CN110885775B

CN110885775B - Nocardia culture method

Info

Publication number: CN110885775B
Application number: CN201911357043.4A
Authority: CN
Inventors: 王文勇
Original assignee: Guangzhou Keli Biotechnology Co ltd
Current assignee: Guangzhou Keli Biotechnology Co ltd
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-06-29
Anticipated expiration: 2039-12-25
Also published as: CN110885775A

Abstract

The invention relates to the field of microbial culture, in particular to a culture method of nocardia. The culture method comprises the steps of preparing a nocardia culture medium, preparing a seed solution and fermenting and culturing; the Nocardia culture medium in the culture method comprises the following raw materials in percentage by weight: 0.5-2% of glucose, 0.5-2% of yeast powder, 0.5-2% of tryptone, 0.3-0.5% of sodium citrate, 0.3-0.5% of sodium pyruvate, 0.02-0.06% of nalidixic acid, 0.2-0.6% of cysteine hydrochloride and the balance of deionized water, and the defoaming agent can also be included, and comprises a composition of polyglycerol fatty acid ester and sucrose fatty acid ester. Compared with the existing shake flask culture technology, the invention has the advantages of high growth speed of the nocardia, shortened culture period, stable content of bacterial cell wall components, suitability for industrial production, cost saving and high popularization value.

Description

Nocardia culture method

Technical Field

The invention relates to the technical field of microbial culture, in particular to a culture method of nocardia.

Background

Nocardia (Nocardia) is a bacterium that is widely found in soil, seawater, fresh water, and dust. It belongs to the bacteria kingdom, Actinomycetes, Actinomycetales, Corynebacterium, Nocardiaceae, Nocardia, and belongs to gram-positive bacteria. Nocardiosis is an infectious disease common to mammals, cetaceans, fish, and humans. Nocardiosis can cause chronic death of fish, reduce the commodity value of adult fish, has great influence on aquaculture industry, five strips can generate serum agglutination antibodies after inoculation of nocardia inactivated vaccines, but can not provide any immune protection effect on virus attack, so that the research and development of efficient nocardia vaccines are imperative. One of the characteristics of the nocardia culture is that the primary generation grows slowly, and the research and development of the vaccine needs to culture the thalli on a large scale, so that how to culture the nocardia thalli on a large scale by using a method with high efficiency, high yield and simple process becomes a problem which needs to be solved urgently.

At present, a mechanical stirring type fermentation tank is mainly adopted in factory production to culture microorganisms. In the closest prior art, a new culture method for red nocardia (patent No. CN201410320363.3) also discloses a mechanical stirring fermentation tank for culturing nocardia, compared with the shake flask culture technology, the method has the advantages of high thallus growth speed and high thallus yield, but the nocardia cultured by the method is easily polluted by mixed bacteria in the growth process, and the fermentation liquor is easily subjected to a large amount of foam during mechanical intense stirring, and the more the thallus is, the more the bubbles generated by metabolism are, so that the production resources are wasted, the opportunity of bacteria infection is increased, and the great harm is caused to the process production, such as reduction of production capacity, influence on the product quality, influence on the normal operation of the production and improvement on the production cost. Meanwhile, the invention discloses a culture medium for culturing Nocardia, which has high separation rate and reliable detection, but the method is mainly used for a solid culture medium and is inconvenient for industrial large-scale culture.

Disclosure of Invention

The invention aims to overcome the defect that the existing shake flask and culture medium do not reach the optimal state, and provides a culture method of nocardia, so that the bacterial cell wall components can be fully expressed and have stable content, and the addition of a defoaming agent shortens the fermentation time, improves the production efficiency and reduces the production cost.

The purpose of the invention is realized by the following technical scheme:

a Nocardia culture method comprises the following steps:

s1, preparing a nocardia culture medium: the paint consists of the following components in percentage by weight: 0.5-2% of glucose, 0.5-2% of yeast powder, 0.5-2% of tryptone, 0.3-0.5% of sodium citrate, 0.3-0.5% of sodium pyruvate, 0.02-0.06% of nalidixic acid, 0.2-0.6% of cysteine hydrochloride and the balance of deionized water, weighing the components according to the proportion to prepare a Nocardia culture medium, and heating the Nocardia culture medium in a microwave oven at the temperature of 65 ℃ for 2min until the solid is dissolved; then adding 2.5% NaOH to adjust pH to 7.0-7.2, sterilizing in a high-pressure sterilizing pot for 30min, and cooling to obtain Nocardia culture medium;

s2, preparing seed liquid: adding 2.5% NaOH into the nocardia culture medium prepared in the step S1 to adjust the pH value to 7.0-7.2, then inoculating an initial bacterial liquid of the nocardia, and then culturing in a constant-temperature shaking table at 280-350 r/min and 28-30 ℃ for 32 hours to obtain a seed liquid;

s3, fermentation culture: adding a liquid culture medium accounting for 65% of the volume of the tank into the mechanical stirring fermentation tank, inoculating the seed liquid prepared in the step S1 and accounting for 5% -10% of the volume of the culture medium into the mechanical stirring fermentation tank, and adjusting the ventilation amount to be 1: 0.8-1.01.

Preferably, the culture medium formula in the nocardia culture method comprises the following components in percentage by weight: 1 to 1.5 percent of glucose, 1 to 1.5 percent of yeast powder, 1 to 1.5 percent of tryptone, 0.4 to 0.45 percent of sodium citrate, 0.4 to 0.45 percent of sodium pyruvate, 0.04 to 0.05 percent of nalidixic acid, 0.2 to 0.6 percent of cysteine hydrochloride and the balance of deionized water.

Preferably, the culture medium formula in the nocardia culture method comprises the following components in percentage by weight: 1% of glucose, 1% of yeast powder, 2% of tryptone, 0.3% of sodium citrate, 0.3% of sodium pyruvate, 0.02% of nalidixic acid, 0.2% of cysteine hydrochloride and the balance of deionized water.

Preferably, the culture medium formula in the nocardia culture method further comprises an antifoaming agent, and the antifoaming agent is a composition of polyglycerol fatty acid ester and sucrose fatty acid ester.

Furthermore, the invention optimizes the weight percentage of the antifoaming agent in the nocardia culture medium formula.

Preferably, the defoamer in the nocardia culture medium formula accounts for 0.01-0.05% by weight.

Preferably, the defoamer in the nocardia culture medium formula is 0.04% by weight.

Still further, the invention preferably selects the weight part ratio of the polyglycerol fatty acid ester and the sucrose fatty acid ester in the defoaming agent.

Preferably, the weight part ratio of the polyglycerol fatty acid ester to the sucrose fatty acid ester in the defoaming agent is 1: 3 to 5.

Preferably, the weight part ratio of the polyglycerol fatty acid ester to the sucrose fatty acid ester in the defoaming agent is 1: 4.

preferably, the method for culturing Nocardia is used for industrial-scale Nocardia fermentation.

According to the invention, through the absorbance of the bacterial liquid obtained in the culture period of the embodiment 3 and the embodiments 7-9, the weight percentage of the defoaming agent in the culture medium formula of the nocardia in the embodiment 9 is 0.04%, the thallus grows faster and more in yield within 48-140 h, and the addition of the defoaming agent can promote the thallus growth and improve the yield of the fermented nocardia.

The cell concentration of the culture medium is measured in the culture period of the embodiment 9-11, and the fact that when the weight ratio of the polyglycerol fatty acid ester to the sucrose fatty acid ester added into the defoaming agent is 1:4 is proved, the cell growth is fast, and the yield is high.

Further, in the invention, the bacterial concentration of the bacterial liquid obtained in the culture period of the embodiment 10 and the comparative examples 1-8 is measured, and it can be seen that the dry weight of the nocardia thallus obtained in the embodiment 10 is heaviest compared with the bacterial liquid without adding sodium citrate, nalidixic acid, cysteine hydrochloride and the defoaming agent, and the dry weight of the nocardia thallus can be obviously improved by adding the sodium citrate, the nalidixic acid, the cysteine hydrochloride and the defoaming agent.

Wherein glucose and sodium citrate in the culture medium provide organic carbon sources for Nocardia, yeast powder and tryptone provide nitrogen sources for Nocardia, and cysteine hydrochloride is used as a nutrition enhancer to promote the absorption of nutrient substances; in the fermentation process of microorganisms, as the production and feeding can increase the chance of mixed bacteria pollution, nalidixic acid can inhibit the growth of gram-negative bacteria, so that the Nocardia can be fermented with high purity; sodium citrate acts as a buffer in addition to providing an organic carbon source; and the defoaming agent in the fermentation tank is used for eliminating bubbles generated by mechanical stirring and metabolism of the nocardia.

Therefore, compared with the prior art, the invention has the beneficial effects that: compared with the existing shake flask culture technology and fermentation tank culture technology, the method has the characteristics of superior culture medium, obviously shortened fermentation period and realization of industrial amplification and industrial production of enterprises; secondly, the bacterial cell wall components obtained by the method are fully expressed and have stable content; and thirdly, the method adds the defoaming agent, reduces the defoaming device, reduces the equipment investment and reduces the production cost.

Drawings

FIG. 1 is a graph showing the change in absorbance of cells of Nocardia bacteria in examples 1 to 6 and comparative examples 1 to 4.

FIG. 2 is a graph showing the change in absorbance of cells of Nocardia bacteria in examples 3 and 7 to 9.

FIG. 3 is a graph showing the change in absorbance of cells of Nocardia bacteria in examples 9 to 11.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

The nocardia used in the examples is nocardia corallina, purchased from Guangdong province microorganism strain preservation center, with the preservation number: GIM4.78

Examples 1 to 8

Examples 1-8 provide a culture method for culturing nocardia, comprising the steps of:

s1, preparing a nocardia culture medium: weighing and preparing the nocardia culture medium according to the proportion of the nocardia culture medium formula shown in the table 1 in percentage by weight, and heating the nocardia culture medium in a microwave oven at the temperature of 65 ℃ for 2min until the solid is dissolved; then adding 2.5% NaOH to adjust pH to 7.0-7.2, sterilizing in a high pressure sterilizing pot for 30min, and cooling to obtain Nocardia culture medium;

s3, fermentation culture: and (3) adding 19.5L of the Nocardia culture medium obtained in the step S1 into a 30L mechanical stirring type fermentation tank, inoculating the seed liquid prepared in the step S1 with the volume of the seed liquid being 5-10% of the volume of the culture medium into the mechanical stirring type fermentation tank, and adjusting the ventilation quantity to be 1: 0.8-1.01.

Table 1 examples 1-8 formulations

Note: "/" indicates no addition, and the blending ratio indicates the blending ratio of the polyglycerin fatty acid ester and the sucrose fatty acid ester

Comparative examples 1 to 8

Comparative examples 1 to 8 provide a culture method for culturing nocardia, comprising the steps of:

s1, preparing a nocardia culture medium: weighing and preparing the nocardia culture medium according to the proportion of the nocardia culture medium formula shown in the table 2 in percentage by weight, and heating the nocardia culture medium in a microwave oven at the temperature of 65 ℃ for 2min until the solid is dissolved; then 2.5 percent NaOH is added to adjust the pH value to 7.0-7.2; then placing into an autoclave, sterilizing at 121 deg.C for 30min, and cooling to obtain Nocardia culture medium;

TABLE 2 comparative example formulation

Test example 1 changes in cell growth concentration during Nocardia fermentation

The bacterial liquid obtained in the culture period of the examples 1 to 6 and the comparative examples 1 to 4 is taken, fully mixed by an electric tissue mixer, 300ul of the mixture is respectively absorbed into a 96-hole enzyme label plate, 3 samples are paralleled, and the bacterial concentration of the bacterial liquid is measured by the Optical Density (OD) of 600nm by using a full-waveband automatic enzyme label instrument, so that the bacterial concentration is shown in figure 1, the bacterial growth is faster within 48 to 140 hours in the example 3, the yield is more, and the fermentation period is obviously shortened compared with the comparative examples 1 to 4.

The bacterial liquid obtained in the culture period of example 3 and examples 7-9 is taken, fully mixed by an electric tissue mixer, 300ul of the mixture is respectively absorbed into a 96-hole enzyme label plate, 3 samples are paralleled, and the bacterial concentration of the bacterial liquid is measured by the Optical Density (OD) of 600nm of the bacterial liquid by using a full-wave band automatic enzyme label instrument, and the result is shown in figure 2, the bacterial growth is faster and the yield is more in the 48-140 h of example 9, which shows that the addition of the antifoaming agent can promote the bacterial growth and improve the yield of the nocardia fermentans.

The bacterial liquid obtained in the culture period of the example 9-11 is taken, fully mixed by an electric tissue mixer, 300ul of the mixture is respectively absorbed into a 96-hole enzyme label plate, 3 samples are paralleled, and the bacterial concentration of the bacterial liquid is measured by the Optical Density (OD) of 600nm by using a full-wave band automatic enzyme label instrument, and the result is shown in figure 1, the bacterial growth is faster and the yield is more in the example 10, which shows that the effect is best when the weight ratio of the polyglycerol fatty acid ester and the sucrose fatty acid ester in the antifoaming agent is 1: 4.

Taking the bacterial liquid obtained in the culture period of the example 10 and the comparative examples 5-8, fully mixing the bacterial liquid by using an electric tissue mixer, sucking 300ul of the bacterial liquid into a 96-hole enzyme label plate, sampling 3 samples in parallel, and measuring the bacterial concentration of the bacterial liquid by using a full-wave band automatic enzyme label instrument, wherein the bacterial concentration is measured by measuring the Optical Density (OD) of the bacterial liquid at 600nm, and the result is shown in Table 1.

TABLE 3 Dry weight of Nocardia cells in each group

Test example 1 Nocardia cell wall content measurement

The bacterial suspension obtained after the nocardia of examples 1-11 and comparative examples 1-8 is fermented for 4 days is added with 5% KOH in a ratio of 1:1, treated in water bath at 95 ℃ for 35min, the bacterial cell wall is broken, freeze-dried to obtain cell wall skeleton powder, and the content of the bacterial cell wall is measured.

a. Analysis of lipid compounds

Weighing cell wall skeleton powder 50mg, adding 2ml of 2.5% NaOH solution, refluxing and hydrolyzing in methanol and benzene (1: 1, v/v)20ml for 2h, neutralizing with 1mol/LHCl, extracting with diethyl ether for three times, concentrating the extractive solution, refining with anhydrous ethanol, and determining the content of lipoid compounds as shown in Table 3.

b. Analysis of polysaccharides

Weighing 50mg of cell wall skeleton powder into a test tube, and adding 1mol/L H₂SO₄5ml, sealing the tube, hydrolyzing in 100 ℃ water bath for 8h, extracting the liquid layer of the hydrolysate to obtain polysaccharide, and determining the polysaccharide content as shown in Table 4.

TABLE 4 Nocardia cell wall content determination

The results show that the content of lipid in the bacterial cell wall of the nocardia prepared in the embodiment is more than 17%, the content of polysaccharide is more than 40%, and the content is far greater than that of the bacterial cell wall obtained in the comparative examples 1-8, which shows that the content of lipid in the bacterial cell wall component obtained by the method is stable, wherein the content of lipid in the bacterial cell wall component of the nocardia obtained in the embodiment 10 is the highest; the content of polysaccharide in the bacterial cell wall component obtained by the method is stable, wherein the content of polysaccharide in the nocardia cell wall component obtained in example 10 is the highest.

c. Analysis of mucin

Weighing 10mg of cell wall skeleton powder, adding 1ml of HCl with the molar concentration of 6mol/L into a tube, hydrolyzing at 105 ℃ for 18h after closing the tube, refining hydrolysate, and analyzing by an amino acid automatic analyzer, wherein main amino acids of the hydrolysate contain alanine, glutamic acid and meso-diaminopimelic acid, amino sugar contains glucosamine and muramic acid, and secondary amino acid components contain leucine, serine, valine, glycine, phenylalanine and lysine, so that the nocardia cell wall component obtained by the method has uniform sticky peptide.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A culture method of Nocardia is characterized by comprising the following steps:

s1, preparing a nocardia culture medium: the paint consists of the following components in percentage by weight: 0.5-2% of glucose, 0.5-2% of yeast powder, 0.5-2% of tryptone, 0.3-0.5% of sodium citrate, 0.3-0.5% of sodium pyruvate, 0.02-0.06% of nalidixic acid, 0.2-0.6% of cysteine hydrochloride and the balance of deionized water, weighing the components according to the proportion to prepare a Nocardia culture medium, heating the Nocardia culture medium in a microwave oven at 65 ℃ for 2min until the solid is dissolved, then adding 2.5% of NaOH to adjust the pH value to 7.0-7.2, sterilizing the Nocardia culture medium in an autoclave for 30min, and cooling the Nocardia culture medium to obtain the Nocardia culture medium;

2. The method for culturing Nocardia bacteria according to claim 1, wherein the Nocardia bacteria culture medium of step S1 comprises the following components in percentage by weight: 1 to 1.5 percent of glucose, 1 to 1.5 percent of yeast powder, 1 to 1.5 percent of tryptone, 0.4 to 0.45 percent of sodium citrate, 0.4 to 0.45 percent of sodium pyruvate, 0.04 to 0.05 percent of nalidixic acid, 0.2 to 0.6 percent of cysteine hydrochloride and the balance of deionized water.

3. The method for culturing Nocardia bacteria according to claim 1, wherein the Nocardia bacteria culture medium of step S1 comprises the following components in percentage by weight: 1% of glucose, 1% of yeast powder, 2% of tryptone, 0.3% of sodium citrate, 0.3% of sodium pyruvate, 0.02% of nalidixic acid, 0.2% of cysteine hydrochloride and the balance of deionized water.

4. The method for culturing Nocardia bacteria according to claim 1, wherein the Nocardia bacteria culture medium of step S1 further comprises an antifoaming agent, and the antifoaming agent is a combination of a polyglycerol fatty acid ester and a sucrose fatty acid ester.

5. The method for culturing Nocardia according to claim 4, wherein the percentage by weight of the antifoaming agent in the culture is 0.01% to 0.05%.

6. The method for culturing Nocardia according to claim 4, wherein the percentage by weight of antifoaming agent in the culture is 0.04%.

7. The method for culturing nocardia according to claim 4, wherein the weight ratio of the polyglycerol fatty acid ester to the sucrose fatty acid ester in the antifoaming agent is 1: 3 to 5.

8. The method for culturing Nocardia according to claim 4, wherein the ratio of the polyglycerin fatty acid ester to the sucrose fatty acid ester in the antifoaming agent is 1:4 by weight.

9. The method for culturing Nocardia bacteria according to any of claims 1 to 8, wherein the method is used for industrial-scale Nocardia fermentation.