阅读说明：本技术 一种微生物发酵产普鲁兰多糖用培养基及其应用 (Culture medium for producing pullulan through microbial fermentation and application of culture medium ) 是由 王圣 余文婷 张潇 于 2021-11-16 设计创作，主要内容包括：本发明公开了一种微生物发酵产普鲁兰多糖用培养基及其应用。该培养基包括水,碳源10～220g/L,氮源0.1～10g/L,镁源0.01～3 g/L,钾源0.01～3 g/L,磷酸盐源0.01～3g/L,通过将水,碳源,氮源,镁源,钾源,磷酸盐源混合后,再用pH调节剂调节pH至3～10。通过对化学合成物质等成分的严苛限制,可以保证生产的普鲁兰多糖产品没有任何危害人体的成分,且完全安全有益,提高有机普鲁兰多糖的产量,使其满足有机贴标的要求,同时有利于工业化放大生产,得到附加值更高的普鲁兰多糖,也有利于高附加值普鲁兰多糖衍生产品的开发。(The invention discloses a culture medium for producing pullulan through microbial fermentation and application thereof. The culture medium comprises water, 10-220 g/L of carbon source, 0.1-10 g/L of nitrogen source, 0.01-3 g/L of magnesium source, 0.01-3 g/L of potassium source and 0.01-3 g/L of phosphate source, and the pH value is adjusted to 3-10 by using a pH regulator after the water, the carbon source, the nitrogen source, the magnesium source, the potassium source and the phosphate source are mixed. By strictly limiting components such as chemical synthetic substances, the produced pullulan polysaccharide product can be ensured to have no components harmful to human bodies, is completely safe and beneficial, the yield of organic pullulan polysaccharide is improved, the organic pullulan polysaccharide meets the requirement of organic labeling, the industrial amplification production is facilitated, the pullulan polysaccharide with higher added value is obtained, and the development of a pullulan derivative product with high added value is facilitated.)

1. A culture medium for producing pullulan through microbial fermentation is characterized by comprising water, 10-220 g/L of carbon source, 0.1-10 g/L of nitrogen source, 0.01-3 g/L of magnesium source, 0.01-3 g/L of potassium source and 0.01-3 g/L of phosphate source, wherein after the water, the carbon source, the nitrogen source, the magnesium source, the potassium source and the phosphate source are mixed, the pH is adjusted to 3-10 through a pH regulator.

2. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the carbon source is one or more of glucose, sucrose, starch, lactose, fructose, dextrin or maltose, and the concentration of the carbon source is 60-160 g/L.

3. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the nitrogen source is one or more of concentrated whey protein, yeast powder, ammonium bicarbonate and ammonium carbonate, and the concentration of the nitrogen source is 2-5 g/L.

4. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the magnesium source is one or a mixture of magnesium chloride and magnesium sulfate, and the concentration of the magnesium source is 0.1-0.3 g/L.

5. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the potassium source is one or more of potassium chloride, potassium iodide, potassium carbonate, potassium citrate and potassium lactate, and the concentration of the potassium source is 0.3-0.7 g/L.

6. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the phosphate source is one or a mixture of calcium hydrogen phosphate and calcium dihydrogen phosphate, and the concentration is 0.1-1 g/L.

7. The culture medium for producing pullulan through microbial fermentation according to claim 1, wherein the pH regulator is one or more of citric acid, lactic acid, tartaric acid and alginic acid, and the pH of the culture medium is adjusted to 6.0-7.0.

8. The application of the culture medium in the production of pullulan through fermentation according to claim 1 is characterized by comprising the following specific steps:

step 1, first-order seed culture

Inoculating the strain blocks with wet surfaces into a seed culture medium, subpackaging the strain blocks into conical flasks according to 10-50% of liquid loading amount, and culturing at 20-35 ℃ and 150-260 rpm for 22-50 h to obtain a primary seed liquid;

step 2, two-stage seed culture

Inoculating the primary seed liquid into a seed culture medium in a seed tank at 0.1-12% (v/v), culturing at 20-35 ℃, at a rotating speed of 80-400 rpm, with an air inflow of 0.5-2 vvm and a tank pressure of 0.1-0.8 bar for 22-50 h to obtain a secondary seed liquid, and performing multi-stage seed culture according to the fermentation scale to obtain a corresponding-stage seed liquid;

step 3, fermentation culture

Inoculating the corresponding-grade seed solution obtained in the step 2 into a fermentation tank in an inoculation amount of 0.1-12% (v/v), and culturing at 20-35 ℃, a rotation speed of 80-400 rpm, an air inflow of 0.5-2 vvm and a tank pressure of 0.1-0.8 bar for 48-120 h to obtain a fermentation liquid;

and 4, centrifuging the fermentation liquor at 8000rpm for 10min, taking supernatant, adding ethanol with the volume twice that of the supernatant, centrifuging at 8000rpm for 10min, taking white precipitate, and drying at 105 ℃ to constant weight to obtain the pullulan polysaccharide.

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a culture medium for producing pullulan through microbial fermentation and application thereof.

Background

The pullulan is an extracellular mucilaginous polysaccharide obtained by fermenting aureobasidium pullulans, has the characteristics of excellent film forming property, water solubility, biocompatibility, degradability and the like, and becomes a high-quality raw material of a natural plant capsule. However, the production level of the product is low in the global scope at present, the pullulan produced by the prior art has low conversion rate, the components of fermentation liquor are complex, and a fermentation formula meeting the requirement of organic labeling is lacked, so that the production cost is high, the additional value is relatively low, and the industrialization process is delayed.

CN103243135A discloses a novel culture medium for producing pullulan and a method for producing pullulan by fermentation, wherein the formula of the culture medium is as follows: the carbon source is sucrose, the concentration of the sucrose is 60-120g/L, the nitrogen source is urea, and the concentration of the urea is 2-4 g/L; the inorganic salt is K₂HPO₄，MgSO₄·7H₂O，FeSO₄·7H₂O，NaCl：2-4g/L，K₂HPO₄The concentration is 3-10g/L, MgSO₄·7H₂The concentration of O is 0.3-0.5g/L, FeSO₄·7H₂The concentration of O is 25-100mg/L, and the pH value is 6-7. The culture medium used in the method has definite components, the components of the fermented product are simple, and the subsequent extraction production cost is reduced; and the used components have wide sources, are easy to purchase and have lower cost. But does not meet the requirement of organic labeling and contains components which are not beneficial to human bodies.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a culture medium for producing pullulan through microbial fermentation and application thereof, which can ensure that the produced pullulan product has no components harmful to human bodies through severe limitation on components such as chemical synthetic substances and the like, is completely safe and beneficial, improves the yield of organic pullulan, meets the requirement of organic labeling, is beneficial to industrial amplification production, obtains the pullulan with higher added value and is also beneficial to development of high added value pullulan derivative products.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a culture medium for producing pullulan through microbial fermentation comprises water, 10-220 g/L of carbon source, 0.1-10 g/L of nitrogen source, 0.01-3 g/L of magnesium source, 0.01-3 g/L of potassium source and 0.01-3 g/L of phosphate source, wherein after the water, the carbon source, the nitrogen source, the magnesium source, the potassium source and the phosphate source are mixed, the pH is adjusted to 3-10 by using a pH adjusting agent.

The improvement is that the carbon source is one or a mixture of more of glucose, sucrose, starch, lactose, fructose, dextrin or maltose, and the concentration is 60-160 g/L.

The improvement is that the nitrogen source is formed by mixing one or more of concentrated whey protein, yeast powder, ammonium bicarbonate and ammonium carbonate, and the concentration is 2-5 g/L.

The improvement is that the magnesium source is one or a mixture of magnesium chloride and magnesium sulfate, and the concentration is 0.1-0.3 g/L.

The improvement is that the potassium source is one or a mixture of potassium chloride, potassium iodide, potassium carbonate, potassium citrate and potassium lactate, and the concentration is 0.3-0.7 g/L.

The improvement is that the phosphate source is one or two of calcium hydrogen phosphate and calcium dihydrogen phosphate, and the concentration is 0.1-1 g/L.

In an improvement, the pH regulator is one or more of citric acid, lactic acid, tartaric acid and alginic acid, and the pH of the culture medium is regulated to be 6.0-7.0.

The application of the culture medium in the production of pullulan through fermentation comprises the following specific steps:

step 1, first-order seed culture

Inoculating the strain blocks with wet surfaces into a seed culture medium, subpackaging the strain blocks into conical flasks according to 10-50% of liquid loading amount, and culturing at 20-35 ℃ and 150-260 rpm for 22-50 h to obtain a primary seed liquid;

step 2, two-stage seed culture

Inoculating the primary seed liquid into a seed culture medium in a seed tank at 0.1-12% (v/v), culturing at 20-35 ℃, at a rotating speed of 80-400 rpm, with an air inflow of 0.5-2 vvm and a tank pressure of 0.1-0.8 bar for 22-50 h to obtain a secondary seed liquid, and performing multi-stage seed culture according to the fermentation scale to obtain a corresponding-stage seed liquid;

step 3, fermentation culture

Inoculating the corresponding-grade seed solution obtained in the step 2 into a fermentation tank in an inoculation amount of 0.1-12% (v/v), and culturing at 20-35 ℃, a rotation speed of 80-400 rpm, an air inflow of 0.5-2 vvm and a tank pressure of 0.1-0.8 bar for 48-120 h to obtain a fermentation liquid;

and 4, centrifuging the fermentation liquor at 8000rpm for 10min, taking supernatant, adding ethanol with the volume twice that of the supernatant, centrifuging at 8000rpm for 10min again, taking white precipitate, and drying at 105 ℃ until the weight is constant to obtain the pullulan polysaccharide.

Has the advantages that:

compared with the prior art, the culture medium for producing pullulan through microbial fermentation and the application thereof can ensure that the produced pullulan product has no components harmful to human bodies through severe limitation on chemical synthetic substances and other components, is completely safe and beneficial, and improves the yield of organic pullulan from 26.7g/L to 58g/L through formula optimization, so that the organic pullulan product meets the requirement of organic labeling, is beneficial to industrial amplification production, obtains pullulan with higher added value, and is also beneficial to development of high added value pullulan derivative products.

Drawings

FIG. 1 shows the effect of example 1 and the absence of dibasic calcium phosphate on pullulan production.

Detailed Description

The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

The aureobasidium pullulans SWP35 has a preservation number of CGMCC NO.11602, and the aureobasidium pullulans SWP35 used in the embodiment of the invention is purchased from China general microbiological culture Collection center.

Example 1

Step 1, first-order seed culture

Inoculating aureobasidium pullulans SWP35 with wet surface into a seed culture medium (the formula of the seed culture medium is 80g/L of glucose, 2.2g/L of whey protein concentrate, 0.7g/L of calcium hydrophosphate, 0.1g/L of magnesium chloride and 0.5g/L of potassium carbonate), mixing the components, adjusting the pH value to 7.0 by using citric acid with the concentration of 0.7g/L, subpackaging the mixture into conical flasks according to the liquid filling amount of 20 percent, and culturing the conical flasks at the temperature of 28 ℃ and the rotating speed of 230 rpm for 36 hours to obtain a primary seed liquid;

step 2, secondary seed culture

Inoculating the primary seed solution into a seed culture medium in a seed tank at 0.5% (v/v), culturing at 28 deg.C, rotation speed of 350rpm, air inflow of 1vvm, and tank pressure of 0.5bar for 42h to obtain secondary seed solution. By analogy, performing multistage seed culture according to the fermentation scale;

step 3, fermentation culture

Inoculating the corresponding-grade seed liquid obtained in the step 2 into a fermentation medium (the formula of the fermentation medium is 130g/L of cane sugar, 3g/L of whey protein concentrate, 0.7g/L of calcium hydrophosphate, 0.15 g/L of magnesium chloride and 0.5g/L of potassium carbonate) in a fermentation tank at 8% (v/v), mixing the components, adjusting the pH value to 6.0 by using 1.35 g/L of citric acid, and culturing at 28 ℃, the rotation speed of 400rpm, the air input of 1vvm and the tank pressure of 0.5 for 96h to obtain a fermentation liquid;

and 4, centrifuging the fermentation liquor at 8000rpm for 10min, taking supernatant, adding ethanol with the volume twice that of the supernatant, centrifuging at 8000rpm for 10min again, taking white precipitate, drying at 105 ℃ until the weight is constant to obtain pullulan, wherein the yield of the pullulan can reach 58 g/L. The comparison example of this example produces pullulan by fermentation without adding calcium hydrogen phosphate, and the results are shown in fig. 1, and it can be seen from fig. 1 that the yield of pullulan is significantly higher after adding calcium hydrogen phosphate than that of the sample without adding calcium hydrogen phosphate.

Example 2

Step 1, first-order seed culture

Inoculating aureobasidium pullulans SWP35 with wet surface into a seed culture medium (the formula of the seed culture medium comprises 60g/L of glucose, 2g/L of whey protein concentrate, 0.1g/L of calcium hydrophosphate, 0.1g/L of magnesium chloride and 0.3g/L of potassium carbonate), mixing the components, adjusting the pH value to 6.5 by using citric acid with the concentration of 0.3g/L, subpackaging the mixture into conical flasks according to the liquid loading of 10%, and culturing at the temperature of 20 ℃ and the rotating speed of 150rpm for 22h to obtain a primary seed liquid.

Step 2, secondary seed culture

Inoculating the primary seed solution into a seed culture medium in a seed tank at 0.1% (v/v), culturing at 20 deg.C, 80rpm, 0.5vvm of air inflow, and 0.1bar of tank pressure for 22h to obtain a secondary seed solution. By analogy, multi-stage seed culture is carried out according to the fermentation scale.

Step 3, fermentation culture

And (3) inoculating the corresponding-grade seed liquid obtained in the step (2) into a fermentation medium (the formula of the fermentation medium is 60g/L of cane sugar, 2g/L of whey protein concentrate, 0.1g/L of calcium hydrophosphate, 0.1g/L of magnesium chloride and 0.3g/L of potassium carbonate) in a fermentation tank at 0.1% (v/v), adjusting the pH value to 6.5 by using 0.3g/L of citric acid, and culturing at 20 ℃, the rotating speed of 80rpm, the air inflow of 0.5vvm and the tank pressure of 0.1bar for 48 hours to obtain a fermentation liquid.

And 4, centrifuging the fermentation liquor at 8000rpm for 10min, taking supernatant, adding ethanol with the volume twice that of the supernatant, centrifuging at 8000rpm for 10min again, taking white precipitate, drying at 105 ℃ until the weight is constant to obtain pullulan, wherein the yield of the pullulan can reach 23 g/L.

Example 3

Step 1, first-order seed culture

Inoculating aureobasidium pullulans SWP35 with wet surface into a seed culture medium (the formula of the seed culture medium is 100g/L of glucose, 5g/L of whey protein concentrate, 1g/L of calcium hydrogen phosphate, 0.3g/L of magnesium chloride and 0.7g/L of potassium carbonate), mixing the components, adjusting the pH value to 6.0 by using citric acid with the concentration of 0.9 g/L, subpackaging the mixture into conical flasks according to 50% of liquid loading amount, and culturing at 35 ℃ and the rotating speed of 260rpm for 50 hours to obtain a primary seed liquid.

Step 2, secondary seed culture

Inoculating the primary seed liquid into a seed culture medium in a seed tank at 12% (v/v); culturing at 35 deg.C, rotation speed of 400rpm, air input of 2vvm, and tank pressure of 0.8bar for 50h to obtain secondary seed solution. By analogy, multi-stage seed culture is carried out according to the fermentation scale.

Step 3, fermentation culture

And (3) inoculating the corresponding-grade seed liquid obtained in the step (2) into a fermentation medium (the formula of the fermentation medium is sucrose 160 g/L, whey protein concentrate 5g/L, calcium hydrogen phosphate 1g/L, magnesium chloride 0.1g/L and potassium carbonate 0.3 g/L) in a fermentation tank at 12% (v/v), mixing the components, adjusting the pH value to 7.0 by using 1.35 g/L citric acid, and culturing at 35 ℃, the rotation speed of 400rpm, the air inflow of 2vvm and the tank pressure of 0.8bar for 120 hours to obtain a fermentation liquid.

And 4, centrifuging the fermentation liquor at 8000rpm for 10min, taking supernatant, adding ethanol with the volume twice that of the supernatant, centrifuging at 8000rpm for 10min again, taking white precipitate, drying at 105 ℃ until the weight is constant to obtain pullulan, wherein the yield of the pullulan can reach 48 g/L.

As shown in figure 1, the yield of the organic pullulan is higher than that of the organic pullulan without the calcium hydrogen phosphate. Meanwhile, according to the formula added with the calcium hydrophosphate component, the yield is increased firstly and then reduced with the increase of the addition amount of the sucrose, and the maximum value of 58g/L is reached when the addition amount of the sucrose is 130 g/L.

In conclusion, the culture medium for producing pullulan through microbial fermentation and the application thereof can ensure that the produced pullulan product has no components harmful to human bodies through severe limitation on chemical synthetic substances and other components, is completely safe and beneficial, improves the yield of organic pullulan, enables the organic pullulan product to meet the requirement of organic labeling, is beneficial to industrial amplification production, obtains the pullulan with higher added value, and is also beneficial to development of high-added-value pullulan derivative products.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.