Mutagenesis cultivation method of strain resistant to high-concentration chromium solution and application of strain in preparation of dry bacterial powder

Abstract

The invention provides a mutagenesis cultivation method of a high-concentration chromium solution-resistant strain and application of the high-concentration chromium solution-resistant strain in preparation of dry bacterial powder, and the high-concentration chromium solution-resistant strain is obtained by firstly cultivating a strain mutagenized by a chemical or physical method in a low-concentration chromium ion environment and then cultivating in a high-concentration chromium ion environment. A bacterial strain capable of converting chromium ions into organic chromium and a bacterial strain resistant to high-concentration chromium ions are screened out respectively, and then the bacterial strain and the bacterial strain are fused through protoplast to obtain the bacterial strain capable of converting the organic chromium and resistant to a high-concentration chromium solution. Compared with the prior art, the mutagenesis cultivation method of the strain resistant to the high-concentration chromium solution has the advantages that the difficulty of mutagenesis cultivation is reduced, and the speed of mutagenesis cultivation is increased.

Description

Technical Field

[0001] The present invention relates to a method for mutagenic cultivation of strains, and particularly to a method for mutagenic cultivation of strains resistant to high-concentration chromium solution and its application in the preparation of dried bacterial powder. Background Art

[0002] In the prior art, since chromium ions with a concentration above 10 mg / L have an obvious inhibitory effect on the growth of strains, chromium ions with a concentration above 10 mg / L are generally considered as high-concentration chromium ions, and chromium ions with a concentration below 5 mg / L have no obvious inhibitory effect on the growth of strains, and chromium ions with a concentration below 5 mg / L are generally considered as low-concentration chromium ions. In order to obtain strains capable of converting organic chromium in a high-concentration chromium ion environment, a method of mutating the strains and then culturing them in a culture medium rich in high-concentration chromium ions is usually adopted. However, using this method, due to the inhibitory effect of high-concentration chromium ions on the growth of strains, the strains generated by mutagenesis need to have the ability to convert chromium ions into organic chromium and the ability to be normally cultivated in high-concentration chromium ions at the same time. This makes the mutagenic cultivation of strains more difficult, and it is necessary to carry out mutagenic cultivation repeatedly for many times, and the mutagenic cultivation period is relatively long. Summary of the Invention

[0003] In order to solve the above technical problem of "the strains generated by mutagenesis need to have the ability to convert chromium ions into organic chromium and the ability to be normally cultivated in high-concentration chromium ions at the same time, which makes the mutagenic cultivation of strains more difficult", the present invention provides a method for mutagenic cultivation of strains resistant to high-concentration chromium solution.

[0004] Another object of the present invention is to provide an application of a method for mutagenic cultivation of strains resistant to high-concentration chromium solution in the preparation of dried bacterial powder.

[0005] In the first aspect of the present invention, a method for mutagenic cultivation of strains resistant to high-concentration chromium solution includes the following steps: Step 1), preparing a suspension of 10 6 -10 7 cells / ml from the original strain Cunninghamella echinulata AS.3.2473 provided by the National Center for Microbial Culture Collection; Step 2), inoculating the suspension prepared in the above step 1) into a liquid medium, and culturing for 2 - 4 hours under the conditions of a pH of 5.5 - 6, a temperature of 28 - 31 °C, and 100 - 200 r / min; Step 3), evenly inoculating the Cunninghamella echinulata cultured in step 2) on a plate, and irradiating with ultraviolet light with a wavelength of 245 - 260 nm for 60 - 220 s to obtain mutant Cunninghamella echinulata; Step 4), divide the mutant Cunninghamella echinulata obtained in step 3) into two equal parts, inoculate one part on a PDA medium for cultivation, and the concentration of chromium chloride in the PDA medium is 1-5 mg / L; reserve the other part; Step 5), divide each mutant Cunninghamella echinulata colony with organic chromium on the PDA medium after cultivation in step 4) into two equal parts to obtain organic chromium mutant Cunninghamella echinulata a and organic chromium mutant Cunninghamella echinulata b; Step 6), transfer the organic chromium mutant Cunninghamella echinulata a obtained in step 5) and the mutant Cunninghamella echinulata reserved in step 4) to a PDA medium containing 10-100 mg / L of chromium chloride for cultivation; Step 7), for the organic chromium mutant Cunninghamella echinulata a after cultivation in step 6), if there are surviving colonies, perform step 8); if there are no surviving colonies, perform step 9); Step 8), verify the stability of the organic chromium mutant Cunninghamella echinulata a obtained in step 7); Step 9), perform protoplast fusion on the surviving mutant Cunninghamella echinulata after cultivation in step 6) and the organic chromium mutant Cunninghamella echinulata b obtained in step 5) to obtain a strain resistant to high-concentration chromium solution.

[0006] Preferably, the liquid medium in step 2) is a potato extract added with a carbon source, a nitrogen source, and inorganic salts.

[0007] Preferably, the liquid medium includes a carbon source of 2-4% by mass, a nitrogen source of 0.1-0.25% by mass, inorganic salts of 0.01-0.07% by mass, and the rest is a potato extract.

[0008] Preferably, the carbon source includes glucose, sucrose, molasses; the nitrogen source includes soybean cake powder, peptone; the inorganic salts include potassium, magnesium, calcium, zinc.

[0009] Preferably, the protoplast fusion in step 9) is specifically as follows, Inoculate the surviving mutant Cunninghamella echinulata and the organic chromium mutant Cunninghamella echinulata b into a seed medium respectively, shake and cultivate at 28 °C for 40-48 h, collect the cells in the logarithmic growth phase, and prepare a bacterial suspension of 10 6 -10 7 cells / ml; Add an equal volume of 1-5 mg / L lysozyme solution to the bacterial suspension, mix well, water bath at 30-40 °C for 1-2 h, filter the enzyme solution through absorbent cotton, collect the filtrate, centrifuge and discard the supernatant, wash the lower layer twice with the preservation solution and then resuspend and mix well in 1 ml of the preservation solution to obtain a protoplast suspension; The obtained protoplast suspension is inactivated by ultraviolet light. It is irradiated with a 25-40w ultraviolet lamp at a distance of 30-40 cm above the petri dish containing the protoplast suspension for 1-2 min; the protoplast suspension of organic chromium mutant Cunninghamella echinulata b is inactivated by heat. The protoplast suspension of organic chromium mutant Cunninghamella echinulata b is placed at 60-80 °C for 10-30 min; The two inactivated protoplasts are mixed in equal volume, a cosolvent is added, and after standing culture at 28-30 °C for 10-20 min, protoplast fusants are obtained. After centrifugation, the supernatant is discarded, the lower layer is washed twice with the preservation solution and then placed in the preservation solution. The fusants are inoculated onto the regeneration medium and cultured at 28-30 °C for 7-10 days to obtain single colonies of fusants.

[0010] In the second aspect of the present invention, the application of a strain resistant to high-concentration chromium solution cultivated by a method for mutagenic cultivation of a strain resistant to high-concentration chromium solution in the preparation of dry bacterial powder, wherein the dry bacterial powder includes linolenic acid, r-linolenic acid, organic chromium, proteoglycan, amino acids and superoxide dismutase.

[0011] The preparation method includes the following steps: Step a), a strain resistant to high-concentration chromium solution cultivated by the method for mutagenic cultivation of a strain resistant to high-concentration chromium solution according to any one of claims 1 to 5 is made into a suspension and inoculated onto a slant medium for activation; Step b), the activated strain resistant to high-concentration chromium solution is transferred to an eggplant bottle for subculture; Step c), the strain resistant to high-concentration chromium solution in the eggplant bottle is transferred to a seed tank for subculture; Step d), the strain resistant to high-concentration chromium solution in the seed tank is transferred to a fermentation tank for subculture; Step e), after collecting the subcultured strains, they are washed with water, and dry bacterial powder is obtained by low-temperature freeze-drying method.

[0012] Preferably, the medium in the seed tank in step c) includes sucrose or glucose with a mass percentage of 2-4%, soybean cake powder or peptone with a mass percentage of 0.1-0.25%, magnesium sulfate with a mass percentage of 0.01-0.03%, potassium chloride with a mass percentage of 0.01-0.03% and zinc sulfate with a mass percentage of 0.0005-0.001%, the PH is 5.5-6, the culture temperature is 28-30 °C, the air volume is 1:0.05-0.3 v / v, the stirring speed is 180-250 rpm, and the culture time is 18-24 hours.

[0013] Preferably, the culture medium in the fermenter in step d) comprises molasses or sucrose at 8-12% by mass, soybean cake powder at 0.8-1.5% by mass, magnesium sulfate at 0.01-0.03% by mass, potassium chloride at 0.01-0.03% by mass, zinc sulfate at 0.0005-0.001% by mass and chromium trichloride at 0.0005-0.001% by mass, with a pH of 5.5-6, a culture temperature of 25-28 °C, an air volume of 0.05 v / v - 0.5 v / v, a stirring speed of 150-180 rpm, and a culture time of 80-96 hours.

[0014] Adopting the technical solution of the present invention, compared with the prior art, the beneficial effects are as follows: The strain mutated by chemical or physical methods is first cultivated in a low-concentration chromium ion environment below 5 mg / L, and then cultivated in a high-concentration chromium ion environment. Strains that convert chromium ions into organic chromium and strains that are resistant to high-concentration chromium ions are respectively screened out. Then, the two are fused by protoplasts to obtain a strain that can convert organic chromium and is resistant to high-concentration chromium solution. The difficulty of mutagenic cultivation is reduced, and thus the speed of mutagenic cultivation is accelerated. Description of the Drawings

[0015] Figure 1 It is a schematic diagram of the overall process of an embodiment of the present invention. Detailed Embodiments Example 1

[0016] As Figure 1 shown, this example provides a method for mutagenic cultivation of a strain resistant to high-concentration chromium solution, which comprises the following steps: Step 1), preparing a suspension of the original strain Cunninghamella echinulata AS.3.2473 provided by the National Center for Microbial Culture Collection at a concentration of 10 6 -10 7 cells / ml; Step 2), inoculating the suspension prepared in step 1) into a liquid culture medium, and culturing for 2-4 hours under the conditions of a pH of 5.5-6, a temperature of 28-31 °C, and a rotation speed of 100-200 r / min; Step 3), evenly inoculating the Cunninghamella echinulata cultured in step 2) on a plate, and irradiating with ultraviolet light with a wavelength of 245-260 nm for 60-220 s to obtain mutant Cunninghamella echinulata; Step 4), dividing the mutant Cunninghamella echinulata obtained in step 3) into two equal parts, inoculating one part on a PDA culture medium for cultivation, and the concentration of chromium chloride in the PDA culture medium is 1-5 mg / L; the other part is reserved; Step 5), divide each mutant Cunninghamella echinulata colony with organic chromium on the PDA medium after the culture in Step 4) into two equal parts on average, obtaining organic chromium mutant Cunninghamella echinulata a and organic chromium mutant Cunninghamella echinulata b; Step 6), transfer the organic chromium mutant Cunninghamella echinulata a obtained in Step 5) and the spare mutant Cunninghamella echinulata in Step 4) to a PDA medium containing chromium chloride at a concentration of 10 - 100 mg / L for culture; Step 7), for the organic chromium mutant Cunninghamella echinulata a after the culture in Step 6), if there are surviving colonies, perform Step 8); if there are no surviving colonies, perform Step 9); Step 8), conduct stability verification on the organic chromium mutant Cunninghamella echinulata a obtained in Step 7); Step 9), perform protoplast fusion on the surviving mutant Cunninghamella echinulata after the culture in Step 6) and the organic chromium mutant Cunninghamella echinulata b obtained in Step 5) to obtain a strain resistant to high - concentration chromium solution, and conduct stability verification.

[0017] Among them, Cunninghamella echinulata is mutagenized by chemical and physical methods, and its morphological characteristics are significantly different from those of the original strain. And it has characteristics such as growth on PDA medium rich in high - concentration chromium trichloride. This strain is cultured in a medium with glucose, sucrose, molasses as carbon sources, peptone, soybean cake powder, and diammonium hydrogen phosphate as nitrogen sources, combined with trace elements such as potassium, magnesium, calcium, zinc and a certain concentration of chromium trichloride.

[0018] Among them, the liquid medium includes 2 - 4% by mass of sucrose or glucose, 0.1 - 0.25% by mass of soybean cake powder or peptone, 0.01 - 0.03% by mass of magnesium sulfate, 0.01 - 0.03% by mass of potassium chloride, and 0.0005 - 0.001% by mass of zinc sulfate.

[0019] Among them, the PDA medium includes 2 - 4% by mass of sucrose or glucose, 0.1 - 0.25% by mass of soybean cake powder or peptone, 0.01 - 0.03% by mass of magnesium sulfate, 0.01 - 0.03% by mass of potassium chloride, and 0.0005 - 0.001% by mass of zinc sulfate.

[0020] Among them, stability verification means that after multiple sub - cultures, the traits of this strain still remain stable. Usually, 3 sub - cultures are sufficient.

[0021] Among them, step 1) specifically involves dissolving the spores of Cunninghamella echinulata in sterile physiological saline containing polysorbate 80, filtering through a bacterial sieve with 350 to 400 meshes, filtering into a conical flask, counting the cells in the filtrate, and diluting it to 10 6 -10 7 cells / ml with physiological saline in a sterile centrifuge tube.

[0022] Among them, protoplast fusion specifically involves inoculating the surviving mutant Cunninghamella echinulata and the organic chromium mutant Cunninghamella echinulata b into the seed medium respectively. The seed medium is 2% (by mass percentage) of mycose, 0.02% (by mass percentage) of diammonium hydrogen phosphate, 0.03% (by mass percentage) of magnesium sulfate, 0.02% (by mass percentage) of potassium chloride, 0.02% (by mass percentage) of zinc sulfate. Shake and culture at 28°C for 40 to 48 h, collect the bacteria in the logarithmic growth phase, and prepare a bacterial suspension of 10 6 -10 7 cells / ml.

[0023] Add an equal volume of 1 - 5 mg / L lysozyme solution to the bacterial suspension, mix well, water bath at 30 - 40°C for 1 - 2 h, filter the enzyme solution through absorbent cotton, collect the filtrate, centrifuge and discard the supernatant. Wash the lower layer twice with the preservation solution and then resuspend and mix evenly in 1 ml of the preservation solution to obtain a protoplast suspension.

[0024] The preservation solution includes: 103 g of sucrose, 0.25 g of K2SO4, 2.02 g of MgCl2·6H2O, 0.05 g of KH2PO4, 3.68 g of CaCl2·2H2O, 2 mL of trace element solution, made up to 800 mL with distilled water, and add 10 mL of 5.73% TES buffer (pH 7.2) to every 80 mL of the solution. Sterilize at 121°C for 20 min.

[0025] The trace element solution includes 0.004% (by mass percentage) of ZnCl2, 0.02% (by mass percentage) of FeCl3·6H2O, 0.001% (by mass percentage) of CaCl2·2H2O, 0.001% (by mass percentage) of MnCl2·4H2O, 0.001% (by mass percentage) of Na2B4O7·10H2O, 0.001% (by mass percentage) of (NH4)6Mo7O24·4H2O.

[0026] Carry out ultraviolet inactivation on the obtained surviving mutant Cunninghamella echinulata protoplast suspension, irradiate with a 25 - 40 w ultraviolet lamp at a distance of 30 - 40 cm above the petri dish containing the protoplast suspension for 1 - 2 min; carry out heat inactivation on the organic chromium mutant Cunninghamella echinulata b protoplast suspension, place the organic chromium mutant Cunninghamella echinulata b protoplast suspension at 60 - 80°C for 10 - 30 min.

[0027] Mix the inactivated two kinds of protoplasts with polyethylene glycol cosolvent in equal volume, and let them stand and culture at 28 - 30 °C for 10 - 20 min to obtain protoplast fusants. Centrifuge to discard the supernatant, wash the lower layer material twice with the preservation solution and then place it in the preservation solution. Inoculate the fusants into the regeneration medium and culture at 28 - 30 °C for 7 - 10 days to obtain single colonies of fusants; the preservation solution is the same as that for preparing the protoplast suspension.

[0028] The regeneration medium includes yeast powder with a mass percentage of 0.4 - 0.5%, peptone with a mass percentage of 0.4 - 0.5%, glucose with a mass percentage of 10.4 - 10.5%, sodium chloride with a mass percentage of 0.1 - 0.15%, agar powder with a mass percentage of 1.5 - 2.0%, valine with a mass percentage of 0.5 - 1.0%, and pH 7.0 - 7.2.

[0029] Carry out stability verification. Specifically: Inoculate the obtained single colonies of fusants into the PDA medium added with 10 mg / L chromium chloride and culture for 7 - 10 days. Then inoculate the cultured strain into a shake flask containing the liquid medium of the PDA medium added with 10 mg / L chromium chloride and culture for 5 - 6 days, which is one subculture. Then repeat the above operations three times for the strains obtained after each subculture to complete the stability verification.

[0030] In some specific embodiments, while carrying out the stability verification, screen the strains that can produce high - content polyunsaturated fatty acids.

[0031] Specifically, after each subculture, screen the strains that can produce high - content polyunsaturated fatty acids for the next subculture.

[0032] In some specific embodiments, the medium in step 2) is the potato extract added with carbon source, nitrogen source and inorganic salts.

[0033] In some specific embodiments, the carbon source includes glucose, sucrose, molasses.

[0034] In some specific embodiments, the nitrogen source includes soybean cake powder, peptone.

[0035] In some specific embodiments, the inorganic salts include potassium, magnesium, calcium, zinc.

[0036] In some specific embodiments, step 3) is replaced by evenly inoculating Cunninghamella echinulata on the plate and performing microwave and nitrosoguanidine compound mutagenesis.

[0037] Specifically, first treat the plate with microwave at 2450 MHz for 5 seconds, then place the plate on ice to cool for 20 s, and repeat the operation until the microwave treatment time reaches 120 s; then put the plate into the phosphate buffer solution containing nitrosoguanidine, mix well, and stand in a water bath for a period of time.

[0038] Exemplarily, the original strain Cunninghamella echinulata AS.3.2473 provided by the National Center for Culture Collection of Microorganisms was made into a suspension of 10 6 -10 7 CFU / ml; the suspension of Cunninghamella echinulata was inoculated into a liquid medium and cultured for 4 hours under the conditions of a pH of 5.5, a temperature of 28 °C, and 100 r / min; the cultured Cunninghamella echinulata was evenly inoculated on a plate and irradiated with ultraviolet light with a wavelength of 245 nm for 220 s to obtain mutant Cunninghamella echinulata; the mutant Cunninghamella echinulata was inoculated on a PDA medium containing 1 mg / L of chromium chloride for culture; each colony of mutant Cunninghamella echinulata with organic chromium on the PDA medium was divided into two parts to obtain organic chromium mutant Cunninghamella echinulata a and organic chromium mutant Cunninghamella echinulata b; the organic chromium mutant Cunninghamella echinulata a and the mutant Cunninghamella echinulata were transferred to a PDA medium containing 10 mg / L of chromium chloride for culture; there were surviving colonies of organic chromium mutant Cunninghamella echinulata a; the obtained organic chromium mutant Cunninghamella echinulata a was subjected to stability verification.

[0039] Exemplarily, the original strain Cunninghamella echinulata AS.3.2473 provided by the National Center for Culture Collection of Microorganisms was made into a suspension of 10 6 -10 7 CFU / ml; the suspension of Cunninghamella echinulata was inoculated into a liquid medium and cultured for 2 hours under the conditions of a pH of 6, a temperature of 31 °C, and 200 r / min; the cultured Cunninghamella echinulata was evenly inoculated on a plate and irradiated with ultraviolet light with a wavelength of 260 nm for 60 s to obtain mutant Cunninghamella echinulata; the mutant Cunninghamella echinulata was inoculated on a PDA medium containing 5 mg / L of chromium chloride for culture; each colony of mutant Cunninghamella echinulata with organic chromium on the PDA medium was divided into two parts to obtain organic chromium mutant Cunninghamella echinulata a and organic chromium mutant Cunninghamella echinulata b; the organic chromium mutant Cunninghamella echinulata a and the mutant Cunninghamella echinulata were transferred to a PDA medium containing 100 mg / L of chromium chloride for culture; there were no surviving colonies of organic chromium mutant Cunninghamella echinulata a; the surviving mutant Cunninghamella echinulata and organic chromium mutant Cunninghamella echinulata b were subjected to protoplast fusion, and the fused mutant Cunninghamella echinulata was subjected to stability verification. Example 2

[0040] This example provides a method for preparing dry bacterial powder of a strain resistant to high-concentration chromium solution, which includes the following steps: Step a): Prepare a suspension of the chromium-resistant solution strain cultivated by the method for mutagenic cultivation of a chromium-resistant solution strain described in Example 1, and inoculate it into a slant medium for activation; Step b): Transfer the activated chromium-resistant solution bacteria to an eggplant bottle for subculture; Step c): Transfer the chromium-resistant solution bacteria in the eggplant bottle to a seed tank for subculture; Step d): Transfer the chromium-resistant solution bacteria in the well-growing seed tank to a fermentation tank for subculture; Step e): After collecting the strains, wash them with water and obtain dry bacterial powder by low-temperature freeze-drying method.

[0041] Among them, the specific process of preparing the suspension in step a) is as follows: The spore powder is dissolved in sterile physiological saline containing polysorbate 80, filtered through a bacterial sieve with 350 to 400 meshes, filtered into a conical flask, the cell count of the filtrate is carried out, and it is diluted to 10 6 -10 7 CFU / ml with physiological saline in a sterile centrifuge tube.

[0042] Among them, the slant medium includes sucrose or glucose with a mass percentage of 2-4%, soybean cake powder or peptone with a mass percentage of 0.1-0.25%, magnesium sulfate with a mass percentage of 0.01-0.03%, potassium chloride with a mass percentage of 0.01-0.03%, and zinc sulfate with a mass percentage of 0.0005-0.001%.

[0043] Among them, the bacterial strain prepared by the method described in Example 1 is subjected to fermentation culture to obtain dry bacterial powder rich in various bioactive substances such as γ-linolenic acid, linoleic acid, and organic chromium.

[0044] Among them, the low-temperature freeze-drying method reduces the oxidation degree of lipids and ensures the product quality.

[0045] Using the method provided in this example, the polyunsaturated fatty acids produced by the mutagenic strain used can reach 12-15% with γ-linolenic acid as an example, and up to 18% at most, calculated by fatty acid content. At the same time, it has the function of being rich in chromium. Experiments prove that a variety of metabolites with physiological functions and their synergistic effects can significantly improve the body's immunity and the efficacy of preventing and treating diseases. The preparation process is simple, the production cycle is short, and the whole production is environmentally friendly and pollution-free. Methods such as variable temperature and variable air are used in the production to increase the content of effective combinations.

[0046] In some specific embodiments, the culture medium in the seed tank in step c) comprises sucrose or glucose at 2-4% by mass, soybean cake powder or peptone at 0.1-0.25% by mass, magnesium sulfate at 0.01-0.03% by mass, potassium chloride at 0.01-0.03% by mass, and zinc sulfate at 0.0005-0.001% by mass, with a pH of 5.5-6, a culture temperature of 28-30 °C, a ventilation rate of 1:0.05-0.3 v / v, a stirring speed of 180-250 rpm, and a culture time of 18-24 hours.

[0047] In some specific embodiments, the culture medium in the fermenter in step d) comprises molasses or sucrose at 8-12% by mass, soybean cake powder at 0.8-1.5% by mass, magnesium sulfate at 0.01-0.03% by mass, potassium chloride at 0.01-0.03% by mass, zinc sulfate at 0.0005-0.001% by mass, and chromium trichloride at 0.0005-0.0001% by mass, with a pH of 5.5-6, a culture temperature of 25-28 °C, a ventilation rate of 0.05 v / v-0.5 v / v, a stirring speed of 150-180 rpm, and a culture time of 80-96 hours.

[0048] Exemplarily, for the preparation method by shake flask fermentation, the slant culture is inoculated into the seed culture medium. The seed culture medium is: glucose at 2% by mass, diammonium hydrogen phosphate at 0.02% by mass, magnesium sulfate at 0.03% by mass, potassium chloride at 0.02% by mass, trace zinc sulfate, and cultured with shaking at 30 °C for 24 hours. The seed liquid is transferred to the fermentation culture medium. The culture medium is: molasses at 10% by mass, diammonium hydrogen phosphate at 0.025% by mass, magnesium sulfate at 0.03% by mass, potassium chloride at 0.03% by mass, trace zinc sulfate, and cultured with shaking at 28 °C for 96 hours. The oil containing γ-linolenic acid and linoleic acid is obtained by the petroleum ether extraction method.

[0049] Exemplarily, for the preparation method using a 2:500-liter fermenter, the slant culture is placed in a flask for culture. The seed liquid is transferred to the fermentation culture medium, and 350 liters of the culture medium is charged into the 500-liter fermenter. The culture medium is: glucose at 8% by mass, peptone at 2% by mass, magnesium sulfate at 0.03% by mass, potassium chloride at 0.03% by mass, trace zinc sulfate, cobalt trichloride at 0.0005% by mass, at a temperature of 25-28 °C, a stirring speed of 200-250 rpm, and a ventilation rate of 1:0.1-0.3 v / v for fermentation for 120 hours. The bacterial cells are collected, washed with water, and freeze-dried to obtain 15 kg of dry bacterial powder, with a fat content of 38% and γ-linolenic acid of 12.2%.

[0050] Exemplarily, for the preparation method of a 2:500-liter fermentation tank, several spore suspensions are prepared by inoculating slant culture bacteria into a 500-liter seed tank with 350 liters of feedstock. The culture medium is as follows: 14 kg of oligosaccharide, 88 g of diammonium hydrogen phosphate, 105 g of magnesium sulfate, 91 g of potassium chloride, with a pH of 5.5 - 6, a culture temperature of 25 - 28°C, an air volume ratio of 1:0.083 v / v, a stirring speed of 150 rpm, and cultured for 36 hours. The well-developed seed liquid is transferred to the fermentation tank. The volume of the fermentation tank is 5 tons, with 3.5 tons of feedstock. The culture medium is 350 kg of molasses, 30 kg of protein powder, 0.88 kg of diammonium hydrogen phosphate, 1.05 kg of magnesium sulfate, 0.91 kg of potassium chloride, 3.5 g of zinc sulfate, and 3 g of chromium trichloride. The culture temperature is 25 - 28°C, the air volume ratio is 1:0.25 v / v, the stirring speed is 180 rpm, and the culture time is 96 hours. The bacteria are collected, washed with water, and freeze-dried to obtain 112 kg of bacterial powder, with a fat content of 35%, γ-linolenic acid of 14.3%, and chromium of 35.2 mg / g. Example 3

[0051] This example provides a dry bacterial powder of a strain resistant to high-concentration chromium solution, which is prepared by the method described in Example 2 and includes linolenic acid, γ-linolenic acid, organic chromium, proteoglycan, amino acids, and superoxide dismutase.

[0052] In some specific embodiments, it also includes fat, crude protein, and amino acids.

[0053] Among them, per 100 grams of dry bacterial powder, it contains 30 - 40 g of fat, 18 - 24 g of crude protein, 3.5 - 60 g of γ-linolenic acid, 8.5 - 150 g of linoleic acid, 300 - 500 mg of chromium, 3.8 - 6.0 g of amino acids, 4 - 8 g of proteoglycan, and 600 - 800 mg / g of superoxide dismutase.

[0054] Among them, γ-linolenic acid (r-Linotelicacid) is an octadecatrienoic acid and is an essential fatty acid for the human body. It is a precursor for synthesizing prostaglandins and has important physiological functions in the body. The lack of γ-linolenic acid in the human body can lead to metabolic disorders and cause various diseases, such as hyperlipidemia, hypertension, diabetes, cancer, etc.

[0055] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for mutagenic cultivation of strains resistant to high-concentration chromium solution, characterized in that, It includes the following steps: Step 1), prepare a suspension of the original strain Cunninghamella echinulata AS.3.2473 provided by the National Center for Microbial Culture Collection at a concentration of 10 6 -10 7 CFU / ml; Step 2), inoculate the suspension prepared in the above step 1) into a liquid medium, and culture it for 2 - 4 hours under the conditions of a pH of 5.5 - 6, a temperature of 28 - 31 °C, and 100 - 200 r / min; Step 3), evenly inoculate the Cunninghamella echinulata cultured in step 2) on a plate, and irradiate it with ultraviolet light with a wavelength of 245 - 260 nm for 60 - 220 s to obtain mutant Cunninghamella echinulata; Step 4), divide the mutant Cunninghamella echinulata obtained in step 3) into two equal parts on average. One part is inoculated on a PDA medium for culture, and the concentration of chromium chloride in the PDA medium is 1 - 5 mg / L; the other part is reserved; Step 5), divide each mutant Cunninghamella echinulata colony with organic chromium on the PDA medium cultured in step 4) into two equal parts on average to obtain organic chromium mutant Cunninghamella echinulata a and organic chromium mutant Cunninghamella echinulata b; Step 6), transfer the organic chromium mutant Cunninghamella echinulata a obtained in step 5) and the mutant Cunninghamella echinulata reserved in step 4) to a PDA medium containing chromium chloride at a concentration of 10 - 100 mg / L for culture; Step 7), for the organic chromium mutant Cunninghamella echinulata a cultured in step 6), if there are surviving colonies, perform step 8); if there are no surviving colonies, perform step 9); Step 8), perform stability verification on the organic chromium mutant Cunninghamella echinulata a obtained in step 7); Step 9), perform protoplast fusion on the surviving mutant Cunninghamella echinulata after culturing in step 6) and the organic chromium mutant Cunninghamella echinulata b obtained in step 5) to obtain a strain resistant to high - concentration chromium solution, and perform stability verification.

2. The method for mutagenic cultivation of a strain resistant to high-concentration chromium solution according to claim 1, characterized in that, The liquid medium in step 2) is a potato extract added with a carbon source, a nitrogen source, and inorganic salts.

3. A method for mutagenic cultivation of a strain resistant to high-concentration chromium solution according to claim 2, characterized in that, The liquid medium includes a carbon source with a mass percentage of 2 - 4%, a nitrogen source with a mass percentage of 0.1 - 0.25%, inorganic salts with a mass percentage of 0.01 - 0.07%, and the rest is potato extract.

4. A method for mutagenic cultivation of a strain resistant to high-concentration chromium solution according to claim 3, characterized in that, The carbon source includes glucose, sucrose, molasses; the nitrogen source includes soybean cake powder, peptone; the inorganic salts include potassium, magnesium, calcium, zinc.

5. A method for mutagenic cultivation of a strain resistant to high-concentration chromium solution according to claim 1, characterized in that, The protoplast fusion in step 9) is specifically as follows, Inoculate the viable mutant Cunninghamella echinulata and the organic chromium mutant Cunninghamella echinulata b into the seed medium respectively, and culture them with shaking at 28 °C for 40 - 48 h. Collect the thalli in the logarithmic growth phase and prepare a bacterial suspension with a concentration of 10 6 -10 7 cells / ml; Add an equal volume of a 1 - 5 mg / L lysozyme solution to the bacterial suspension, mix evenly, water - bath at 30 - 40 °C for 1 - 2 h, filter the enzyme - digested solution through absorbent cotton, collect the filtrate, centrifuge to discard the supernatant, wash the lower layer substance twice with the preservation solution, and then resuspend and mix it evenly in 1 ml of the preservation solution to obtain a protoplast suspension; Perform ultraviolet inactivation on the obtained protoplast suspension. Irradiate it with a 25 - 40 w ultraviolet lamp at a distance of 30 - 40 cm above the petri dish containing the protoplast suspension for 1 - 2 min; perform heat inactivation on the organic chromium mutant Cunninghamella echinulata b protoplast suspension by placing the organic chromium mutant Cunninghamella echinulata b protoplast suspension at 60 - 80 °C for 10 - 30 min; Mix the inactivated two kinds of protoplasts in equal volume, add a solubilizer, and let it stand and culture at 28 - 30 °C for 10 - 20 min to obtain protoplast fusants. Centrifuge to discard the supernatant, wash the lower layer material twice with the preservation solution and then place it in the preservation solution. Inoculate the fusants into the regeneration medium and culture at 28 - 30 °C for 7 - 10 days to obtain single colonies of fusants.

6. Application of the strain resistant to high - concentration chromium solution cultivated by the method for mutagenic cultivation of the strain resistant to high - concentration chromium solution according to any one of claims 1 to 5 in the preparation of dry bacterial powder, wherein the dry bacterial powder comprises linolenic acid, γ - linolenic acid, organic chromium, proteoglycan, amino acids and superoxide dismutase.

7. Use of a strain resistant to high-concentration chromium solution according to claim 6 in the preparation of dry bacterial powder, characterized in that, The preparation method comprises the following steps: Step a), make the strain resistant to high - concentration chromium solution cultivated by the method for mutagenic cultivation of the strain resistant to high - concentration chromium solution according to any one of claims 1 to 5 into a suspension, and inoculate it onto the slant medium for activation; Step b), transfer the activated strain resistant to high - concentration chromium solution to an eggplant bottle for sub - culture; Step c), transfer the strain resistant to high - concentration chromium solution in the eggplant bottle to a seed tank for sub - culture; Step d), transfer the strain resistant to high - concentration chromium solution in the seed tank to a fermentation tank for sub - culture; Step e), after collecting the sub - cultured strains, wash them with water and obtain dry bacterial powder by low - temperature freeze - drying method.

8. A method for preparing a dry bacterial powder of a strain resistant to high-concentration chromium solution according to claim 7, characterized in that, The medium in the seed tank in step c) comprises sucrose or glucose with a mass percentage of 2 - 4%, soybean cake powder or peptone with a mass percentage of 0.1 - 0.25%, magnesium sulfate with a mass percentage of 0.01 - 0.03%, potassium chloride with a mass percentage of 0.01 - 0.03%, and zinc sulfate with a mass percentage of 0.0005 - 0.001%, the pH is 5.5 - 6, the culture temperature is 28 - 30 °C, the air volume is 1:0.05 - 0.3 v / v, the stirring speed is 180 - 250 rpm, and the culture time is 18 - 24 hours.

9. The preparation method of the dry bacterial powder of the strain resistant to high-concentration chromium solution according to claim 7, characterized in that, The medium in the fermentation tank in step d) comprises molasses or sucrose with a mass percentage of 8 - 12%, soybean cake powder with a mass percentage of 0.8 - 1.5%, magnesium sulfate with a mass percentage of 0.01 - 0.03%, potassium chloride with a mass percentage of 0.01 - 0.03%, zinc sulfate with a mass percentage of 0.0005 - 0.001% and chromium trichloride with a mass percentage of 0.0005 - 0.001%, the pH is 5.5 - 6, the culture temperature is 25 - 28 °C, the air volume is 0.05 v / v - 0.5 v / v, the stirring speed is 150 - 180 rpm, and the culture time is 80 - 96 hours.

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