Mutagenesis screening method and application of L-sorbose fermentation high-temperature-resistant strain

Abstract

The invention relates to the technical field of bioengineering, in particular to a mutagenesis screening method and application of an L-sorbose fermentation high-temperature-resistant strain, and the mutagenesis screening method comprises the following steps: (1) mutagenizing a Gluconobacter oxydans strain to obtain a mutagenesis strain; (2) carrying out enlarged culture on the mutant strain to obtain a single colony of the mutant strain; (3) carrying out amplification culture on the single colony of the mutant strain to obtain a mutant strain lawn; (4) carrying out enlarged culture on the mutagenesis strain lawn to obtain a mutagenesis strain culture solution; and (5) carrying out fermentation shake-flask screening on the mutant strain culture solution to obtain the L-sorbose fermentation high-temperature-resistant strain. According to the scheme, the strain with the fermentation temperature increased by 6 DEG C compared with the original strain can be bred, the production technology is simple, the process is environmentally friendly, efficiency is high, and the method has remarkable advantages in the aspect of L-sorbose production.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a mutagenesis screening method for a thermostable L-sorbose fermentation strain and its application. Background Art

[0002] Vitamin C is an essential water-soluble vitamin for the human body. It not only possesses potent antioxidant activity, scavenging free radicals, promoting collagen biosynthesis, and enhancing immune function, but also acts as a coenzyme in various redox reactions. Due to its unique physical and chemical properties, it is widely used in diverse applications, including medicine, the food industry, cosmetic formulations, and animal husbandry.

[0003] The current mainstream production process of vitamin C is a two-step fermentation method. The first step is to catalyze the conversion of D-sorbitol into L-sorbose by Gluconobacter oxydans; the second step is to use a mixed bacteria fermentation system, in which positive Bacillus (Bacillus spp.) and common ketogenic gulonic acid bacteria (KetoguLonicigeniumvuLgare) work together to convert L-sorbose into 2-keto-L-gulonic acid, the precursor of vitamin C.

[0004] The first step is aerobic fermentation, during which a large amount of bioheat is generated by bacterial metabolism. The temperature needs to be precisely controlled through the cooling water circulation system to maintain the catalytic activity of Gluconobacter oxidans. After the first fermentation step is completed, the L-sorbose fermentation broth needs to be pasteurized to completely inactivate the remaining production bacteria. Otherwise, the residual bacteria will interfere with the substrate selectivity of the second fermentation step. This process characteristic leads to a contradiction in energy consumption: in the early stage, cooling water is relied upon to remove heat, and in the later stage, steam heating is required for sterilization. The alternating hot and cold cycles increase the overall energy consumption. If the temperature resistance of the bacteria can be improved, the amount of cooling water and the demand for sterilization steam can be reduced simultaneously, thereby significantly reducing production energy consumption.

[0005] Current production strains used in the first fermentation step perform poorly when the fermentation temperature is increased, resulting in a decrease in the conversion efficiency of L-sorbose. There is an urgent need to screen for thermotolerant strains through strain improvement to achieve energy conservation. Traditional strain selection techniques are limited by the high toxicity of chemical mutagens, high wastewater treatment costs, and significant operational risks. For example, CN 102660599 B discloses a method for producing L-sorbose by fermentation using self-cloned engineered bacteria, belonging to the field of optimizing fermentation processes using metabolic engineering control strategies. The present invention utilizes metabolic engineering to obtain a self-cloned engineered strain of Gluconobacter oxydans, which serves as the production strain. Through fermentation process optimization, the activity of sorbitol dehydrogenase is increased by 1.33 times. Using sorbitol as the sole carbon source, after 36 hours of fermentation, L-sorbose production reaches 225 g / L, shortening the fermentation time by 12 hours compared to the starting strain. However, the fermentation temperature is only 30°C, which does not alleviate the aforementioned problems. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a mutagenesis screening method and application of a high-temperature-resistant strain for L-sorbose fermentation, which can select a strain whose fermentation temperature is 6°C higher than that of the starting strain. The production process is simple, the process is environmentally friendly, and the efficiency is high, which has significant advantages in L-sorbose production.

[0007] To achieve the above object, the technical solution of the present invention is: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In the step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing the Gluconobacter oxidans strain into a bacterial suspension of 105 cfu / mL to 106 cfu / mL, subjecting the suspension to ionization mutagenesis at a temperature of 25°C to 35°C, and placing the suspension in physiological saline after mutagenesis, shaking the suspension, and obtaining the mutagenized strain; The ionization mutagenesis process is as follows: atomizing the bacterial suspension under a helium atmosphere, with a radio frequency power input power of 80W-100W, a treatment time of 10s-20s, a lethality of 85%-98%, and then condensing to obtain a mutagenized bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and cultured for 36 hours under the condition of pH 4.8-5.8 in stages with temperature control, and the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The process of the segmented temperature-controlled culture is as follows: the culture temperature is controlled at 27° C. to 36° C. from 0 h to 16 h; and the culture temperature is controlled at 36° C. to 42° C. from 16 h to 36 h.

[0008] To achieve better mutagenesis, this approach pioneered the atomization of the bacterial suspension, which was done to make the electric field distribution more uniform and improve the mutagenesis efficiency. At the same time, ionization was carried out in helium, primarily to protect the strain. During the atomization ionization process, the surfaces of the atomized droplets have the same charge, which creates a certain repulsive effect and improves the uniformity of ionization mutagenesis. During the condensation process, the charge on the surface of the atomized droplets will be carried away by the wall of the condensation container. Traditional ionization times are generally over 40 seconds. This approach can reduce radio power and shorten ionization time by atomizing the bacterial suspension.

[0009] Preferably, in step (2), the process of expanding the culture of the induced strain is as follows: applying the induced strain obtained in step (1) to a solid culture medium, culturing for 24 h to 96 h at pH 4.8 to 5.8 and 27° C. to 37° C., and growing the induced strain into a single colony; The culture medium formula comprises: 50 mg / mL to 80 mg / mL of D-sorbitol, 0.1 mg / mL to 5 mg / mL of corn steep liquor, 0.1 mg / mL to 5 mg / mL of yeast extract, 0.1 mg / mL to 5 mg / mL of peptone, 0.1 mg / mL to 5 mg / mL of calcium carbonate, 0.001 mg / mL to 0.05 mg / mL of defoaming agent, and 10 mg / mL to 20 mg / mL of agar.

[0010] Preferably, in step (3), the process of expanding the culture of the single colony of the induced strain is as follows: the single colonies of the induced strain obtained in step (2) are inoculated into the proliferation culture medium one by one, and expanded and cultured for 24 hours to 72 hours at pH 4.8 to 5.8 and 27° C. to 37° C. to obtain a bacterial lawn of the induced strain; The proliferation culture medium comprises: 80 mg / mL to 150 mg / mL of D-sorbitol, 3 mg / mL to 8 mg / mL of corn steep liquor, 3 mg / mL to 8 mg / mL of yeast extract, 3 mg / mL to 8 mg / mL of peptone, 3 mg / mL to 8 mg / mL of calcium carbonate, 0.01 mg / mL to 0.3 mg / mL of defoaming agent, and 10 mg / mL to 20 mg / mL of agar.

[0011] Preferably, in step (4), the process of expanding the culture of the induced strain lawn is as follows: inoculating the induced strain lawn obtained in step (3) into a proliferation culture solution, expanding the culture at pH 4.8 to 5.8, 27° C. to 37° C. for 8 h to 48 h to obtain a culture solution of the induced strain; The proliferation culture fluid has a formula of 150 mg / mL to 200 mg / mL of D-sorbitol, 5 mg / mL to 10 mg / mL of corn steep liquor, 5 mg / mL to 10 mg / mL of yeast extract, 5 mg / mL to 10 mg / mL of peptone, 5 mg / mL to 10 mg / mL of calcium carbonate, and 0.05 mg / mL to 0.1 mg / mL of defoaming agent.

[0012] Preferably, in step (5), the formula of the fermentation culture medium is: D-sorbitol 200 mg / mL to 300 mg / mL, corn steep liquor 8 mg / mL to 10 mg / mL, yeast extract 8 mg / mL to 10 mg / mL, peptone 8 mg / mL to 10 mg / mL, calcium carbonate 8 mg / mL to 10 mg / mL, and defoaming agent 0.08 mg / mL to 0.1 mg / mL.

[0013] This scheme designs culture materials according to different culture purposes. For example, steps (2) and (3) are mainly for preliminary screening, so culture medium is selected and the concentration of prepared nutrients is also relatively low; steps (4) and (5) are mainly for expanding the number of strains, especially step (5) requires further screening of the expanded strains, so the nutrient concentration is also relatively high.

[0014] Preferably, the concentration of the bacterial suspension after atomization is 2 to 3 mg / m3.

[0015] The atomized concentration of the bacterial suspension should not be too low, as too low a concentration will result in a high lethality rate and difficulty in selecting the target bacteria; too low a concentration will result in a poor mutagenic effect.

[0016] Preferably, the bacterial suspension comprises a food-grade nonionic surfactant at a concentration of 0.001 mg / mL-0.004 mg / mL.

[0017] The main function of food-grade non-ionic surfactants is to stabilize the atomized droplets and adjust the electric field distribution of the droplets. Polysorbate 80 and the like can be used.

[0018] This proposal also proposes a mutagenesis screening method for the above-mentioned thermostable L-sorbose fermentation strain, which is applied to the preparation of an L-sorbose solution, comprising the following steps: Step (a): preparing a thermostable L-sorbose fermentation strain according to a mutagenesis screening method for thermostable L-sorbose fermentation strains; Step (b): inoculating the L-sorbose fermentation thermostable strain into a proliferation culture medium, and expanding the culture at pH 4.8-5.8, 27°C-37°C, for 8h-48h to obtain a seed culture medium; Step (c): transferring the inoculum solution to the culture medium of the fermentation tank, culturing the culture medium at pH 4.8-5.8 for 36 hours under staged temperature control, and obtaining the L-sorbose dissolved solution after pasteurization.

[0019] Preferably, in step (c), the formula of the fermentation tank culture medium is: D-sorbitol 200mg / mL~300mg / mL, corn steep liquor 8mg / mL~10mg / mL, yeast extract 8mg / mL~10mg / mL, peptone 8mg / mL~10mg / mL, calcium carbonate 8mg / mL~10mg / mL, and defoaming agent 0.08mg / mL~0.1mg / mL.

[0020] Preferably, in step (c), the process of segmented temperature-controlled culture is as follows: the culture temperature is controlled at 27°C to 36°C from 0h to 16h; and the culture temperature is controlled at 36°C to 42°C from 16h to 36h.

[0021] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention provides a method for screening mutagenized strains of Gluconobacter oxydans using plasma mutagenesis equipment, using high-temperature fermentation. This method effectively increases the probability of obtaining highly effective strains. The optimal strain of Gluconobacter oxydans obtained through screening increased the fermentation temperature by 6°C, saving 15% of cooling water consumption and 12.5% of steam consumption during the production process. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the examples of implementation of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained based on the embodiments of the present invention without making creative work in the art are within the scope of protection of the present invention.

[0023] Overall embodiment: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing the Gluconobacter oxidans strain into a bacterial suspension of 105 cfu / mL to 106 cfu / mL, subjecting the suspension to ionization mutagenesis at a temperature of 25°C to 35°C, and placing the suspension in physiological saline after mutagenesis, shaking the suspension, and obtaining the mutagenized strain; The ionization mutagenesis process is as follows: in a helium atmosphere, the bacterial suspension is atomized to a concentration of 2 to 3 mg / m 3 , RF power input power 80W-100W, treatment time 10s-20s, lethality 85%-98%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and cultured in sections under the condition of pH 4.8-5.8 for 36 hours, and the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The process of staged temperature-controlled culture is as follows: the culture temperature is controlled at 27℃~36℃ from 0h to 16h; the culture temperature is controlled at 36℃~42℃ from 16h to 36h; In step (2), the process of expanding the culture of the induced strain is as follows: the induced strain obtained in step (1) is spread on a solid culture medium, cultured at pH 5.2, 30°C for 24 hours, and grown into a single colony of the induced strain; the culture medium formula is 60 mg / mL of D-sorbitol, 3 mg / mL of corn steep liquor, 3 mg / mL of yeast extract, 3 mg / mL of peptone, 3 mg / mL of calcium carbonate, 0.01 mg / mL of defoaming agent, and 20 mg / mL of agar; In step (3), the process of expanding the culture of the single colony of the induced strain is as follows: the single colonies of the induced strain obtained in step (2) are inoculated into the proliferation culture medium one by one, and expanded and cultured at pH 5.2, 32°C for 48 hours to obtain the induced strain moss; The proliferation medium comprises 100 mg / mL of D-sorbitol, 5 mg / mL of corn steep liquor, 5 mg / mL of yeast extract, 5 mg / mL of peptone, 5 mg / mL of calcium carbonate, 0.05 mg / mL of defoaming agent, and 20 mg / mL of agar. In step (4), the process of expanding the culture of the induced strain moss is as follows: the induced strain moss obtained in step (3) is inoculated into the proliferation culture medium, and expanded and cultured for 36 hours under the conditions of pH 5.4 and 35°C to obtain the induced strain culture medium; The formula of the proliferation culture medium is: D-sorbitol 180 mg / mL, corn steep liquor 8 mg / mL, yeast extract 8 mg / mL, peptone 8 mg / mL, calcium carbonate 8 mg / mL, defoamer 0.08 mg / mL; In step (5), the formula of the fermentation culture medium is: D-sorbitol 250 mg / mL, corn steep liquor 9 mg / mL, yeast extract 9 mg / mL, peptone 9 mg / mL, calcium carbonate 9 mg / mL, and defoaming agent 0.09 mg / mL.

[0024] Example 1: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , RF power input power 90W, treatment time 15s, lethality 95%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0025] Example 2: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of 105 cfu / mL of Gluconobacter oxidans, ionizing and mutagenizing at a temperature of 30°C, and then placing the suspension in physiological saline and shaking to obtain a mutagenized strain; the bacterial suspension includes food-grade polysorbate 80 at a concentration of 0.003 mg / mL; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , RF power input power 90W, treatment time 15s, lethality 97%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0026] Example 3: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing the Gluconobacter oxidans strain into a bacterial suspension of 106 cfu / mL, ionizing and mutagenizing the suspension at a temperature of 32°C, and then placing the suspension in physiological saline and shaking the suspension to obtain a mutagenized strain; the bacterial suspension includes food-grade polysorbate 80 at a concentration of 0.004 mg / mL; The ionization mutagenesis process is as follows: in a helium atmosphere, the bacterial suspension is atomized to a concentration of 2 mg / mL.3 , RF power input power 95W, treatment time 10s, lethality 98%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.4, the culture temperature is controlled at 30°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation temperature-resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0027] Comparative Example 1: The difference from Example 1 is that, without ionization mutagenesis: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans at a concentration of 105 cfu / mL, placing the suspension in physiological saline, and shaking the suspension to obtain a mutagenized strain; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , and then condense to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0028] Comparative Example 2: The difference from Example 1 is that it is not atomized: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: in a helium atmosphere, the bacterial suspension is spread on a glass slide at a density of 2.5 mg / m 2 , RF power input power 90W, treatment time 15s, lethality 75%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0029] Comparative Example 3: The difference from Example 1 is that step (5) is not cultured in segmented temperature control: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , RF power input power 90W, treatment time 15s, lethality 95%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, under the condition of pH 5.2, the culture temperature is controlled at 42°C from 0h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0030] Comparative Example 4: The difference from Example 1 is that the temperature of the segmented temperature control culture in step (5) is too high: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , RF power input power 90W, treatment time 15s, lethality 95%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 48°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0031] Comparative Example 5: The difference from Example 1 is that the temperature of the segmented temperature-controlled culture in step (5) is too low: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: the bacterial suspension is atomized in a helium atmosphere, and the concentration of the bacterial suspension after atomization is 2.5 mg / m 3 , RF power input power 90W, treatment time 15s, lethality 95%, then condensation to obtain the mutagenic bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 28°C from 0h to 16h; and the culture temperature is controlled at 33°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation temperature-resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0032] Comparative Example 6: The difference from Example 1 is that the droplet concentration after atomization is low and the radio frequency is too high: A method for mutagenesis screening of a thermostable L-sorbose fermentation strain comprises the following steps: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing a bacterial suspension of Gluconobacter oxidans with a concentration of 105 cfu / mL, subjecting it to ionization mutagenesis at a temperature of 30°C, and placing it in physiological saline after mutagenesis, shaking it, to obtain a mutagenized strain; The ionization mutagenesis process is as follows: in a helium atmosphere, the bacterial suspension is atomized to a concentration of 1.5 mg / m 3 , the RF power input power is 120W, the treatment time is 30s, the lethality rate is 99.9%, and then the mutagenic bacterial solution is obtained by condensation; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and under the condition of pH 5.2, the culture temperature is controlled at 32°C from 0h to 16h; and the culture temperature is controlled at 42°C from 16h to 36h, the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The culture medium and culture solution formula used in steps (2) to (5) are the same as those in the general embodiment.

[0033] Experimental test: The test subjects were the starting strain (i.e., the unmutated Glucose Bacillus oxydans strain) and the optimal strains screened by mutagenesis in Examples 1-3 and Comparative Examples 1-6; 1. Mutagenesis effect detection: The mutagenesis screening method of the thermostable L-sorbose fermentation strains of Examples 1-3 and Comparative Examples 1-6 was applied to prepare L-sorbose solution, and the results were used to characterize the mutagenesis effect, comprising the following steps: Step (a): preparing a thermostable L-sorbose fermentation strain according to a mutagenesis screening method for thermostable L-sorbose fermentation strains; Step (b): inoculating the L-sorbose fermentation thermostable strain into a proliferation culture medium, and expanding the culture at pH 5.2 and 30°C for 36 hours to obtain a seed culture medium; Step (c): transferring the inoculum solution to a fermentation tank culture medium, culturing at pH 5.2 under staged temperature control for 36 hours, and pasteurizing to obtain an L-sorbose dissolved solution; The formula of the proliferation culture medium is: D-sorbitol 180 mg / mL, corn steep liquor 8 mg / mL, yeast extract 8 mg / mL, peptone 8 mg / mL, calcium carbonate 8 mg / mL, defoamer 0.08 mg / mL; The fermentation broth formula is: D-sorbitol 250 mg / mL, corn steep liquor 10 mg / mL, yeast extract 10 mg / mL, peptone 10 mg / mL, calcium carbonate 10 mg / mL, defoamer 0.1 mg / mL; The process of segmented temperature-controlled culture is as follows: the culture temperature is controlled at 32°C from 0h to 16h; the culture temperature is controlled at 42°C from 16h to 36h; 2. High temperature resistance test: The L-sorbose fermentation thermotolerant strains obtained by screening in Examples 1-3 and Comparative Examples 1-6 were spread on solid culture medium and cultured at pH 5.2, 36°C / 39°C / 42°C for 24 h, and the number of colonies grown was compared; the culture medium formula was 60 mg / mL of D-sorbitol, 3 mg / mL of corn steep liquor, 3 mg / mL of yeast extract, 3 mg / mL of peptone, 3 mg / mL of calcium carbonate, 0.01 mg / mL of defoamer, and 20 mg / mL of agar for comparison.

[0034] The test results are shown in Table 1.

[0035] Table 1

[0036] The fermentation temperature of the strains 1-3 screened by mutagenesis was 6° C. higher than that of the starting strain, saving 15% of cooling water consumption and 12.5% of steam consumption in the production process.

[0037] Comparative Example 1 was not ionized and mutagenized, and was essentially the same as the starting bacteria.

[0038] The difference between Comparative Example 2 and Example 1 is that the atomization treatment was not performed. This may be due to the uneven mutagenesis caused by coating the glass slide with the bacterial suspension, low lethality, insufficient screening pressure, and low mutant diversity, resulting in the fermentation efficiency not being as good as Example 1. However, the starting efficiency is higher than that of Example 1 and the high temperature resistance is better.

[0039] The difference between Comparative Example 3 and Example 1 is that the high temperature culture throughout the process may have led to early growth of the bacteria, resulting in insufficient bacterial quantity and limited L-sorbose synthesis, resulting in fermentation efficiency lower than that of Example 1.

[0040] The difference between Comparative Example 4 and Example 1 is that the temperature in the latter stage is too high, resulting in a small amount of viable bacteria and failure to achieve efficient conversion.

[0041] The difference between Comparative Example 5 and Example 1 is that the segmented temperature control culture is too low, and the 33°C during the 16h to 36h culture period cannot screen high-temperature resistant strains, the strains have poor adaptability, and the efficiency of the high-temperature stage is low.

[0042] The difference between Comparative Example 6 and Example 1 is that the atomization concentration is low, the radio frequency is too strong, the lethality is too high, the number of live bacteria is small, and effective mutants are not necessarily obtained.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for mutagenesis screening of thermotolerant L-sorbose fermentation strains, characterized in that: The following steps are involved: Step (1), mutagenizing the Gluconobacter oxidans strain to obtain a mutagenized strain; Step (2), expanding the culture of the induced strain to obtain a single colony of the induced strain; Step (3), expanding and culturing a single colony of the induced strain to obtain a bacterial lawn of the induced strain; Step (4), expanding the culture of the mutant strain to obtain a culture solution of the mutant strain; Step (5), screening the culture broth of the induced strain by fermentation in shake flasks to obtain a thermostable strain capable of fermenting L-sorbose; In the step (1), the method for mutagenizing the Gluconobacter oxidans strain is as follows: preparing the Gluconobacter oxidans strain into a bacterial suspension of 105 cfu / mL to 106 cfu / mL, subjecting the suspension to ionization mutagenesis at a temperature of 25°C to 35°C, and placing the suspension in physiological saline after mutagenesis, shaking the suspension, and obtaining the mutagenized strain; The ionization mutagenesis process is as follows: atomizing the bacterial suspension under a helium atmosphere, with a radio frequency power input power of 80W-100W, a treatment time of 10s-20s, a lethality of 85%-98%, and then condensing to obtain a mutagenized bacterial solution; In step (5), the fermentation shake flask screening method is as follows: the culture fluid of the induced strain obtained in step (4) is respectively inoculated into the fermentation culture fluid, and cultured for 36 hours under the condition of pH 4.8-5.8 in stages with temperature control, and the conversion rate of L-sorbose in each fermentation culture fluid is calculated, and the fermentation culture fluid with high conversion efficiency is selected to obtain the L-sorbose fermentation high-temperature resistant strain; The process of the segmented temperature-controlled culture is as follows: the culture temperature is controlled at 27° C. to 36° C. from 0 h to 16 h; and the culture temperature is controlled at 36° C. to 42° C. from 16 h to 36 h.

2. The method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to claim 1, wherein: In step (2), the process of expanding the culture of the induced strain is as follows: applying the induced strain obtained in step (1) onto a solid culture medium, culturing at pH 4.8 to 5.8 and 27°C to 37°C for 24 to 96 hours, and growing the induced strain into a single colony; The culture medium formula comprises: 50 mg / mL to 80 mg / mL of D-sorbitol, 0.1 mg / mL to 5 mg / mL of corn steep liquor, 0.1 mg / mL to 5 mg / mL of yeast extract, 0.1 mg / mL to 5 mg / mL of peptone, 0.1 mg / mL to 5 mg / mL of calcium carbonate, 0.001 mg / mL to 0.05 mg / mL of defoaming agent, and 10 mg / mL to 20 mg / mL of agar.

3. The method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to claim 1, wherein: In the step (3), the process of expanding the culture of the single colony of the induced strain is as follows: the single colonies of the induced strain obtained in the step (2) are inoculated into the proliferation culture medium one by one, and expanded and cultured for 24 hours to 72 hours at a pH of 4.8 to 5.8 and a temperature of 27° C. to 37° C. to obtain a bacterial lawn of the induced strain; The proliferation culture medium comprises: 80 mg / mL to 150 mg / mL of D-sorbitol, 3 mg / mL to 8 mg / mL of corn steep liquor, 3 mg / mL to 8 mg / mL of yeast extract, 3 mg / mL to 8 mg / mL of peptone, 3 mg / mL to 8 mg / mL of calcium carbonate, 0.01 mg / mL to 0.3 mg / mL of defoaming agent, and 10 mg / mL to 20 mg / mL of agar.

4. The method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to claim 1, wherein: In the step (4), the process of expanding the culture of the mutant strain lawn is as follows: the mutant strain lawn obtained in the step (3) is inoculated into the proliferation culture solution, and expanded and cultured for 8 hours to 48 hours at pH 4.8 to 5.8 and 27° C. to 37° C. to obtain the mutant strain culture solution; The proliferation culture fluid has a formula of 150 mg / mL to 200 mg / mL of D-sorbitol, 5 mg / mL to 10 mg / mL of corn steep liquor, 5 mg / mL to 10 mg / mL of yeast extract, 5 mg / mL to 10 mg / mL of peptone, 5 mg / mL to 10 mg / mL of calcium carbonate, and 0.05 mg / mL to 0.1 mg / mL of defoaming agent.

5. The method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to claim 1, wherein: In step (5), the formula of the fermentation culture medium is: D-sorbitol 200mg / mL~300mg / mL, corn steep liquor 8mg / mL~10mg / mL, yeast extract 8mg / mL~10mg / mL, peptone 8mg / mL~10mg / mL, calcium carbonate 8mg / mL~10mg / mL, and defoaming agent 0.08mg / mL~0.1mg / mL.

6. The method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to claim 1, wherein: The concentration of bacterial suspension after atomization is 2-3 mg / m 3 .

7. The method for mutagenesis screening of thermotolerant L-sorbose fermentation strains according to claim 1, wherein: The bacterial suspension includes a food-grade nonionic surfactant with a concentration of 0.001 mg / mL-0.004 mg / mL.

8. An application of the method for mutagenesis screening of a thermostable L-sorbose fermentation strain according to any one of claims 1 to 7, characterized in that: The method is applied to prepare L-sorbose solution, comprising the following steps: Step (a): preparing a thermostable L-sorbose fermentation strain according to a mutagenesis screening method for thermostable L-sorbose fermentation strains; Step (b): inoculating the L-sorbose fermentation thermostable strain into a proliferation culture medium, and expanding the culture at pH 4.8-5.8, 27°C-37°C, for 8h-48h to obtain a seed culture medium; Step (c): transferring the inoculum solution to the culture medium of the fermentation tank, culturing the culture medium at pH 4.8-5.8 for 36 hours under staged temperature control, and obtaining the L-sorbose dissolved solution after pasteurization.

9. Use of the method for mutagenesis screening of thermostable L-sorbose fermentation strains according to claim 8, characterized in that: In step (c), the formula of the fermentation tank culture medium is: D-sorbitol 200mg / mL~300mg / mL, corn steep liquor 8mg / mL~10mg / mL, yeast extract 8mg / mL~10mg / mL, peptone 8mg / mL~10mg / mL, calcium carbonate 8mg / mL~10mg / mL, and defoaming agent 0.08mg / mL~0.1mg / mL.

10. Use of the method for mutagenesis screening of thermostable L-sorbose fermentation strains according to claim 8, characterized in that: In step (c), the process of staged temperature-controlled culture is as follows: the culture temperature is controlled at 27° C. to 36° C. from 0 h to 16 h; and the culture temperature is controlled at 36° C. to 42° C. from 16 h to 36 h.

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