阅读说明：本技术 细胞重复利用生产普鲁兰多糖的方法及应用 (Method for producing pullulan polysaccharide by cell recycling and application ) 是由 魏培莲 吕磊磊 陈林杰 肖功年 鲍文娜 柳永 刘士旺 于 2021-08-02 设计创作，主要内容包括：本发明提供了一种细胞重复利用发酵生产普鲁兰多糖的方法及应用,包括如下步骤：将出芽短梗霉种子液接种到发酵培养基进行发酵；发酵培养7d后,用盐酸或氢氧化钠调节发酵液中的pH值至稳定后,滴加絮凝剂和助凝剂,振荡混匀后静置分层,菌液分离,得到含有普鲁兰多糖的发酵液清液和絮凝沉淀的出芽短梗霉菌体,收集含有普鲁兰多糖的发酵液清液,将出芽短梗霉菌体用新鲜发酵液重新悬浮并继续发酵。本发明通过采用絮凝法回收并重复利用细胞发酵的方式进行普鲁兰多糖的发酵生产,提高普鲁兰多糖的产量；减少发酵生产过程中的种子培养基配制、种子培养及接种等环节,节约发酵时间,降低原料成本,并进一步提高生产效率。(The invention provides a method for producing pullulan polysaccharide by cell recycling fermentation and application thereof, comprising the following steps: inoculating the aureobasidium pullulans seed liquid to a fermentation culture medium for fermentation; after fermentation culture for 7 days, adjusting the pH value in the fermentation liquor to be stable by using hydrochloric acid or sodium hydroxide, dropwise adding a flocculating agent and a coagulant aid, shaking, uniformly mixing, standing, layering, separating bacterial liquid to obtain a fermentation liquor clear liquid containing pullulan polysaccharide and flocculation-precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan polysaccharide, resuspending the Aureobasidium pullulan thallus by using a fresh fermentation liquor, and continuing fermentation. According to the invention, the fermentation production of the pullulan is carried out by adopting a flocculation method to recover and repeatedly utilize a cell fermentation mode, so that the yield of the pullulan is improved; the links of seed culture medium preparation, seed culture, inoculation and the like in the fermentation production process are reduced, the fermentation time is saved, the raw material cost is reduced, and the production efficiency is further improved.)

1. The method for producing the pullulan polysaccharide by cell recycling fermentation is characterized by comprising the following steps:

1) inoculating a prepared aureobasidium pullulans seed solution into a fermentation culture medium for fermentation culture;

2) after fermentation culture for one week, adjusting the pH value in the fermentation liquor by using hydrochloric acid or sodium hydroxide;

3) after the pH value in the fermentation liquor is stable, adding a flocculating agent and a coagulant aid dropwise into the fermentation liquor, and uniformly mixing by oscillation;

4) standing the fermentation liquor after oscillation and uniform mixing until the bacterial liquor is layered, pouring to separate the bacterial liquor to obtain a fermentation liquor clear liquid containing pullulan and flocculently precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan to extract pullulan, suspending the flocculently obtained Aureobasidium pullulan with a fresh culture medium again, continuing to ferment, and continuing to prepare the pullulan.

2. The method for producing pullulan through cell recycling and fermentation according to claim 1, wherein the inoculation amount of the aureobasidium pullulans seed solution in step 1) is 6%, the time for culturing the seed solution is 2-3 days, and the culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.

3. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the culture medium for preparing the seed liquid in step 1) comprises the following components in percentage by weight: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH₂PO₄ 0.5%，MgSO₄•7H₂0.04% of O, 0.1% of NaCl and the balance of water.

4. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the fermentation medium in the step 1) comprises the following components in percentage by weight: glucose 12%, NH₄NO₃ 0.1%，KH₂PO₄ 0.01%，KCl 0.05%，MgSO₄•7H₂0.02% of O, the balance of water, and the fermentation culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.

5. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the concentration of hydrochloric acid or sodium hydroxide in step 2) is 2mol/L, and the pH value of the fermentation broth is adjusted to 5.0 to 5.5.

6. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the flocculating agent in step 3) is 10g/L chitosan solution prepared from 1% acetic acid solution and autoclaved, and the dropping amount is 0.03-0.04 mL/mL; the coagulant aid is 10g/L sodium alginate solution prepared by ultrapure water and autoclaved, and the dropping amount is 0.02-0.04 mL/mL.

7. The method for producing pullulan through cell recycling and fermentation according to claim 1, wherein the oscillating and mixing condition of the fermentation liquid in the step 3) is 28 ℃, 180r/min and the time is 20-30 min.

8. The method for producing pullulan through cell recycling fermentation according to claim 1, wherein the calculation formula of the flocculation rate of the cells after the flocculation treatment of the fermentation liquid is as follows:(ii) a The calculation formula of the pullulan retention rate is as follows: retention rate。

9. The method for producing pullulan by cell recycling fermentation according to claims 1 to 8 should be used in the production of pullulan.

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a method for producing pullulan polysaccharide by cell recycling and application of the pullulan polysaccharide.

Background

The pullulan is a colorless and tasteless dextro-glucoside polymer, is powdery, can give off slight sweet taste when dissolved in water, has the obvious advantages of no toxicity, biological safety, easy degradability, good edibility, good film coating property and the like, and has huge application potential in the fields of medicines, cosmetics, environment, food and the like. The microbial fermentation method has the advantages of mild reaction conditions, high purity of target products and little pollution, shows greater production advantages and has huge development prospect. Aureobasidium pullulans: (Aureobasidium pullulans) Is a yeast-like fungus and is a main pullulan production strain. The current strategies for efficiently producing the pullulan by using a microbial fermentation method mainly comprise breeding high-yield strains, optimizing culture conditions, controlling metabolic processes and the like, however, the future demand space of the pullulan is large, and the strategies cannot realize large-scale production of the pullulan.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for producing pullulan by cell recycling and application thereof, wherein the method can maintain the continuous fermentation performance of cells, reduce the product inhibition, save the fermentation time, reduce the production cost and improve the total yield of fermentation products.

The invention is realized by the following technical scheme:

the method for producing the pullulan polysaccharide by cell recycling fermentation is characterized by comprising the following steps:

1) inoculating a prepared aureobasidium pullulans seed solution into a fermentation culture medium for fermentation culture;

2) after fermentation culture for one week, adjusting the pH value in the fermentation liquor by using hydrochloric acid or sodium hydroxide;

3) after the pH value in the fermentation liquor is stable, adding a flocculating agent and a coagulant aid dropwise into the fermentation liquor, and uniformly mixing by oscillation;

4) standing the fermentation liquor after oscillation and uniform mixing until the bacterial liquor is layered, pouring to separate the bacterial liquor to obtain a fermentation liquor clear liquid containing pullulan and flocculently precipitated Aureobasidium pullulan thallus, collecting the fermentation liquor clear liquid containing pullulan to extract pullulan, suspending the flocculently obtained Aureobasidium pullulan with a fresh culture medium again, continuing to ferment, and continuing to prepare the pullulan.

Preferably, the inoculation amount of the aureobasidium pullulans seed solution in the step 1) is 6%, the culture time of the seed solution is 2-3 days, and the culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.

Preferably, the culture medium for preparing the seed liquid in the step 1) comprises the following components in percentage by weight: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH₂PO₄ 0.5%，MgSO₄•7H₂0.04% of O, 0.1% of NaCl and the balance of water.

Preferably, the fermentation medium in step 1) comprises the following components in percentage by weight: glucose 12%, NH₄NO₃0.1%，KH₂PO₄ 0.01%，KCl 0.05%，MgSO₄•7H₂0.02% of O, the balance of water, and the fermentation culture conditions are as follows: the culture temperature is 28 ℃, and the rotating speed of the shaking table is 180 r/min.

Preferably, the concentration of the hydrochloric acid or the sodium hydroxide in the step 2) is 2mol/L, and the pH value of the fermentation liquor is adjusted to 5.0-5.5.

Preferably, the flocculating agent in the step 3) is 10g/L chitosan solution prepared by 1% acetic acid solution and autoclaved, and the dropping amount is 0.03-0.04 mL/mL; the coagulant aid is 10g/L sodium alginate solution prepared by ultrapure water and autoclaved, and the dropping amount is 0.02-0.04 mL/mL.

Preferably, the condition of shaking and mixing the fermentation liquor in the step 3) is 28 ℃, 180r/min, and the time is 20-30 min.

Preferably, the calculation formula of the cell flocculation rate after the flocculation treatment of the fermentation liquor is as follows:(ii) a General purposeThe formula for calculating the retention rate of the luran polysaccharide is as follows: retention rate。

The method for producing the pullulan by repeatedly utilizing the cells through fermentation should be used in the production of the pullulan.

According to the method, a flocculation method is adopted, the fermentation production of the pullulan is carried out in a cell fermentation mode, after a fermentation period is finished, the bacterial cells are recovered through flocculation treatment, a fresh culture medium is supplemented for resuspension and continuous fermentation, products are discharged in time, the product inhibition is reduced, meanwhile, metabolic waste is continuously transferred out, the toxic hazard to the cell growth is avoided, and the yield of the pullulan is effectively improved; the method for producing the pullulan polysaccharide by cell recycling fermentation reduces the links of seed culture medium preparation, seed culture, inoculation and the like in the fermentation production process, saves the fermentation time, reduces the raw material cost and further improves the production efficiency. After the fermentation liquor is flocculated, the cell flocculation rate is over 80 percent, the retention rate of the pullulan is over 95 percent, and the yield of the pullulan is improved by over 45 percent.

Detailed Description

The present invention will be described in further detail with reference to specific examples to better understand the technical solution.

Example 1

The method for producing the pullulan polysaccharide by cell recycling fermentation comprises the following steps:

1) selecting a well-cultured slant strain, selecting 2 rings, inoculating the strain into a seed culture medium, filling 50mL of the seed culture medium into a triangular flask with the liquid filling amount of 250mL, and culturing for 2d at the temperature of 180r/min in a shaking table;

the seed culture medium comprises: glucose 5%, peptone 0.1%, yeast extract 0.1%, KH₂PO₄ 0.5%，MgSO₄•7H₂0.04% of O, 0.1% of NaCl and the balance of water;

2) fermentation culture: inoculating the prepared seed liquid into a fermentation culture medium according to the proportion of 6 percent, filling 50mL of the seed culture medium into a triangular flask with the liquid filling amount of 250mL, and culturing for 7d at the temperature of 180r/min by a shaking table;

3) dropwise adding HCl into the fermentation liquor fermented in the step 2), adjusting the pH of the fermentation liquor to 5.1, respectively dropwise adding 2mL of flocculant and 2mL of coagulant aid, and then uniformly mixing for 20 min;

4) standing the fermentation liquid treated in the step 3) for 2 hours, pouring out the supernatant, adding a fresh fermentation medium with the same volume again for suspension, and continuing fermentation culture for 7 days;

through determination, the cell flocculation rate is 84.8%, the pullulan retention rate is 96.1%, and the pullulan yield is improved by 46.4%.

Wherein, the calculation formula of the cell flocculation rate is as follows:(ii) a The calculation formula of the pullulan retention rate is as follows: retention rate。

Example 2

This example is the same as the process step of example 1, except that the amount of flocculant added dropwise in step 3) was 1.5 mL.

The final determination in the example shows that the cell flocculation rate is 80.3%, the pullulan retention rate is 95.1%, and the yield of the pullulan is improved by 45.1%.

Example 3

This example is the same as the procedure of example 1 except that the coagulant aid was added in an amount of 1.5mL in step 3).

The final determination in the example shows that the cell flocculation rate is 81.4%, the pullulan retention rate is 95.3%, and the yield of the pullulan is improved by 45.9%.

Example 4

This example is identical to the procedure of example 1, except that in step 3) the shaking and homogenisation time is 30 min.

The final determination in the example shows that the cell flocculation rate is 83.7%, the pullulan retention rate is 95.6%, and the yield of the pullulan is improved by 46.2%.