阅读说明：本技术 一种产黄青霉的固定化方法及其应用 (Immobilization method of penicillium chrysogenum and application thereof ) 是由 应汉杰 王芳娟 陈勇 丁赛 刘丽 俞莹 张德力 于 2019-11-21 设计创作，主要内容包括：本发明公开了一种产黄青霉的固定化方法及其应用,将电晕改性后的塑料介质作为固定化载体,将产黄青霉活化后接入固定化发酵培养基中培养,使菌株在培养的过程中吸附于载体上此外。本发明中以塑料介质为惰性材料,实现了产黄青霉的高密度发酵,降低发酵液的粘度,发酵液的黏度适中,传质效果很好。在与游离发酵相比,产物的效价大大提高,青霉素的效价达到1672.65U/mL,有效缩短发酵总周期从21天缩短到18天。(The invention discloses an immobilization method of penicillium chrysogenum and application thereof, wherein a plastic medium subjected to corona modification is used as an immobilization carrier, the penicillium chrysogenum is activated and then inoculated into an immobilized fermentation culture medium for culture, and a strain is adsorbed on the carrier in the culture process. The plastic medium is used as an inert material, so that the high-density fermentation of the penicillium chrysogenum is realized, the viscosity of the fermentation liquor is reduced, the viscosity of the fermentation liquor is moderate, and the mass transfer effect is good. Compared with free fermentation, the titer of the product is greatly improved, the titer of penicillin reaches 1672.65U/mL, and the total fermentation period is effectively shortened from 21 days to 18 days.)

1. A method for immobilizing penicillium chrysogenum is characterized in that a plastic medium subjected to corona modification is used as an immobilized carrier, the penicillium chrysogenum is activated and inoculated into an immobilized fermentation culture medium for culture, and a strain is adsorbed on the carrier in the culture process.

2. The immobilization method according to claim 1, wherein said Penicillium chrysogenum is ATCC 48271.

3. The method of claim 1, wherein the plastic medium is polymethylmethacrylate.

4. The immobilization method according to claim 1, wherein the plastic medium is added in an amount of 15-20 g/L to the immobilized fermentation medium.

5. The method of claim 1, wherein the corona modification comprises the steps of:

(1) cleaning a plastic medium in deionized water, extracting the cleaned plastic medium in acetone for 15-30h, and drying;

(2) and (3) performing corona treatment on the plastic medium treated in the step (1), and storing for later use.

6. The fixing method according to claim 5, wherein in the step (2), the current intensity of the corona is 6-14A; the corona time is 10-30 min.

7. The immobilization method according to claim 1, wherein the immobilized fermentation medium comprises, by mass, 4.65% of soybean cake powder, 1% of calcium carbonate, 3% of lactose, 0.5% of potassium dihydrogen phosphate, 0.4% of ammonium sulfate, 0.15% of sodium sulfate, 0.03% of salad oil, 2.5% of phenylacetic acid, and water as a solvent, wherein the pH is 5.8.

8. The immobilization method according to claim 1, wherein the culture is carried out at 25 ℃ for 3 days.

9. Immobilized Penicillium chrysogenum prepared by a process according to any of claims 1 to 8.

10. The use of the immobilized Penicillium chrysogenum of claim 9 for the production of penicillin, wherein the immobilized Penicillium chrysogenum is inoculated into a liquid-change immobilized fermentation medium and cultured at 20-30 ℃ and 150-300 rpm for 70-96 hours;

the liquid-changing immobilized fermentation medium comprises, by mass, 4.65% of soybean cake powder, 1% of calcium carbonate, 3% of lactose, 0.5% of monopotassium phosphate, 0.4% of ammonium sulfate, 0.15% of sodium sulfate, 0.03% of salad oil and 2.5% of phenylacetic acid, and the pH value is 5.8.

Technical Field

The invention belongs to the technical field of industrial biology, and particularly relates to an immobilization method of penicillium chrysogenum and application thereof.

Background

As early as 1929, Alexander Fleming discovered a substance that inhibited staphylococcus aureus on the plates, later named penicillin. Penicillin (Penicillin) can be named by a number of names, such as: penicillin G, penicillin sodium, penicillin potassium, and many of the antibiotics currently used for therapy are derived from the β -lactam antibiotics found in fleming. Penicillin is one of antibiotics, and refers to a class of antibiotics which contain penam in molecules extracted from a penicillium culture solution and can destroy the cell wall of bacteria and kill bacteria in the propagation period of the bacteria cells. Penicillin antibiotics are a generic term for a large group of antibiotics in the beta-lactam class. Penicillin is still one of clinically important antibiotics as an antibiotic with low toxin and good treatment effect.

Penicillium chrysogenum belongs to the subphylum Ascomycotina, Ascomycetes, Ascomycoaceae, Penicillium. Penicillium chrysogenum is a mold that is widely found in nature, most commonly in moist food and environments. The optimum growth temperature of the penicillium chrysogenum is different from 25 ℃ to 28 ℃, the requirement on nutrition is not strict, and therefore, the penicillium chrysogenum is easy to culture. The first bacteria used for producing penicillin is penicillium notatum, but currently penicillium chrysogenum is used, according to the state of hypha, penicillium chrysogenum can be divided into filamentous bacteria and coccoid, and the filamentous bacteria and coccoid can be further divided into different bacteria according to the color of spores, while the penicillium chrysogenum is mainly green spore filamentous bacteria which is currently used for producing penicillin. The colony morphology of P.chrysogenum is easily recognized because of its colony morphology and the unique color of spores. However, in industrial production, the fermentation period is prolonged greatly due to the problem of long seed culture time, the fermentation liquid is viscous, and the cells are easily aged without separation of the product.

Accordingly, many attentions have been paid to the immobilization technology, and the technology has become mature, and thus the technology is widely used in various fields of biotechnology production. The thallus is contacted with some materials suitable for immobilized fermentation, and the thallus is adsorbed on the carrier, so that the concentration of the fermentation liquor is reduced, and the transfer of nutrient substances is facilitated.

At present, the cell immobilization methods mainly include: the embedding method and the adsorption method are mainly adopted in the immobilization of the cells in China. However, little is known about immobilization of P.chrysogenum, and immobilization of P.chrysogenum by calcium alginate has been reported before, but this immobilization of cells introduces by-products which are not advantageous for the later isolation of the product and also leads to a loss of penicillin yield. Based on the method for immobilizing aspergillus niger, the invention also discloses a method for immobilizing fermentation of penicillium chrysogenum, namely a carrier suitable for growth of the penicillium chrysogenum is searched in a naturally existing inert medium, so that the fermentation efficiency can be improved, the production cost is reduced, and the problem to be solved in the process is solved.

With the development of the technology, various novel inert materials have appeared in many years, however, in the process of immobilization, we find that the plastic medium is used as a medium for immobilized fermentation, which is not only non-toxic, but also has stable process embodiment, the inert materials are easily obtained, the product can be recycled after separation, the production cost is greatly reduced, and more importantly, a certain protection effect is exerted on active cells, so the effect on immobilized fermentation is very significant, however, penicillium is used as a filamentous bacterium, and the bacterium grows fast because of the long mycelium, so few people adopt an adsorption method for immobilized fermentation.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing an immobilization method of penicillium chrysogenum, which ensures that the penicillium chrysogenum is simple and convenient to immobilize, and the immobilization process is stable and efficient.

In order to solve the technical problem, the invention discloses an immobilization method of penicillium chrysogenum, which takes a plastic medium subjected to corona modification as an immobilization carrier, activates the penicillium chrysogenum, then inoculates the activated penicillium chrysogenum into an immobilized fermentation culture medium for culture, and enables a strain to be adsorbed on the carrier in the culture process.

Wherein, the penicillium chrysogenum is ATCC 48271.

Wherein, the plastic medium is polymethyl methacrylate.

Wherein the plastic medium is placed in the immobilized fermentation medium in an addition amount of 15-20 g/L (preferably 15 g/L).

Wherein, the corona modification comprises the following steps:

(1) cleaning a plastic medium in deionized water for 1-5 times (2-10 min), extracting the cleaned plastic medium in acetone for 15-30h, removing oil stains and impurities on the surface, and drying in an oven at 45 ℃ for 30-60 min;

(2) and (3) performing corona treatment on the plastic medium treated in the step (1), and storing for later use.

In the step (1), the extraction is circular extraction, and 50mL/0.1g of acetone serving as a purification solvent is directly added at one time.

In the step (2), the equipment for corona treatment is SDCD 16-3-20; the current intensity of the corona is 6-14A (preferably 8A); the corona time is 10-30min (preferably 15 min); the principle of corona is to discharge continuously on the surface of the medium, and the invention adopts corona to change the radical property so as to make the medium more hydrophilic.

The immobilized fermentation medium comprises, by mass, 4.65% of soybean cake powder, 1% of calcium carbonate, 3% of lactose, 0.5% of monopotassium phosphate, 0.4% of ammonium sulfate, 0.15% of sodium sulfate, 0.03% of salad oil and 2.5% of phenylacetic acid, and the pH value is 5.8.

The soybean cake powder is used as an organic nitrogen source, but is more suitable for immobilization of penicillium chrysogenum than common corn steep liquor, and the source is wide and easy to obtain; compared with the traditional corn oil, the salad oil serving as a mixture of various vegetable oils can reduce air bubbles more effectively, and the substances contained in the salad oil are more suitable for fermentation of penicillium.

Wherein the culture is carried out for 3 days at 25 ℃.

The immobilized penicillium chrysogenum prepared by the method is also within the protection scope of the invention.

The use of the immobilized Penicillium chrysogenum described above for the production of penicillin is within the scope of the present invention.

Wherein, the application is as follows: inoculating immobilized penicillium chrysogenum into a liquid-changing immobilized fermentation culture medium, and culturing for 72-96h at 20-30 ℃ and 150-300 rpm in a shaking table;

the liquid-changing immobilized fermentation medium comprises, by mass, 4.65% of soybean cake powder, 1% of calcium carbonate, 3% of lactose, 0.5% of monopotassium phosphate, 0.4% of ammonium sulfate, 0.15% of sodium sulfate, 0.03% of salad oil and 2.5% of phenylacetic acid, and the pH value is 5.8.

Preferably, the culture is carried out for 72-96h at 25 ℃ and 220rpm by shaking.

Wherein, the specific fermentation process comprises the following steps:

(1) the Penicillium chrysogenum, which had been frozen and preserved at-80 ℃ in a refrigerator, was streaked and activated on a plate and cultured at 25 ℃ for 3 days.

(2) After 3 days, collecting spores by using a spore scraping solution (0.9% NaCl + Tween 80), inoculating the spores into a 500mL shake flask, wherein the liquid loading amount is 100mL, carrying out shake culture at 25 ℃ and 220rmp for 72-96h, measuring the concentration of phenylacetic acid in fermentation liquor, ending a batch of fermentation when the concentration is lower than 0.01%, and then removing the fermentation liquor.

(3) Immobilized repeated batch fermentation: 1L of fresh culture medium was prepared. Then 100mL of the mixture was dispensed into 500mL Erlenmeyer flasks, and sterilized at 115 ℃ for 20 min. After the sterilization is finished, the first batch of bacteria liquid is poured out, and then fresh culture liquid is poured into the super clean bench. This was repeated for 9 batches.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. in the invention, the plastic medium adopted by the method is an inert material, has chemical property and stability, does not produce toxic action on cells in the fermentation process, has high mechanical strength, can not be decomposed and metabolized, has good uniformity after being modified, and can not cause excessive growth and uniform distribution of the penicillium chrysogenum on the carrier.

2. In the invention, in the fermentation process, the process is in a dynamic balance state due to the falling of dead cells and the adsorption of living cells, thereby reducing the damage to the cells in the fermentation process, greatly reducing the total fermentation time and the fermentation cost, and effectively shortening the total fermentation period from 21 days to 18 days.

3. The optimal plastic medium is selected, and has a small contact angle, so that the number of immobilized cells in unit volume is large, and the immobilized cells have high density, so that the high-density fermentation of the penicillium chrysogenum is realized, and in addition, the viscosity of the fermentation liquid is reduced, the viscosity of the fermentation liquid is moderate, and the mass transfer effect is good. Compared with free fermentation, the titer of the product is greatly improved, the titer of penicillin reaches 1672.65U/mL, and the reduction of viscosity is beneficial to the subsequent separation of the product.

4. The plastic medium is used for immobilizing penicillium chrysogenum to produce penicillin, 9 batches of penicillin are continuously produced, the yield of the penicillin is not obviously reduced, compared with the prior art, the modified plastic medium is used for immobilizing fermentation, the process is quite stable, and the carrier can be recycled.

Drawings

Fig. 1 is a contact angle of the supports prepared in examples 1 to 6.

Fig. 2 is a contact angle after the activated carbon treatment of comparative example 1.

FIG. 3 shows the contact angle of the PMMA of comparative example 2 without corona treatment.

FIG. 4 shows the data of fermentation using the vectors obtained in examples 1, 3 and 5 as media.

FIG. 5 shows the data for immobilized fermentations with different amounts of free and added carrier.

Figure 6 variation of contact angles for different corona treatments, an optimal conditional contact angle map was determined.

FIG. 7 comparison of free and immobilized fermentations.

FIG. 8 shows the amount of delayed biofilm.

FIG. 9 is the amount of biofilm in the logarithmic growth phase.

FIG. 10 shows the amount of biofilm in stationary phase.

FIG. 11 is an electron micrograph of a biofilm at the decay phase.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

The method described in the present invention can be applied to all penicillium, and in the following embodiment, the penicillium chrysogenum strain we have selected is ATCC 48271.

In many plastic media, we find by comparison that the surfaces of the plastic media have certain hydrophobicity, and in the case of the filamentous fungus Penicillium chrysogenum, the hypha has stronger hydrophilicity, and in order to make the hypha uniformly adsorb on the carrier, the carrier is correspondingly pretreated so that the hydrophilicity can be enhanced, thereby promoting the adsorption of the hypha in the seed solution and the surfaces of the carrier to be more uniform, and the specific implementation mode is as follows:

(1) cleaning plastic medium in deionized water for 2-10min, extracting the cleaned plastic medium in acetone for 15-30h, removing oil stain and impurities on surface, and oven drying at 45 deg.C for 30-60min

(2) And (3) placing the treated plastic medium on a corona treatment device of the SDCD16-3-20 type to perform corona treatment with the current intensity of 6-14A and the time of 10-30 min.