Enterococcus faecium strain with high lactic acid yield and application of enterococcus faecium strain

Abstract

The invention discloses an enterococcus faecium strain with high lactic acid yield and application of the enterococcus faecium strain, and belongs to the technical field of biology. According to the invention, ultraviolet mutagenesis, diethyl sulfate mutagenesis, single mutagenesis of 5-brorouracil and compound mutagenesis of enterococcus faecium are adopted to finally obtain an enterococcus faecium strain with better acid production performance and stable genetic performance, the enterococcus faecium strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 2, 2024, and the preservation number of the strain is CGMCC No.31810. The pH value of the silage can be reduced, so that the growth of other microorganisms including pathogenic bacteria is inhibited, the nutritional ingredients of the silage are maintained, the palatability of the feed is improved, and the livestock yield is increased; meanwhile, the quality of the silage is ensured, so that the enterococcus faecium strain has relatively high application value in the fields of animal husbandry and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a high-lactic acid-producing Enterococcus faecium strain and application thereof. Background Art

[0002] Enterococcus faecium is one of the normal intestinal flora. It produces acid extracellularly and can cooperate with other intestinal probiotics to maintain the normal physiological functions of the host. As a type of enterococcus, Enterococcus faecium has good growth performance, strong stress resistance, and the ability to produce antibacterial substances. It is included in my country's "Feed Additive Variety Catalog (2023)". Most of the research directions on Enterococcus faecium in the existing technology are strain isolation and identification, exploring its probiotic properties, or studying its influence and role in actual production, while there are few studies on improving its lactic acid production performance through single mutagenesis and compound mutagenesis. After improving the lactic acid production performance of microorganisms, the pH value of silage can be quickly reduced, thereby inhibiting the growth of other microorganisms including pathogens, maintaining the nutritional content of silage, increasing the palatability of feed, and increasing livestock production; at the same time, ensuring the quality of silage, so the relevant microorganisms have a high application value in animal husbandry and other fields. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a high lactic acid producing Enterococcus faecium strain and application thereof.

[0004] The object of the present invention is to achieve the following:

[0005] The present invention provides a high-lactic acid-producing Enterococcus faecium DLWHY-24, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 2, 2024, with a strain deposit number of CGMCC No.31810; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Another aspect of the present invention provides a high-efficiency fermentation agent for preparing silage, which is prepared by fermenting the above-mentioned high-lactic acid-producing Enterococcus faecium DLWHY-24.

[0007] Based on the above technical scheme, further, the preparation process of the efficient fermentation agent for preparing silage comprises the following steps: inoculating the Enterococcus faecium DLWHY-24 in a fermentation medium at an inoculation rate of 1-5%, culturing at 25-45°C and pH 5-8 for 12-48 hours to obtain a fermentation liquid.

[0008] Based on the above technical scheme, further, the carbon source in the fermentation medium includes glucose, sucrose, lactose, maltose, fructose, β-cyclodextrin, starch, chitosan, dextrin, rhamnose, and the nitrogen source includes peptone, tryptone, yeast extract powder, beef extract, urea, ammonium chloride, and soybean meal.

[0009] Based on the above technical scheme, further, the fermentation medium is composed of: glucose (carbon source) 5-20 g / L, trypsin (nitrogen source) 25-45 g / L, Tween-80 0.5-2 mL / L, diammonium hydrogen citrate 1-3 g / L, magnesium sulfate heptahydrate 0.5-0.9 g / L, manganese sulfate 0.03-0.07 g / L, dipotassium hydrogen phosphate 1-3 g / L, sodium acetate 4-6 g / L, and the balance is water.

[0010] Another aspect of the present invention provides the use of the above-mentioned Enterococcus faecium DLWHY-24 and the high-efficiency fermentation agent for preparing silage in the process of preparing silage.

[0011] Based on the above technical solution, further, the specific process of the application is: spraying the bacterial suspension of Enterococcus faecium DLWHY-24 or the high-efficiency fermentation agent for preparing silage onto the silage preparation raw materials (corn stalks, etc.), sealing, and culturing for more than 40 days to promote the silage preparation process and inhibit the growth of other microorganisms including pathogens.

[0012] Based on the above technical solution, further, the spraying amount of the bacterial suspension of Enterococcus faecium DLWHY-24 or the high-efficiency fermentation agent for preparing silage is 10-100 mL / kg (bacterial content is 1-9×10 8 cfu / mL); the moisture content of silage preparation was maintained at 65-70%.

[0013] Based on the above technical solution, further, the culture conditions are cultured at 25-45°C and pH 5-8.

[0014] The beneficial effects of the present invention compared with the prior art are as follows:

[0015] The present invention uses mutagenesis and optimization, and uses the pH drop value or the lactic acid content in the fermentation liquid as the detection standard, rather than a single microbial biomass as the detection standard, to screen out a high lactic acid-producing Enterococcus faecium DLWHY-24; under optimized fermentation conditions, its lactic acid production performance is significantly improved compared with that before mutagenesis, the acid production is increased by more than 50%, and its various tolerances are also well improved. Therefore, the use of the mutagenic strain in silage preparation can rapidly reduce the pH value in the silage, thereby inhibiting the growth of other microorganisms including pathogens, maintaining the nutritional components of silage, increasing the palatability of feed, and increasing animal husbandry production; while ensuring the quality of silage, the Enterococcus faecium strain has a high application value in the fields of animal husbandry and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0017] Figure 1 It shows the growth of test microorganisms in culture medium over time.

[0018] Figure 2 The results of the effect of DES on the lethality of the test microorganisms are shown in Figure 1. a shows the effect of different concentrations of DES on the lethality of the test microorganisms, and b shows the effect of different DES treatment times on the lethality of the test microorganisms.

[0019] Figure 3 The effect of UV irradiation time on the lethality of the strain.

[0020] Figure 4 This is the effect of 5-bromouracil (5-BU) concentration on the lethality of the strain.

[0021] Figure 5 is the effect of lithium chloride (LiCl) concentration on the lethality of the strain.

[0022] Figure 6 The standard curve for determining lactic acid concentration by UV spectrophotometry.

[0023] Figure 7 To test the genetic stability of the strain.

[0024] Figure 8 is the lethality of the strain in culture medium containing different glucose concentrations.

[0025] Fig. 9 It is the lethality rate of the strain in the culture medium with pH values ​​of 2, 3, 4, 9 and 10.

[0026] Fig.10 It is the mortality rate of the strain after treatment at 50℃, 60℃, 70℃ and 80℃.

[0027] Fig.11 is the lethality rate of the strain in culture medium containing different ethanol concentrations.

[0028] Fig.12 Figure 3 shows the effects of carbon source and nitrogen source on the lactic acid production of Enterococcus faecalis DLWHY-24, where a is the effect of different carbon sources on the lactic acid production of Enterococcus faecalis DLWHY-24, b is the effect of glucose concentration on the lactic acid production of Enterococcus faecalis DLWHY-24, c is the effect of different nitrogen sources on the lactic acid production of Enterococcus faecalis DLWHY-24, and d is the effect of trypsin concentration on the lactic acid production of Enterococcus faecalis DLWHY-24.

[0029] Fig.13 is the inoculum size for the OD of Enterococcus faecium DLWHY-24 600nm impact.

[0030] Fig.14 Effects of temperature (a) and pH (b) on lactic acid production by Enterococcus faecium DLWHY-24. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0032] 1. Culture medium

[0033] ①MRS liquid culture medium (fermentation medium): each liter of distilled water contains 20g of glucose, 10g of peptone, 5g of yeast extract powder, 10g of beef extract, 2g of diammonium hydrogen citrate, 0.7g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 1mL of Tween-80, natural pH (if MRS solid culture medium is to be prepared, add 20g of agar powder on this basis), sterilize at 115℃ for 30min.

[0034] ② Solid culture medium containing different concentrations of 5-bromouracil (5-BU) for mutagenesis: Different amounts of 5-BU were added to the MRS solid culture medium, so that the final concentrations of 5-BU were 0 μg / mL (control group), 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL.

[0035] ③ Slant preservation medium: Prepare MRS solid culture medium, sterilize at 115℃ for 30min, and then pour 5mL of culture medium into a sterile test tube (specification: 16×100mm, capacity: about 14.5mL) while it is still hot.

[0036] 2. Reagents

[0037] ① Phosphate buffer solution (PB solution for short, 0.2 mol / L PB solution in this test, pH value is 7.0): first prepare 0.2 mol / L disodium hydrogen phosphate solution, that is, accurately weigh 7.16 g Na2HPO4·12H2O and dissolve it in 100 mL distilled water; then prepare 0.2 mol / L sodium dihydrogen phosphate solution, that is, accurately weigh 3.12 g NaH2PO4·2H2O and dissolve it in 100 mL distilled water; finally, mix 0.2 mol / L disodium hydrogen phosphate solution and 0.2 mol / L sodium dihydrogen phosphate solution in a ratio of 62:38 to prepare 0.2 mol / L PB solution with a pH value of 7, and sterilize at 115℃ for 30 min.

[0038] ②25% sodium thiosulfate solution: Accurately weigh 25 g of sodium thiosulfate, dilute to 100 mL with distilled water, and sterilize at 115°C for 30 min.

[0039] ③ Physiological saline: weigh 0.85-0.9 g of NaCl, dissolve it in 100 mL of distilled water, and sterilize it at 115°C for 30 min.

[0040] ④0.2% FeCl3 solution: Accurately weigh 0.3g of FeCl3 and make up to 100mL with distilled water.

[0041] ⑤ A series of lactic acid solutions containing different concentrations: Pipette 1 mL of lactic acid (ρ=1.2 g / mL), place it in a 10 mL volumetric flask, dilute to the mark with distilled water to obtain a 109 g / L stock solution, and then dilute with distilled water to obtain a series of lactic acid solutions containing different concentrations.

[0042] Example 1

[0043] 1. Preparation before the test

[0044] ① Determine the growth curve: After the strain (a strain of Enterococcus faecium screened from a sample of cattle rumen contents and cattle feces mixed) was inoculated from the slant preservation medium to the MRS liquid medium, it was placed in a 30°C static incubator for 24 hours, and then 2 mL of the bacterial solution was inoculated into 100 mL of new MRS liquid medium (i.e., the inoculation amount was 2%), and statically cultured at 30°C. Using the MRS liquid medium without bacterial solution as a control, 1 mL of bacterial solution was taken every 2 hours, 4 mL of distilled water was added, and after sufficient mixing, the OD value was measured at 600 nm using a visible light spectrophotometer.

[0045] The results are as follows Figure 1As shown, the results show that when the inoculation time is 4 to 12 hours, the microorganisms are in the logarithmic growth phase, at which time the microorganisms begin to multiply rapidly, the cell number increases rapidly, and then gradually enters the stable phase.

[0046] ② Preparation of bacterial suspension: Take a certain amount of fermentation broth (in the logarithmic growth phase), centrifuge at 8000r / min for 10min; wash the precipitate twice with saline, and finally resuspend it with saline to obtain a certain concentration of bacterial suspension (the concentration of bacterial suspension in this test is generally maintained at OD 600nm =0.8±0.3).

[0047] ③ Determine the optimal conditions for diethyl sulfate (DES) mutagenesis: take 1 mL of bacterial suspension and add it to 9 mL of PB solution, then add different volumes of DES solution to make the DES concentrations 0 μL / mL, 5 μL / mL, 10 μL / mL, 15 μL / mL, and 20 μL / mL, respectively, shake for 0 to 100 min, then add 1 mL of 25% sodium thiosulfate solution to terminate the mutagenesis reaction; after appropriate dilution, take 100 μL and spread it on an MRS solid plate, incubate at 30°C for a period of time, count the colonies, and calculate the lethality. The results are shown in Figure 2 .

[0048]

[0049] The optimal mutagenic dose is a lethality of about 70%. At this time, there are more positive mutants, but fewer strains that significantly increase acid production. Figure 2 It can be seen that when using DES to mutate the test strain, the optimal conditions are determined to be: 10μL / mL DES solution shaken for 60 minutes. Then 1mL of 25% sodium thiosulfate solution was added to terminate the mutagenesis reaction. After dilution and plating, the colonies grown on the plate were taken with a sterile inoculation loop and inoculated into MRS liquid culture medium. After static culture at 35℃ for 20 hours, the pH change value in the fermentation broth was measured.

[0050] ④ Determine the optimal conditions for ultraviolet (UV) mutagenesis: turn on the ultraviolet lamp (20W) in advance and preheat for about 15 minutes; draw 10-15mL of bacterial suspension into a sterile plate, place the plate on a magnetic stirrer, turn on the preheated ultraviolet lamp, and irradiate while stirring at a vertical distance of 30cm from the ultraviolet lamp for 30s, 60s, 90s, 120s, 150s, and 180s respectively, with the unirradiated bacterial suspension as a control. After the irradiation, dilute appropriately, spread on an MRS solid plate, and incubate in a dark environment at 30℃ for a period of time, then count the colonies and calculate the lethality. The results are shown in the table. Figure 3 .

[0051] Depend on Figure 3It can be seen that 120s should be selected as the optimal UV irradiation time for this strain, and the mutagenic dose at this time is conducive to forward mutation.

[0052] ⑤ Determine the optimal conditions for 5-bromouracil (5-BU) mutagenesis: Take a certain amount of bacterial suspension, dilute it appropriately, take 100 μL and spread it on solid culture medium containing different concentrations of 5-BU (5-BU concentrations are 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL), incubate at 30°C for a period of time, count the colonies and calculate the lethality. The results are shown in the table. Figure 4 .

[0053] Depend on Figure 4 It can be seen that a 5-BU concentration of 300 μg / mL should be selected as the optimal 5-BU mutagenesis concentration for this strain.

[0054] ⑥ Determine the optimal conditions for UV-LiCl composite mutagenesis: LiCl is an alkali metal halide, and its mutagenesis mechanism is Li + Changing the environment outside the cells, increasing the destructiveness of radiation to cells, + It has a repressive effect on some enzymes in the gene repair system. Its mutagenic effect is poor when used alone, and it is a weak mutagen. However, ultraviolet radiation is non-ionizing radiation, and the two together can produce a synergistic effect and improve the efficiency of mutagenesis.

[0055] Under the determined optimal UV mutagenesis conditions (UV irradiation time of 120s), the optimal UV-LiCl composite mutagenesis conditions were determined. The UV-treated bacterial suspension was gradiently diluted with sterile physiological saline, and 100 μL was spread on a solid culture medium containing a certain concentration of LiCl (LiCl concentrations were: 0g / L, 3g / L, 6g / L, 9g / L, 12g / L, 15g / L, 18g / L, 20g / L, 30g / L). After culturing at 30°C for a period of time, the control group with a LiCl concentration of 0g / L was used to count the colonies and calculate the lethality. The results are shown in Figure 5 .

[0056] Depend on Figure 5 It can be seen that 15g / L lithium chloride should be selected as the optimal lithium chloride concentration for UV-LiCl composite mutagenesis.

[0057] ⑦ Determination of lactic acid content in fermentation broth by ultraviolet spectrophotometry: First, a standard curve was constructed. The prepared lactic acid solution (50 μL) of different concentrations was mixed with 2 mL of FeCl3 solution, and the reaction was fully reacted. The standard curve was drawn based on the relationship between the absorbance of the reaction solution and the change in lactic acid concentration. The results are shown in Figure 6 .

[0058] Depend on Figure 6 It can be seen that the regression equation of the lactic acid standard curve is: y = 0.1236x + 0.0396, R 2 =0.9936.

[0059] Then centrifuge the fermentation liquid (8000r / min, 10min), discard the precipitate, take 50μL of the supernatant, add it to 2mL FeCl3 solution, mix thoroughly, and measure the OD value at 390nm on a UV spectrophotometer. 390nm The lactic acid content of the fermentation broth can be obtained by substituting the value into the standard curve.

[0060] Example 2 Strain UV mutagenesis and screening

[0061] Turn on the ultraviolet lamp (20W) in advance and preheat for about 15 minutes; draw 10-15mL of the above-mentioned Enterococcus faecalis suspension into a sterile plate, put the plate on a magnetic stirrer, turn on the preheated ultraviolet lamp, irradiate while stirring at a vertical distance of 30cm from the ultraviolet lamp, after irradiation for 120s, dilute appropriately with physiological saline, spread on an MRS solid plate, incubate in a dark environment at 30℃ for a period of time, use a sterile inoculation loop to take the colonies grown on the plate, inoculate them into MRS liquid culture medium, incubate at 35℃ for 20h, and measure the pH change in the fermentation liquid.

[0062] Table 1 Comparison of pH drop values ​​of UV-induced strains

[0063] serial number Acid production (pH drop) 10-UV1a 1.28 10-UV1b 1.26 10-UV2a 1.21 10-UV2b 1.25 10-UV3a 1.26 10-UV3b 1.31

[0064] As shown in Table 1, after UV mutagenesis, the pH drop value of the fermentation broth of some strains increased. A strain with the highest acid production was selected, numbered 10-UV3b, and its acid production (i.e., the pH drop value in the fermentation broth) reached 1.31, which was 4.8% higher than that of the starting strain. This strain was preserved by inoculating it on a slant culture medium, culturing it at 30°C for a period of time, and then temporarily storing it in a refrigerator at 4°C after colonies were formed.

[0065] Example 3 5-bromouracil mutagenesis and screening of bacterial strains

[0066] A certain amount of the above-mentioned Enterococcus faecium suspension was taken, and after appropriate dilution, 100 μL was taken and spread on a mutagenic solid culture medium containing 300 μg / mL 5-BU. After incubation at 30°C for a period of time, a sterile inoculation loop was used to take the colonies grown on the plate, and the colonies were inoculated into MRS liquid culture medium. After incubation at 35°C for 20 hours, the pH change in the fermentation broth was measured.

[0067] Table 2 Comparison of pH decrease values ​​of 5-bromouracil-induced strains

[0068] serial number Acid production (pH drop) 10-5BU1a 1.30 10-5BU1b 1.10 10-5BU2a 1.25 10-5BU2b 1.24 10-5BU3a 1.27 10-5BU3b 1.29

[0069] As shown in Table 2, after mutagenesis by 5-bromouracil, the pH drop value of the fermentation broth of some strains increased. Among them, a strain with the highest acid production was screened out, numbered 10-5BU1a, and its acid production (i.e., the pH drop value in the fermentation broth) reached 1.30, which was 4% higher than that of the starting strain. This strain was preserved on a slant.

[0070] Example 4 Diethyl sulfate mutagenesis and screening of strains

[0071] 1 mL of the above-mentioned Enterococcus faecium suspension was taken and added to 9 mL of PB solution, followed by addition of 0.1 mL of diethyl sulfate solution to make the diethyl sulfate concentration in the mixed solution reach 10 μL / mL. After shaking for 60 min, 1 mL of 25% sodium thiosulfate solution was added to terminate the mutagenesis reaction, and the mixture was appropriately diluted with physiological saline and spread on an MRS solid plate. After incubation at 30°C for a period of time, a sterile inoculation loop was used to take the colonies grown on the plate and inoculated into MRS liquid culture medium. After incubation at 35°C for 20 h, the pH change in the fermentation broth was measured.

[0072] Table 3 Comparison of pH drop values ​​of diethyl sulfate induced strains

[0073] serial number Acid production (pH drop) Strain number Acid production (pH drop) 10-DES1a 1.26 10-DES5a 1.06 10-DES1b 1.33 10-DES5b 1.46 10-DES2a 1.22 10-DES6a 1.3 10-DES2b 1.28 10-DES6b 1.25 10-DES3a 1.28 10-DES7a 1.33 10-DES3b 1.40 10-DES7b 1.35 10-DES4a 1.10 10-DES8a 1.31 10-DES4b 1.09 10-DES8b 1.40

[0074] As shown in Table 3, after mutagenesis with diethyl sulfate, the pH drop value of the fermentation broth of most strains increased. Among them, a strain with the highest acid production was screened out, numbered 10-DES1b, and its acid production (i.e., the pH drop value in the fermentation broth) reached 1.46, which was 16.8% higher than that of the starting strain. This strain was preserved on a slant.

[0075] Example 5: UV-LiCl composite mutagenesis and screening of strains

[0076] Turn on the ultraviolet lamp (20W) in advance and preheat for about 15 minutes; draw 10-15mL of the above-mentioned Enterococcus faecalis suspension into a sterile plate, put the plate on a magnetic stirrer, turn on the preheated ultraviolet lamp, irradiate while stirring at a vertical distance of 30cm from the ultraviolet lamp, after irradiation for 120s, dilute appropriately with physiological saline, spread on a solid culture medium plate containing 300μg / mL LiCl, incubate in a dark environment at 30℃ for a period of time, take the colonies grown on the plate with a sterile inoculation loop, inoculate into MRS liquid culture medium, incubate at 35℃ for 20h, and measure the pH change in the fermentation liquid.

[0077] Table 4 Comparison of pH drop values ​​of UV-LiCl composite mutagenesis strains

[0078] serial number Acid production (pH drop) Strain number Acid production (pH drop) 10-UL1a 1.60 10-UL6a 1.37 10-UL1b 1.57 10-UL6b 1.35 10-UL2a 1.48 10-UL7a 1.44 10-UL2b 1.47 10-UL7b 1.41 10-UL3a 1.50 10-UL8a 1.37 10-UL3b 1.35 10-UL8b 1.43 10-UL4a 1.29 10-UL9a 1.26 10-UL4b 1.28 10-UL9b 1.34 10-UL5a 1.33 10-UL10a 1.45 10-UL5b 1.40 10-UL10b 1.39

[0079] As shown in Table 4, after UV-lithium chloride combined mutagenesis, the pH drop value of the fermentation broth of most strains increased. Among them, a strain with the highest acid production was screened out, numbered 10-UL1a, and its acid production (i.e., the pH drop value in the fermentation broth) reached 1.60, which was 28% higher than that of the starting strain. This strain was preserved on a slant.

[0080] Example 6 Diethyl sulfate-5-bromouracil composite mutagenesis and screening of strains

[0081] 1 mL of the above-mentioned Enterococcus faecium suspension was taken and added to 9 mL of PB solution, followed by addition of 0.1 mL of diethyl sulfate solution to make the diethyl sulfate concentration in the mixed solution reach 10 μL / mL. After shaking for 60 min, 1 mL of 25% sodium thiosulfate solution was added to terminate the mutagenesis reaction, and the mixture was appropriately diluted with saline and spread on a solid plate containing 300 μg / mL 5-BU. After incubation at 30°C for a period of time, a colony grown on the plate was taken with a sterile inoculation loop and inoculated into MRS liquid culture medium. After incubation at 35°C for 20 h, the pH change in the fermentation broth was measured.

[0082] Table 5 Comparison of pH drop values ​​of diethyl sulfate-5-bromouracil compound mutagenesis strains

[0083]

[0084]

[0085] As shown in Table 5, after the combined mutagenesis of diethyl sulfate and 5-bromouracil, the pH drop value of the fermentation broth of most strains increased. Among them, a strain with the highest acid production (numbered 10-DB2a) was screened out and named Enterococcus faecium DLWHY-24. Its acid production (i.e., the pH drop value in the fermentation broth) reached 1.75, which was 40% higher than that of the starting strain. This strain was preserved on a slant.

[0086] Example 7 Genetic stability test of high lactic acid producing strains

[0087] From the above, it can be seen that the strain with the best mutagenesis effect has an acid production of 1.75 (pH drop value of the fermentation liquid), named Enterococcus faecium DLWHY-24, and inoculated into MRS liquid culture medium, and continuously subcultured for 7 generations for stability testing. The specific operation is as follows: After static culture at 35℃ for 20h, take a certain amount of fermentation liquid, centrifuge and discard the precipitate, take 50μL of supernatant, add it to 2mL FeCl3 solution, mix thoroughly, measure the OD value at 390nm on a UV spectrophotometer, and set the OD 390nm The value can be put into the standard curve to get the lactic acid content in the fermentation broth. Figure 7 shown.

[0088] Depend on Figure 7 It can be seen that this mutant strain is relatively stable in inheritance and can be used as a subsequent test object. After seven generations of inheritance, the lactic acid content in the fermentation broth was 8.2g / L, which was more than 50% higher than that of the starting strain (the lactic acid content in the fermentation broth of the starting strain was 5.27g / L after 20h of culture under the same conditions).

[0089] Example 8 Comparison of changes in strain tolerance before and after mutagenesis

[0090] ① Glucose tolerance comparison: MRS liquid culture medium containing 50g / L, 100g / L, 150g / L, and 200g / L glucose was prepared in advance and sterilized at 115℃ for 30min before use. The activated bacterial liquid was inoculated with 2% inoculation amount (of Enterococcus faecium DLWHY-24 and the original strain) in MRS liquid culture medium with different glucose concentrations, and the bacterial liquid inoculated in the original MRS liquid culture medium (i.e., the glucose concentration was 20g / L) was used as the control. The culture was statically cultured at 35℃ for 24h, and the OD of each group of bacterial liquid was measured. 600nm， Calculate the mortality rate of each test group. Figure 8 shown.

[0091] Depend on Figure 8 It can be seen that with the increase of glucose concentration in the culture medium, the lethality of the two strains in this test increased, but it is obvious that under the condition of the same glucose concentration, the glucose tolerance of Enterococcus faecium DLWHY-24 is significantly higher than that of the original strain.

[0092] ② pH tolerance comparison: The pH of the MRS liquid culture medium was adjusted to 2, 3, 4, 9, and 10, and sterilized at 115℃ for 30 minutes before use. The activated bacterial liquid (Enterococcus faecium DLWHY-24 and the original strain) was inoculated at 2% in the MRS liquid culture medium with different pH values, and the bacterial liquid inoculated in the original MRS culture medium (i.e., the pH value was natural, about 5.7) was used as the control. The culture was statically cultured at 35℃ for 24 hours, and the OD of each group of bacterial liquid was measured. 600nm , calculate the mortality rate of each test group. Fig. 9 shown.

[0093] Depend on Fig. 9 It can be seen that when the pH value is in the range of 2 to 9, as the initial pH value of the culture medium decreases, the mortality rate of the two strains in this test increases; when the pH value is 2, the mortality rate of the two strains reaches nearly 100%, but under the same pH conditions, the mortality rate of Enterococcus faecium DLWHY-24 is lower than that of the original strain, and the pH tolerance is improved.

[0094] ③ Temperature tolerance comparison: The constant temperature water bath was set to 50℃, 60℃, 70℃, and 80℃ respectively. After a period of time, 10mL of activated bacterial solution (Enterococcus faecium DLWHY-24 and original strain) was placed in the constant temperature water bath. After 15 minutes of treatment, 100μL was appropriately diluted and spread on the MRS solid plate. After 24 hours of static culture at 35℃, the control group was placed at room temperature for 15 minutes, and the colony count was performed to calculate the mortality rate. The results are as follows Fig.10 shown.

[0095] Depend on Fig.10 It can be seen that with the increase of treatment temperature, the mortality rate of the two strains in this test increased. When the treatment temperature was 80℃, the mortality rate of the two strains reached nearly 100%. However, under the same conditions, the mortality rate of Enterococcus faecium DLWHY-24 was lower than that of the original strain, and its temperature tolerance was improved.

[0096] ④Comparison of ethanol tolerance: MRS liquid culture medium was prepared in advance, sterilized at 115℃ for 30min, and then different volumes of anhydrous ethanol (filtered through a 0.22μm sterile filter) were added to make the ethanol concentration in the culture medium reach 10mL / L, 20mL / L, 30mL / L, 40mL / L, 50mL / L, 60mL / L, 80mL / L, and 100mL / L. The activated bacterial liquid (Enterococcus faecium DLWHY-24 and the original strain) was inoculated at 2% in MRS liquid culture medium with different ethanol concentrations, and the bacterial liquid inoculated in the original MRS liquid culture medium (i.e., without anhydrous ethanol) was used as the control. The culture was statically cultured at 35℃ for 24h, and the OD of each group of bacterial liquid was measured. 600nm , calculate the mortality rate of each test group. Fig.11 .

[0097] Depend on Fig.11 It can be seen that with the increase of ethanol concentration in the culture medium, the mortality rate of the two strains in this test slowly increased. When the ethanol concentration in the culture medium reached 100mL / L, the mortality rate of the two strains reached nearly 100%. However, under the same conditions, the mortality rate of Enterococcus faecium DLWHY-24 was lower than that of the original strain, and its ethanol tolerance was improved.

[0098] Example 9 Optimization of fermentation process conditions of Enterococcus faecium DLWHY-24

[0099] ① Optimization of culture medium components: When other components in MRS culture medium remain unchanged, different carbon sources (glucose, sucrose, lactose, maltose, fructose, β-cyclodextrin, starch, chitosan, dextrin, rhamnose) are used to replace the glucose (concentration of 20 g / L) in MRS culture medium, and the lactic acid content in the fermentation broth (static culture at 35°C for 24 hours) is used as an indicator to screen the optimal carbon source; the concentrations of the optimal carbon source are set to 0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L, respectively, to investigate the effect of carbon source concentration on the lactic acid production of the strain. The original nitrogen source (peptone + beef extract + yeast extract, total concentration 25g / L) in the MRS medium was replaced with different nitrogen sources (peptone, tryptone, yeast extract, beef extract, urea, ammonium chloride, soybean meal), and the original MRS medium was used as a control to screen the best nitrogen source; the optimal nitrogen source concentration was set to 5g / L, 15g / L, 25g / L, 35g / L, 45g / L, and 55g / L to investigate the effect of nitrogen source concentration on lactic acid production by the strain. The results are as follows Fig.12 shown.

[0100] Depend on Fig.12 From a, we can see that when glucose is used as the carbon source, the lactic acid content in the fermentation broth is the highest, followed by sucrose. Fig.12 b It can be seen that as the glucose concentration of the culture medium increases, the lactic acid content in the fermentation broth slowly decreases, and the optimal glucose concentration is 10g / L; Fig.12 c shows that the strain has the best utilization effect on tryptone, followed by beef extract and peptone, while the utilization of other nitrogen sources is lower than that of the tryptone group; Fig.12 It can be seen that when the trypsin concentration is 35 g / L, the lactic acid content in the fermentation broth is the highest, which is 9.27 g / L.

[0101] ② Optimization of culture conditions: Based on the above optimal culture medium for lactic acid fermentation, inoculate the seed liquid into the fermentation medium at 0.5%, 2%, 4%, and 6% inoculation rates, and culture at 35°C. Take samples every two hours to measure OD 600nm value to determine the optimal inoculation size; inoculate the seed liquid into the fermentation medium at the optimal inoculation size, place them in constant temperature incubators at 25°C, 30°C, 35°C, 40°C, and 45°C, respectively, and culture them statically for 24 hours, then take samples, measure the lactic acid content in the fermentation liquid, and determine the optimal culture temperature; prepare fermentation medium with different pH values ​​(the pH values ​​are 3, 4, 5, 6, 7, and 8, respectively), inoculate the seed liquid into the fermentation medium at the optimal inoculation size, place them at the optimal culture temperature, and culture them statically for 24 hours, then take samples, measure the lactic acid content in the fermentation liquid, and determine the optimal pH value.

[0102] Depend on Fig.13 It can be seen that the inoculation amount has little effect on the lactic acid production of the strain, and 2% inoculation amount is selected as the optimal inoculation amount for subsequent experiments; Fig.14 As shown in a, with the increase of culture temperature, the lactic acid production capacity of the strain gradually increased. The lactic acid content in the fermentation broth was the highest at 40°C. Then, as the temperature continued to rise, the lactic acid production capacity of the strain decreased. The optimal culture temperature was 40°C. Fig.14 b It can be seen that the lactic acid content in the fermentation broth showed a trend of first increasing and then decreasing when the pH value ranged from 3 to 8, and when the initial pH was 6 and 7, the difference in lactic acid content in the fermentation broth was not very obvious, indicating that the strain was relatively stable under neutral conditions and the optimal pH value was 7.

[0103] ③ The seed liquid was inoculated into the above-mentioned optimal fermentation medium (i.e. 10 g of glucose, 35 g of tryptone, 1 mL of Tween-80, 2 g of diammonium citrate, 0.7 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, and 1 L of deionized water). The culture conditions were also the above-mentioned optimal culture conditions (i.e., the inoculation amount was 2%, the culture temperature was 40°C, and the initial pH value of the culture medium was 7). After static culture for 24 hours, samples were taken and the lactic acid concentration in the fermentation broth was measured to be 9.54 g / L, which was 16% higher than that before optimization (the lactic acid content before optimization was about 8.2 g / L).

[0104] Enterococcus faecium DLWHY-24 was deposited in the General Microbiology Center of China Microbiological Culture Collection on September 2, 2024, and the strain deposit number is CGMCC No.31810; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0105] Example 10 Application of Enterococcus faecium DLWHY-24 in silage preparation

[0106] (1) Enterococcus faecium DLWHY-24 was inoculated into 100 mL of seed culture medium at a 2% inoculum concentration, and cultured at pH 7 and 40°C for 24 h;

[0107] (2) 20 mL of the bacterial culture solution cultured in step (1) was inoculated into 1 L of fermentation medium, pH 7, 40° C., and cultured for 24 h;

[0108] (3) The fermentation liquid obtained in step (2) or the seed liquid obtained in step (1) is uniformly sprayed on the silage feed raw material, with a spraying amount of 10 to 50 mL / kg (the number of bacteria is 10 8 cfu / mL), the moisture of silage was maintained at 65-70%, covered with plastic cloth, and cultured at 30-45°C for 45 days.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high lactic acid producing strain of Enterococcus faecium DLWHY-24, characterized in that: It was deposited in the General Microbiology Center of China Microbiological Culture Collection on September 2, 2024, and the strain deposit number is CGMCC No.31810; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A highly efficient fermentation agent for preparing silage, characterized in that: The method is prepared by fermenting the high lactic acid producing Enterococcus faecium DLWHY-24 according to claim 1.

3. The efficient fermentation agent for preparing silage according to claim 2, characterized in that: The preparation process of the high-efficiency fermentation agent comprises the following steps: inoculating the Enterococcus faecium DLWHY-24 in a fermentation medium at a rate of 1-5%, culturing at 25-45°C and pH 5-8 for 12-48 hours to obtain a fermentation liquid.

4. The efficient fermentation agent for preparing silage according to claim 3, characterized in that: The carbon sources in the fermentation medium include glucose, sucrose, lactose, maltose, fructose, beta-cyclodextrin, starch, chitosan, dextrin and rhamnose, and the nitrogen sources include peptone, tryptone, yeast extract powder, beef extract, urea, ammonium chloride and soybean meal.

5. The efficient fermentation agent for preparing silage according to claim 4, characterized in that: The fermentation medium comprises: 5-20 g / L of carbon source, 25-45 g / L of nitrogen source, 0.5-2 mL / L of Tween-80, 1-3 g / L of diammonium hydrogen citrate, 0.5-0.9 g / L of magnesium sulfate heptahydrate, 0.03-0.07 g / L of manganese sulfate, 1-3 g / L of dipotassium hydrogen phosphate, 4-6 g / L of sodium acetate, and the balance is water.

6. Use of the Enterococcus faecium DLWHY-24 according to claim 1 and the high-efficiency fermentation agent for preparing silage according to any one of claims 2 to 5 in the process of preparing silage.

7. The use according to claim 6, characterized in that: The specific process of the application is: spraying the bacterial suspension of Enterococcus faecium DLWHY-24 or the high-efficiency fermentation agent for preparing silage onto the silage preparation raw materials, sealing, and culturing for more than 40 days to promote the silage preparation process and inhibit the growth of other microorganisms including pathogenic bacteria.

8. The use according to claim 7, characterized in that: The spraying amount of the bacterial suspension of Enterococcus faecium DLWHY-24 or the high-efficiency fermentation agent for preparing silage is 10-100 mL / kg; the water content of the prepared silage is maintained at 65%-70%.

9. The use according to claim 7, characterized in that: The bacterial content of the bacterial suspension of Enterococcus faecium DLWHY-24 or the high-efficiency fermentation agent for preparing silage is 1 to 9×10 8 cfu / mL, culture conditions are 25-45°C, pH 5-8.

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