阅读说明：本技术 一种赤藓糖醇发酵生产方法 (Erythritol fermentation production method ) 是由 杨顺保 王瑞昌 张培红 卢佩龙 付松 崔怀成 于 2019-09-23 设计创作，主要内容包括：本发明属于赤藓糖醇生产技术领域,具体公开了一种赤藓糖醇发酵生产方法。该赤藓糖醇发酵生产方法采用一次性投料方式将发酵培养基投入发酵罐内,之后高温灭菌,降温后向发酵罐内再移入种子,发酵培养生产赤藓糖醇。采用一次性投料发酵,有效降低了发酵罐染菌风险,有利于发酵培养的菌种生长,提高了发酵转化率,缩短了发酵周期,还可以提高赤藓糖醇产率。并且通过发酵过程中发酵培养温度、pH值等条件的控制,达到了缩短发酵周期、提高赤藓糖醇产率及转化率的效果。(The invention belongs to the technical field of erythritol production, and particularly discloses an erythritol fermentation production method. The erythritol fermentation production method adopts a one-time feeding mode to put a fermentation medium into a fermentation tank, then high-temperature sterilization is carried out, seeds are transferred into the fermentation tank after cooling, and erythritol is produced by fermentation culture. The method has the advantages that the one-time feeding fermentation is adopted, the risk of contamination of a fermentation tank is effectively reduced, the growth of strains cultured by fermentation is facilitated, the fermentation conversion rate is improved, the fermentation period is shortened, and the yield of erythritol can be improved. And the effects of shortening the fermentation period and improving the yield and the conversion rate of the erythritol are achieved by controlling conditions such as fermentation culture temperature, pH value and the like in the fermentation process.)

1. A fermentation production method of erythritol is characterized in that a fermentation medium is fed into a fermentation tank in a one-time feeding mode, then high-temperature sterilization is carried out, seeds with OD values of 0.5-0.9 are transferred into the fermentation tank after cooling, and erythritol is produced through fermentation culture;

The formula of the fermentation medium is as follows: 30-40% of glucose, 0.9-1.3% of yeast extract, 0.12-0.18% of urea, 0.0015-0.0025% of ferrous sulfate heptahydrate and 0.0015-0.0025% of zinc sulfate heptahydrate; the pH value is 6.0-7.0;

The conditions of fermentation culture are as follows: the culture temperature is 28-32 ℃, the pressure is 0.08-0.1 Mpa, the pH value is 5.0-7.0, and the ventilation rate is 4000-5000 m³/h；

Wherein the inoculation amount of the seeds is 8-12% of the weight of the fermentation medium.

2. The erythritol fermentation production method according to claim 1, wherein the seed is cultured by: pumping the seed culture medium into an empty seed tank, sterilizing at high temperature, cooling, inoculating strains according to the weight ratio of 8-12%, culturing until the OD value is 0.5-0.9, and finishing the culture.

3. The erythritol fermentation production method according to claim 2, wherein the seed culture medium formula is: 37-43 g/L of glucose, 4-6 g/L of yeast extract and 1-3 g/L of urea; the pH value is 6.0-7.0.

4. The method for producing erythritol through fermentation according to claim 2, wherein the seed is cultured under the conditions: the culture temperature is 30-36 ℃, the pH value is 6.0-7.0, the pressure is 0.1-0.15 MPa, the ventilation volume is 0.3-0.8V/V.m, and the dissolved oxygen is 30-60%.

5. The method for producing erythritol by fermentation according to claim 2, wherein the culturing is terminated when the OD value is 0.6 to 0.7.

6. The method for producing erythritol through fermentation according to claim 2, wherein the strain is candida lipolytica.

7. The method for producing erythritol through fermentation according to claim 1, wherein the glucose concentration in the fermentation medium is 35-40%.

8. the erythritol fermentation production method according to claim 1, wherein the erythritol concentration in the fermentation liquid in the fermentation tank is higher than 14g/dl, the residual reducing sugar content is lower than 0.5% by mass, and the fermentation culture is completed.

9. The method for the fermentative production of erythritol according to claim 1, wherein the conditions of the fermentation culture are: the culture temperature is 30-32 ℃, and the pH value is 5.5-6.5.

10. The method for producing erythritol through fermentation according to claim 1, wherein the seed is inoculated in an amount of 10% by weight based on the fermentation medium.

Technical Field

The invention relates to the technical field of erythritol production, and particularly relates to an erythritol fermentation production method.

Background

erythritol, also called erythritol, is a novel sugar alcohol food sweetener. The erythritol has the characteristics of good crystallinity, low calorie, high stability, harmonious sweet taste, no hygroscopicity, low freezing point, no dental caries, no gastrointestinal discomfort and the like, does not participate in sugar metabolism, is suitable for diabetics to eat, and can be used as a substitute sweetener for the diabetics; in addition, erythritol is very stable to heat and acid, and almost no browning or decomposition occurs under common food processing conditions.

At present, in the international sweetener market, erythritol has strong competitiveness as a novel functional food additive. In the aspect of food processing, erythritol has the following advantages: erythritol has high sweetness and can be used as a sweetener for food and beverage; the erythritol has small molecular weight, and the solution with the same concentration has higher osmotic pressure and lower water activity, and can be used for processing and preserving low-water-activity food; erythritol has a large heat of dissolution, so that solid foods and candies made from erythritol have a relatively obvious cooling feeling; the erythritol has a fresh and clean sweet taste, and can effectively mask the aftertaste of other high-sweetness sweeteners such as aspartame, stevioside and the like when being compounded with the sweeteners; erythritol is low in calories and therefore can be used to produce low-calorie foods and beverages; erythritol can also promote the combination of ethanol molecules and water molecules in the solution, thereby reducing the off-flavor and sensory stimulation of alcohol in alcoholic beverages, and improving the quality of distilled liquor and wine.

Erythritol also has wide application in pharmaceutical and chemical industries and the like. In the pharmaceutical industry, erythritol can be used as a flavoring agent and an excipient of tablets of a medicine, and can effectively improve the taste of the medicine. The erythritol is used as raw material to synthesize various medicines, such as dideoxy erythritol generated after erythritol is deoxidized and has anti-HIV activity, and nitroerythritol generated after erythritol is nitrified can be used for treating cardiovascular and cerebrovascular diseases and asthma. In the chemical industry, erythritol is used as an intermediate for organic synthesis, and can be used for synthesis of alkyd resin, polyester and polyether, manufacture of paint and explosive and the like. In the aspect of cosmetics, erythritol can be added instead of part of glycerin, so that the cosmetics can be prevented from deteriorating. With the technical progress, new application of erythritol is continuously developed.

Erythritol can be produced by three process technologies, extraction, chemical synthesis and biological fermentation, respectively. The erythritol content in certain mushroom varieties and seaweeds can reach 5 percent, so that the erythritol can be produced by a direct extraction method, but generally, the mushrooms and the seaweeds also contain mannitol and other polyols, and the problems of effective separation of the polyols with similar properties, economy and the like in the production of the erythritol by the extraction method are solved. The chemical synthesis method can be synthesized by reacting butylene glycol and hydrogen peroxide, and can also be used for generating erythritol by oxidizing and cracking starch as a raw material with periodic acid, but the process method for producing erythritol by chemical synthesis has the defects of long flow, high cost, high requirement on conditions, poor product safety and the like. The fermentation method is characterized in that glucose is used as a raw material, erythritol is produced by fermentation of a hyperosmotic-resistant yeast strain, and a high-purity erythritol product is obtained by separation, extraction and refining.

At present, a common fermentation production method of erythritol is a continuous fermentation process, wherein a strain for producing erythritol is cultured by a multi-stage seed tank and then is connected to a fermentation tank, a sugar source, a natural killer and the like are fed into the fermentation tank for culture, and after the culture reaches a required stable yield, the obtained fermentation liquid is further extracted to obtain the erythritol. The continuous fermentation production method has the disadvantages of longer fermentation period, easy bacterial contamination of a fermentation tank, lower fermentation yield and higher production cost.

Disclosure of Invention

The invention mainly solves the technical problem of providing the erythritol fermentation production method, which can shorten the production period and improve the yield by one-time feeding fermentation.

In order to solve the technical problems, the invention adopts a technical scheme that: a fermentation production method of erythritol comprises the steps of putting a fermentation medium into a fermentation tank in a one-time feeding mode, then sterilizing at high temperature, cooling to 32-37 ℃, then transferring seeds with OD values of 0.5-0.9 into the fermentation tank, and carrying out fermentation culture to produce erythritol;

The formula of the fermentation medium is as follows: 30-40% of glucose, 0.9-1.3% of yeast extract, 0.12-0.18% of urea, 0.0015-0.0025% of ferrous sulfate heptahydrate and 0.0015-0.0025% of zinc sulfate heptahydrate; the pH value is 6.0-7.0;

The conditions of fermentation culture are as follows: the culture temperature is 28-32 ℃, the pressure is 0.08-0.1 Mpa, the pH value is 5.0-7.0, and the ventilation rate is 4000-5000 m³/h；

Wherein the inoculation amount of the seeds is 8-12% of the weight of the fermentation medium.

As a preferred embodiment, the seed is cultured by the following method: pumping the seed culture medium into an empty seed tank, sterilizing at high temperature, cooling to 32-36 ℃, inoculating strains according to the weight ratio of 8-12%, culturing until the OD value is 0.5-0.9, and finishing the culture to obtain the cultured seeds. Alternatively, the bacterial species may be candida lipolytica.

Preferably, the seed culture medium formula is as follows: the seed culture medium comprises the following components in percentage by weight: 37-43 g/L of glucose, 4-6 g/L of yeast extract, 1-3 g/L of urea and water as a solvent; the pH value is 6.0-7.0.

Preferably, the seed is cultured under conditions selected from: the culture temperature is 30-36 ℃, the pH value is 6.0-7.0, the pressure is 0.1-0.15 MPa, the ventilation volume is 0.3-0.8V/V.m, and the dissolved oxygen is 30-60%.

Preferably, the seed culture is terminated when the culture is carried out until the OD value is 0.6-0.7.

Preferably, the concentration of glucose in the fermentation medium is 35-40%.

Preferably, the yield concentration of the erythritol in the fermentation liquid in the fermentation tank is more than 14g/dl, the mass percentage content of the residual reducing sugar is less than 0.5%, and the yield concentration tends to be stable, namely, the culture can be stopped.

Preferably, the conditions of the fermentation culture are: the culture temperature is 30-32 ℃, and the pH value is 5.5-6.5.

Preferably, the seed is inoculated in an amount of 10% by weight of the fermentation medium.

The erythritol fermentation method provided by the invention mainly aims at the existing fermentation and adopts continuous fed-batch feeding, and the continuous fed-batch fermentation process is complex and has high control difficulty; in the continuous fed-batch material fermentation process, the fed-batch material can cause unstable control of sugar concentration, and simultaneously can bring heat to cause that the temperature of a fermentation environment is higher than the temperature required by the growth of a strain to inhibit the growth of the strain, and the strain growth can be inhibited by overhigh temperature or overhigh sugar concentration, so that the fermentation period is longer, the capability of generating erythritol is reduced, the conversion rate is reduced, more residues in the fermentation liquor are not beneficial to extraction, and the extraction yield is reduced; the continuous fed-batch fermentation process is easy to be infected with bacteria, and the generated foam quantity is large, so that the continuous fed-batch fermentation process is not beneficial to fermentation. The invention adopts a one-time feeding fermentation process, namely, fermentation culture medium required by fermentation is put into a fermentation tank at one time, and then the fermentation tank is sterilized at high temperature, and cultured seeds are transferred into the fermentation tank after the temperature is reduced, so that erythritol is produced by fermentation culture. Adding sugar source, nitrogen source, inorganic salt and the like required by fermentation into a fermentation tank at one time, sterilizing and cooling, inoculating seeds for further fermentation culture, and adding materials in a flowing manner during the fermentation process. The method has the advantages that the one-time feeding fermentation is adopted, the risk of contamination of a fermentation tank is effectively reduced, the growth of strains cultured by fermentation is facilitated, the fermentation conversion rate is improved, the fermentation period is shortened, and the yield of erythritol can be improved. Of course, the fermentation process also needs to control proper environmental conditions to achieve good fermentation effect. Through the selection of the components of the fermentation medium and the control of the conditions of air quantity, fermentation culture temperature, pH value and the like in the fermentation process, the effects of shortening the fermentation period and improving the yield and the conversion rate of the erythritol are achieved.

According to the erythritol fermentation method provided by the invention, when the yield concentration of the fermentation liquid in the fermentation tank is more than 14g/dl, the residual reducing sugar is less than 0.5% (W/W), and the yield concentration tends to be stable, the culture can be stopped, and the erythritol can be obtained by further extracting the fermentation liquid. By adopting the fermentation method, the fermentation period is less than 100 hours, the conversion rate is more than 50 percent, the erythritol yield is more than 14 percent, and the unexpected effects of short fermentation period and high yield are obtained.

drawings

FIG. 1 is a graph showing the effect of pH of a fermentation broth on erythritol content in the present invention;

FIG. 2 is a graph of the effect of different seed inoculations on erythritol content in the present invention.

Detailed Description

The technical solution of the present invention will be explained in detail below.

The erythritol fermentation production method provided by the invention is characterized in that one-time feeding fermentation is adopted, namely a fermentation medium is fed into a fermentation tank in a one-time feeding mode, then high-temperature sterilization is carried out, sterilization is carried out for 30min at 121 ℃, then seeds are transferred into the fermentation tank after the temperature is reduced to 35 ℃, and erythritol is produced through fermentation culture.

In the research process, factors such as fermentation temperature, fermentation pH value, seed inoculation amount and the like are respectively considered.

The formula of the adopted fermentation medium is as follows: the concentration of glucose is 35 percent, the concentration of yeast extract is 1.1 percent, the concentration of urea is 0.15 percent, the concentration of ferrous sulfate heptahydrate is 0.002 percent, and the concentration of zinc sulfate heptahydrate is 0.002 percent; the pH value is 6.0-7.0. The percentages (%) are mass percentages.

the seed culture method comprises the following steps: pumping a seed culture medium into an air-digestion seeding tank, sterilizing the materials in the seeding tank at the temperature of 123-125 ℃ for 30min, cooling to 36 ℃, inoculating a strain according to the proportion of 10 percent (by mass percentage), wherein the strain is candida lipolytica and is purchased from Shandong province food fermentation industry research design institute. Culturing until OD value is 0.6, and finishing culturing to obtain cultured seeds. Wherein the formula of the seed culture medium is as follows: 40g/L glucose, 5g/L yeast extract and 2g/L urea, and tap water is used as a solvent, and the pH value is 6.0. The culture conditions of the seeds in the seed tank are as follows: the culture temperature is 30-33 ℃, the pH value is 6.0-7.0, the pressure is 0.1MPa, the ventilation volume is 0.3-0.8V/V.m, and the dissolved oxygen is 30-60%.

The yield concentration of the erythritol in the fermentation liquid in the fermentation tank is more than 14g/dl, the mass percentage content of the residual reducing sugar is less than 0.5%, the yield concentration tends to be stable, and the culture is stopped.

First, the fermentation temperature was examined.

First, fermentation temperature

The fermentation process must provide a suitable growth temperature for the microorganisms, and it is also apparent that the activity of the enzyme is affected by the temperature. In the range of the growth metabolism temperature suitable for the enzyme, the enzyme activity is improved along with the rise of the temperature, the fermentation reaction speed is increased, the growth metabolism of the strain is accelerated, the fermentation period can be shortened, but the enzyme is inactivated due to overhigh temperature, the strain is aged and autolyzed in advance, the yield is reduced, and the conversion rate is reduced; temperature also affects the direction of production and synthesis of the cells and the metabolic regulation of the cells. Therefore, a suitable fermentation temperature is favorable for erythritol production.

Selecting a 50L fermentation tank, controlling the fermentation pressure within the range of 0.08-0.1 Mpa, controlling the pH value to be 6.0-6.5, and controlling the ventilation quantity to be 4000-4500 m³In the range of/h. When the cultured seeds are transferred into the fermentation tank, the inoculation amount of the cultured seeds is 10 percent of the weight of the fermentation medium.

The effects of different temperature controls at the initial, middle and late stages of fermentation were examined and the results are shown in table 1.

TABLE 1

From the above table experimental results it can be derived: the temperature is controlled at 30 ℃ in the initial stage of fermentation, so that the rapid growth of thalli can be promoted, the adaptability of strains to the fermentation environment is shortened, and the period is shortened; the temperature in the middle stage of fermentation is controlled at 32 ℃, so that the dissolved oxygen in the fermentation liquor can be improved, and the rapid growth and proliferation of the strains are accelerated; and in the later fermentation stage, the fermentation temperature is continuously controlled at 32 ℃, the synthesis of erythritol is improved, the fermentation speed is accelerated, and the fermentation is completed as soon as possible. Therefore, the preferred conditions for the fermentation culture are: the fermentation temperature is controlled to be 30-32 ℃.

II, fermentation pH value

The pH value is also an obvious index of the growth and metabolism activity of the microorganism, has great influence on the growth of the thalli and the accumulation of products, simultaneously the change of the pH value reflects different stages of the growth and metabolism process of the strains, particularly the pH value of the fermentation liquor is controlled at the initial stage, the optimum pH value is selected, the growth and the synthesis of the strains are facilitated, and the fermentation yield is improved.

Selecting a 50L fermentation tank, controlling the fermentation pressure within the range of 0.08-0.1 Mpa and the ventilation quantity within the range of 4000-4500 m³The fermentation temperature is controlled to be 30-32 ℃ within the range of the/h. When the cultured seeds are transferred into the fermentation tank, the inoculation amount of the cultured seeds is 10 percent of the weight of the fermentation medium.

Different pH values are selected within the range of 4.5-7 for experiments, and the influence curve of the pH value of the obtained fermentation liquor on the erythritol content is shown in figure 1.

From fig. 1, it can be seen that: the optimum pH value for production is 5.5-6.5, the yield of erythritol is highest when the pH value is 6.0, and the yield is reduced along with the gradual increase of the pH value. The analysis reason is that the initial fermentation stage is mainly the strain growth adaptation period, so that the growth and the propagation of the strain are accelerated; when the pH value is too low, the strains do not enter a mass propagation stage, so that the number of the strains is small, the strains grow insufficiently, and the yield is low; when the pH value is too high, the growth and metabolism of the fermentation strain are synthesized by various enzyme catalysis in vivo, the activity of the enzyme is obviously influenced by the pH value, the enzyme activity is reduced along with the increase of the pH value, the fermentation reaction speed is reduced, the growth of the strain is slowed, even the strain dies, and the yield is reduced. The preferred conditions for the fermentation culture are: the pH value is controlled to be 5.5-6.5.

Third, the seed inoculation amount

The growth speed of the saccharomycetes in the fermentation tank is influenced by the size of the seed inoculation amount, the growth period of thalli can be shortened, the fermentation conversion is advanced, the inoculation amount is excessive, the thalli often grow too fast, the fermentation liquor becomes thick, the insufficient dissolved oxygen is often caused, the thalli are further declined in advance, and the thalli are autolyzed; on the contrary, if the inoculation amount is too small, the fermentation period is prolonged, the seed activity is poor, and the bacterial contamination is easy, so that the fermentation yield is reduced. Therefore, the amount of erythritol must be investigated in order to increase the yield.

The experiment is carried out by inoculating seeds with a period of 20 hours according to different inoculation amounts, fermenting for 100 hours in a 50L fermentation tank, controlling the fermentation pressure within the range of 0.08-0.1 Mpa, the pH value within the range of 6.0-6.5 and the ventilation volume within the range of 4000-4500 m³In the range of/h. The fermentation temperature is controlled to be 30-32 ℃.

When the cultured seeds are transferred into the fermentation tank, the inoculation amount of the cultured seeds is 4% -13%, experiments are carried out, and the influence curve of the obtained different seed inoculation amounts on the erythritol content is shown in figure 2.

As can be seen from FIG. 2, the erythritol content increased and then decreased with increasing inoculation, and the erythritol content was the highest at an inoculation amount of 10%. The reason is that the inoculation amount is less, the strain amount is less, the time from the growth and the propagation to the strain amount required by normal fermentation is longer, the passage is more, and the fermentation content is lower; and the inoculation amount is too much, the growth, the propagation, the consumption and the metabolism of the strain are fast, the nutrition of the culture medium is insufficient, the fermentation period of the seeds is short, and the content is low. Preferably, the amount of the cultured seeds added is 10% by weight of the fermentation medium.

One specific example is provided below.