC166 of the recombinant bacterium YC166 is a wild type Escherichia coli, and the performance of the recombinant bacterium YC166 is obviously different from that of Escherichia coli commonly used in laboratories. Earlier studies found that YC166, when used in tyrosol fermentation, had an optimal carbon source other than glucose, but maltose. When YC166 is fermented by using pure maltose as a carbon source, the yield of tyrosol can reach more than 4.2g/L, and the fermentation period is still 24 h. Pure maltose is not suitable for being used as a fermentation raw material due to high price, and the fermented tyrosol is low in yield and not obvious in application value due to the fact that the maltose syrup prepared by the traditional methods such as fungal amylase, beta-amylase and the like is possibly insufficient in purity and the like.

Disclosure of Invention

The invention aims to overcome the defects and provides a preparation method and application of maltose syrup, which provides a specific carbon source for producing tyrosol by recombinant escherichia coli YC166 fermentation, and the fermentation can obtain higher tyrosol yield.

The technical scheme of the invention is that the preparation method of the maltose syrup uses yeast amylase SfA to hydrolyze corn starch to obtain the maltose syrup taking maltose and maltotriose as main components.

Further, the steps are as follows: taking corn starch as a raw material, adding water to prepare a starch suspension with the mass concentration of 8-9%, heating to 78-82 ℃ to fully gelatinize the starch, and naturally cooling to below 40 ℃ after gelatinization; accurately adding yeast amylase SfA into the gelatinized starch solution according to the amount of 0.055-0.065U enzyme activity unit added into each gram of starch, and then reacting for 11-12h at 39-41 ℃ to obtain the maltose syrup.

Further, the yeast amylase SFA is a recombinant amylase prepared by fermenting recombinant kluyveromyces lactis YW 07.

The application of the maltose syrup prepared by the method is applied to recombinant escherichia coli fermentation.

Further, the recombinant Escherichia coli is specifically recombinant Escherichia coli YC 166.

Further, firstly, preparing a fermentation medium of a fermentation tank by adopting maltose syrup; preparing a corn starch suspension, adding medium-temperature alpha-amylase and fungal amylase into the starch suspension to prepare a fungal amylase hydrolysate, and then adding yeast powder to prepare a supplemented medium; and fermenting the recombinant Escherichia coli YC166 by adopting a fermentation tank culture medium, and supplementing materials by adopting a supplementing culture medium to finally prepare the tyrosol.

Further, the preparation process of the fermentation medium of the fermentation tank is as follows: adding maltose syrup according to 40-60% of the total volume of the initial fermentation liquid, sequentially adding 16-18g/L of disodium hydrogen phosphate dodecahydrate, 1-2g/L of monopotassium phosphate, 3-5g/L of ammonium sulfate, 5-8g/L of yeast powder and 1-2g/L of magnesium sulfate heptahydrate, and stirring for dissolving; adding water to 35-45g/L of the original starch amount; sterilizing at 110-.

Further, the preparation process of the fungal amylase hydrolysate is as follows: firstly, preparing starch suspension with the mass concentration of 40-60%, adding medium-temperature alpha-amylase according to the amount of 0.4-0.6U per gram of starch, and heating in a water bath kettle at 74-76 ℃ until the starch is completely liquefied; cooling to 50 deg.C, adding fungal amylase at an amount of 0.12-0.13U/g starch, and reacting for 3.3-3.4 hr to obtain fungal amylase hydrolysate.

Further, yeast powder with the concentration of 180-.

Further, the fermentation process specifically comprises: adding fermentation medium into the fermentation tank. Shake-flask culturing recombinant Escherichia coli YC166 with LB liquid culture medium, culturing at 30 deg.C for 24h, inoculating new LB liquid culture medium according to 1% -2% inoculum size, and continuously culturing for 2-3h to obtain seed culture solution; inoculating the seed culture solution into an initial culture medium of a fermentation tank according to the inoculation amount of 10% for fermentation; in the fermentation process, ammonia water is used for controlling the pH value to be 6.5 +/-0.5, the residual sugar of the culture medium is detected at intervals of 4 hours, when the residual sugar content of the culture medium is lower than 20g/L, the supplemented culture medium is supplemented until the total sugar concentration is 30-40g/L, and the tyrosol is prepared by fermentation.

The invention has the beneficial effects that: the raw material for producing tyrosol by fermenting escherichia coli YC166 provided by the invention is mainly corn starch, the price is relatively low, the obtained tyrosol has higher fermentation level, the fermentation period is shorter than that of the obtained tyrosol, and the tyrosol has good industrial application prospect and industrial value.

Biological material sample preservation: tyrosol-producing recombinant Escherichia coli YC166 has been disclosed in patents (longyichen, zhangmawen, shenwei, Chengyuan, yanhaiquan, quality of summer, Chenyili, a tyrosol-producing recombinant Escherichia coli with shortened fermentation period and its application, chinese invention patent application, 202110598376.7), during which the strain YC166 has been deposited as a patent strain and is deposited in the chinese typical culture collection, wuhan university, the classification of the strain is named as Escherichia coli YC166(Escherichia coli YC166), the deposition date is 2021 year, 4 months and 12 days, the deposition number is CCTCC NO: 2021358.

kluyveromyces lactis YW07, a strain producing yeast amylase SfA, has been disclosed in the literature (Kuo Yuwang, Shenwei, Wang Yi Tian, Yanfan, Wang Zheng Xiang. Fujian. the expression and recombinase properties of the Saccharomyces cerevisiae alpha-amylase gene in Kluyveromyces lactis. Industrial microorganisms 2014,44(2): 25-29).

Drawings

FIG. 1 shows the course of tyrosol fermentation from malt syrup.

Detailed Description

The tyrosol standards used for liquid phase detection in the examples below are available from Sigma. The starch is corn starch, and is a product of Hengren Engineers Limited in Shandong. Both moderate alpha-amylase (also known as moderate amylase) and fungal amylase are products of Nanning Pompe bioengineering GmbH. In order to ensure the stability of the enzyme action, the enzyme activity needs to be re-measured within 24 hours before use, and the method is the same as the literature (Zingiber officinale, et al, New Enzymatic preparation practical technical Manual 2002, Beijing, light industry Press), and the alpha-amylase measurement method is carried out under the optimal conditions of the mesophilic amylase and the yeast amylase SfA respectively. The enzyme activity is defined as: the amount of enzyme required to liquefy 1g of starch in one hour is one unit of enzyme activity, expressed as 1U). The saccharifying enzyme is a product (powerful Powermix2.0) of Xiusheng biological technology Limited company, and the enzyme activity is directly used according to the product label when in use. The yeast powder is yeast extract powder, and is a product of Qingdao Haibo biotechnology limited company. Other reagents were purchased from the national pharmaceutical group chemical agents corporation.

EXAMPLE 1 preparation of maltose syrup

(1) Taking a proper amount of corn starch, and adding common tap water to prepare starch suspension with the mass concentration of 8%. Heating to 80 deg.C to fully gelatinize starch, and naturally cooling to below 40 deg.C after gelatinization. Adding yeast amylase SfA into the gelatinized starch solution according to the amount of adding 0.06U of enzyme activity unit into each gram of starch; reacting at 40 ℃ for 11-12h to obtain maltose syrup;

the yeast amylase SfA is a recombinant amylase prepared by fermenting recombinant kluyveromyces lactis YW 07. The recombinant strain Kluyveromyces lactis YW07 and the method for obtaining the recombinase SfA by fermenting the recombinant strain are disclosed in the literature. (Guoyangwang, Shenwei, Wangyibian, Yanxiang, Wangzhengxiang, Fujianfermenta yeast alpha-amylase gene expression and recombinase property in Kluyveromyces lactis. Industrial microorganism 2014,44(2): 25-29; Guoyangwang. yeast alpha-amylase gene cloning, expression and application thereof in preparation of maltotriose-A Master thesis 2014, Wuxi, Jiangnan university). Specifically, the fermentation is carried out according to the optimized 5L fermentation tank fermentation method of the YW07 strain introduced in the section of '3.1.6 recombinant yeast tank-in-tank fermentation experiment' in Kuo Yuwang yeast alpha-amylase, gene cloning, expression and application thereof in maltotriose preparation, Master academic paper, 2014, Wuxi, Jiangnan university), and the fermentation liquid can be directly used for hydrolysis of starch after being centrifuged to remove thalli. The enzyme activity of SfA fermentation broth obtained by other fermentation methods may be slightly lower but the hydrolysis effect is basically the same as long as the activity of the enzyme used per gram of starch is equivalent. The same effect is obtained by using concentrated or purified SfA.

The preparation of the syrup needs to be carried out strictly according to conditions, and the increase and decrease of the enzyme activity or the change of the enzymolysis time can cause the tyrosol obtained by final fermentation to be greatly reduced. For example, hydrolysis of starch or dextrin with SfA under conditions as disclosed in the above-mentioned publication, or in another related publication (Shen, Linlizhen, Wen and Wen, Guo Yu Wan, Chen Xiang, Yan you, Wang Zheng Xiang, heterologous expression and recombinant enzyme properties of an acid fungal alpha-amylase) results in a decrease in tyrosol production at the end of fermentation and a significant increase in fermentation time.

Example 2 preparation of fungal amylase hydrolysate for feed and preparation of feed medium

Firstly, preparing 50% corn starch suspension, adding 0.5U of medium-temperature alpha-amylase per gram of starch, heating in a 75 ℃ water bath until the starch is completely liquefied, and quickly heating the liquefied starch liquid in an electric furnace or a microwave oven to above 95 ℃ after the starch is liquefied and maintaining for 5 min. Cooling to 50 deg.C, adding fungal amylase at an amount of 0.12-0.13U/g starch, reacting for 3.3-3.4 hr, reacting for 4 hr to obtain fungal amylase hydrolysate, and rapidly heating the hydrolysate to 90 deg.C for 5min after hydrolysis. And adding yeast powder into the hydrolysate to reach the concentration of 200g/L to obtain the supplemented medium.

This step also necessitates a strict control of the degree of starch hydrolysis according to the above-mentioned methods, and insufficient or excessive liquefaction of the mesophilic amylase or hydrolysis of the fungal amylase leads to a significant reduction in the final fermented tyrosol yield.

The fungal amylase hydrolysate is basically maltose syrup taking maltose or maltooligosaccharide as a main component, but the specific components are quite different from the SfA hydrolysate, and the tyrosol yield obtained by fermentation is quite low and is generally below 3g/L when the fungal amylase hydrolysate is used for replacing the maltose syrup obtained in the example 1 to prepare the fermentation liquor initial culture medium. For the purpose of the present invention, a hydrolysate obtained by hydrolyzing corn starch under specific conditions with SfA is referred to as a maltose syrup, and a starch hydrolysate obtained by further hydrolyzing starch with fungal amylase after liquefying the starch with a mesophilic amylase is referred to as a fungal amylase hydrolysate.

EXAMPLE 3 5L fermenter fermentation of tyrosol

The detection method used in this example is as follows:

the cell concentration was measured as follows: detecting the cell density OD600 at 600nm by using an ultraviolet spectrophotometer; the detection of the dry weight of the cells is carried out according to the following method: taking 1mL of bacterial liquid to be detected, centrifuging at 12000r/min for 10min, discarding the supernatant, repeatedly washing with deionized water for 3 times, and drying in a drying oven to constant weight; weighing, according to the previous study result, the ratio of YC166 cell dry weight DCW (y) to OD600(x) is: 0.382:1.

Detecting the content of tyrosol in the fermentation liquor by an HPLC method:

sample treatment: centrifuging the fermentation liquid to remove cells, heating at 100 deg.C for 10min, centrifuging, collecting supernatant, and filtering with microporous membrane.

Liquid phase conditions: using 1mL/min 80% of 0.1% formic acid and 20% pure methanol as mobile phases; the chromatographic column is a Bornaagel C18 chromatographic column (4.6X 250mm, the aperture is 5 μm); the column temperature is 30 ℃; the detection wavelength is 276nm of an ultraviolet detector, and the sample injection amount is 10 mu L.

And (3) standard curve determination: and (3) diluting the standard sample by a multiple proportion, detecting under the conditions, and determining to obtain a standard curve of the tyrosol concentration (g.L-1) by taking the tyrosol concentration as an abscissa and the HPLC peak area of the tyrosol standard as an ordinate: y 7054x 564.5(R2 0.9998).

The detection instrument of HPLC is as follows: high performance liquid chromatography Agilent, model 1260 HPLC.

Method for detecting residual sugar in fermentation liquor

Taking out the fermentation liquor, centrifuging for 5min at 8000r/min, taking supernatant, adding 10U saccharifying enzyme into each ml of fermentation liquor, mixing, reacting for 20min at 60 ℃, and detecting the total amount of reducing sugar as the residual sugar content of the fermentation liquor by the conventional DNS method.

Fermentation of recombinant Escherichia coli YC 166: recombinant E.coli YC166 was fermented in a 5L fermentor as follows:

(1) preparation of initial medium: the maltose syrup prepared in example 1 above, typically 1.5L, was added to a 5L beaker followed by the sequential addition of: 17.1g/L disodium hydrogen phosphate dodecahydrate, 1.5g/L potassium dihydrogen phosphate, 4g/L ammonium sulfate, 6g/L yeast powder and 1.2g/L magnesium sulfate heptahydrate, and stirring for dissolving. Water was added to a predetermined volume (typically 3L). For fermentation experiments, 2.5L of the mixture was typically sterilized in a 5L fermentor at 115 ℃ for 15 min. Thus obtaining the initial culture medium of the fermentation tank.

(2) Preparing and inoculating strains: shake-flask culturing recombinant Escherichia coli YC166 with LB liquid culture medium, culturing at 30 deg.C for 24 hr, inoculating new LB liquid culture medium according to 1% inoculum size, and culturing for 2-3 hr to obtain seed culture solution with culture solution concentration of OD₆₀₀About 0.25; inoculating the seed culture solution into the initial culture medium of the 5L fermentation tank obtained in the previous step according to the inoculation amount of 10% for fermentation;

(3) the tyrosol fermentation process comprises the following steps: ammonia water is used as a neutralizer in the fermentation process, and the constant pH value is 6.5 +/-0.5; the dissolved oxygen is controlled at 20-30%, the dissolved oxygen is mainly controlled at the stirring speed, the dissolved oxygen is controlled by the ventilation quantity, the same effect is achieved, and the influence on the fermentation result is not obvious. The fermentation temperature is 30 ℃ in the whole process.

Sampling every 4h in the fermentation process, and measuring the residual sugar concentration during sampling. According to the residual sugar detection result, the feed medium described in example 2 was supplemented to a total sugar concentration of 30-40g/L when the total sugar content of the medium was less than 20 g/L. According to the process, the final yield of tyrosol produced by YCC166 fermentation is about 4.3g/L, and the fermentation time is 20 h.

FIG. 1 shows a typical tyrosol fermentation process, where the end point is reached at 20 hours and the tyrosol yield is 4.3 g/L. Similar results were obtained for multiple experiments with data errors within 5%. The most critical factor for maintaining this result is the preparation of maltose syrup, and the activity of yeast amylase SfA must be strictly detected before preparation to ensure that the components of the syrup are basically unchanged.

In the research of preparing tyrosol by a microbial fermentation method, the highest level of fermentation of the obtained tyrosol is 5.06g/L, which is the highest level of fermentation seen so far, based on the YMG5A strain reported in the morning and the like (in the morning of the same day, Zhang Hai Bing, Zhang Li Hua, and the like. high-density fermentation of Escherichia coli produces tyrosol [ EB/OL ]. Beijing: Chinese scientific and technical paper on line, [2021-03-26]. http:// www.paper.edu.cn/releaseper/content/202103-.

The fermentation level of tyrosol in the method established by the invention is 4.3g/L, and the fermentation time is shortened to 20h although the fermentation level is not as good as the above reports. Compared with the study on the strain YC166 in early stages such as the morning, the fermentation level obtained by the method is obviously improved, the fermentation time is shortened from 24h to 20h, the production intensity and the equipment utilization rate are further improved, and the risk of bacterial contamination in the fermentation process is reduced. The raw material used by the invention is corn starch, and the price is relatively low. Therefore, the tyrosol fermentation method taking YC166 as the strain has certain industrial application value.