Propionibacterium capable of producing propionic acid and method for fermenting propionic acid by using propionibacterium

Abstract

The invention discloses propionibacterium capable of obtaining high-yield propionic acid through compound mutation screening and a method for fermenting propionic acid by using the propionibacterium. The high-yield propionic acid strain YCBS010 (CGMCC No.31160) is obtained by carrying out compound mutagenesis on acid-producing propionibacterium CGMCC 1.2232 serving as an original strain through microwave-ultrasonic combined mutagenesis and compound normal-pressure and room-temperature plasma (ARTP) mutagenesis means. According to the invention, YPA15 is used as a starting strain, microwave and ultrasonic combined mutagenesis is carried out for 100 seconds to screen a bacterial strain with increased propionic acid yield, then ARTP mutagenesis is carried out for 90 seconds to screen out a high-yield mutant strain YCBS010, fermentation in a 1-ton tank verifies that the highest propionic acid yield reaches 65.87 g / L and the fermentation intensity is 0.73 g / L.h after fermentation is carried out for 90 hours, and the bacterial strain has better industrial fermentation potential.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and specifically to obtaining a strain of Propionibacterium acidipropionici that produces high propionic acid by efficient screening through microwave and ultrasonic composite mutagenesis and superimposed atmospheric pressure room temperature plasma (ARTP) mutagenesis. Background Art

[0002] Propionic acid (PA) is an organic acid with three carbon atoms and one carboxylic acid. As a compound module molecule, it can participate in the synthesis of valuable products such as n-propanol, propylene, vitamin B12, antibacterial compounds (including diketopiperazine, linear and cyclic peptides, 3-phenyllactic acid), etc. At the same time, propionic acid (PA) and calcium propionate are widely used as preservatives in food and feed industry, medicine, and cosmetics. About 50% of the propionic acid produced globally is used for feed, and its use in the preservation of baked goods is another major demand in the recent market. In 2021, the global propionic acid market sales reached US$770 million, and it is expected to reach US$960 million in 2028, with a compound annual growth rate (CAGR) of 3.1% (2022-2028). In 2019, the global PA production was about 470,000 tons, and it is expected to reach 550,000 tons in 2026. The Chinese market has changed rapidly in the past few years. In 2015, the market supply of calcium acetate and calcium propionate in my country was 330,100 tons. In 2019, it was 430,000 tons, an increase of 2.58% over 2018.

[0003] The world's major propionic acid manufacturers include BASF, Dow, Perstorp and Eastman, and the top four manufacturers account for more than 70% of the market share. Currently, the Asia-Pacific region is the world's largest propionic acid market, accounting for more than 33% of the market share, followed by the US market, which accounts for nearly 30%.

[0004] Currently, the worldwide demand for propionic acid and its derivatives is met by petroleum-based conversion, which has serious consequences for climate change. Alternatives to produce PA propionic acid using renewable raw materials provide a sustainable approach. Petroleum-based propionic acid costs about $1 / kg, while the cost of propionic acid obtained through microbial fermentation is between $1.5 and $2 / kg. The higher cost of the microbial fermentation route is attributed to the high cost of fermentation raw materials, low fermentation efficiency and high extraction energy consumption. In Propionibacterium acidogenicum ATCC 4965, Coral J et al. used sodium lactate as a carbon source and obtained a propionic acid yield of 15.06 g / L and a production intensity of 0.113 g / Lh; Liu Y et al. used a mixed carbon source of glucose and glycerol to obtain a propionic acid yield of 21.90 g / L and a production intensity of 0.152 g / Lh. Propionibacterium acidigenicum ATCC 4875, Yang H et al. used fructose as a carbon source and achieved a propionic acid yield of 26.5 g / L and a production intensity of 0.159 g / Lh; Zhen L et al. used high-density culture and cell recycling to increase the propionic acid yield to 75.9 g / L and a production intensity of 0.32 g / Lh; Ma Yongliang used Propionibacterium acidigenicum FS1171 and obtained a propionic acid fermentation yield of 86.65 g / L and a production intensity of 0.52 g / Lh in 166 hours under batch feeding optimization conditions. Summary of the invention

[0005] The invention uses Propionibacterium acidipropionici CGMCC1.2232 as a starting strain, performs microwave 100 seconds and ultrasound 9 seconds combined mutagenesis to screen strains with improved propionic acid production, then performs ARTP mutagenesis for 90 seconds, screens out a high-yield mutant YCBS010, and verifies by fermentation in a 1-ton tank for 90 hours of fermentation, the highest propionic acid production reaches 65.87 g / L, and the fermentation intensity is 0.73 g / Lh, indicating that the strain has good industrial fermentation potential.

[0006] To achieve the above purpose, the present invention discloses the following technical contents: The present invention first discloses a high-yield propionic acid plant ( Propionibacterium acidipropionic i) YCBS010, characterized in that the Propionibacterium acidophilum YCBS010 is deposited in the General Microbiological Center (GCMCC) of China Culture Collection Administration, with the deposit number being CGMCC No. 31160.

[0007] By using multiple mutagenesis combined with an efficient screening method, a strain with greatly improved fermentation performance, Propionibacterium sp., was obtained. Propionibacterium sp. YCBS010 (CGMCC No. 31160) with propionic acid production was obtained. The deposit number is CGMCC No. 31160, and the deposit institution is General Microbiological Center of China General Microbiological Cultuer Collection Centre. The deposit date is July 3, 2024, and the classification name is Propionibacterium sp. It is deposited at the General Microbiological Center of China General Microbiological Cultuer Collection Centre, and the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0008] The physicochemical properties of the high-propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160) are as follows: This strain belongs to the genus Propionibacterium, is a Gram-positive, non-motile, facultative anaerobic bacterium, generally rod-shaped, non-spore-forming, and can produce propionic acid using a variety of carbon sources (glucose, fructose, mannose, glycerol, lactic acid), but cannot grow using polysaccharides (cellulose, starch, hemicellulose, etc.). For microscopic observation of this strain, please see Figure 7 The characteristics of its colonies are: the colonies are round, 0.1-1.2mm, milky white, smooth, opaque, and have neat edges; microscopic observation shows that the bacterial cells are rod-shaped, single, arranged in pairs or "V" shape, and of uneven size, 0.5-0.8×0.9-1.6 µm.

[0009] The present invention also discloses a screening method of a strain of Propionibacterium CGMCC No. 31160 with high propionic acid production obtained by composite mutagenesis screening as follows: (1) Dilute the mutagenic bacterial suspension by 10 4 Take 100 μL and apply it on the preliminary screening medium. The formula of the preliminary screening medium is: glucose 3%~4%, yeast powder 1%, peptone 0.5%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 0.05%~0.2%, pH 7.5; (2) Preliminary screening method: Culture in an anaerobic box at 32°C for 120-144 hours, measure the diameter of the colony and the diameter of the transparent zone of the same colony, and select strains with a ratio of transparent zone diameter to colony diameter greater than 3; (3) Rescreening method: The strains preliminarily screened were subjected to two rounds of anaerobic fermentation in serum bottles to finally obtain strains with high propionic acid production; the rescreening culture medium: 4% to 6% glucose, 1% yeast powder, 0.7% peptone, 0.05% magnesium sulfate, 0.15% dipotassium hydrogen phosphate, 1.5% to 2.0% calcium carbonate, pH 7; cultured in an anaerobic box at 32°C for 120 to 144 hours, and the content of propionic acid in the fermentation broth was determined.

[0010] The present invention further discloses the application of a propionic acid bacteria strain with high propionic acid production obtained by composite mutagenesis screening in the industrial production of high-quality propionic acid in ton-level tank fermentation. The fermentation medium includes: 6% to 8% glucose, 0.2% to 0.5% yeast powder, 1% to 1.5% corn steep liquor, 0.1% peptone, 0.07% magnesium sulfate, 0.25% dipotassium hydrogen phosphate, pH 7; fermentation conditions: calcium hydroxide is automatically added during the fermentation process to adjust the pH to 6.0 to 6.5, the fermentation tank is ventilated with N2 gas to maintain an anaerobic environment, and the fermentation is carried out for 90 hours, and the maximum propionic acid production reaches 65.87 g / L. The experimental results show that the high-yield strain reaches 65.87 g / L of propionic acid in a relatively short time of 90 hours, the fermentation intensity is 0.62 g / Lh, the propionic acid production rate is accelerated, the cost of producing propionic acid is greatly reduced, and it is possible to realize large-scale industrial production of propionic acid.

[0011] The present invention is described in more detail as follows: Determination of mutagenesis method Microwave mutagenesis method and its mutagenic dose The starting strain was Propionibacterium acidipropionici CGMCC1.2232, the microwave power was 800W, taken out every 8 seconds, and ice bathed for 15 seconds to eliminate the thermal effect. The microwave treatment time was set to 40 seconds, 60 seconds, 80 seconds, 100 seconds, 120 seconds, 150 seconds and 200 seconds. The bacterial suspension after mutagenesis was diluted and spread on the basic culture medium and cultured anaerobically for 72 hours. The microwave mutagenesis lethality curve was obtained with the number of surviving colonies as an indicator. Figure 1 As the mutagenesis time increased, the lethality rate showed an upward trend, reaching 80% at 100 seconds. Considering continuous mutagenesis, the microwave mutagenesis dose of 100 seconds was selected.

[0012] Ultrasonic mutagenesis method and its mutagenic dose The starting strain was Propionibacterium acidipropionici CGMCC1.2232, which was placed in a low-frequency ultrasonic treatment of 40 kHz, and was taken out every 9 seconds and placed in an ice bath for 5 seconds to eliminate the thermal effect. The bacteria were treated with ultrasonic waves for 9 seconds, 18 seconds, 27 seconds, 36 seconds, 45 seconds and 54 seconds, respectively. The diluted solution after mutagenesis was spread on the basic culture medium and cultured under anaerobic conditions for 72 hours. The number of colonies was used as an indicator to obtain the lethality curve as shown in the figure. Figure 2The strain Propionibacterium acidipropionici CGMCC1.2232 was extremely sensitive to ultrasonic mutagenesis, and 90% lethality was achieved after 9 seconds of ultrasonic treatment. Based on the above lethality results, microwave mutagenesis for 100 seconds and ultrasonic mutagenesis for 9 seconds were finally selected for microwave-ultrasound combined mutagenesis in order to obtain better mutants.

[0013] ARTP mutagenesis method and mutagenesis dose The operation process of the ARTP biological breeding instrument was adopted, with high-purity helium as the gas source, the power set to 100 W, the gas flow rate 10 seconds LM, the distance between the slide and the airflow port was 2 mm, and the metal slide was immediately placed in 1 mL of sterile saline after the mutagenesis was completed, and it was protected from light. The ARTP mutagenesis time was 15 seconds, 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds and 120 seconds respectively. The strain after mutagenesis was diluted and spread on the basic culture medium and cultured under anaerobic conditions for 72 hours. The lethality curve of the ARTP-induced propionic acid bacteria was obtained using the number of colonies as an indicator. Figure 3 . 90 seconds was selected as the ARTP mutagenesis treatment time.

[0014] Screening of mutagenic strains Microwave-ultrasound combined mutagenesis and strain screening The starting strain was Propionibacterium acidipropionici CGMCC1.2232. After combined mutagenesis by microwave for 100 seconds and ultrasound for 9 seconds, the mutagenic bacterial suspension was diluted 10 4 Take 100 μl and spread it on the primary screening medium.

[0015] The formula of the primary screening culture medium is: glucose 3%~4%, yeast powder 1%, peptone 0.5%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 0.05%~0.2%, pH 7.5; Preliminary screening method: culture in an anaerobic box at 32℃ for 120-144h, measure the diameter of the colony and the diameter of the transparent circle of the same colony, and select strains with a ratio of transparent circle diameter to colony diameter greater than 3 as shown in Figure 4 and Table 1: Table 1 The diameter ratio of Propionibacterium acidipropionici induced by microwave and ultrasound

[0016] We selected 13 strains with a circle diameter ratio larger than that of the starting strain Propionibacterium acidipropionici CGMCC1.2232, namely: LH1, LH2, LH3, LH5, LH37, LH38, LH41, LH44, LH45, LH52, LH54, LH56, and LH57. The 13 strains initially screened were rescreened and fermented, and finally a strain with high propionic acid production was obtained.

[0017] The secondary screening medium was composed of 4% to 6% glucose, 1% yeast powder, 0.7% peptone, 0.05% magnesium sulfate, 0.15% potassium dihydrogen phosphate, 1.5% to 2.0% calcium carbonate, and pH 7. The culture was carried out in an anaerobic box at 32°C for 96 to 168 hours, and the content of propionic acid in the fermentation broth was determined. The results are shown in Figure 5. Compared with the starting strain Propionibacterium acidipropionici CGMCC1.2232, all 11 strains were superior to the starting strain, with a positive screening rate of 85%, indicating that the plate transparent circle screening method has good applicability. Among them, the strain LH54 produced 14.54% more propionic acid than the starting strain. Finally, LH54 with the highest propionic acid production was selected for subsequent ARTP mutagenesis in order to obtain a more advantageous strain.

[0018] Obtaining high-yielding propionic acid bacteria by superimposing ARTP mutagenesis The LH54 strain was used as the starting strain and the mutagenesis time was 90 seconds. The strain after mutagenesis was diluted and spread on the basic culture medium. The mutagenesis bacterial suspension was diluted 10 4 Take 100 μl and spread it on the primary screening medium. ARTP mutagenesis plate screening The formula of the primary screening culture medium is: glucose 3%~4%, yeast powder 1%, peptone 0.5%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 0.05%~0.2%, pH 7.5; Preliminary screening method: culture in an anaerobic box at 32℃ for 120~144h, measure the diameter of the colony and the diameter of the transparent circle of the same colony, and select strains with a ratio of transparent circle diameter to colony diameter greater than 3 as shown in Table 2: Table 2 Circle diameter ratio of ARTP-induced strains

[0019] After 90 seconds of ARTP mutagenesis of the LH54 strain, 45 strains were spotted on the primary screening plate, named DLZ1-DLZ44, and spotted at the same time as the starting strain LH54. The strain circle diameter ratio was recorded at 120 hours. The results are shown in Table 2.

[0020] Fifteen strains with a circle diameter ratio larger than the starting strain and vigorous growth were selected for the next screening, namely: DLZ10, DLZ11, DLZ15, DLZ19, DLZ23, DLZ24, DLZ27, DLZ32, DLZ37, DLZ38, DLZ39, DLZ41, DLZ42, DLZ44, and DLZ45.

[0021] ARTP induced 15 strains to be screened again. The 15 strains obtained through the initial screening had a larger circle diameter ratio than the starting strain LH54 strain, and the liquid screening fermentation experiment was carried out. The screening medium: glucose 4%~6%, yeast powder 1%, peptone 0.7%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.15%, calcium carbonate 1.5%~2.0%, pH 7; cultured in an anaerobic box at 32℃ for 96~168h, and the content of propionic acid in the fermentation liquid was determined. The results are shown in the figure. Figure 6 shown.

[0022] Compared with the starting strain LH54, the mutant strain DLZ15 increased its propionic acid production by 16.79%. The DLZ15 strain was verified for stability through subculture and transferred to slant for 10 generations. There was no difference in propionic acid production by slant fermentation of the 0th, 2nd, 4th, 6th, 8th and 10th generations of bacteria. It was decided to select the DLZ15 strain with the highest acid production as the high-yield strain screened by multiple mutagenesis and named it YCBS010 (CGMCC No.31160) for pilot fermentation test.

[0023] Fermentation test of Propionibacterium acyltransferase YCBS010 (CGMCC No.31160) with high propionic acid production A 1-ton tank fermentation test was conducted using the high-propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160); Fermentation medium: glucose 6%~8%, yeast powder 0.2%~ 0.5%, corn steep liquor 1%~1.5%, peptone 0.1%, magnesium sulfate 0.07%, dipotassium hydrogen phosphate 0.25%, pH 7 Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH to 6.0-6.5, and N2 gas was passed through the fermentation tank to maintain an anaerobic environment. After 90 hours of fermentation, the maximum propionic acid production reached 65.87 g / L.

[0024] Compared with the prior art, the propionic acid-producing propionibacterium and the method for fermenting propionic acid disclosed in the present invention have the following positive effects: the high-yield strain can reach 65.87 g / L of propionic acid in a relatively short time of 90 h, with a fermentation intensity of 0.73 g / Lh, thereby accelerating the propionic acid production rate and significantly reducing the cost of producing propionic acid, thus providing the possibility for large-scale industrial production of propionic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Microwave-induced lethality curve; Figure 2 : Ultrasound-induced lethality curve; Figure 3 : ARTP-induced lethality curve; Figure 4 Screening method for transparent zone produced by Propionibacterium acidipropionici and its zone diameter ratio; Figure 5 Microwave-ultrasound combined mutagenesis and fermentation results of multiple screening strains; Figure 6 : ARTP mutagenesis re-screening strain fermentation results; Figure 7 : Morphological observation of propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160). DETAILED DESCRIPTION

[0026] The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also fall within the scope of protection of the present invention. The fermentation medium raw materials described in the present invention are all commercially available; the starting strain of Propionibacterium CGMCC1.2232 is a known strain, which is deposited in the National Culture Collection Center. Example

[0027] A 1-ton tank fermentation test was conducted using the high-propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160); Fermentation medium: glucose 6%, yeast powder 0.5%, corn steep liquor 1%, peptone 0.1%, magnesium sulfate 0.07%, dipotassium hydrogen phosphate 0.25%, pH 7; Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH to 6.0-6.5, and the fermentation tank was ventilated with N2 gas. Maintaining an anaerobic environment, the maximum propionic acid production reached 64.46 g / L after 90 hours of fermentation. Example

[0028] A 1-ton tank fermentation test was conducted using the high-propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160); Fermentation medium: glucose 7%, yeast powder 0.4%, corn steep liquor 1.2%, peptone 0.1%, magnesium sulfate 0.07%, dipotassium hydrogen phosphate 0.25%, pH 7; Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH to 6.0-6.5, and the fermentation tank was ventilated with N2 gas. Maintaining an anaerobic environment, the maximum propionic acid production reached 65.21 g / L after 90 hours of fermentation. Example

[0029] A 1-ton tank fermentation test was conducted using the high-propionic acid-producing Propionibacterium YCBS010 (CGMCC No.31160); Fermentation medium: glucose 8%, yeast powder 0.5%, corn steep liquor 1.5%, peptone 0.1%, magnesium sulfate 0.07%, dipotassium hydrogen phosphate 0.25%, pH 7; Fermentation conditions: Calcium hydroxide was automatically added during the fermentation process to adjust the pH to 6.0-6.5, and the fermentation tank was ventilated with N2 gas. Maintaining an anaerobic environment, the maximum propionic acid production reached 65.87 g / L after 90 hours of fermentation.

Claims

1. A strain with high propionic acid production obtained by composite mutagenesis screening ( Propionibacterium sp )YCBS010, characterized in that The Propionibacterium acidobacterium YCBS010 is deposited in the General Microbiological Center (GCMCC) of the China Culture Collection Administration, with the deposit number being CGMCC No. 31160.

2. The screening method of the Propionibacterium strain with high propionic acid production obtained by composite mutagenesis screening according to claim 1, with the deposit number CGMCC No. 31160, is as follows: (1) Dilute the mutagenic bacterial suspension by 10 4 Take 100 μL and apply it on the preliminary screening medium. The formula of the preliminary screening medium is: glucose 3%~4%, yeast powder 1%, peptone 0.5%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 0.05%~0.2%, pH 7.5; (2) Preliminary screening method: Culture in an anaerobic box at 32°C for 120-144 h, measure the colony diameter and the diameter of the transparent zone of the same colony, and select strains with a ratio of transparent zone diameter to colony diameter greater than 3; (3) Rescreening method: The strains preliminarily screened were subjected to two rounds of anaerobic fermentation in serum bottles to finally obtain strains with high propionic acid production; the rescreening culture medium: 4% to 6% glucose, 1% yeast powder, 0.7% peptone, 0.05% magnesium sulfate, 0.15% dipotassium hydrogen phosphate, 1.5% to 2.0% calcium carbonate, pH 7; cultured in an anaerobic box at 32°C for 120 to 144 hours, and the content of propionic acid in the fermentation broth was determined.

3. The application of a Propionibacterium strain with high propionic acid production obtained by composite mutagenesis screening in the industrial production of high-quality propionic acid by ton-scale tank fermentation.

4. The use according to claim 4, wherein the fermentation medium comprises: 6% to 8% glucose, 0.2% to 0.5% yeast powder, 1% to 1.5% corn steep liquor, 0.1% peptone, 0.07% magnesium sulfate, 0.25% dipotassium hydrogen phosphate, pH 7; fermentation conditions: during the fermentation process, calcium hydroxide is automatically added to adjust the pH to 6.0 to 6.5, the fermentation tank is ventilated with N2 gas to maintain an anaerobic environment, and the fermentation is carried out for 90 hours, the maximum propionic acid production reaches 65.87 g / L, and the fermentation intensity is 0.73 g / Lh.

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