Method for efficiently culturing red nocardia and application thereof

Abstract

The present application belongs to the field of biological medicine and microbial culture technology, and particularly relates to a method for efficiently culturing red Nocardia and application thereof. The method for efficiently culturing red Nocardia comprises the steps of inoculating a red Nocardia fermentation strain into a fermentation medium and performing fermentation culture by using an air-lift fermenter. By optimizing the fermentation medium and using the air-lift fermentation culture mode with reduced shear force and improved mass transfer capacity to control air flow, a culture process suitable for high-yield cell mycelium of the strain is established, the synthesis capacity and cell amount of the red Nocardia mycelium cells are greatly improved, the harvested mycelium cells are mainly rod-shaped cells, which is more conducive to the crushing of the mycelium cells, the extraction and preparation steps of the cell wall skeleton are simplified, and thus a basis is provided for the large-scale preparation of N-CWS.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microbial culture technology, and in particular relates to a method for efficiently culturing Nocardia rubrum and its application. Background Technology

[0002] Nocardia rubrum cell wall cytoskeleton (N-CWS) is a high-molecular-weight polymer with a natural network structure composed of nocardic acid, arabinogalactan, and peptidoglycan. It is refined from Nocardia rubrum through fermentation, cell disruption, enzymatic treatment, and solvent extraction. Studies have shown that the Nocardia rubrum cell wall cytoskeleton can activate the body's immune function, enhance the activity of various immune cells such as macrophages, T cells, and NK cells, exhibit mitogenic activity against human lymphocytes, stimulate T lymphocyte DNA synthesis, induce the expression of cytokines such as intercellular adhesion molecule-1, IL-1, and interferon, induce the release of NO, induce the growth of LAK cells and tumor necrosis factor, significantly inhibit tumor cell growth, and significantly promote hemolysin formation. Furthermore, N-CWS is a biological response modifier, characterized by strong immunomodulatory activity and fewer side effects compared to similar biological agents. Clinically, it has been widely used in the treatment of immune diseases, tumors, in vitro inflammation, gynecological diseases, HPV infection, and herpes virus infection.

[0003] Nocardia redissus isolated and screened from Kunming soil by the Fujian Provincial Institute of Microbiology Nocardia rubra Nocardia rubra PO-8, as confirmed by research, has the following properties: Nocardia rubra Cell Wall Skeleton (N-CWS) possesses significant immunomodulatory and antitumor effects and has been clinically applied as a novel biological product in the immunotherapy of cancer, making it a practical antitumor drug. However, the yield of the original Nocardia rubrum PO-8 producing strain is low. For example, the highest yield of Nocardia rubrum in shake flasks disclosed in Chinese patent CN1094288A is only 2.5%, with a culture period of 3-5 days; similarly, the highest yield of Nocardia rubrum under optimized conditions in a 5L tank disclosed in Chinese patent CN104099271A is only 2.67%, which still cannot meet the demand for large-scale industrial production of N-CWS. Currently, many technicians have attempted to enhance the industrial utilization of the strain through strain domestication or improve fermentation conditions to enhance the biosynthetic capacity of the strain, but the results have not been ideal. Therefore, finding and solving the technical bottlenecks in the large-scale production of Nocardia rubrum to achieve efficient culture of Nocardia rubrum and optimization of downstream processes is of positive significance for the widespread development and promotion of N-CWS. Summary of the Invention

[0004] To this end, the technical problem to be solved by the present application is to provide a method for efficiently culturing red Nocardia bacterial cells, so as to solve the problem of low yield of red Nocardia bacterial cells in the prior art.

[0005] The second technical problem to be solved by the present application is to provide a preparation method of N-CWS.

[0006] To solve the above technical problems, the method for efficiently culturing red Nocardia bacteria according to the present application comprises the steps of inoculating a red Nocardia fermentation strain into a fermentation medium and performing fermentation culture in a gas-lift fermenter.

[0007] Specifically, in the method for efficiently culturing red Nocardia bacteria, the conditions of the fermentation culture step include controlling the tank pressure at 0.04±0.02 Mpa, the aeration rate at 0.1-0.5 vvm, and performing fermentation culture at 30℃±2℃ for 24-56h.

[0008] Specifically, in the method for efficiently culturing red Nocardia bacteria, the aeration rate control of the fermentation culture step includes controlling the aeration rate at 0.1-0.2 vvm in the early fermentation stage, and increasing the aeration rate to 0.3-0.5 vvm when the bacterial cell concentration reaches 8.0-9.0%.

[0009] More preferably, the conditions of the fermentation culture step are as follows: inoculating at an inoculation amount of 10%, controlling the tank pressure at 0.03 Mpa, controlling the aeration at 0.1-0.2 vvm in the early fermentation stage, and increasing the aeration to 0.3-0.4 vvm when the bacterial cell concentration reaches 8.5%.

[0010] Specifically, in the method for efficiently culturing red Nocardia bacteria, the fermentation medium comprises the following components in the following mass contents: glucose 1.0-5.0%, yeast extract powder 2.0-3.5%, peptone 0.8-1.5%, antifoaming agent 0.05-1.0%, and pH value is adjusted to 6.8-7.8.

[0011] Preferably, the antifoaming agent comprises THIX-298.

[0012] Preferably, the fermentation medium comprises the following components in the following mass contents: glucose 2.5%, yeast extract powder 2.5%, peptone 1.0%, THIX-298 0.1%, and pH value is adjusted to 7.0-7.4, and tap water is used for preparation.

[0013] Specifically, in the method for efficiently culturing red Nocardia bacteria, the method further comprises the step of inoculating the red Nocardia fermentation strain into a seed culture medium for seed liquid culture.

[0014] The seed culture medium comprises the following components with the following mass content: glucose 0.5-1.5%, yeast extract 1.0-2.0%, THIX-298 0.05-1.0%, and the pH value is adjusted to 6.8-7.8;

[0015] Preferably, the seed culture medium comprises the following components with the following mass content: glucose 1.0%, yeast extract 1.5%, THIX-298 0.1%, and the pH value is adjusted to 7.0-7.4, and tap water is used for preparation.

[0016] Preferably, the seed liquid is inoculated into the fermentation medium at an inoculation amount of 2-20% for fermentation culture.

[0017] Specifically, the method for efficiently culturing the red Nocardia sp. further comprises the step of inoculating the red Nocardia sp. fermentation strain into a nutrient agar solid slant medium for activation culture.

[0018] The seed liquid culture step comprises the following conditions: under sterile conditions, 1 ring of spores is scraped from the bacterial layer on a fresh slant with an inoculation loop, inoculated into a shake flask containing the seed culture medium, and cultured at 27-29°C with a controlled rotation speed of 150-200 r / min for 28-40 h to obtain a shake flask seed culture liquid.

[0019] Preferably, the shake flask seed liquid culture step comprises the following conditions: under sterile conditions, 1 ring of spores is scraped from the bacterial layer on a fresh slant with an inoculation loop, inoculated into the seed culture medium, and cultured at 28°C with a rotation speed of 180 r / min for 36 h to obtain a shake flask seed culture liquid.

[0020] The seed tank seed liquid culture step comprises the following conditions: the cultured shake flask seed culture liquid is inoculated into a seed tank containing the seed culture medium at an inoculation amount of 0.2-1.0%, the temperature is controlled at 27-29°C, the tank pressure is controlled at 0.02-0.04 Mpa, the aeration amount is controlled at 0.8-1vvm, the rotation speed is controlled at 150-200 r / min, and the fermentation culture is performed for 24-36 h to obtain the required seed tank seed liquid.

[0021] Preferably, the seed tank culture step comprises the following conditions: the cultured shake flask seed liquid is inoculated into the seed tank containing the seed culture medium at an inoculation amount of 0.5%, the culture temperature is 28°C, the tank pressure is 0.03 Mpa, the aeration amount is 1vvm, the rotation speed is controlled at 180 r / min during the culture period, and the culture is performed for 30 h until the seed matures.

[0022] Specifically, the method for efficiently culturing the red Nocardia sp. further comprises the step of inoculating the red Nocardia sp. fermentation strain into a nutrient agar solid slant medium for activation culture.

[0023] The solid slant culture medium comprises the following components with the following mass content: glucose 0.05-0.15%, proteose peptone 0.8-1.2%, beef extract 0.2-0.4%, NaCl 0.3-0.7%, agar 1.8-2.0%, pH value is adjusted to 7.0-7.4, and tap water is used for preparation.

[0024] Preferably, the solid slant culture medium comprises the following components with the following mass content: glucose 0.1%, proteose peptone 1.0%, beef extract 0.3%, NaCl 0.5%, agar 2.0%, pH value is adjusted to 7.0-7.4, and tap water is used for preparation.

[0025] Specifically, the method for efficiently culturing the red Nocardia comprises a red Nocardia fermentation strain, and the red Nocardia fermentation strain comprises red Nocardia PO-8, which is classified as red Nocardia rubra. Nocardia rubra The red Nocardia PO-8 has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 0187.

[0026] The application further discloses a method for preparing N-CWS, which comprises the steps of culturing red Nocardia according to the method, collecting the red Nocardia cells, and preparing N-CWS.

[0027] Specifically, the method for preparing N-CWS comprises the following steps:

[0028] (1) culturing and collecting the red Nocardia cell according to the method;

[0029] (2) mixing the cell with water, performing ultrasonic crushing and stepwise centrifugation to obtain red card cell wall fragments; preferably, the stepwise centrifugation comprises the steps of collecting supernatant by low-speed centrifugation and collecting red card cell wall fragments by high-speed centrifugation of the supernatant;

[0030] (3) adding phosphate buffer, trypsin and chymotrypsin into the red card cell wall, mixing and performing enzymolysis, collecting the enzymolysis product and performing centrifugation to obtain red card cell wall precipitate;

[0031] (4) adding Tris-HCl buffer containing pronase into the red card cell wall precipitate to perform enzymolysis, collecting the enzymolysis product and performing centrifugation to obtain red card wall skeleton crude product;

[0032] (5) performing purification treatment on the red card wall skeleton crude product to obtain white-like N-CWS.

[0033] Specifically, the method for preparing N-CWS comprises the following steps:

[0034] Pure water is added to the bacterial cells in a ratio of 1:2-6 of cells to water, and after stirring and dissolving, ultrasonic crushing is performed, and the cell crushing condition is observed under a microscope. The supernatant is collected by centrifugation, and the red cell wall fragments are harvested;

[0035] In the red cell wall, 0.07M pH7.6-7.8 phosphate buffer is added in a ratio of 1:3-8, and trypsin and chymotrypsin are added. The temperature is controlled at 25℃, and the enzyme hydrolysis is stirred for 24-36 hours. After high-speed centrifugation and discarding the supernatant, the collected red cell wall precipitate is washed once with P.B.S and once with 0.1M pH7.2-7.5 Tris-HCl solution. The red cell wall precipitate is harvested by high-speed centrifugation;

[0036] In the red cell wall precipitate, 20-40mg of streptomyces protease (810U) is added to the Tris-HCl buffer in a ratio of 1:3-8, and the temperature is controlled at 37℃. The enzyme hydrolysis is stirred for 24-36 hours. After high-speed centrifugation and discarding the supernatant, the red cell wall skeleton crude product is washed with Tris-HCl solution, N.S and pure water, and harvested by high-speed centrifugation;

[0037] In the red cell wall skeleton crude product, ethanol, chloroform and petroleum ether are added in a ratio of 1:3-7, and oscillation extraction is performed. After centrifugation and washing with ethanol, the product is dried under reduced pressure at 60℃, and ground to obtain a white N-CWS.

[0038] The method for efficiently culturing red Nocardia bacteria cells provided by the present application optimizes the fermentation medium, especially selects a suitable amount of defoaming agent, and controls the air flow by using a gas-lift fermentation culture method that reduces shear force and improves mass transfer capacity, thereby establishing a culture process suitable for high-yield cell bacteria of the strain, promoting rapid growth of the bacteria, and greatly improving the synthesis capacity and cell amount of the red Nocardia bacteria cells. In particular, the method optimizes the culture fermentation method, improves the yield of microbial bacteria cells, and the harvested bacteria cells are mainly rod-shaped, which is more conducive to the crushing of the bacteria cells, simplifies the extraction and preparation steps of the cell wall skeleton, improves the preparation yield of the cell wall skeleton, and thus provides a basis for the large-scale preparation of N-CWS.

[0039] The method for efficiently culturing red Nocardia bacteria cells provided by the present application optimizes the fermentation medium, especially selects a suitable amount of defoaming agent, and controls the air flow by using a gas-lift fermentation culture method that reduces shear force and improves mass transfer capacity, thereby establishing a culture process suitable for high-yield cell bacteria of the strain, promoting rapid growth of the bacteria, and greatly improving the synthesis capacity and cell amount of the red Nocardia bacteria cells. In particular, the method optimizes the culture fermentation method, improves the yield of microbial bacteria cells, and the harvested bacteria cells are mainly rod-shaped, which is more conducive to the crushing of the bacteria cells, simplifies the extraction and preparation steps of the cell wall skeleton, improves the preparation yield of the cell wall skeleton, and thus provides a basis for the large-scale preparation of N-CWS. Nocardia rubra PO-8 fermentation process, it is surprisingly found that when the morphology of the cultured bacteria is rod-shaped, it is especially conducive to the crushing of the bacteria cells in the downstream process, simplifies the extraction and preparation steps of the cell wall skeleton, improves the yield of the cell wall skeleton, has the advantages of simple operation, low cost and easy large-scale industrial production, and has important significance for the industrialization research of N-CWS preparation.

[0040] The method for efficiently culturing red Nocardia bacteria cells provided by the present application optimizes the fermentation medium, especially selects a suitable amount of defoaming agent, and controls the air flow by using a gas-lift fermentation culture method that reduces shear force and improves mass transfer capacity, thereby establishing a culture process suitable for high-yield cell bacteria of the strain, promoting rapid growth of the bacteria, and greatly improving the synthesis capacity and cell amount of the red Nocardia bacteria cells. In particular, the method optimizes the culture fermentation method, improves the yield of microbial bacteria cells, and the harvested bacteria cells are mainly rod-shaped, which is more conducive to the crushing of the bacteria cells, simplifies the extraction and preparation steps of the cell wall skeleton, improves the preparation yield of the cell wall skeleton, and thus provides a basis for the large-scale preparation of N-CWS.

[0041] Nocardia rubra

[0042] During the PO-8 fermentation process, through optimization of the fermentation medium and fermentation method, the yield of synthesized cell cells reached 114.9 g / L in a 60L airlift fermenter fermentation experiment, which greatly improved the cell synthesis capacity and thus provided a foundation for the large-scale preparation of Nocardia rubrum cell cells and its extracts. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0044] Figure 1 The strain in Example 3 of this invention Nocardia rubra Morphological images of different batches of PO-8 cells fermented in a 60L airlift fermenter;

[0045] Figure 2 The strain in Comparative Example 3 of this invention Nocardia rubra Morphological images of different batches of PO-8 cells fermented in a 50L stirred fermenter. Detailed Implementation

[0046] To better understand the present invention, the present invention will be further illustrated by several specific embodiments below, but these do not constitute any limitation on the scope of protection of the present invention.

[0047] In the following embodiments of the present invention, the strain involved in the fermentation culture of Nocardia rubrum is Nocardia rubrum. Nocardia rubra PO-8 is deposited at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 0187 and deposit date of January 11, 1993.

[0048] Example 1: Selection of antifoaming agent for Nocardia rubrum culture medium

[0049] The preserved strains Nocardia rubra PO-8 was cultured on nutrient agar solid medium at 28°C for 7 days to obtain the strain. Nocardia rubra Fresh slope of PO-8.

[0050] The strain was inoculated using an inoculation loop under aseptic conditions. Nocardia rubra One loopful of spores was scraped from the bacterial layer on a fresh PO-8 slant and inoculated into a shake flask seed culture medium. The culture was then incubated at 28°C and 200 rpm for 36 h to obtain the shake flask seed culture solution.

[0051] The cultivated shake flask seed liquid was inoculated into the shake flask fermentation medium containing different concentrations of THIX-298 at an inoculation amount of 5%, the influence of the defoaming agent on the growth of the red Nocardia bacteria was investigated, the bottle was placed at 28°C and 200 r / min for 72 h, the bacterial cells were collected and weighed, and the results are shown in Table 1 below.

[0052] The seed culture medium preparation method is as follows: 10 g of glucose, 15 g of yeast extract powder, 1 g of THIX-298, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0053] The fermentation medium preparation method is as follows: 25 g of glucose, 25 g of yeast extract powder, 10 g of peptone, 0-15 g of THIX-298, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0054] Table 1 Influence of THIX-298 on bacterial yield

[0055]

[0056] The results in Table 1 show that the selected defoaming agent THIX-298 has a positive dose-effect relationship with the strain Nocardia rubra PO-8 within a certain concentration range, and can promote bacterial growth and increase bacterial cell yield while defoaming. Therefore, 0.05-1.0% THIX-298 is selected as the appropriate dosage, which provides a basis for subsequent pilot fermentation.

[0057] Example 2 Shake flask culture of red Nocardia bacteria

[0058] The preserved strain Nocardia rubra PO-8 was cultured on a nutrient agar solid culture medium at 28°C for 7 days to obtain a fresh slant of the strain Nocardia rubra PO-8.

[0059] Under sterile conditions, 1 ring of spores of the bacterial layer on the fresh slant of the strain Nocardia rubra PO-8 was scraped off with an inoculation loop and inoculated into a shake flask seed culture medium, which was cultured at 28°C and 200 r / min for 36 h to obtain a shake flask seed liquid.

[0060] The cultivated shake flask seed liquid was inoculated into the shake flask fermentation medium containing different concentrations of THIX-298 at an inoculation amount of 5%, the influence of the defoaming agent on the growth of the red Nocardia bacteria was investigated, the bottle was placed at 28°C and 200 r / min for 72 h, the bacterial cells were collected and weighed, and the results are shown in Table 1 below.

[0061] The seed culture medium preparation method is as follows: 10 g of glucose, 15 g of yeast extract powder, 1 g of THIX-298, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0062] The preparation method of the fermentation medium is as follows: 25 g of glucose, 25 g of yeast extract powder, 10 g of peptone, 1 g of THIX-298, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0063] Comparative Example 1

[0064] The culture method of the fermentation red Nocardia bacterium cell in the present comparative example is the same as that in Example 1, and the only difference is that the shake flask fermentation medium does not contain the THIX-298.

[0065] The preparation method of the fermentation medium is as follows: 25 g of glucose, 25 g of yeast extract powder, 10 g of peptone, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0066] Comparative Example 2

[0067] The culture method of the fermentation red Nocardia bacterium cell in the present comparative example is the same as that in Example 1, and the only difference is that the shake flask fermentation medium uses the fermentation medium in Chinese patent CN104099271A.

[0068] The preparation method of the fermentation medium is as follows: 10 g of glucose, 10 g of yeast powder, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0069] After the shake flask fermentation in the above Example 2 and Comparative Examples 1-2, the cell amount is determined, and the morphology of the cell is observed, and the results are shown in Table 2.

[0070] Table 2 Comparison of cell yield and morphology of shake flask fermentation in Example and Comparative Examples

[0071]

[0072] As shown in Table 2, the detection results show that the average yield of the red Nocardia bacterium cell in Example 2 is 106.5 g / L, and the main cell morphology is short rod shape; the average yield of the red Nocardia bacterium cell in Comparative Example 1 is only 86.2 g / L, and the main cell morphology is spherical shape, and the average yield of the red Nocardia bacterium cell in Comparative Example 2 is 27.3 g / L, and the main cell morphology is spherical shape.

[0073] It can be seen that, by optimizing the fermentation medium, the synthesis capacity of the red Nocardia bacterium cell can be significantly improved, the yield is increased by 290.1% compared with the scheme in Comparative Example 2, and the main morphology of the cultured cell is rod shape, which is more beneficial to the separation of the red Nocardia bacterium extract and the preparation of N-CWS in the later stage.

[0074] Example 3 Red Nocardia bacterium culture in 60 L air-lift fermentor

[0075] The preserved strain Nocardia rubra PO-8 was cultured on a nutrient agar solid culture medium at 28°C for 7 days to obtain a strain Nocardia rubra A fresh slope of PO-8.

[0076] The strain Nocardia rubra PO-8 was scraped off from the bacterial layer on the fresh slope with a loop under sterile conditions and inoculated into a seed culture medium in a shake flask, and cultured at 28°C and 200 r / min for 36 hours to obtain a seed culture solution in the shake flask.

[0077] The seed culture medium and the preparation method are as follows: 10 g of glucose, 15 g of yeast extract powder, 1 g of THIX-298, and 1 L of tap water, and the pH value is adjusted to 7.2.

[0078] The seed culture solution cultured in the shake flask was inoculated into a 10 L seed tank containing the seed culture medium at an inoculation amount of 0.8%, the seed tank culture medium was filled to 7 L, the temperature was controlled at 28°C, the tank pressure was controlled at 0.02-0.04 Mpa, the aeration was 1 vvm, the rotating speed was controlled at 200 r / min, and the culture was carried out for 32 hours to obtain the required seed solution.

[0079] The seed culture medium and the preparation method are as follows: 70 g of glucose, 105 g of yeast extract powder, 7 g of THIX-298, and tap water, and the pH value is adjusted to 7.2, and the culture medium is filled to 7 L after sterilization.

[0080] The seed solution cultured above was inoculated into a 60 L air-lift type fermentation tank at an inoculation amount of 5%, the fermentation tank culture medium was filled to 45 L, the fermentation temperature was 28°C, the tank pressure was 0.03 Mpa, the aeration amount was controlled at 0.2 vvm in the early stage of fermentation, and when the cell content reached 8.5%, the aeration amount was adjusted to 0.4 vvm, the fermentation was stopped at 56 hours, the cell was collected after the tank was discharged, and the cell morphology was observed.

[0081] The fermentation culture medium and the preparation method are as follows: 1125 g of glucose, 1125 g of yeast extract powder, 450 g of peptone, 45 g of THIX-298, and tap water, and the pH value is adjusted to 7.2, and the culture medium is filled to 45 L after sterilization.

[0082] Comparative Example 3

[0083] The preserved red Nocardia Nocardia rubra PO-8 was cultured on a nutrient agar solid culture medium at 28°C for 7 days to obtain a red Nocardia Nocardia rubra A fresh slope of PO-8.

[0084] The red Nocardia Nocardia rubraThe spores scraped from the bacterial layer on the fresh slope of PO-8 were inoculated into a seed culture medium in a shake flask, and cultured at 28°C and 200 r / min for 36 h to obtain a seed culture solution in the shake flask.

[0085] The seed culture medium had the following components and preparation method: 10 g of glucose, 15 g of yeast extract powder, 1 g of THIX-298, and 1 L of tap water, and the pH value was adjusted to 7.2.

[0086] The seed culture solution cultured in the shake flask was inoculated into a 10 L seed tank containing the seed culture medium at an inoculation amount of 0.8%, the seed tank had a medium loading capacity of 7 L, the temperature was controlled at 28°C, the tank pressure was 0.02-0.04 Mpa, the aeration was 1 vvm, the rotating speed was controlled at 200 r / min, and the culture was performed for 32 h to obtain the required seed solution.

[0087] The seed culture medium had the following components and preparation method: 70 g of glucose, 105 g of yeast extract powder, 7 g of THIX-298, and tap water, and the pH value was adjusted to 7.2, and the medium had a capacity of 7 L after sterilization.

[0088] The seed solution cultured above was inoculated into a 50 L (with stirring blades) fermentation tank at an inoculation amount of 5%, the fermentation tank had a medium loading capacity of 35 L, the fermentation temperature was 28°C, the tank pressure was 0.03 Mpa, the aeration amount was controlled at 0.8-1.5 vvm, the rotating speed was 150-200 r / min, the fermentation was terminated at 72 h, the bacterial cells were collected after the tank was opened, and the morphology of the bacterial cells was observed.

[0089] The fermentation medium had the following components and preparation method: 875 g of glucose, 875 g of yeast extract powder, 350 g of peptone, 35 g of THIX-298, and tap water, and the pH value was adjusted to 7.2, and the medium had a capacity of 35 L after sterilization.

[0090] Comparative Example 4

[0091] The preserved red Nocardia rubra PO-8 was cultured on a nutrient agar solid medium at 28°C for 7 days to obtain red Nocardia rubra PO-8. Nocardia rubra The preserved red Nocardia rubra PO-8 was cultured on a nutrient agar solid medium at 28°C for 7 days to obtain red Nocardia rubra PO-8. Nocardia rubra The fresh slope of PO-8.

[0092] The red Nocardia rubra PO-8 was inoculated into a seed culture medium in a shake flask under sterile conditions by using an inoculation loop, and cultured at 28°C and 200 r / min for 36 h to obtain a seed culture solution in the shake flask. Nocardia rubra The spores scraped from the bacterial layer on the fresh slope of PO-8 were inoculated into a seed culture medium in a shake flask, and cultured at 28°C and 200 r / min for 36 h to obtain a seed culture solution in the shake flask.

[0093] The seed culture medium had the following components and preparation method: 10 g of glucose, 15 g of yeast extract powder, 1 g of THIX-298, and 1 L of tap water, and the pH value was adjusted to 7.2.

[0094] The prepared shake-flask seed culture solution was inoculated at a rate of 0.8% into a 10L seed tank containing seed culture medium (7L). The temperature was controlled at 28℃, the tank pressure at 0.02-0.04 MPa, the aeration rate at 1 vvm, and the rotation speed at 200 r / min. The culture was carried out for 32 hours to obtain the desired seed solution.

[0095] The above seed culture medium components and preparation method are as follows: 70g glucose, 105g yeast extract, 7g THIX-298, add tap water, adjust the pH to 7.2, and the volume of culture medium after sterilization is 7L.

[0096] The above-cultured seed culture was inoculated into a 100L (with stirring blade) fermenter at an inoculation rate of 5%. The fermenter was filled with 70L of culture medium, fermented at 28℃, with a pressure of 0.03Mpa. The fermentation aeration rate was controlled at 0.8-1.5vvm, and the rotation speed was 150-200r / min. Fermentation was terminated after 72h. The fermentation was then discharged from the tank, the bacterial cells were collected, and the morphology of the cells was observed.

[0097] The above fermentation medium composition and preparation method are as follows: 1750g glucose, 1750g yeast extract, 700g peptone, 70g THIX-298, add tap water, adjust the pH to 7.2, and the volume of the culture medium after sterilization is 70L.

[0098] Comparative Example 5

[0099] Preserved Nocardia redis Nocardia rubra Nocardia rubra was obtained by culturing PO-8 on nutrient agar solid medium at 28°C for 7 days. Nocardia rubra Fresh slope of PO-8.

[0100] Nocardia rubrum was inoculated using an inoculation loop under aseptic conditions. Figure 1-2 One loopful of spores was scraped from the bacterial layer on a fresh PO-8 slant and inoculated into a shake flask seed culture medium. The culture was then incubated at 28°C and 200 rpm for 36 h to obtain the shake flask seed culture solution.

[0101] The above seed culture medium composition and preparation method are as follows: 10g glucose, 15g yeast extract, 1g THIX-298, 1L tap water, and pH adjusted to 7.2.

[0102] The prepared shake-flask seed culture solution was inoculated at a rate of 0.8% into a 10L seed tank containing seed culture medium (7L). The temperature was controlled at 28℃, the tank pressure at 0.02-0.04 MPa, the aeration rate at 1 vvm, and the rotation speed at 200 r / min. The culture was carried out for 32 hours to obtain the desired seed solution.

[0103] The above seed culture medium composition and preparation method: 70g glucose, 105g yeast extract, 7g THIX-298, add tap water, adjust the pH to 7.2, and the volume of culture medium after sterilization is 7L.

[0104] The above-cultured seed culture was inoculated into a 100L (with stirring blade) fermenter at an inoculation rate of 5%. The fermenter was filled with 70L of culture medium, fermented at 28℃, with a pressure of 0.03Mpa. The fermentation aeration rate was controlled at 0.8-1.5vvm, and the rotation speed was 150-200r / min. Fermentation was terminated after 72h. The fermentation was then discharged from the tank, the bacterial cells were collected, and the morphology of the cells was observed.

[0105] The fermentation medium described above uses the fermentation medium from Chinese patent CN104099271A, whose components and preparation method are as follows: 700g glucose, 700g yeast powder, add tap water, adjust the pH value to 7.2, and the volume of the culture medium after sterilization is 70L.

[0106] After fermentation in Examples 3 and 3-5, the cell count and morphology of the cultured cells were measured. The results are shown in Table 3. The morphology of the cultured cells in Examples 3 and 3 are shown in the attached figures. Figure 1-2 As shown, (a) and (b) represent the morphological results of two randomly selected batches of bacteria.

[0107] Table 3 Comparison of cell yield and morphology in fermentation tanks between the examples and control examples

[0108]

[0109] As shown in Table 3 and appendix Nocardia rubra The results showed that the average yield of Nocardia rubes somatic cells in the three batches of fermentation culture in Example 3 was 114.9 g / L, with a calculated yield of 2.05 g / L. -1 .h -1 Its main cell morphology is long rods; while in Comparative Example 3, the average yield of Nocardia rubes somatic cells in three batches of fermentation culture was 80.9 g / L, with a calculated yield of 1.12 g / L. -1 .h -1 The bacterial cell morphology is spherical or short rod-shaped; in Comparative Example 4, the average yield of Nocardia rubes cell culture from three batches of fermentation culture was 80.7 g / L, and the calculated yield was 1.12 g / L. -1 .h -1 The main morphology of the bacteria is spherical or short rod-shaped; the average yield of Nocardia rubes cells in the three batches of fermentation culture in Comparative Example 5 was 28.2 g / L, and the calculated yield was 0.39 g / L. -1 .h -1 .

[0110] The above results show that the present application cultivates the red Nocardia strain by optimizing the fermentation medium and controlling the air flow of the air-lift fermenter, reduces the shearing effect in the fermentation process, improves the oxygen transfer capacity, promotes the rapid growth of the strain, and the yield is 3 times (307%) higher and the yield is 4 times (424%) higher than those of the comparative example 5. The main morphology of the cultivated strain cells is long rod, which is more beneficial to the cell breaking in the later stage, and can be used for efficiently separating and preparing N-CWS and other red Nocardia extracts.

[0111] Example 4 Preparation of N-CWS

[0112] Under sterile conditions, the strain was inoculated with an inoculation ring Nocardia rubra PO-8 The spores of 1 ring of the bacterial layer on the fresh slope were inoculated into a seed culture medium in a shake flask, and cultured at 28 DEG C and 200 r / min for 36 h to obtain a seed culture solution in a shake flask.

[0113] The above seed culture medium components and preparation method: glucose 10 g, yeast extract powder 15 g, THIX-298 1 g, tap water 1 L, and the pH value was adjusted to 7.2.

[0114] The cultured seed culture solution in a shake flask was inoculated into a 10 L seed tank containing the seed culture medium at an inoculation amount of 0.8%, the seed tank culture medium was filled with 7 L, the temperature was controlled at 28 DEG C, the tank pressure was controlled at 0.02-0.04 Mpa, the aeration was 1 vvm, the rotation speed was controlled at 200 r / min, and the seed solution was obtained after 32 h of culture.

[0115] The above seed culture medium components and preparation method: glucose 70 g, yeast extract powder 105 g, THIX-298 7 g, tap water, and the pH value was adjusted to 7.2, and the culture medium was 7 L after sterilization.

[0116] The above cultured seed solution was inoculated into a 60 L air-lift fermenter at an inoculation amount of 5%, the fermentation tank culture medium was filled with 45 L, the fermentation temperature was 28 DEG C, the tank pressure was 0.03 Mpa, the aeration amount was controlled at 0.2 vvm in the early stage of fermentation, and the aeration amount was adjusted to 0.4 vvm when the strain cells reached 8.5% with the growth of the strain cells, the fermentation was stopped after 56 h of fermentation, and the strain cells were collected.

[0117] The above fermentation medium components and preparation method: glucose 1125 g, yeast extract powder 1125 g, peptone 450 g, THIX-298 45 g, tap water, and the pH value was adjusted to 7.2, and the culture medium was 45 L after sterilization.

[0118] Pure water was added to the bacterial cells at a ratio of 1 :4 of cells to water, and after stirring and dissolving, ultrasonic crushing was performed (VCX1600 American sonics ultrasonic crusher, 30-60 min). The cell crushing was observed under a microscope. Low-speed centrifugation (Allegra X-15R American BECKMAN centrifuge, 800*g, 15-20 min) was performed to collect the supernatant. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min) was performed to harvest the red cell wall fragments.

[0119] In the red cell wall, 0.07M pH7.6-7.8 phosphate buffer (P.B.S) was added at a ratio of 1 :5, and 60mg of trypsin (chymotrypsin, not less than 800u / mg) and chymotrypsin (1:250, i.e. 250u / mg) were added. The temperature was controlled at 25°C, and the enzyme was stirred for 30 hours. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min) was performed, and the red cell wall precipitate was washed once with P.B.S and once with 0.1M pH7.2-7.5 Tris-HCl solution. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min) was performed to harvest the red cell wall precipitate.

[0120] In the red cell wall precipitate, 400ml of Tris-HCl buffer containing 30mg of pronase (810U) was added at a ratio of 1 :5, and the temperature was controlled at 37°C. The enzyme was stirred for 30 hours, and high-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min) was performed. The supernatant was discarded, and the red cell wall skeleton was washed with Tris-HCl solution, N.S and pure water, respectively. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min) was performed to harvest the red cell wall skeleton.

[0121] In the red cell wall skeleton, ethanol, chloroform and petroleum ether were added at a ratio of 1 :5, respectively, and oscillation extraction was performed. Centrifugation (Allegra X-15R American BECKMAN centrifuge, 15°C, 3000 rpm*10-15 min) was performed. After washing with ethanol, the product was dried at 60°C under reduced pressure, and was ground to obtain a white N-CWS.

[0122] Comparative Example 6

[0123] Under sterile conditions, the red Nocardia bacteria were inoculated with a inoculation loop ​PO-8 The spores of a ring of the mycelium layer on the fresh slope were inoculated into a seed culture medium in a shake flask, and the seed culture solution was obtained by culturing at 28°C and 200 r / min for 36 h.

[0124] The seed culture medium and the preparation method were as follows: 10 g of glucose, 15 g of yeast extract powder, 8 g of THIX-298, and 1 L of tap water, and the pH value was adjusted to 7.2.

[0125] The seed culture solution was inoculated into a 10 L seed tank containing the seed culture medium at an inoculation amount of 0.8%, the seed tank culture medium was filled to 7 L, the temperature was controlled at 28°C, the tank pressure was 0.02-0.04 Mpa, the aeration was 1 vvm, the rotating speed was controlled at 200 r / min, and the seed solution was obtained by culturing for 32 h.

[0126] The seed culture medium and the preparation method were as follows: 70 g of glucose, 105 g of yeast extract powder, 7 g of THIX-298, tap water was added, the pH value was adjusted to 7.2, and the culture medium was filled to 7 L after sterilization.

[0127] The seed solution was inoculated into a 50 L (with stirring blades) fermentation tank at an inoculation amount of 5%, the fermentation tank culture medium was filled to 35 L, the fermentation temperature was 28°C, the tank pressure was 0.03 Mpa, the aeration amount was controlled at 0.8-1.5 vvm, the rotating speed was 150-200 r / min, the fermentation was stopped after 72 h, and the bacterial cells were collected.

[0128] The seed culture medium and the preparation method were as follows: 875 g of glucose, 875 g of yeast extract powder, 350 g of peptone, 35 g of THIX-298, tap water was added, the pH value was adjusted to 7.2, and the culture medium was filled to 35 L after sterilization.

[0129] Pure water was added to the bacterial cells at a ratio of 1:4 of cells to water, and after stirring and dissolving, ultrasonic crushing was performed (VCX1600 American sonics ultrasonic crusher, 30-60 min), and the cell crushing was observed by microscope tracking; the supernatant was collected by low-speed centrifugation (Allegra X-15R American BECKMAN centrifuge, 800*g, 15-20 min), and the red cell wall fragments were harvested by high-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000 rpm*15-20 min).

[0130] In the red card cell wall, 0.07M pH7.6-7.8 phosphate buffer (P.B.S) was added at a ratio of 1:5, 60mg of trypsin (chymotrypsin, not less than 800u / mg) and chymotrypsin (1:250, i.e. 250u / mg) were added, the temperature was controlled at 25°C, and the enzyme hydrolysis was stirred for 30 hours. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000rpm*15-20min) was performed, the supernatant was discarded, and the collected red card cell wall precipitate was washed once with P.B.S and once with 0.1M pH7.2-7.5 Tris-HCl solution. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000rpm*15-20min) was performed, and the red card cell wall precipitate was harvested.

[0131] In the red card cell wall precipitate, 400ml of Tris-HCl buffer containing 30mg of pronase (810U) was added at a ratio of 1:5, the temperature was controlled at 37°C, and the enzyme hydrolysis was stirred for 24-36 hours. High-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000rpm*15-20min) was performed, and the supernatant was discarded. Tris-HCl solution, N.S and pure water were used for washing, high-speed centrifugation (Avanti J-26XPI American BECKMAN centrifuge, 16000rpm*15-20min) was performed, and the red card wall skeleton crude product was harvested.

[0132] In the red card wall skeleton crude product, ethanol, chloroform and petroleum ether were added at a ratio of 1:5, respectively, and oscillation extraction was performed. Centrifugation (Allegra X-15R American BECKMAN centrifuge, 15°C, 3000rpm*10-15min) was performed, and the ethanol was washed at 60°C under reduced pressure. Grinding obtained the white N-CWS.

[0133] The cell bodies of Example 4 and Comparative Example 6 were collected in a tank, the cell disruption and cell wall fragments were collected, and the red card wall skeleton crude product was obtained after the cell wall was hydrolyzed and the solvent was extracted. After drying and grinding, the white N-CWS was obtained, and the test results are shown in Table 4.

[0134] Table 4 Comparison of cell morphology and disruption rate and N-CWS yield of examples and control examples

[0135]

[0136] *Yield is the amount of N-CWS prepared from 100g of wet cells

[0137] As shown in Table 4, the average breakage rate of the three batches of fermented red Nocardia bacterium cell of Example 4 is 90.7%, and 9.21g of N-CWS can be prepared from 100g of wet red Nocardia bacterium cell on average, while the average breakage rate of the red Nocardia bacterium cell of Comparative Example 6 is less than 38.4%, and only 3.63g of N-CWS can be prepared from 100g of wet red Nocardia bacterium cell on average.

[0138] In summary, the method of the present application can efficiently produce red Nocardia bacterium cell by optimizing the fermentation medium and controlling the air flow of the air-lift fermenter, the cell growth is fast, the culture period is short, the cell yield is greatly improved, the long rod shape of the cell is more conducive to cell breakage in the later stage, the N-CWS yield prepared from the cell is greatly improved, which is conducive to the large-scale preparation of N-CWS, lays a foundation for improving the market competitiveness of N-CWS.

[0139] Obviously, the above examples are only examples for clearly illustrating, but not limit the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for efficiently culturing Nocardia rubra, characterized by, The process includes inoculating a Nocardia rubrum fermentation strain into a fermentation medium and then using an airlift fermenter for fermentation culture. The fermentation medium comprises the following components in the indicated mass percentages: glucose 1.0-5.0%, yeast extract 2.0-3.5%, peptone 0.8-1.5%, and defoamer 0.05-1.0%, with the pH adjusted to 6.8-7.

8.

2. The method for efficiently culturing red Nocardia according to claim 1, wherein The conditions for the fermentation culture step include: controlling the tank pressure at 0.04±0.02 MPa, the aeration rate at 0.1-0.5 vvm, and fermenting at 30℃±2℃ for 24-56 h.

3. The method for efficiently culturing red Nocardia according to claim 2, characterized by, The aeration control in the fermentation culture step includes: controlling the aeration rate at 0.1-0.2 vvm in the early stage of fermentation, and increasing the aeration rate to 0.3-0.5 vvm when the cell concentration reaches 8.0-9.0%.

4. The method for efficiently culturing red Nocardia according to claim 1, wherein The defoamer includes THIX-298.

5. The method for efficiently culturing red Nocardia according to claim 1, wherein The method further includes the step of inoculating the Nocardia rubra fermentation strain into a seed culture medium for seed liquid culture. The seed culture medium comprises the following components in the indicated mass percentages: glucose 0.5-1.5%, yeast extract 1.0-2.0%, and THIX-298 0.05-1.0%, with the pH adjusted to 6.8-7.8; The seed culture is inoculated into the fermentation medium at an inoculation rate of 2-20% for fermentation culture.

6. The method for efficiently culturing red Nocardia according to claim 5, wherein The seed culture step includes shake flask seed culture and seed tank seed culture steps; The steps of the shake flask seed culture include: under aseptic conditions, scraping one loopful of spores from the bacterial layer on a fresh slant using an inoculation loop, inoculating it into a shake flask containing the seed culture medium, and culturing it at 27-29℃ and a rotation speed of 150-200 r / min for 28-40 h to obtain the shake flask seed culture solution; The conditions for the seed culture in the seed tank include: inoculating the cultured shake flask seed culture solution into a seed tank containing the seed culture medium at an inoculation rate of 0.2-1.0%; controlling the temperature at 27-29℃, the tank pressure at 0.02-0.04 MPa, the aeration rate at 0.8-1 vvm, and the rotation speed at 150-200 r / min; and fermenting for 24-36 hours to obtain the desired seed culture in the seed tank.

7. The method for efficiently culturing red Nocardia according to claim 1, wherein The method further includes the step of inoculating the Nocardia rubra fermentation strain onto a nutrient agar solid slant medium for activation culture; The solid slant culture medium comprises the following components in the indicated mass percentages: glucose 0.05-0.15%, peptone 0.8-1.2%, beef extract 0.2-0.4%, NaCl 0.3-0.7%, and agar 1.8-2.0%, with pH adjusted to 7.0-7.

4.

8. The method for efficient cultivation of red Nocardia according to any one of claims 1 to 7, characterized in that, The red Nocardia fermentation strain includes red Nocardia PO-8, which is classified as red Nocardia Nocardia rubra and has been preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 0187.

9. A method of preparing N-CWS, characterized by, The method includes the steps of culturing Nocardia rubra cells according to any one of claims 1-8, and collecting the Nocardia rubra cells and preparing N-CWS.

10. The method of claim 9, wherein the N-CWS is prepared by, Includes the following steps: (1) The Nocardia rubrum cells are cultured and collected according to the method of any one of claims 1-8; (2) The red Nocardia cell is mixed with water, and is subjected to ultrasonic wave breaking and stepwise centrifugation to obtain cell wall fragments of the red Nocardia; (3) The cell wall fragments are mixed with a phosphate buffer, trypsin and chymotrypsin, and are subjected to enzymatic hydrolysis, and the hydrolyzate is collected and centrifuged to obtain cell wall precipitate of the red Nocardia; (4) The cell wall precipitate is mixed with a Tris-HCl buffer containing pronase, and is subjected to enzymatic hydrolysis, and the hydrolyzate is collected and centrifuged to obtain a crude product of the cell wall skeleton of the red Nocardia; (5) The crude product of the cell wall skeleton of the red Nocardia is subjected to purification to obtain a white-like N-CWS.

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