CN115873754A - Enteromorpha clotrimaca RS804 and application thereof - Google Patents
Enteromorpha clotrimaca RS804 and application thereof Download PDFInfo
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Abstract
The invention discloses a Wei-arrowroot-causing enterobacter clotrimacae RS804 and application thereof. The enterobacter coagulans RS804 capable of efficiently utilizing the beet sucrose is provided, has certain temperature resistance and acid resistance, has good enzyme activity of polysaccharide hydrolases such as cellulase, xylanase, amylase and the like, and can be used for saccharification and fermentation to produce the L-lactic acid. In addition, the enterogenous coagulans Werkmann RS804 can metabolize to generate a certain content of bacteriocin, can effectively inhibit certain competitive strains such as Primedes megateria, bacillus subtilis and the like, and is favorable for preventing and treating fermentation contamination. The enterogenous Wegener clotrimaca RS804 disclosed by the invention has important significance for reducing the production cost of L-lactic acid fermentation and improving the L-lactic acid yield.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a bacillus coagulans RS804 derived from intestinal tract and application thereof; in particular to a bacillus coagulans RS804 capable of producing L-lactic acid by utilizing sugar beet fermentation and application thereof.
Background
Lactic acid (also known as alpha-hydroxypropionic acid) of the formula CH 3 CHOHCOOH has a relative molecular mass of 900.8, is a relatively common organic acid with a simple structure, and widely exists in human bodies, animals, plants and microorganisms. Lactic acid has optical activity due to the presence of an asymmetric carbon atom in the molecule, and is L-lactic acid (D-form), D-lactic acid (L-form), DL-lactic acid (racemic form). As an important daily chemical product, the organic acid is one of three major organic acids recognized in the world at present, and is widely applied to various industrial fields of medicines, foods, chemical industry, textiles, cosmetics, environmental protection and the like. In particular, polylactic acid (PLA) prepared from high-optical purity L-lactic acid is a novel non-petroleum material with biodegradability and biocompatibility, and is considered to be a promising recyclable polymer.
At present, the production method of L-lactic acid is mainly a fermentation method, wherein microorganisms of Lactobacillus and Weizmann (Weizmann) are mostly used, and the Wezmann bacterium coagulans (old called Bacillus coagulans) is recognized as one of the most excellent strains for L-lactic acid biological fermentation because of the advantages of strong stress resistance, developed amylase system, fast growth speed, high optical purity of products and the like because of sporulation. Lactic acid is mainly produced by microorganisms through metabolic fermentation by using glucose, starch or sucrose and the like as carbon sources, and how to reduce the production cost of lactic acid has become a research hotspot recently. Because the carbon source raw material accounts for a large proportion of the production cost of the L-lactic acid, if the refined saccharide is used for producing the L-lactic acid, although the cost of the product post-treatment is reduced to a certain extent, the price of the refined saccharide is generally higher, not only is the overall cost increased, but also the situation of 'competing for grains with people' is caused by the fact that the refined saccharide from the traditional food crops is used as a substrate for carrying out biological refining on the lactic acid, so the method is not economical. Beet is an inexhaustible renewable resource as a renewable energy crop. The sugar beet (mainly sucrose) is used as the lactic acid fermentation substrate, so that the problems of limited raw material source and high raw material cost can be solved, and the sugar beet byproduct, namely sugar beet molasses, can be fully utilized, so that the L-lactic acid fermentation yield and the utilization rate of the ground source raw material are improved. Based on the advantages, the fermentation production of L-lactic acid by utilizing sugar production of beet is a good development direction for industrial production of lactic acid.
The L-lactic acid fermentation strain taking the Wegener clotrimaran as the main stream has the defects of long fermentation period, easy staining of other spore mixed bacteria, low production efficiency, low optical purity and the like although the yield is high. How to breed the strains which can efficiently utilize sugar beet to produce sugar products, can tolerate the osmotic pressure of high-concentration substrates and final products, maintain the cell growth and the enzyme activity and obtain high productivity is the key of L-lactic acid strain breeding research.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide Weizmannia coagulans capable of efficiently fermenting L-lactic acid using sugar beet, and an application thereof in the fermentation production of L-lactic acid.
In order to realize the purpose, the invention adopts the technical scheme that: a Weizmannia coerulans RS804 is characterized in that the Weizmannia coerulans RS804 is preserved in Guangdong province microorganism strain preservation center at 8 months and 10 days in 2022, the preservation number is GDMCC No. 62692, and the preservation address is No. 59 building 5 of Michelia Torresiae 100 of Guangzhou city.
The Wegener clotrimaran RS804 provided by the invention is obtained by performing ARTP mutagenesis breeding on protoplast of an initial strain W103 separated from excrement of healthy lactating sows. The L-lactic acid has the advantages of rapid growth, stable genetic character, certain amylase, xylanase and cellulase synthesis capability, and high efficiency of utilizing beet sugar as a carbon source for fermentation production of the L-lactic acid.
Further, the invention also claims the application of the Weissemnia coagulans RS804 in fermentation.
The enterogenous West arrowhead bacterium RS804 provided by the invention has certain amylase, xylanase and cellulase synthesis capacity, so that the characteristics of the enterogenous West arrowhead bacterium RS804 can be utilized in various corresponding fermentation production.
As a preferred embodiment of the present invention, the enterogenous West arrowhead bacterium is used for fermentation production of L-lactic acid.
As a preferred embodiment of the invention, the process for producing the L-lactic acid by fermenting the Wegener welchii clotted by the intestines comprises the following steps: and inoculating the intestinal source Wei-arrowhead bacterium coagulates in a fermentation culture medium which takes beet sucrose as a main carbon source, and producing the L-lactic acid by fermentation.
The specific method takes beet sugar as a carbon source, screens excellent strains which can resist high temperature and produce substances with antibacterial performance, can effectively reduce the risk of contamination in the fermentation process, and the high-temperature fermentation condition is favorable for reducing the fermentation energy consumption, saving the cost and fermenting the high-yield lactic acid. Under the condition of the invention, the enterogenous West arrowhead bacteria RS804 has good L-lactic acid fermentation production capacity which can reach 194.8g/L. The yield is improved by about 21.3 percent relative to the yield of the grown bacteria.
As a preferred embodiment of the invention, the fermentation medium taking beet sucrose as a main carbon source comprises the following components in percentage by mass and volume: 0.25% of yeast extract, 5% of beet molasses, 0.05% of dipotassium hydrogen phosphate, 0.025% of monopotassium phosphate, 0.18% of ammonium dihydrogen phosphate, 0.5% of ammonium sulfate, 0.01% of zinc sulfate, 0.02% of folic acid, 0.02% of biotin and 10.0% of calcium carbonate, and the following components in percentage by volume: beet liquid sugar 75%; and deionized water is used for complementing the balance.
As a preferred embodiment of the present invention, the process for producing L-lactic acid by fermentation comprises: standing and culturing at 50-55 deg.C for 40-48h.
The fermentation condition is simple, the requirement on equipment is low, and the production cost is further saved because stirring is not needed.
Further, the fermentation process also comprises the processes of strain activation, shake flask seed culture and seed amplification culture.
As a preferred embodiment of the present invention, the process of activating the strain is: inoculating the enterogenous West arrowhead bacterium to an improved MRS agar culture medium slant, and culturing for 48h at 50-55 ℃; washing the slant strain with sterile normal saline to prepare bacterial suspension, transferring into a culture medium containing improved MRS culture medium, and culturing at 50-55 deg.C for 24h.
The process of shake flask seed culture is as follows: adding bacteria obtained by activation culture into sterile water for elution to prepare bacterial suspension, transferring into a shake flask filled with improved MRS culture medium, performing shake culture at 50-55 deg.C for 12-18h at 100-150r/min, and then performing static culture at 50-55 deg.C for 12-18h.
The process of seed amplification culture comprises the following steps: transferring seed jar containing seed culture medium according to 20% of inoculum size, culturing at 50-55 deg.C under 0.06Mpa for 6-10 hr under stirring at 100-150r/min, stopping stirring, maintaining positive pressure of jar body, and standing at 50-55 deg.C for 12-18 hr, wherein the strain transferring standard is colony count greater than 10 9 CFU/mL；
The improved MRS culture medium contains the following components in percentage by mass and volume: 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.06% magnesium sulfate, 0.025% manganese sulfate, 0.6% calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar and 0.1% tween-80; adjusting the pH value to about 6.5; supplementing the balance of deionized water;
the improved MRS agar culture medium is prepared by adding the following components in percentage by mass and volume on the basis of the improved MRS culture medium: 2% agar powder; supplementing the balance of deionized water;
the seed culture medium comprises the following components in percentage by mass and volume: 1.5 percent of yeast extract, 0.2 percent of ammonium dihydrogen phosphate, 0.5 percent of ammonium sulfate, 1.0 percent of light calcium carbonate, 0.02 percent of magnesium sulfate, 0.01 percent of zinc sulfate and the following components in percentage by volume: 75% beet pulp sugar; adjusting pH to 6.0-6.5, and adding deionized water to make up the balance.
The enzyme activity and the flora number of the enterogenous Werkmann bacillus coagulans RS804 can be improved through strain activation, shake flask seed culture and seed expansion culture, and favorable support is provided for subsequent inoculation in a fermentation tank for large-scale fermentation.
As a preferred embodiment of the present invention, the fermentation conditions are: transferring the seed culture solution with the transfer amount of 20-50% to a fermentation tank filled with a fermentation culture medium, controlling the ventilation amount of the fermentation tank to be 150-200mL/min, controlling the concentration of initial total sugar to be 100-160g/L, controlling the pH value to be 5.5-6.0, and standing and culturing at 50-55 ℃; maintaining the total sugar content in the fermentation tank at 16-20g/L, and fermenting for 48h under positive pressure in the fermentation tank.
In the initial stage of fermentation culture, the aeration amount of the fermentation tank is controlled to be 150-200mL/min, and the stirring is not started to control a certain amount of dissolved oxygen mainly for the propagation of the thallus at the early stage. After the fermentation is finished, the residual sugar content in the fermentation liquor is less than 5g/L after the fermentation is finished.
More preferably, the method of adjusting the pH during fermentation is controlled by controlled feeding of 25% lime milk.
The enterobacter winkle RS804 capable of efficiently fermenting and producing L-lactic acid by utilizing sugar beet has certain temperature resistance and acid resistance, has better enzyme activity of polysaccharide hydrolases such as cellulase, xylanase, amylase and the like, and can carry out saccharification and fermentation to produce the L-lactic acid. In addition, the enterogenous West arrowhead bacteria RS804 can also be metabolized to generate a certain content of bacteriocin, can effectively inhibit certain competitive strains such as Priesia megaterium (Priesia megaterium), bacillus subtilis (Bacillus subtilis) and the like, and is beneficial to preventing and controlling fermentation contamination. The enteron-derived Wegener condensation RSs 804 disclosed by the invention has important significance for reducing the production cost of L-lactic acid fermentation and improving the yield of L-lactic acid.
Drawings
FIG. 1 shows the results of the detection of the enzyme production characteristics of Weissemnia coagulans RS804 according to the present invention.
FIG. 2 is a phylogenetic tree of the RS804 strain of Enterobacter coagulans based on 16s rDNA sequences according to the present invention.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
EXAMPLE 1 selection of starting Strain W103 producing L-lactic acid
1. Pretreatment and strain separation of healthy lactating sow fecal sample
Pretreatment: in a sterile operating table, 10g of healthy lactating sow feces are taken at different time periods, added into a triangular flask containing 50mL of sterile physiological saline (pH value is 2.5, pH is adjusted by adding 3mol/L hydrochloric acid), sufficiently vortexed, filtered by gauze, the filtrate is taken to a new sterilized triangular flask, and the pH value is adjusted to 5.5-6.0 by using sterilized 20% sodium hydroxide. To enable specific selection of strains that can tolerate a certain temperature, the flask containing the contents was continued to oscillate in a water bath at 80 ℃ at 100rpm for 10min.
After the pretreatment is finished, the water bath solution is diluted to 10 times of gradient by adopting sterile normal saline -5 Spreading the diluted solution on a separate culture medium plate containing 1.5% calcium carbonate and 1.5% calcium lactate (i.e. adding 1.5% (w/w) calcium carbonate and 1.5% calcium lactate into the separate culture medium), and culturing at 55 deg.C for more than 48 hrUntil a single colony grows.
Wherein the separation medium composition comprises the following components in mass-volume concentrations: 10g/L tryptone, 10g/L yeast extract powder, 10g/L beef extract and K 2 HPO 4 2g/L、MgSO 4 0.2 g/L、MnSO 4 0.1g/L、NaCl 0.03g/L、FeSO 4 0.01g/L and 20g/L of agar powder, and the following components in percentage by volume: beet juice sugar 75% (v/v), 0.1% Tween-80, pH6.5 + -0.2, deionized water to make up the balance. Sterilizing at 121 deg.C for 15min.
The beet juice sugar is prepared into a concentrated solution with the total sugar content of about 20 percent, wherein the sucrose content is about 18.0 percent, and the monosaccharide content is about 2.0 percent according to the literature method of' Guoguoyu, modern beet sugar-making technology [ M ]. China light industry Press, 2015.
2. Specific screening of strains
(1) Selective prescreening
Selecting 10 strains which have the advantages of fast growth of the colonies, larger colonies, milky white color, raised surface, obvious calcium-dissolving rings and gram-positive staining on a separation culture medium plate, repeatedly scribing on the separation culture medium plate, culturing at 50-55 ℃, purifying the strains, and storing. Meanwhile, respectively inoculating the purified strains in enzyme-producing screening culture media containing different substrates, culturing at 50-55 ℃ for 24-36H, respectively adding 0.1% Congo red or iodine solution to determine the activities of cellulase (CMC), xylanase (XYL) and Amylase (AMY), and recording the ratio (H/C) of the diameter (H) of an enzymolysis ring of the strains to the diameter (C) of a colony.
Wherein, the enzyme production screening culture medium is prepared by respectively adding the following components in percentage by mass and volume on the basis of an improved MRS agar culture medium: selective screening was performed with 0.5% sodium carboxymethylcellulose (cellulase screening) or 0.5% xylan (xylanase screening) or 1.0% soluble starch (amylase screening).
The improved MRS agar culture medium contains the following components in percentage by mass: 2% of agar powder, 1% of peptone, 1% of beef extract, 0.5% of yeast extract, 0.2% of diammonium hydrogen citrate, 0.5% of sodium acetate, 0.2% of dipotassium hydrogen phosphate, 0.06% of magnesium sulfate, 0.025% of manganese sulfate, 0.6% of calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar and 0.1% tween-80; the balance of deionized water is complemented, and the pH value is about 6.5.
According to the difference of enzyme production capacities of different strains, selecting bacteria capable of forming an obvious enzymolysis loop on various substrates, and primarily sequencing the enzyme production capacities of the bacteria according to the ratio of the diameter of the enzymolysis loop formed by the bacteria to the diameter of a bacterial colony, wherein the larger the ratio is, the stronger the enzyme activity is. The results of the enzyme production screen are shown in Table 1.
TABLE 1 comparison of the enzyme-producing capacities of different strains
| Strain numbering | CMC H/C | XYL H/C | AMY H/C |
| W01 | 1.0 | 1.0 | 1.0 |
| W12 | 1.0 | 1.21 | 1.0 |
| W23 | 1.20 | 1.0 | 1.0 |
| W29 | 1.16 | 1.12 | 1.0 |
| W58 | 1.22 | 1.0 | 1.0 |
| W77 | 1.0 | 1.0 | 1.21 |
| W103 | 1.45 | 1.30 | 1.25 |
| W109 | 1.11 | 1.0 | 1.10 |
| W116 | 1.15 | 1.0 | 1.10 |
| W121 | 1.20 | 1.0 | 1.0 |
The results of the preliminary screening of the enzyme-producing fermentations from Table 1 show that: the polysaccharide hydrolysis ability of the strains obtained by primary screening is not strong. However, compared with other strains, the W103 strain has the enzyme activities of cellulase, xylanase and amylase at the same time, and shows that the strain has better polysaccharide hydrolysis and utilization capability.
(2) Shaking flask fermentation double screen
Comparing the L-lactic acid synthesis ability of the strains with different enzyme production abilities and determining the bacteriostatic property of the fermentation liquor by adopting a shake flask fermentation mode:
transferring the strains with different substrate hydrolysis abilities to test tube slant (containing modified agar MRS culture medium, 18 × 180mm test tube), culturing at 50-55 deg.C for 24 hr, adding 5mL sterile physiological saline, eluting, and preparing bacterial suspension (colony count is 1 × 10) 8 CFU/mL), and finally transferring the bacterial suspension to a shake flask for fermentation culture, wherein the shake flask fermentation conditions are as follows: 100mL of shake flask fermentation medium is filled in a 250mL triangular flask, and standing culture is carried out for 48h at 55 ℃.
The shake flask fermentation medium comprises the following components in percentage by mass and volume: 0.25 percent of yeast extract, 5 percent of beet molasses, 0.05 percent of dipotassium hydrogen phosphate, 0.025 percent of monopotassium phosphate, 0.18 percent of ammonium dihydrogen phosphate, 0.5 percent of ammonium sulfate, 0.01 percent of zinc sulfate, 0.02 percent of folic acid, 0.02 percent of biotin and 10.0 percent of calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar; and deionized water is used for complementing the balance. Autoclaving at 121 deg.C for 15min.
The above molasses, containing 45% (w/v) of sucrose, 0.5% (w/v) of nitrogen and 2.6% of betaine, was purchased from Xinjiang Kuntze sugar Mill (Akosonle 169, N.Q.M.).
After the fermentation was completed, the fermentation supernatant was collected by centrifugation at 10000r/min for 2min, and the content of L-lactic acid in the fermentation medium was quantitatively analyzed by an enzymatic reaction using SBA-40D type biosensor analyzer (purchased from Jinan Industriaceae laboratory instruments Co., ltd.).
In addition, the agar diffusion method is adopted to detect the bacteriostatic performance of the shake flask fermentation supernatant: regulating pH of the supernatant to 5.5-6.0 with 10% sodium hydroxide solution, removing interference of acid on subsequent determination, preparing bacteriostatic flat plate with Primesteria megaterium as indicator, measuring diameter of bacteriostatic circle, and determining supernatant size according to the size of bacteriostatic circleHas strong and weak inhibiting effect on indicator bacteria. The bacteriostatic flat plate is prepared by heating sterilized LB solid culture medium, standing at room temperature, cooling to 45-55 deg.C, adding appropriate amount of Listeria megaterium or Bacillus subtilis suspension (final concentration of indicator bacteria is 1 × 10) 5 CFU/mL), mixing, pouring into a plate, punching the plate by using a sterile puncher with the diameter of 6.0mm, removing an agar block, adding 50 mu L of fermentation liquor into the sample hole, standing at 4 ℃ for about 1h, then culturing in an incubator at 37 ℃ for 24h, taking out the plate, and measuring the diameter of a bacteriostatic zone.
Zone of inhibition diameter = zone of inhibition external diameter-aperture (6.0 mm). The results of shake flask fermentation rescreening are shown in Table 2.
TABLE 2 shake flask fermentation re-screening results
| Strain numbering | L-lactic acid (%) | Diameter of bacteriostatic circle (mm) |
| W01 | 21.0 | 2.5mm |
| W12 | 11.9 | 2.2mm |
| W23 | 32.6 | 2.3mm |
| W29 | 11.6 | 1.8mm |
| W58 | 22.2 | 1.6mm |
| W77 | 21.0 | 1.5mm |
| W103 | 44.5 | 2.6mm |
| W109 | 11.1 | 1.1mm |
| W116 | 11.5 | 2.0mm |
| W121 | 22.0 | 1.4mm |
As can be seen from table 2, the shake flask fermentation of the W103 strain has the strongest ability to produce L-lactic acid, and can metabolize to produce certain bacteriostatic active substances, which have certain inhibitory activity, and effectively reduce the risk of contamination by bacillus that can also produce spores and utilize fermentation raw materials competitively. And performing slant preservation and glycerol preservation on the strain W103 with the number of the strain, namely obtaining the starting strain W103 with high L-lactic acid yield by separation.
Example 2 Breeding of Excellent mutant Strain Wegener condensation RS804
1. Protoplast preparation
(1) Activation of the Strain
Taking the W103 strain obtained by the separation as an initial strain, inoculating glycerol strain of the strain on a slope of an improved MRS agar culture medium, culturing for 48h at 50-55 ℃, taking a ring of strains from the initial strain, streaking the strains into another fresh improved MRS slope culture medium after the culture is finished, and culturing for 24h at 50-55 ℃, so as to further strengthen the strain activity and rejuvenate the strains and achieve the aim of strain activation.
(2) Preparation of a suspension of the Strain
Washing the strain with sterile normal saline to prepare slant strain suspension, and fully whirling, oscillating and mixing uniformly. Centrifuging at 8000r/min at 4 deg.C for 5min to collect thallus cells, washing with sterile normal saline twice, washing with hypertonic solution SMM twice, centrifuging to remove supernatant, suspending thallus in SMM solution to obtain thallus suspension, and adjusting colony number to 2.0 × 10 6 CFU/mL (composition of SMM hypertonic solution comprising sucrose 0.5mol/L, mgCl 2 ·6H 2 0.02mol/L of O and 0.02mol/L of maleic acid, pH7.0, 121 ℃, sterilizing 22min, and storing at 4 ℃ for later use).
(3) Preparation of protoplasts
The preparation of the protoplast refers to firstly carrying out slant activation of a strain, then preparing a thallus suspension, and finally carrying out enzymolysis wall breaking under certain conditions to prepare the protoplast.
Enzymolysis of the cells is carried out in the form of combined enzymes. The method specifically comprises the following steps: the method comprises the steps of carrying out combined digestion by adopting lysozyme and muramidase, taking the thallus suspension, respectively adding a certain amount of enzyme mother liquor (prepared by SMM solution) after filtration and sterilization to prepare thallus suspension containing enzyme, placing the thallus suspension in a shaking table, carrying out 80r/min reaction, carrying out microscopic examination once every 5min, observing the formation condition of protoplast, when about 90% of cells are converted into the protoplast, centrifugally collecting the cells at 2000rpm, washing and centrifuging twice by using SMM, and carrying out heavy suspension by using a proper amount of SMM. Wherein, the using concentration of the lysozyme (purchased from Biotechnology engineering (Shanghai) GmbH) is 0.05-0.5%, and the optimal concentration is 0.05-0.15%; the use concentration of the muramidase (purchased from institute of microorganisms in Guangdong province) is 0.01-0.1%, and the optimal concentration is 0.01-0.05%; the enzymolysis temperature range is 24-35 ℃, and the optimum enzymolysis temperature is 26-30 ℃; the enzymolysis time is 10-90minThe optimal enzymolysis time is 10-30min. Under optimized conditions, the final protoplast concentration reaches 1.9 multiplied by 10 6 The protoplast formation rate of each mL is 95.0%, and the regeneration rate of the protoplast is preferably 20.5%.
2. ARTP mutagenesis of protoplasts
(1) Determination of ARTP mutagenesis parameters
The ARTP mutagenesis breeding is realized by Atmospheric pressure Room Temperature Plasma (ARTP) mutagenesis breeding machine (No-tin source Qingtian Wood Biotech Co., ltd.), the breeding machine is acted on microorganisms by generating Plasma jet with the Temperature of 25-40 ℃ and high active particle (including helium atom, oxygen atom, nitrogen atom, OH free radical and the like in an excited state) concentration under Atmospheric pressure, the structure and permeability of the microorganism wall/membrane can be changed, gene damage is caused, the microorganism gene sequence and the metabolic network thereof are obviously changed, and finally mutation is caused to the microorganisms.
The optimal conditions for the protoplast mutation breeding by adopting the mutation breeding machine are as follows: taking 10-20 mu L of protoplast suspension, uniformly coating the protoplast suspension on the upper surface of a metal slide, drying, and transferring the protoplast suspension slide to a stage by using tweezers. High-purity helium gas is used as working gas of plasma, the power supply power is set to be 80W, the irradiation distance is set to be 4mm, the temperature of the plasma is 26-30 ℃, the gas flow is 10L/min, the bacterial slide is treated, different treatment groups are set, the treatment time of each group is respectively 0 (contrast), 10, 20, 30, 40, 50 and 60s, and each group is set to be repeated for three times. And after treatment, transferring the slide glass into an EP tube containing SMM solution, shaking and eluting to form a new protoplast suspension, coating a regeneration plate, then placing the regeneration plate into an incubator at 50-55 ℃ to culture until a single bacterial colony grows out, and counting. The mortality rate was calculated as follows:
mortality% = (number of colonies not mutagenized-number of colonies mutagenized)/number of colonies not mutagenized × 100%
By counting the fatality rate of each treatment group, the irradiation treatment time with the fatality rate of about 90% is selected for formal experiments, so that certain mutation abundance is ensured, and certain survival rate is provided for subsequent screening.
The results show that: taking a strain which is not treated by ARTP (treatment time is 0 s) as a control, after the bacterial suspension of W103 is subjected to ARTP mutagenesis, the lethality rates are respectively 54.1 percent (treatment time is 10 s) and 79.8 percent (treatment time is 20 s); 89.8 percent (the treatment time is 30 s), and the lethality reaches more than 99.9 percent after the treatment for more than 40s, and the cell survival rate is basically 0. Therefore, subsequent screening was carried out while ensuring the mutagenic lesion and a certain cell survival rate, and ARTP treatment was carried out under the condition of selecting a lethal rate of about 90%, i.e., the most effective treatment time of ARTP using the W103 strain was determined to be 30 seconds.
(3) Screening of mutagenized strains
Primary screening with a 96-pore plate: coating the protoplast suspension after the ARTP mutagenesis on a regeneration plate containing 1% bromocresol green-methyl red, selecting 245 colonies with a light blue to light red ring from the plate according to the size of a colony color ring in the plate, transferring the colonies into a 96 micro-porous plate containing a micro-porous plate screening culture medium, standing and culturing for 48h at 50-55 ℃, observing the color change of the culture medium every 2h after 24h, and selecting 10 bacteria with fast color change and deep red of the culture solution for further storage for later use.
Wherein, the bromocresol green-methyl red is prepared according to the following method: accurately weighing 0.1g of bromocresol green and 0.06g of methyl red, dissolving in 95% ethanol, and diluting to 100mL with 95% ethanol.
The preparation method of the regeneration plate culture medium comprises the following steps: weighing 20g of glucose, 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 2g of diammonium hydrogen citrate, 1g of NaCl and K 2 HPO 4 2g、MgSO 4 0.6g、MnSO 4 0.25g, sucrose 171g, caCl 2 2.5g、MgCl 2 6H 2 O4.066 g and maleic acid 2.32g, and measuring Tween-80 mL and bromocresol green-methyl red solution 2mL, diluting with deionized water to volume of 1L, and autoclaving at 121 deg.C for 20min for use.
The preparation method of the microplate screening medium comprises the following steps: weighing 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 2g of diammonium citrate, 5g of NaCl and K 2 HPO 4 2g、MgSO 4 0.6g、MnSO 4 0.25g and 10g of cane sugar, measuring Tween-80 mL, 2mL of bromocresol green-methyl red solution and beet liquid sugar with the volume percentage content of 75 percent, supplementing deionized water to 1000ml, and carrying out autoclaving at 121 ℃ for 20min for standby.
Further performing shake flask fermentation and re-screening on the obtained strains: selecting 9 mutant strains which are primarily screened and an original strain W103, transferring the mutant strains and the original strain W103 to slant activation, preparing a bacterial suspension, inoculating the bacterial suspension into a triangular flask containing 150mL of shake flask fermentation medium, standing and culturing at 50-55 ℃ for 48h, and detecting the content of L-lactic acid by adopting a biosensor analyzer after the fermentation is finished. The results of shake flask fermentation rescreening are shown in Table 3.
TABLE 3 Shake flask fermentation rescreening of ARTP mutagenized strains
As can be seen from the results in Table 3, the L-lactic acid production of RS804 strain was 62.5g/L, which is significantly higher than that of the other strains. The RS804 strain is subjected to slant preservation and glycerol preservation, and the number is RS804.
RS804 with the highest acid yield is selected to be used together with the original strain, and the enzyme production characteristic is verified by adopting a point-cloning method, wherein the result is shown in figure 1 (AMY is amylase, CMC is cellulase, and XYL is xylanase). As can be seen from FIG. 1, the colony size of RS804 strain was significantly larger than that of the starting strain W103 on the enzyme-producing screening plates containing different substrates, and the enzyme-producing characteristics (AMY, CMC and XYL enzyme activities) were also significantly better than that of the starting strain W103.
Example 3 molecular characterization
Firstly, extracting the genome of the strain RS804 with better L-lactic acid production capacity separated and screened by using a bacterial genome DNA extraction kit (purchased from Biotechnology engineering (Shanghai) Co., ltd.), and amplifying a 16S rDNA partial sequence by adopting a universal primer, wherein the universal primer has the following sequence:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO：1)
1492R：5'-TACGGCTACCTTGTTACGACTT-3'(SEQ ID NO：2)
the reaction system is as follows:
pre-mix Ex Taq (Takara corporation) 12.5. Mu.L, 27F primer (10. Mu. Mol/L) 1. Mu.L, 1492R primer (10. Mu. Mol/L) 1. Mu.L, LA18 genomic DNA template 0.5. Mu.L, and ultrapure water 10. Mu.L.
PCR conditions of pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ 40s, annealing at 57 ℃ 40s, extension at 72 ℃ for 90s, 30 cycles, final extension at 72 ℃ for 10min, and storage at 4 ℃. After obtaining the specific amplification product, a PCR recovery kit (purchased from Biotechnology engineering (Shanghai) GmbH) is adopted to recover the target fragment, and the target fragment is sent to the Biotechnology engineering (Shanghai) GmbH for sequencing. The sequence of the RS804 strain is shown as SEQ ID NO:3, respectively.
Sequencing results Blast analysis and alignment was performed in the NCBI nucleic acid bank (https:// Blast. NCBI. Nlm. Nih. Gov/Blast. Cgi), and the results showed: the closest genetic relationship to the RS804 strain is that of Wenzmann' S of Bacillaceae (Bacillus; weizmania), and the 16S rDNA sequence of the homologous strain with higher similarity is selected for phylogenetic analysis, and a phylogenetic evolutionary tree is constructed by the N-J method (Neighbor-Joining method) in MEGA4.0, as shown in FIG. 2.
RS804 clustered into a large population with strains of Bacillus and were closest in branching evolution to the congealing Werkinjania, indicating that RS804 and the congealing Werkinjania were evolutionarily related species. By combining the morphological characteristics of the colonies and the thalli, the RS804 is determined to be Weizmannia coagulousnea (Weiizmannia coagulousns), the number of the Weiizmannia coagulousns RS804 is the Weizilla coagulousnea RS804, and the Weizia coagulousnea RS804 is simply called as Weizilla coagulans RS804.
Example 4 genetic stability verification
The excellent mutant strain obtained above: performing subculture on the congealing Werkinjmann bacterium RS804, performing subculture once every 3 days for 15 generations, performing shake flask fermentation every other generation, measuring the content of L-lactic acid in fermentation liquor, and inspecting the stability of the strain in the subculture process. The results show that: the L-lactic acid content in the fermentation liquor of the Wegener condensation RS804 has no obvious change in the passage process, and the Wegener condensation RS has good genetic stability.
The obtained genetically stable excellent mutant strain of the Wenzmann clotrimacae RS804 is preserved in the microbial strain preservation center (GDMCC for short; address: no. 59 building 5 of Michelia Torresiae No. 100, guangzhou, microbiological research institute, guangdong, zip code: 510075) in 8.11.2022, and the preservation number of the RS804 strain is GDMCC No. 62692.
Example 5 fermentation application of Wegener RS804 coagulates
1. Seed culture
(1) Activation of bacterial strains
Firstly, inoculating Wegener RS804 glycerobacteria to an improved MRS agar culture medium slant (18 multiplied by 180 mm), and culturing for 48h at 55 ℃ in an incubator, namely F1 generation.
Then washing the slant strain with 5mL of sterile normal saline to prepare a bacterial suspension, transferring the bacterial suspension to an eggplant bottle culture medium containing an improved MRS culture medium, and culturing for 24 hours at 55 ℃ in an incubator to obtain the F2 generation activated strain.
The improved MRS culture medium is an agar-free improved MRS agar culture medium.
(2) Seed culture in shake flasks
And adding 20mL of sterile water into the eggplant bottle strain subjected to activation culture for elution to prepare a bacterial suspension, transferring the bacterial suspension into a 5L shake flask filled with 2000mL of improved MRS culture medium, performing shake culture at 100r/min and 55 ℃ for 12h in a constant-temperature shake incubator, and then performing standing culture at 55 ℃ for 12h to further improve the total number of bacterial colonies and strengthen the activity of the bacterial strain.
(3) Seed expansion culture
Adopting a seeding tank to carry out the amplification culture of the seed in the shake flask, transferring the seed shake flask to the seeding tank containing the seed culture medium according to the inoculation amount of 20 percent, and controlling the process as follows: and (3) tank pressure: 0.06Mpa, pot temperature: 55 ℃, initial stirring: 100r/min, naturally reducing dissolved oxygen, culturing for 6h, stopping stirring, maintaining positive pressure in the tank, continuously standing and culturing at 55 deg.C for 12h, and transplanting with colony count > 10 9 CFU/mL。
The seed culture medium comprises the following components in percentage by mass and volume: 1.5 percent of yeast extract, 0.2 percent of ammonium dihydrogen phosphate, 0.5 percent of ammonium sulfate, 1.0 percent of light calcium carbonate, 0.02 percent of magnesium sulfate, 0.01 percent of zinc sulfate and the following components in percentage by volume: 75% beet pulp sugar; supplementing the balance with deionized water; adjusting the pH value to 6.0-6.5. Autoclaving at 121 deg.C for 20min.
2. Fermentation to produce L-lactic acid
Transferring the seed culture solution to a fermentation tank filled with a fermentation culture medium, wherein the seed transferring amount is 50%. In the initial stage of fermentation culture, the aeration amount of the fermentation tank is controlled to be 150-200mL/min, the stirring is not started, a certain amount of dissolved oxygen is mainly controlled to propagate thalli at the early stage, the temperature is controlled to be 50-55 ℃, the initial total sugar concentration is controlled to be 100-160g/L, and the dissolved oxygen is controlled to be natural. In the whole process, the pH value of the fermentation liquor is controlled to be 5.5-6.0 by controlling the feeding of 25% lime milk, and the total sugar content in the fermentation tank is maintained to be 16-20g/L by adding concentrated beet liquid sugar (40-50%) solution. Maintaining the positive pressure in the fermentation tank, fermenting for 48h, and stopping fermentation. When the fermentation is finished, the content of residual sugar in the fermentation liquid is less than 5g/L, and the content of L-lactic acid in the fermentation medium is quantitatively analyzed by using an SBA-40D type biosensing analyzer (purchased from Jinan research laboratory instruments, ltd.) through an enzymatic reaction.
The fermentation medium comprises the following components in percentage by mass and volume: 0.25% of yeast extract, 5% of beet molasses, 0.05% of dipotassium hydrogen phosphate, 0.025% of monopotassium phosphate, 0.18% of ammonium dihydrogen phosphate, 0.5% of ammonium sulfate, 0.01% of zinc sulfate, 0.02% of folic acid, 0.02% of biotin and 10.0% of calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar; and d, supplementing the balance of deionized water. The medium was autoclaved at 121 ℃ for 15min.
3. Fermentation of control strains
The starting strain W103 was fermented under the same conditions as those for RS804, and the L-lactic acid production was measured by the same method.
4. Comparison of L-lactic acid production
The results show that: the L-lactic acid content in the RS804 strain fermentation liquor reaches 194.8g/L, the L-lactic acid yield of the original strain W103 is only 160.6g/L, the excellent mutant strain RS804 has better L-lactic acid synthesis and accumulation capacity, the L-lactic acid yield is respectively improved by 21.3 percent compared with the original strain, and the excellent L-lactic acid fermentation capacity is reflected.
Example 6
This example is the same as example 5 except for the following references:
the culture temperature for the culture and the fermentation is 50 ℃;
in the process of the shake flask seed culture, the bacterial suspension is transferred into a shake flask filled with the improved MRS culture medium, is subjected to shake culture at 150r/min for 18 hours, and then is subjected to standing culture for 18 hours.
In the process of seed amplification culture, the stirring culture condition is 150r/min for stirring culture for 10 hours, and the standing culture condition is culture for 18 hours.
In the fermentation process of producing the L-lactic acid, the seed transferring amount is 20 percent, and the culture time is 40 hours.
The RS804 strain is effective in converting sugar beet sucrose in the culture medium into L-lactic acid under the conditions of the example.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The enterogenous Werkinjia welchii (Weizmania coagulans) RS804 is characterized in that the enterogenous Werkinjia welchii RS804 is preserved in Guangdong province microorganism strain preservation center at 2022, 8 months and 10 days, and the preservation number is GDMCC No. 62692, and the preservation address is Michelia Torresiana No. 100 Dazhou No. 59 building 5 building in Guangzhou city.
2. The use of the enterogenic Wegener clotted bacterium of claim 1 in fermentation.
3. The use of claim 2, wherein the enterogenous Wegener clotrimaca is used for fermentative production of L-lactic acid.
4. Use according to claim 3, characterized in that it comprises the following steps: and inoculating the enterogenous West arrowhead bacterium in a fermentation culture medium which takes beet sucrose as a main carbon source, and producing the L-lactic acid by fermentation.
5. The use according to claim 4, wherein the fermentation medium with beet sucrose as the main carbon source comprises the following components in percentage by mass and volume: 0.25% of yeast extract, 5% of beet molasses, 0.05% of dipotassium hydrogen phosphate, 0.025% of monopotassium phosphate, 0.18% of ammonium dihydrogen phosphate, 0.5% of ammonium sulfate, 0.01% of zinc sulfate, 0.02% of folic acid, 0.02% of biotin and 10.0% of calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar; and d, supplementing the balance of deionized water.
6. The use according to claim 4, wherein the fermentative production of L-lactic acid comprises: standing and culturing at 50-55 deg.C for 40-48h.
7. The use according to claim 4, further comprising the processes of strain activation, shake flask seed culture and seed expansion culture.
8. The use as claimed in claim 7, wherein the activation of the strain is carried out by: inoculating the enterogenous Werkinje into an improved MRS agar culture medium slant, and culturing for 48h at 50-55 ℃; washing the slant strain with sterile normal saline to prepare bacterial suspension, transferring into a culture medium containing improved MRS culture medium, and culturing at 50-55 deg.C for 24h.
The process of the shake flask seed culture comprises the following steps: adding bacteria obtained by activation culture into sterile water for elution to prepare bacterial suspension, transferring into a shake flask filled with improved MRS culture medium, performing shake culture at 50-55 deg.C for 12-18h at 100-150r/min, and then performing static culture at 50-55 deg.C for 12-18h.
The process of seed scale-up culture comprises: transferring seed jar containing seed culture medium according to 20% of inoculum size, culturing at 50-55 deg.C under 0.06Mpa for 6-10 hr under stirring at 100-150r/min, stopping stirring, maintaining positive pressure of jar body, and standing at 50-55 deg.C for 12-18 hr, wherein the strain transferring standard is colony count greater than 10 9 CFU/mL；
The improved MRS culture medium contains the following components in percentage by mass and volume: 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.06% magnesium sulfate, 0.025% manganese sulfate, 0.6% calcium carbonate, and the following components in percentage by volume: 75% beet pulp sugar and 0.1% tween-80; the pH value is about 6.5, and the balance is made up by deionized water;
the improved MRS agar culture medium is prepared by adding the following components in percentage by mass and volume on the basis of the improved MRS culture medium: 2% agar powder;
the seed culture medium comprises the following components in percentage by mass and volume: 1.5 percent of yeast extract, 0.2 percent of ammonium dihydrogen phosphate, 0.5 percent of ammonium sulfate, 1.0 percent of light calcium carbonate, 0.02 percent of magnesium sulfate, 0.01 percent of zinc sulfate and the following components in percentage by volume: 75% beet pulp sugar; adjusting pH to 6.0-6.5, and adding deionized water to balance.
9. Use according to claim 8, wherein the fermentation process is: transferring the seed culture solution with the transfer amount of 20-50% to a fermentation tank filled with a fermentation culture medium, controlling the ventilation amount of the fermentation tank to be 150-200mL/min, controlling the concentration of initial total sugar to be 100-160g/L, controlling the pH value to be 5.5-6.0, and standing and culturing at 50-55 ℃; maintaining the total sugar content in the fermentation tank at 16-20g/L, and fermenting for 48h under positive pressure in the fermentation tank.
10. Use according to claim 9, wherein the method of adjusting the pH during fermentation is controlled by controlled feeding of 25% lime milk.
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