Lactobacillus mui Q2 producing ferulic acid esterase, bacterial suspension, preparation method and application

Abstract

The present application relates to the technical field of microbial application, and specifically discloses a mucilaginibacter sp. Q2 producing ferulic acid esterase, a bacterial suspension, a preparation method and an application, wherein the mucilaginibacter sp. is preserved in the China Center for Type Culture Collection on January 29, 2024, and the preservation number is CCTCC NO: M 2024254. The mucilaginibacter sp. Q2 obtained by separation has the characteristic of producing ferulic acid esterase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial application, and particularly relates to a mucilaginibacter sp. Q2 producing ferulic acid esterase, a bacterial suspension, a preparation method and application. BACKGROUND

[0002] Ruminants are important sources of roughage, and improving the digestibility of roughage is of great significance to the production performance of ruminants. The digestible energy of forage is largely dependent on the digestibility of dry matter or fiber. Therefore, improving the quality and digestibility of roughage has been an important issue of attention in animal husbandry.

[0003] Straw roughage has always been a commonly used raw material in animal husbandry, but due to its high content of lignocellulose, it has certain limitations in subsequent enzymatic digestion or rumen digestion, and shows a low digestibility in ruminants, which has an adverse effect on the production performance of ruminants.

[0004] A large number of studies have shown that phenolic substances in plant cell walls are the main factors inhibiting the digestibility of forage. Ferulic acid is one of the main phenolic components widely existing in plant cell walls. It is mainly covalently cross-linked with polysaccharides in the form of ester bonds, and is covalently cross-linked with lignin components in the form of ether bonds or lipid bonds, forming ferulic acid ester bonds.

[0005] The existing methods for increasing the digestibility of lignocellulose include physical and mechanical processing, chemical treatment and biological treatment. Mechanical processing is a key step in feed production, because it affects the physical properties of feed, easily makes livestock have a significant feeling of satiety, and limits the feed intake. Many studies have reported the effects of mechanical processing on the particle size of dairy rations such as hay, straw or silage feed. The particle size of feed is crucial in livestock feed, because the feed must contain enough physically effective neutral detergent fiber, which can stimulate livestock to chew and secrete saliva, reduce the feeling of satiety in the gastrointestinal tract, but does not reduce the digestibility. Chemical treatment mainly directly destroys the structure of lignocellulose through chemical reactions. It mainly includes acid treatment and alkali treatment. Biological treatment includes adding exogenous enzymes and bacterial inoculation treatment.

[0006] Exogenous cellulase is applied in the fibrous diet before feeding, part of neutral detergent fiber and acid detergent fiber is hydrolyzed and releases sugar and free monomer hydroxyl cinnamic acid, which helps to improve in vitro fiber digestibility. Ferulic acid esterase (FAE) has been used to improve the performance of exogenous cellulase in ruminant feed in recent years. When ferulic acid is cross-linked with arabinoxylan and lignin through ester bond and ether bond respectively, the degree of digestion of lignocellulose will be significantly reduced. Esterase is a new enzyme required to release ferulic acid from cell wall by hydrolyzing ester bond, which is generally produced by fungi and rarely exists in rumen environment. For example, there are strains with high enzyme production efficiency such as mold and bacillus in the prior art, but as exogenous microorganisms, it is difficult to colonize in the rumen. Therefore, it is of practical significance to screen new microorganisms that can adapt to the rumen environment and produce ferulic acid esterase from the environment. SUMMARY

[0007] In order to obtain a microorganism producing ferulic acid esterase, the present application provides a lactobacillus mucosus Q2 producing ferulic acid esterase, a bacterial suspension and a preparation method and application. The lactobacillus mucosus Q2 obtained by separation has the characteristics of producing ferulic acid esterase.

[0008] The present application provides a lactobacillus mucosus Q2 producing ferulic acid esterase, which is preserved in China Center for Type Culture Collection on January 29, 2024, and the preservation number is CCTCC NO: M2024254.

[0009] The lactobacillus mucosus Q2 is isolated from rumen, so the possibility of colonization in rumen is much higher than that of other exogenous strains. Therefore, it is of great significance.

[0010] The present application also provides a bacterial suspension containing the lactobacillus mucosus Q2.

[0011] The present application also provides a preparation method of the bacterial suspension, which comprises the following steps: picking up a single colony of lactobacillus mucosus Q2 into MRS liquid culture medium, carrying out anaerobic culture at 37-38 DEG C for 12-16 h, centrifuging at 7000-8000 r / min for 5-10 min, collecting bacterial bodies, washing with sterile normal saline and resuspending in deionized water to obtain the bacterial suspension.

[0012] Further, the centrifugation condition is: 8000 r / min for 5 min.

[0013] The present application also provides a fermentation liquor obtained by fermentation of the bacterial suspension.

[0014] The present application also provides a preparation method of the fermentation liquor, which comprises the following steps: inoculating the bacterial suspension into liquid fermentation enzyme production culture medium according to an inoculation amount of 2-3 %, carrying out anaerobic fermentation at 37-38 DEG C for 2-2.5 d to obtain the fermentation liquor.

[0015] The liquid enzyme production medium is: MRS medium without glucose, sterilization and cooling to 58-62℃, adding methyl formate solution containing dimethyl formamide.

[0016] Further, the liquid enzyme production medium is: MRS medium without glucose, sterilization and cooling to about 60℃, adding 50mL / L methyl formate solution containing 1% W / V dimethyl formamide.

[0017] Further, the inoculation amount of the bacterial suspension is 2%, and the fermentation condition is anaerobic fermentation at 37℃ for 2d.

[0018] The application also provides a method for producing ferulic acid esterase, wherein the Lactobacillus mui Q2 is cultured, centrifuged, resuspended with deionized water to obtain a bacterial suspension, inoculated into a liquid enzyme production medium, and anaerobically fermented to obtain a fermentation liquor, and the supernatant obtained after centrifugation is a crude enzyme liquor.

[0019] The application also provides application of the Lactobacillus mui Q2 producing ferulic acid esterase, the bacterial suspension or the fermentation liquor in the preparation of ferulic acid esterase.

[0020] The application also provides application of the Lactobacillus mui Q2 producing ferulic acid esterase, the bacterial suspension or the fermentation liquor in the field of food technology.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] 1. The Lactobacillus mui Q2 is isolated and screened, and the preservation number is CCTCC NO: M2024254. The Lactobacillus mui Q2 is cultured on a solid plate to form a transparent circle with a diameter of 7.94±1.60mm. It is found through detection that the transparent circle is produced ferulic acid esterase. The Lactobacillus mui Q2 is fermented to obtain a crude enzyme liquor, and it is found through detection that the ferulic acid esterase activity is 0.50±0.10mU / mL.

[0023] The Lactobacillus mui Q2 is isolated and obtained, is a lactic acid bacteria, and is isolated from rumen. Therefore, the possibility of colonization in rumen is much higher than that of other exogenous strains. The lactic acid bacteria can play a balancing role as an intestinal probiotic. The ferulic acid esterase produced by the lactic acid bacteria can act on the ingested feed, and can play a role in regulating the intestinal tract and promoting digestion.

[0024] 2. The Lactobacillus mui Q2 has the function of producing ferulic acid esterase. Therefore, the ferulic acid esterase produced by the bacteria can be used to ferment agricultural waste such as straw, so as to obtain ferulic acid in the agricultural waste. The ferulic acid has antioxidant properties, and can be used in the manufacture of cosmetics, health care products and the like.

[0025] 3. Based on the structural characteristics of lignocellulose in plant cell walls, ferulic acid esterase can break the ferulic acid ester bonds, thereby disrupting the cell wall and improving the digestibility of ruminants, achieving the goal of efficient utilization of roughage. Therefore, the *Lactobacillus mucilaginosus* Q2 of this invention can be applied in the following two ways:

[0026] ① Spray the strain onto roughage (such as straw), and create an anaerobic environment through sealing or other means. After a certain period of fermentation, the strain can improve feed quality and increase the utilization rate of roughage by leveraging the dual characteristics of lactobacillus and ferulic acid esterase.

[0027] ② The strain was directly fed to animals to take advantage of its rumen isolation, allowing it to colonize the rumen and exert its effects, thereby improving the animals' digestibility and rumen health.

[0028] Information on the Preservation of Biological Materials

[0029] Q2, referred to in this application as *Lactobacillus mucosae* Q2, was deposited on January 29, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024254. The address of the depository is Wuhan University, Wuhan, China, 430072, China. The classification name is *Lactobacillus mucosae* Q2. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a streak plate diagram of strain Q2 in this invention.

[0032] Figure 2 The Oxford cup transparent ring of strain Q2 in the present invention in a petri dish.

[0033] Figure 3 This is the growth curve of strain Q2 in this invention. Detailed Implementation

[0034] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0035] Example 1: Isolation, screening and identification of a mucosa mucosa lactobacillus strain producing ferulic acid esterase.

[0036] I. Experimental materials and methods

[0037] 1. Sample and culture medium

[0038] (1) MRS liquid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, sodium acetate anhydrous 5 g, diammonium hydrogen citrate 2 g, potassium phosphate dibasic 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.17 g, Tween-80 1 mL, distilled water 1 L.

[0039] MRS solid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, sodium acetate anhydrous 5 g, diammonium hydrogen citrate 2 g, potassium phosphate dibasic 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.17 g, Tween-80 1 mL, agar 14 g, distilled water 1 L.

[0040] (2) Screening medium: MRS solid medium without glucose, sterilized and cooled to about 60°C, then 15 mL / L filtered and sterilized ethyl ferulate solution was added and shaken immediately.

[0041] Liquid fermentation enzyme production medium: MRS medium without glucose, sterilized and cooled to about 60°C, then 50 mL / L methyl ferulate solution (1% W / V dimethylformamide) was added.

[0042] Ethyl ferulate solution preparation: 10% (W / V) ethyl ferulate was dissolved in N,N-dimethylformamide and filtered to remove bacteria.

[0043] (3) The sample was the rumen fluid of an adult Kazakh ram in vitro.

[0044] 2. Primary screening of ferulic acid esterase-producing strains

[0045] Inoculate the rumen fluid into MRS liquid medium at an inoculation amount of 1%, and incubate at 37°C anaerobically for 12h, then inoculate into the screening medium by cross streaking, and incubate at 37°C for 72h, and repeat the streaking inoculation until transparent (transparent band or transparent ring) appears, and it can be preliminarily considered that Lactobacillus mucosus producing ferulic acid esterase is obtained.

[0046] 3. Re-screening of the ferulic acid esterase-producing strain

[0047] Transparent ring on the plate: inoculate the strain obtained by the preliminary screening into MRS solid medium by streaking, and then use an inoculation needle to dip the transparent bacterial group to the screening medium, and incubate at 37°C for 72h, and observe whether a transparent ring appears on the plate. The strain producing a transparent ring can be preliminarily determined as producing ferulic acid esterase (FAE).

[0048] Oxford cup transparent ring: for the strain producing a transparent ring, further determine the relationship between the enzyme activity and the size of the transparent ring. After the strain is incubated in MRS liquid medium overnight, centrifuge at 8000r / min for 5min to collect the bacterial cells. Wash the bacterial cells with sterile normal saline for 3 times, and resuspend in deionized water to obtain a bacterial suspension. Place an Oxford cup on the screening medium, and inoculate 200μL of the bacterial suspension, and anaerobically incubate for 3d. Observe the size of the transparent ring.

[0049] 4. Preservation

[0050] Select single colonies producing transparent rings on the screening medium, and continuously purify for more than three generations, and pick single colonies with stable traits into MRS liquid medium, and incubate at 37°C anaerobically, and OD 600 When OD is 0.6-0.8, preserve the bacteria with 25% glycerol, and store at -80°C.

[0051] 5. Main biochemical reactions

[0052] Inoculate a loop of bacterial suspension into a bacterial micro biochemical reaction tube, and observe the discoloration after incubation at 37°C for 2-3d.

[0053] 6. Determination of the growth curve of the strain

[0054] Use the Ningbo Xinzhi MGC-200 microbial growth curve analyzer to determine the growth curve of the strain. Place a 96-well plate in an anaerobic environment, seal, and add 200uL of medium to each well, inoculate 2.5uL of bacterial liquid, and incubate at 37°C for 48h.

[0055] 7. Determination of the ferulic acid esterase enzyme activity

[0056] (1) Preparation of the crude enzyme solution

[0057] Strains were cultured overnight (a single colony was inoculated into MRS liquid medium, 37°C, 12h, anaerobic culture), centrifuged at 8000r / min for 5min, and the bacterial cells were collected. The bacterial cells were washed with sterile normal saline for 3 times, resuspended in deionized water to obtain a bacterial suspension. The bacterial suspension was inoculated (2% inoculation amount) into a liquid fermentation enzyme production medium, and 37°C anaerobic fermentation was carried out for 2d to obtain a fermentation broth. The fermentation broth was centrifuged at 8000r / min for 5min, and the supernatant was obtained, that is, a crude enzyme solution was obtained.

[0058] (2) Enzyme activity calculation

[0059] Yang Hongjian's invention patent "A detection method for ferulic acid esterase activity" was used with slight modification. 100uL of the crude enzyme solution diluted 6.25 times was added to a 96-well plate, preheated at 39°C for 15min, 200uL of preheated methyl ferulate solution was added to the blank group, and 200uL of MOPS (100mM) was added to the reaction group, and reacted at 39°C for 30min. The OD was measured before and after the reaction using an enzyme marker. 340 .

[0060] The following formula was used for calculation:

[0061]

[0062] Among them, the meanings of various parameters are as follows:

[0063] OD 反应起始 : OD value at the beginning of the reaction;

[0064] OD 反应终止 : OD value at the end of the reaction;

[0065] OD 空白起始 : OD value at the beginning of the reaction without enzyme;

[0066] OD 空白终止 : OD value at the end of the reaction without enzyme;

[0067] V 体系 = 300uL; l ε阿魏酸甲酯 = 9.467L / mM; l ε阿魏酸 = 2.049L / mM; V 样品 = 100uL;

[0068] Sample dilution multiple = 6.25; reaction time = 30min.

[0069] 8, Identification

[0070] The strain stored at -80℃ was thawed at room temperature, inoculated into MRS liquid medium (1% inoculation amount) and incubated at 37℃ anaerobically for 12h, and 1.5mL was taken and centrifuged for storage, and sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for 16s rDNA full sequence analysis.

[0071] II. Experimental results

[0072] 1. Plate streaking screening results

[0073] The MRS liquid medium of the overnight culture was streaked into the screening medium, and one strain capable of producing a transparent condition was found on the screening medium, which was designated as Q2, and the streak culture plate is shown in Figure 1 .

[0074] 2. Oxford cup transparent circle screening results

[0075] The size of the transparent circle produced by strain Q2 was further determined by the Oxford cup transparent circle test, and the transparent circle produced after light-free culture is shown in Figure 2 .

[0076] The diameter of the Oxford cup transparent circle was recorded (minus the diameter of the Oxford cup), and the results are shown in Table 1.

[0077] Table 1 Size of Oxford cup transparent circle produced by strain

[0078] Strain No. Transparent zone diameter (mm) Q2 7.94±1.60

[0079] 3. Main biochemical reactions of the strain

[0080] Table 2 Main biochemical reactions

[0081]

[0082] Note: "+" is positive reaction, "-" is negative reaction.

[0083] As shown in Table 2, according to GB 4789.35-2023, strain Q2 has positive reactions to maltose, sucrose and raffinose, and negative reactions to mannitol and sorbitol. Since strain Q2 is not listed in the standard, but its main biochemical reactions are consistent with those of Lactobacillus reuteri in the standard, and both strains belong to the dominant lactobacillus in the intestine, it can be considered that the results are consistent with the sequencing results.

[0084] 4. Determination of growth curve of the strain

[0085] As shown in Figure 3 , the strain Q2 grew slowly and was in the stationary phase during 0-2h of culture; it entered the logarithmic growth phase during 2-13h of culture, and the growth rate was the fastest; after 13h of culture, the growth of the strain entered the gentle phase, and the growth gradually stabilized.

[0086] 5. Feruloyl esterase activity determination of the crude enzyme solution of the feruloyl esterase producing strain Q2

[0087] According to the determination of the microplate reader, the feruloyl esterase activity of the strain Q2 was calculated using the formula, and the results are shown in Table 3. The feruloyl esterase activity of the strain Q2 was 0.50 ± 0.10 mU / mL.

[0088] Table 3 Feruloyl esterase activity of the strain

[0089] Strain No. Enzyme activity (mU / mL) Q2 0.50±0.10

[0090] 6. Sequence comparison results of the strain

[0091] According to the sequence comparison results, it was found through sequence alignment that the similarity of the strain Q2 with Lactobacillus mucosae PP916665 in Genbank was 100%. Therefore, the strain Q2 was identified as Lactobacillus mucosae.

[0092] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0093] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A Lactobacillus mucosus Q2 strain producing feruloyl esterase, characterized in that, The Lactobacillus muciapparius is preserved in China Center for Type Culture Collection on January 29, 2024, and the preservation number is CCTCC NO: M2024254.

2. A bacterial suspension, characterized in that, The bacterial suspension contains the Lactobacillus muciapparius Q2 of claim 1.

3. A method for the preparation of the bacterial suspension according to claim 2, characterized in that, The Lactobacillus muciapparius Q2 single colony is picked into MRS liquid culture medium, and is cultured anaerobically at 37-38℃ for 12-16h, centrifuged at 7000-8000r / min for 5-10min, and then the bacterial cells are collected, washed with sterile normal saline, and resuspended in deionized water to obtain the bacterial suspension.

4. The method of claim 3, wherein the bacteria suspension is prepared by, The centrifugation condition is 8000r / min for 5min.

5. A fermentation broth, characterized in that, The bacterial suspension of claim 2 is fermented to obtain.

6. A process for the preparation of the fermentation broth of claim 5, characterized in that, The bacterial suspension is inoculated into liquid enzyme production fermentation medium at an inoculation amount of 2-3%, and is fermented anaerobically at 37-38℃ for 2-2.5d to obtain the fermentation broth. The liquid enzyme production fermentation medium is MRS medium without glucose, and the ferulic acid methyl ester solution containing dimethylformamide is added after sterilization and cooling to 58-62℃.

7. The method of claim 6, wherein the fermentation broth is prepared by the steps of: The inoculation amount of the bacterial suspension is 2-3%, and the fermentation condition is anaerobic fermentation at 37-38℃ for 2d.

8. A method for producing ferulic acid esterase, characterized by, The Lactobacillus muciapparius Q2 of claim 1 is cultured, centrifuged, resuspended in deionized water to obtain the bacterial suspension, and then the bacterial suspension is inoculated into liquid enzyme production fermentation medium to obtain the fermentation broth by anaerobic fermentation, and the supernatant after centrifugation is the crude enzyme solution.

9. Use of the Lactobacillus muciapparius Q2 producing ferulic acid esterase of claim 1, the bacterial suspension of claim 2, or the fermentation broth of claim 5 in the preparation of ferulic acid esterase.

10. Use of the Lactobacillus muciapparius Q2 producing ferulic acid esterase of claim 1, the bacterial suspension of claim 2, or the fermentation broth of claim 5 in the field of food technology.

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