Application of Nocardia rubra to preparation of haematochrome and method for producing haematochrome at high yield

Abstract

In particular to application of red nocardia to preparation of haematochrome, and further discloses a method for producing haematochrome at high yield based on the red nocardia. The nocardia rubra PO-8 strain is used as a haematochrome fermentation strain, and a surfactant and adsorption resin are added into a fermentation culture medium, so that the transfer resistance of nutrient substances and metabolites is reduced, the transportation of the nutrient substances and the secretion of products are promoted, the influence of accumulation of the metabolites on the growth of thalli is eliminated, and the haematochrome yield is increased; meanwhile, the extraction is performed by utilizing different partition coefficients of the haematochrome in the upper phase and the lower phase of the aqueous two-phase, so that the preparation yield is improved, and a high-yield haematochrome process suitable for the culture of the nocardia rubra strain and a rapid extraction preparation technology thereof are established.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and fermentation engineering, and particularly relates to the use of Nocardia rubra for preparing red pigment, and further discloses a method for high-yield production of red pigment based on Nocardia rubra. Background Art

[0002] Pigment is a raw material that can endow products with color and is widely used in the food industry, cosmetics production, textile and daily necessities production, bringing good added value to products. However, traditional synthetic pigments have potential hazards such as carcinogenicity and teratogenicity, which also makes their applications increasingly rejected, while natural pigments are more and more favored by people. In recent years, people have been committed to finding alternative natural pigments, and related research has also increased. As one of the three basic pigments, red pigment has a high "gold content". It can be blended with natural pigments of blue and yellow systems into a variety of tones, making the colors of natural pigments more abundant. Moreover, some natural red pigments also have a variety of physiological functions and can be widely used in the fields of medicine, food and cosmetics. Therefore, the research and development of natural red pigments have broad prospects and development potential.

[0003] At present, microbial pigments have become an important way to obtain natural pigments. Research shows that some microorganisms produce pigments during growth, which not only reproduce quickly, are easy to culture, but also can achieve simple large-scale industrial production. More importantly, their safety, scope of application and maximum dosage have obvious advantages over synthetic pigments.

[0004] Nocardia is widely distributed in nature and has a variety of biological activities. Some can cause nocardiosis in humans and animals, and some are also producers of various antibiotics. In addition, it also plays a certain role in petroleum dewaxing and sewage purification. Nocardia rubra PO-8 isolated and screened from Kunming soil by Fujian Institute of Microbiology has been confirmed through research that the Nocardia rubra Cell Wall Skeleton (N-CWS) prepared by it is a high-molecular polymer with a natural network structure composed of nocardic acid, arabinogalactan and mucopeptide, which can significantly improve the immune activity and antitumor function, and has been successfully developed as a new biological product of class II for immunotherapy of tumor diseases, and is a practical anti-tumor drug. Therefore, this field expects to develop more new applications of Nocardia rubra, which has positive significance for the extensive development and promotion of Nocardia rubra and the full utilization of resources. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide the use of Nocardia rubra for fermenting and preparing red pigment;

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing high-yield red pigment based on Nocardia rubra, which can effectively improve the ability of Nocardia rubra to produce red pigment and effectively improve the efficiency of its red pigment production, so as to solve the problem of low efficiency in culturing Nocardia rubra to prepare red pigment in the prior art.

[0007] To solve the above technical problem, the present invention relates to the use of a Nocardia rubra for fermentative preparation of red pigment, characterized in that the Nocardia rubra includes Nocardia rubra PO-8, which is taxonomically named Nocardia rubra and has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 0187.

[0008] The present invention also discloses a method for preparing high-yield red pigment, which includes the step of inoculating a Nocardia rubra fermentation strain into a fermentation medium for fermentation culture to produce red pigment;

[0009] The Nocardia rubra includes Nocardia rubra PO-8, which is taxonomically named Nocardia rubra and has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 0187.

[0010] Specifically, for the method for preparing high-yield red pigment, the fermentation medium includes the following components by mass content: glucose 1-4%, yeast extract powder 0.2-2.0%, and pH value 6.5-7.2;

[0011] Preferably, the fermentation medium includes the following components by mass content: glucose 1-4%, yeast extract powder 0.2-2.0%, sucrose 2-4%, soy peptone 0.5-3.5%, and pH value 6.5-7.2;

[0012] Preferably, the fermentation medium includes the following components by mass content: glucose 3.5%, yeast extract powder 0.5%, sucrose 3.5%, soy peptone 2.5%, and pH value 6.5-7.2;

[0013] Preferably, the fermentation medium further includes 0.03-1.0% antifoaming agent, preferably the addition amount is 0.05%, and preferably GPE antifoaming agent.

[0014] Specifically, for the method for preparing high-yield red pigment, the fermentation medium includes the following components by mass content: glucose 2.0-4.0%, sucrose 2.0-4.0%, peptone 0.5-3.5%, yeast extract powder 0.2-2.0%, surfactant 0.5-1.5%, adsorption resin 0.2-3.0%, antifoaming agent 0.02-1.0%, and pH value 6.0-7.5.

[0015] Preferably, the fermentation medium comprises the following components by mass content: glucose 3.5%, sucrose 3.5%, soy peptone 2.5%, yeast extract powder 0.5%, surfactant 1.0%, adsorption resin 0.8%, antifoaming agent 0.05%, adjust the pH value to 6.5, and prepare with tap water.

[0016] Specifically, for the method for preparing the high-yield red pigment, in the fermentation medium:

[0017] The surfactant includes polyethylene glycol, preferably includes liquid PEG, and preferably includes at least one of PEG200, PEG400 or PEG600; and / or,

[0018] The adsorption resin includes non-polar or weakly polar macroporous resins, preferably XR919C resin, or non-polar macroporous resin HZ816.

[0019] Specifically, for the method for preparing the high-yield red pigment, the conditions of the fermentation culture step include: controlling the tank pressure at 0.04 ± 0.02 Mpa, the ventilation rate at 0.1 - 0.5 vvm, and performing fermentation culture at 28 - 32 °C for 24 - 48 h.

[0020] Preferably, the conditions of the fermentation culture step are: inoculating with an inoculum size of 2 - 5%, controlling the tank pressure at 0.04 Mpa, the ventilation rate at 0.3 vvm, and performing fermentation culture at 28 - 32 °C for 43 h.

[0021] Specifically, for the method for preparing the high-yield red pigment, the method further includes a step of inoculating the Nocardia rubra fermentation strain into a seed medium for seed liquid culture;

[0022] The seed medium comprises the following components by mass content: glucose 0.5 - 1.5%, yeast extract powder 0.5 - 2.0%, GPE 0.02 - 1.0%, adjust the pH value to 6.0 - 7.5;

[0023] Preferably, the seed medium comprises the following components by mass content: glucose 1.0%, yeast extract powder 1.5%, GPE 0.05%, adjust the pH value to 7.0, and prepare with tap water;

[0024] Preferably, the seed liquid is inoculated into the fermentation medium for fermentation culture with an inoculum size of 1 - 15%.

[0025] Preferably, the seed liquid culture step includes a shake flask seed liquid culture step and a seed tank seed liquid expansion culture step;

[0026] Preferably, the conditions for the shake flask seed culture step include: washing the bacterial lawn on the fresh slant under sterile conditions with N.S., inoculating it into the shake flask of the seed medium at a ratio of 0.1 - 0.5%, and culturing at 28 - 32°C with a controlled rotation speed of 150 - 230 r / min for 20 - 36 h to obtain the shake flask seed culture solution;

[0027] Preferably, the culture step of the shake flask seed solution is: washing the bacterial lawn on the fresh slant under sterile conditions with N.S., inoculating it into the seed medium at a ratio of 0.2%, and culturing at 30°C and 230 r / min for 32 h to obtain the shake flask seed culture solution;

[0028] Preferably, the conditions for the seed tank seed culture step include: inoculating the cultured shake flask seed culture solution into the seed tank of the seed medium at an inoculation amount of 0.2 - 1.0%, controlling the tank pressure at 0.02 - 0.04 Mpa, the ventilation volume at 0.8 - 1 vvm, the rotation speed at 150 - 200 r / min, and the temperature at 28 - 32°C, and fermenting for 20 - 36 h to obtain the required seed tank seed solution;

[0029] Preferably, the culture step of the seed tank is: inoculating the cultured shake flask seeds into the seed tank of the seed medium at an inoculation amount of 0.5%, culturing at a temperature of 30°C, a tank pressure of 0.03 Mpa, and a ventilation of 0.8 vvm, controlling the rotation speed at 180 r / min during the culture, and culturing for 30 h until the seeds are mature.

[0030] Specifically, for the method for preparing the high - yield red pigment, the method further includes the step of inoculating the Nocardia rubra fermentation strain onto a modified nutrient agar slant medium for activation culture;

[0031] The modified nutrient agar slant medium comprises components with the following mass contents: peptone 0.8 - 1.2%, beef extract 0.2 - 0.4%, corn steep powder 0.05 - 0.15%, glucose 0.1 - 0.5%, NaCl 0.3 - 0.6%, agar 1.8 - 2.0%, and adjusting the pH value to 7.0 - 7.4.

[0032] Preferably, the modified nutrient agar slant medium comprises components with the following mass contents: peptone 1.0%, beef extract 0.3%, corn steep powder 0.1%, glucose 0.3%, NaCl 0.5%, agar 2.0%, adjusting the pH value to 7.2, and preparing with tap water.

[0033] Specifically, for the method for preparing the high - yield red pigment, the method further includes the step of extracting the red pigment; the red pigment extraction step includes:

[0034] (1) Collecting the bacterial residue of the Nocardia rubra, adding ethanol for extraction, and collecting the primary extract and concentrating it for the first time to obtain the ethanol concentrate;

[0035] (2) Add the ethanol concentrate to a 15-35 wt% ammonium sulfate solution and mix. After the first extraction, collect the red pigment extract.

[0036] (3) Continue to add ethyl acetate for the second extraction. After collecting and concentrating the extract, wash, centrifuge, and dry to obtain the product.

[0037] Specifically, the method for preparing the high-yield red pigment is as follows:

[0038] In the step (1), the first concentration step includes vacuum scraping film concentration; and / or,

[0039] In the step (2), the concentration of the ammonium sulfate solution is 15-35 wt%; and / or,

[0040] In the step (3), the washing step includes washing with water.

[0041] Specifically, the method for preparing the high-yield red pigment includes the following steps:

[0042] (1) Cultivate Nocardia rubra according to the method, and centrifuge to collect the Nocardia rubra bacterial residue; soak the bacterial residue in ethanol by ultrasonic vibration at a ratio of 1:3-9 for 1-3 times, combine and collect the initial ethanol extract; and concentrate the initial extract by vacuum scraping film at 50-60 °C to obtain an ethanol concentrate.

[0043] (2) Add the concentrate to a 15-35% ammonium sulfate solution, mix well, let stand for extraction for 30 min - 2 h, and harvest the red pigment extract.

[0044] (5) Extract the extract with ethyl acetate at a ratio of 1:0.3-0.8, concentrate by vacuum rotary evaporation at 40-50 °C, wash with water, centrifuge at high speed, and dry in vacuum to obtain a red pigment sample. Send it for inspection, and determine the red pigment content with carotenoid as a reference and calculate the yield.

[0045] Preferably, the method for preparing the red pigment includes the following steps:

[0046] (1) Cultivate Nocardia rubra according to the method, and centrifuge to collect the Nocardia rubra bacterial residue; soak the bacterial residue in ethanol by ultrasonic vibration at a ratio of 1:6 for 2 times, combine and collect the initial ethanol extract; concentrate the initial extract by vacuum scraping film at 55 °C to obtain an ethanol concentrate.

[0047] (2) Add the concentrate to a 25% ammonium sulfate solution, mix well, let stand for extraction for 45 min, and harvest the red pigment extract.

[0048] (3) Extract the extract with ethyl acetate at a ratio of 1:0.5, concentrate it by rotary evaporation under vacuum at 45°C, wash it with water, centrifuge at high speed, and dry it under vacuum to obtain a red pigment sample. Submit it for inspection, and use carotenoids as a reference. Determine the content of the red pigment by spectrophotometry and calculate the yield.

[0049] The present invention also discloses the use of Nocardia rubra for fermenting and preparing red pigment. The Nocardia rubra includes Nocardia rubra PO-8, which is taxonomically named Nocardia rubra and has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and its deposit number is CGMCC No. 0187.

[0050] In the research of the present invention, it is found that the cells of Nocardia rubra PO-8 strain can produce red pigment, mainly carotenoids. Applying it to the production of natural red pigment can effectively enhance its industrial utilization rate, which has positive significance for the extensive development and promotion of Nocardia rubra and the full utilization of resources.

[0051] The method for culturing Nocardia rubra with high yield of red pigment according to the present invention uses Nocardia rubra PO-8 strain as the red pigment fermentation strain. By adding surfactants and adsorption resins to the fermentation medium, the transfer resistance of nutrients and metabolites is reduced, the transportation of nutrients and the secretion of products are promoted, the influence of metabolite accumulation on cell growth is eliminated, and the yield of red pigment is increased. At the same time, extraction is carried out by using the different partition coefficients of the red pigment in the upper and lower phases of the aqueous two-phase system to improve the preparation yield. A process for culturing Nocardia rubra strain with high yield of red pigment and its rapid extraction and preparation technology are established, which has the advantages of simple operation, high efficiency and low energy consumption, and is convenient for large-scale industrial production, and has important significance for the industrial research of preparing natural red pigment.

[0052] The method for culturing Nocardia rubra with high yield of red pigment according to the present invention includes inoculating the Nocardia rubra fermentation strain into the fermentation medium and controlling and optimizing the parameters during cultivation. By improving the fermentation conditions, the biosynthesis ability of the strain is enhanced, and the high-efficiency production of red pigment by Nocardia rubra and the rapid preparation of red pigment products can be realized, effectively solving the technical bottleneck of the high-efficiency large-scale production of red pigment by Nocardia rubra and its separation and purification.

[0053] The present invention starts from the culture process of Nocardia rubra, optimizes the composition of the medium by adding surfactants and adsorption resins to the medium, purifies by using the different partition coefficients of compounds in the biphasic water, improves the biosynthesis ability, shortens the fermentation cycle, reduces the separation time, improves the extraction efficiency, thereby reducing the production cost and enhancing the comprehensive competitiveness. Description of the Drawings

[0054] To make the content of the present invention easier to be clearly understood, the following further elaborates and explains the present invention in detail according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein,

[0055] Figure 1 It is the single colony morphology diagram of the strain Nocardia rubra PO-8 used in the present invention;

[0056] Figure 2 It is the cell morphology diagram of the strain Nocardia rubra PO-8 used in the present invention. Specific embodiments

[0057] To better understand the present invention, the following further elaborates and explains the present invention through several specific embodiments, but it does not constitute any limitation to the protection scope of the present invention.

[0058] In the following embodiments of the present invention, the strain involved in the fermentation culture of Nocardia rubra is Nocardia rubra PO-8, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its preservation number is CGMCC No. 0187, and the preservation date is January 11, 1993.

[0059] In the following embodiments of the present invention, the determination of the red pigment content in the fermentation product adopts the spectrophotometer method. An appropriate amount of the collected fermentation broth is centrifuged and washed, and the collected wet bacterial cells (bacterial residues) are added with sufficient ethanol for extraction 2-3 times. The extraction solutions are combined and the absorbance value of the extraction solution is measured at 470 nm. Taking carotenoid as a reference, the red pigment content is calculated according to the following formula:

[0060]

[0061] In the formula: A is the absorbance value of carotenoid at the maximum wavelength; D is the dilution factor; V1 is the total volume of the extraction solvent used, mL; 0.16 is the molar extinction coefficient; W is the mass of the wet bacterial cells used for determination, g; c is the wet bacterial cell mass concentration in the fermentation broth, g / L.

[0062] Example 1 Shake flask fermentation

[0063] The preserved Nocardia rubra PO-8 is cultured on the improved nutrient agar slant medium at 28 °C for 7 d to obtain a fresh slant of Nocardia rubra PO-8. Among them, the improved nutrient agar slant medium includes the following components with mass contents: peptone 1.0%, beef extract 0.3%, corn steep liquor powder 0.1%, glucose 0.3%, NaCl 0.5%, agar 2.0%, adjust the pH value to 7.2, and prepare with tap water.

[0064] Under aseptic conditions, wash the bacterial lawn on the fresh slant with N.S., inoculate it into the shake flask seed medium at a rate of 0.2 wt%, and culture it at 30 °C and 230 r / min for 36 h to obtain the shake flask seed culture solution. Among them, the preparation method of the seed medium is as follows: 10 g of glucose, 15 g of yeast extract powder, 0.5 g of GPE, 1 L of tap water, and adjust the pH value to 7.0.

[0065] Inoculate the cultured shake flask seed liquid into the shake flask fermentation medium at an inoculation amount of 10%, culture it at 30 °C and 230 r / min for 36 h, then empty the flask, and collect the red bacterial residue. Among them, the preparation method of the fermentation medium is as follows: 35 g of glucose, 35 g of sucrose, 25 g of soy peptone, 5 g of yeast extract powder, 10 g of PEG200, 8 g of XR919C resin, 0.5 g of GPE, 1 L of tap water, and adjust the pH value to 6.5.

[0066] In this example, the colony morphology diagram of the strain Nocardia rubra PO-8 is as shown in the appendix Figure 1 shown, and its electron microscope cell morphology diagram is as shown in the appendix Figure 2 shown.

[0067] Example 2

[0068] The cultivation method for the fermentation of Nocardia rubra to produce red pigment in this example is the same as that in Example 1, the only difference being that the preparation method of the fermentation medium is as follows: 35 g of glucose, 35 g of sucrose, 25 g of soy peptone, 5 g of yeast extract powder, 0.5 g of GPE, 1 L of tap water, and adjust the pH value to 6.5.

[0069] Example 3

[0070] The cultivation method for the fermentation of Nocardia rubra to produce red pigment in this example is the same as that in Example 1, the only difference being that the preparation method of the fermentation medium is as follows: 10 g of glucose, 10 g of yeast extract powder, 1 L of tap water, and adjust the pH value to 7.2.

[0071] According to the method for measuring the red pigment content described above, measure the red pigment content in the fermentation broths of Examples 1-3. After emptying the shake flask fermentation, collect the bacterial residue and weigh it, detect the amount of red pigment by spectrophotometry, and calculate the pigment yield. The results are shown in Table 1 below.

[0072] Table 1 Comparison of the bacterial residue amount, pigment yield, and productivity in the shake flask fermentation of Examples 1-3

[0073]

[0074] As shown in the results of Table 1, the average yield of the red bacterial residue in the three batches of fermentation cultures in Example 1 was 256.3 g·L -1 , and the red pigment yield was 8.2 g·L-1 , the pigment yield was calculated to be 5.5 g·L -1 .d -1 ; while in Example 2, the average yield of the three batches of fermented red bacterial residues was 117.9 g·L -1 , the red pigment yield was 3.4 g·L-1, and the pigment yield was calculated to be 2.3 g·L -1 .d -1 ; in Example 3, the average yield of the three batches of fermented red bacterial residues was 47.2 g·L -1 , the red pigment yield was 1.4 g·L -1 , and the pigment yield was calculated to be 0.9 g·L -1 .d -1 .

[0075] It can be seen that based on Nocardia rubra as the fermentation strain, through the optimization of the fermentation medium, the present invention obtained a 117% and 443% increase in the yield of bacterial residues, and a 141% and 485% increase in the amount of red pigment, respectively. It can be seen that the method of the present invention can significantly improve the red pigment synthesis ability by optimizing the fermentation of the fermentation medium.

[0076] Example 4 Fermentation in a 100 L fermenter

[0077] The preserved Nocardia rubra PO-8 was cultured on a modified nutrient agar solid medium (the same as in Example 1) at 28 °C for 7 d to obtain a fresh slant of Nocardia rubra PO-8.

[0078] Under aseptic conditions, the bacterial lawn on the fresh slant was washed off with N.S. and inoculated into the shake flask seed medium at a rate of 0.2%, and cultured at 30 °C and 200 r / min for 36 h to obtain the shake flask seed culture solution. The composition and preparation method of the above seed medium: 10 g of glucose, 15 g of yeast extract powder, 0.5 g of GPE, 1 L of tap water, and the pH value was adjusted to pH 7.0.

[0079] The cultured shake flask seed culture solution was inoculated into a 10 L seed tank at an inoculation amount of 0.5%. The volume of the seed tank medium was 7 L, the tank pressure was 0.02 - 0.04 Mpa, the aeration was 0.8 vvm, the rotation speed was 200 r / min, the temperature was controlled at 30 °C, and cultured for 30 h to obtain the required seed solution. The composition and preparation method of the above seed medium: 70 g of glucose, 105 g of yeast extract powder, 3.5 g of GPE, added with tap water, the pH value was adjusted to 7.0, and the volume of the sterilized medium was 7 L.

[0080] Inoculate the above-prepared seed liquid into a 100 L fermenter at an inoculation amount of 10%. The medium filling volume in the fermenter is 70 L. Control the tank pressure at 0.03 Mpa, the fermentation temperature at 30 °C, and control the aeration during the fermentation process at 0.8 - 1.0 vvm. Terminate the fermentation after 36 h, and discharge the tank to collect the red bacterial residue. The components and preparation method of the above fermentation medium: 2450 g of glucose, 2450 g of sucrose, 1750 g of soy peptone, 350 g of yeast extract powder, 700 g of PEG200, 560 g of XR919C resin, 35 g of GPE. Add tap water, adjust the pH value to 6.5, and the volume of the medium after sterilization is 70 L.

[0081] Example 5

[0082] The culture method for fermenting Nocardia rubra to produce red pigment in this example is the same as that in Example 4, except that the preparation method of the fermentation medium is as follows: 2450 g of glucose, 2450 g of sucrose, 1750 g of soy peptone, 350 g of yeast extract powder, 35 g of GPE. Add tap water, adjust the pH value to 6.5, and the volume of the medium after sterilization is 70 L.

[0083] Example 6

[0084] The culture method for fermenting Nocardia rubra to produce red pigment in this example is the same as that in Example 4, except that the preparation method of the fermentation medium is as follows: 700 g of glucose, 700 g of yeast extract powder. Add tap water, adjust the pH value to 7.2, and the volume of the medium after sterilization is 70 L.

[0085] According to the method for measuring the content of red pigment described above, measure the content of red pigment in the fermentation broth of Examples 4 - 6. After discharging the 100 L tank fermentation, collect the bacterial residue and weigh it. Detect the amount of red pigment by spectrophotometry and calculate the pigment yield. The results are shown in Table 2 below.

[0086] Table 2 Comparison of the bacterial residue amount, pigment yield, and productivity in the shake flask fermentation of Examples 4 - 6

[0087]

[0088] As shown in the results of Table 2, the average yield of the red bacterial residue in the three batches of fermentation culture in Example 4 is 262.6 g·L -1 , the red pigment yield is 9.4 g·L -1 , and the calculated pigment productivity is 5.6 g·L -1 .d -1 ; while in Example 5, the average yield of the red bacterial residue in the three batches of fermentation culture is 122.8 g·L -1 , the red pigment yield is 3.7 g·L-1, and the calculated pigment productivity is 2.4 g·L -1 .d -1 ; in Example 6, the average yield of the red bacterial residue in the three batches of fermentation culture is 47.6 g·L-1 , the yield of red pigment is 1.5 g / L -1 , calculate the pigment productivity as 1.0 g / L -1 .d -1 .

[0089] It can be seen that based on Nocardia rubra as the fermentation strain, through the optimization of the fermentation medium, the yields of its bacterial residues are increased by 118% and 451% respectively, and the amounts of red pigment are increased by 127% and 460% respectively. This indicates that the method described in the present invention can significantly improve the red pigment synthesis ability through optimizing the fermentation of the fermentation medium and scale-up cultivation in the tank.

[0090] Example 7 Fermentation in a 1t fermenter

[0091] The preserved Nocardia rubra PO-8 was cultured on the improved nutrient agar solid medium (the same as in Example 1) at 28 °C for 7 days to obtain a fresh slant of Nocardia rubra PO-8.

[0092] Under sterile conditions, the bacterial lawn on the fresh slant was washed off with N.S. and inoculated into the shake flask seed medium at a rate of 0.2%, and cultured at 30 °C and 200 r / min for 36 h to obtain the shake flask seed culture solution. The composition and preparation method of the above seed medium: 100 g of glucose, 150 g of yeast extract powder, 5 g of GPE, 10 L of tap water, and the pH value was adjusted to pH 7.0.

[0093] The cultured shake flask seed culture solution was inoculated into a 100 L seed tank at an inoculation amount of 0.5%. The volume of the seed tank medium was 70 L, the tank pressure was 0.02 - 0.04 Mpa, the aeration was 0.8 vvm, the rotation speed was 200 r / min, the temperature was controlled at 30 °C, and cultured for 30 h to obtain the required seed solution. The composition and preparation method of the above seed medium: 700 g of glucose, 1050 g of yeast extract powder, 35 g of GPE, added with tap water, the pH value was adjusted to 7.0, and the volume of the sterilized medium was 70 L.

[0094] The above cultured seed solution was inoculated into a 1t fermenter at an inoculation amount of 10%. The volume of the fermenter medium was 700 L, the tank pressure was controlled at 0.03 Mpa, the fermentation temperature was 30 °C, the aeration was controlled at 0.8 - 1.0 vvm during the fermentation process, the fermentation was terminated at 36 h, and the fermenter was emptied to collect the red bacterial residue. The composition and preparation method of the above fermentation medium: 24.5 kg of glucose, 2.45 kg of sucrose, 17.5 kg of soy peptone, 3.5 kg of yeast extract powder, 7.0 kg of PEG200, 5.6 kg of XR919C resin, 0.35 kg of GPE, added with tap water, the pH value was adjusted to 6.5, and the volume of the sterilized medium was 700 L.

[0095] Example 8

[0096] The culture method for producing red pigment by Nocardia rubra in this example is the same as that in Example 7, with the only difference being the preparation method of the fermentation medium: 24.5 kg of glucose, 2.45 kg of sucrose, 17.5 kg of soy peptone, 3.5 kg of yeast extract powder, 0.35 kg of GPE, add tap water, adjust the pH value to 6.5, and the volume of the medium after sterilization is 700 L.

[0097] Example 9

[0098] The culture method for producing red pigment by Nocardia rubra in this example is the same as that in Example 7, with the only difference being the preparation method of the fermentation medium: 7 kg of glucose, 7 kg of yeast extract powder, 350 g of GPE, add tap water, adjust the pH value to 7.2, and the volume of the medium after sterilization is 700 L.

[0099] According to the method for measuring the content of red pigment described above, measure the content of red pigment in the fermentation broth of Examples 7 - 9. After the fermentation in the 1 t tank is finished, collect the bacterial residue and weigh it, detect the amount of red pigment by spectrophotometry, and calculate the pigment yield. The results are shown in Table 3 below.

[0100] Table 3 Comparison of the amount of bacterial residue, pigment yield and productivity in Examples 7 - 9 of 1 t tank

[0101]

[0102]

[0103] As shown in the results of Table 3, the average yield of the red bacterial residue in three batches of fermentation culture in Example 7 is 296.2 g.L -1 , the red pigment yield is 12.7 g.L -1 , and the calculated pigment productivity is 8.5 g.L -1 .d -1 ; the average yield of the red bacterial residue in three batches of fermentation culture in Example 8 is 134.2 g.L -1 , the red pigment yield is 5.3 g.L -1 , and the calculated pigment productivity is 3.5 g.L -1 .d -1 ; the average yield of the red bacterial residue in three batches of fermentation culture in Example 9 is 52.8 g.L -1 , the red pigment yield is 2.2 g.L -1 , and the calculated pigment productivity is 1.4 g.L -1 .d -1 .

[0104] It can be seen that the present invention is based on Nocardia rubra as the fermentation strain. Through the optimization of the fermentation medium, the yields of its bacterial residues are increased by 121% and 460% respectively, and the amounts of red pigment are increased by 140% and 477% respectively. This further demonstrates that the method described in the present invention can significantly improve the ability to synthesize red pigment by optimizing the fermentation of the fermentation medium.

[0105] In summary, the method described in the present invention uses the optimized fermentation medium to culture Nocardia rubra for the biosynthesis of red pigment, adds surfactants and adsorption resins, reduces the transfer resistance of nutrients and metabolites, promotes the transport of nutrients and the secretion of products, eliminates the impact of metabolite accumulation on the growth of the bacteria, promotes the rapid growth of the bacteria, and improves the yield of red pigment.

[0106] Example 10 Preparation of Red Pigment

[0107] The preserved Nocardia rubra PO-8 was cultured on a modified nutrient agar solid medium at 28 °C for 7 d to obtain a fresh inclined plane of Nocardia rubra PO-8.

[0108] Under sterile conditions, the bacterial colonies on the fresh inclined plane were washed off with N.S. and inoculated into the shake flask seed medium at a ratio of 0.2%, and cultured at 30 °C and 200 r / min for 36 h to obtain the shake flask seed culture solution. The composition and preparation method of the above seed medium: 10 g of glucose, 15 g of yeast extract powder, 0.5 g of GPE, 1 L of tap water, and the pH value was adjusted to pH 7.0.

[0109] The cultured shake flask seed culture solution was inoculated into a 10 L seed tank at an inoculation amount of 0.5%. The loading volume of the seed tank medium was 7 L, the tank pressure was 0.02 - 0.04 Mpa, the ventilation was 0.8 vvm, the rotation speed was 200 r / min, the temperature was controlled at 30 °C, and cultured for 30 h to obtain the required seed solution. The composition and preparation method of the above seed medium: 70 g of glucose, 105 g of yeast extract powder, 3.5 g of GPE, added with tap water, the pH value was adjusted to 7.0, and the volume of the medium after sterilization was 7 L.

[0110] The above cultured seed solution was inoculated into a 100 L fermentation tank at an inoculation amount of 10%. The loading volume of the fermentation tank medium was 70 L, the tank pressure was controlled at 0.03 Mpa, the fermentation temperature was 30 °C, the ventilation was controlled at 0.8 - 1.0 vvm during the fermentation process, the fermentation was terminated at 36 h, and the fermentation tank was emptied to collect the red bacterial residue. The composition and preparation method of the above fermentation medium: 2450 g of glucose, 2450 g of sucrose, 1750 g of soy peptone, 350 g of yeast extract powder, 700 g of PEG200, 560 g of XR919C resin, 35 g of GPE, added with tap water, the pH value was adjusted to 6.5, and the volume of the medium after sterilization was 70 L.

[0111] Collect the red bacterial residue and soak it in ethanol with ultrasonic vibration at a ratio of 1:6 for 2 times. Combine and collect the primary ethanol extract, and concentrate it by vacuum scraping film at 55°C to obtain the ethanol concentrate; add the ethanol concentrate to a 25% ammonium sulfate solution, mix well, let it stand for extraction for 75 minutes, harvest the red pigment extract, extract it with ethyl acetate at a ratio of 1:0.5, concentrate it by rotary evaporation at 45°C, wash it with water, centrifuge it, and dry it under reduced pressure to obtain the red pigment sample.

[0112] Detect by the above spectrophotometer method, and the purity of its red pigment is detected to be 93%, and the yield is 87.6%.

[0113] It can be seen that the method for preparing red pigment by highly cultivating Nocardia rubra described in the present invention uses aqueous two-phase extraction for red pigment, and its purification yield reaches more than 85%, thus providing a basis for the large-scale preparation of red pigment by fermentation of Nocardia rubra.

[0114] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing red pigment by fermentation of Nocardia rubrum, characterized in that: The rubra Nocardia includes rubra Nocardia PO-8, which is classified and named as rubra Nocardia, and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC No.0187.

2. A method for preparing a high-yield red pigment, characterized in that: The method comprises the steps of inoculating a Nocardia rubrum fermentation strain into a fermentation medium for fermentation and culturing to produce red pigment; The Nocardia rubra includes Nocardia rubra PO-8, which is classified and named as Nocardia rubra and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC No.0187.

3. The method for preparing a high-yield red pigment according to claim 2, characterized in that: The fermentation medium comprises the following components by mass content: 1-4% glucose, 0.2-2.0% yeast extract powder, and a pH value of 6.5-7.2; Preferably, the fermentation medium comprises the following components by mass content: glucose 1-4%, yeast extract powder 0.2-2.0%, sucrose 2-4%, soy peptone 0.5-3.5%, pH value 6.5-7.2; Preferably, the fermentation medium further comprises 0.03-1.0% of a defoamer, preferably a GPE defoamer.

4. The method for preparing a high-yield red pigment according to claim 2 or 3, characterized in that: The fermentation medium comprises the following components by mass content: 2.0-4.0% glucose, 2.0-4.0% sucrose, 0.5-3.5% peptone, 0.2-2.0% yeast extract powder, 0.5-1.5% surfactant, 0.2-3.0% adsorption resin, 0.02-1.0% defoamer, and a pH value of 6.0-7.

5.

5. The method for preparing a high-yield red pigment according to claim 4, characterized in that: In the fermentation medium: The surfactant comprises polyethylene glycol, preferably liquid PEG, preferably at least one of PEG200, PEG400 or PEG600; and / or, The adsorption resin includes a non-polar or weakly polar macroporous resin, preferably XR919C resin or a non-polar macroporous resin HZ816.

6. The method for preparing a high-yield red pigment according to any one of claims 2 to 5, characterized in that: The conditions of the fermentation culture step include: controlling the tank pressure to 0.04±0.02 MPa, the ventilation volume to 0.1-0.5 vvm, and fermenting and culturing at 28-32° C. for 24-48 hours.

7. The method for preparing a high-yield red pigment according to any one of claims 2 to 6, characterized in that: The method further comprises the step of inoculating the Nocardia rubrum fermentation strain into a seed culture medium for seed liquid culture; Preferably, the seed culture medium comprises the following components by mass content: glucose 0.5-1.5%, yeast extract powder 0.5-2.0%, GPE 0.02-1.0%, and the pH value is adjusted to 6.0-7.5; Preferably, the seed solution culture step includes the steps of shake flask seed solution culture and seed tank seed solution expansion culture; Preferably, the conditions of the shake flask seed solution culture step include: culturing at 28-32°C and controlling the rotation speed to 150-200 r / min for 20-40 hours; Preferably, the conditions of the seed tank seed liquid expansion step include: controlling the tank pressure to 0.02-0.04Mpa, the ventilation volume to 0.8-1vvm, the rotation speed to 150-200r / min, the temperature to 28-32°C, and fermentation for 16-36h.

8. The method for preparing a high-yield red pigment according to any one of claims 2 to 7, characterized in that: The method further comprises the step of inoculating the Nocardia rubrum fermentation strain into a modified nutrient agar slant medium for activation culture; The improved nutrient agar slant culture medium comprises the following components by mass content: 0.8-1.2% of peptone, 0.2-0.4% of beef extract, 0.05-0.15% of corn steep liquor, 0.1-0.5% of glucose, 0.3-0.6% of NaCl, 1.8-2.0% of agar, and the pH value is adjusted to 7.0-7.

4.

9. The method for preparing a high-yield red pigment according to any one of claims 2 to 8, characterized in that: The method further comprises the step of extracting red pigment; The red pigment extraction step comprises: (1) collecting the bacterial residue of the Nocardia rubrum, adding ethanol to extract, collecting the primary extract, and performing a first concentration to obtain an ethanol concentrate; (2) adding the ethanol concentrate to a 15-35 wt % ammonium sulfate solution, mixing, and performing a first extraction to collect a red pigment extract; (3) Continue to add ethyl acetate for a second extraction, collect the extract, concentrate it, wash it, centrifuge it, and dry it to obtain the product.

10. The method for preparing a high-yield red pigment according to claim 9, characterized in that: In the step (1), the first concentration step comprises vacuum scraping thin film concentration; and / or, In the step (2), the concentration of the ammonium sulfate solution is 15-35wt%; and / or, In the step (3), the washing step includes washing with water.

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