Streptococcus bovis q6 producing ferulic acid esterase, bacterial suspension, preparation method and application thereof

Abstract

The present application relates to the technical field of microbial application, and specifically discloses a Streptococcus bovis Q6 producing ferulic acid esterase, a bacterial suspension, a preparation method and application, wherein the Streptococcus bovis Q6 is preserved in the China Center for Type Culture Collection on January 29, 2024, and the preservation number is CCTCC NO: M 2024253. The Streptococcus bovis Q6 obtained by separation has the characteristic of producing ferulic acid esterase.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a ferulic acid esterase-producing bovine Streptococcus Q6, a bacterial suspension, a preparation method and an application thereof. Background Art

[0002] Roughage is an important feed source for ruminants, and improving its digestibility is crucial for ruminant performance. The digestible energy of forage depends largely on the digestibility of its dry matter or fiber. Therefore, improving the quality and digestibility of roughage has always been a key concern in the livestock industry.

[0003] Straw roughage has always been a commonly used raw material in animal husbandry, but because it contains a large amount of lignocellulose, it has certain limitations in the subsequent enzymatic digestion or rumen digestion process, which manifests as a low digestibility in ruminants and has an adverse effect on the production performance of ruminants.

[0004] Numerous studies have shown that phenolic compounds in plant cell walls are a major factor inhibiting forage digestibility. Ferulic acid is one of the main phenolic components found widely in plant cell walls. Within the cell wall, it primarily covalently cross-links with polysaccharides via ester bonds and with lignin components via ether or lipid bonds, forming ferulic acid ester bonds.

[0005] 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 the feed, which can easily make livestock feel full and limit their feed intake. Many studies have reported the effects of mechanical processing on the particle size of dairy cow diets such as hay, straw or silage. Feed particle size is crucial in livestock diets because the feed must contain sufficient physically effective neutral detergent fiber, which can stimulate livestock to chew and secrete saliva, reduce gastrointestinal fullness, but do not reduce digestibility. Chemical treatment mainly directly destroys the structure of lignocellulose through chemical reactions. It is mainly divided into acid treatment and alkaline treatment. Biological treatment includes the addition of exogenous enzymes and bacterial inoculation.

[0006] When exogenous cellulase is added to a fiber diet before feeding, some neutral detergent fiber and acid detergent fiber are hydrolyzed, releasing sugars and free monomeric hydroxycinnamic acid, which helps improve fiber digestibility in vitro. In recent years, ferulic acid esterase (FAE) has been used to improve the performance of exogenous cellulase in ruminant feeds. When ferulic acid cross-links arabinoxylan and lignin through ester and ether bonds, respectively, the digestibility of lignocellulose is significantly reduced. Esterase is a new enzyme required to hydrolyze ester bonds and release ferulic acid from the cell wall. It is generally produced by fungi and is rarely found in the rumen environment. For example, there are strains of molds or Bacillus with high enzyme production efficiency in the prior art, but as exogenous microorganisms, they are difficult to colonize in the rumen. Therefore, it is of practical significance to isolate and screen new microorganisms from the environment that can adapt to the rumen environment and produce ferulic acid esterase. Summary of the Invention

[0007] In order to obtain a microorganism that produces feruloyl esterase, the present invention provides a feruloyl esterase-producing Streptococcus bovis Q6, a bacterial suspension, a preparation method and an application. The Streptococcus bovis Q6 isolated and obtained by the present invention has the characteristic of producing feruloyl esterase.

[0008] The present invention provides a ferulic acid esterase-producing Streptococcus bovis Q6. The Streptococcus bovis Q6 was deposited in the China Center for Type Culture Collection on January 29, 2024, with a deposit number of CCTCC NO: M2024253.

[0009] The bovine Streptococcus Q6 is isolated from the rumen, and therefore has a much higher possibility of colonization in the rumen than other exogenous strains. Therefore, it is of great significance.

[0010] The present invention also provides a method for preparing a bacterial suspension, comprising picking a single colony of the bovine Streptococcus Q6 and placing it in an MRS liquid culture medium, culturing the culture anaerobically at 37° C. to 38° C. for 12 to 16 hours, and centrifuging the culture at 7000 to 8000 r / min for 5 to 10 minutes. The bacterial bodies are collected, washed with sterile physiological saline, and then resuspended in deionized water to obtain a bacterial suspension.

[0011] Furthermore, the centrifugation condition is: 8000 r / min for 5 min.

[0012] The present invention also provides a bacterial suspension, which is prepared by the bacterial suspension preparation method.

[0013] The present invention also provides a fermentation liquid obtained by fermenting the bacterial suspension.

[0014] The present invention also provides a method for preparing the fermentation broth, comprising inoculating the bacterial suspension into a liquid fermentation enzyme production medium at an inoculation rate of 2% to 3%, and performing anaerobically fermentation at 37° C. to 38° C. for 2 to 2.5 days to obtain the fermentation broth;

[0015] The liquid fermentation enzyme production medium is as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 5 g of anhydrous sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.17 g of manganese sulfate, 1 mL of Tween-80, 14 g of agar, 50 mL of methyl ferulate solution, and 1 L of distilled water;

[0016] The methyl ferulate solution is prepared by dissolving methyl ferulate in dimethylformamide at a mass volume ratio of 1%.

[0017] Furthermore, the inoculation amount of the bacterial suspension is 2% to 3%, and the fermentation condition is anaerobic fermentation at 37° C. to 38° C. for 2 days.

[0018] Furthermore, the bacterial suspension inoculation amount is 2%, and the fermentation condition is anaerobic fermentation at 37° C. for 2 days.

[0019] The present invention also provides a method for producing ferulic acid esterase, comprising the steps of centrifuging the bovine Streptococcus Q6 culture and resuspending the culture in deionized water to obtain a bacterial suspension, inoculating the bacterial suspension into a liquid fermentation enzyme production medium for anaerobic fermentation to obtain a fermentation liquid, and centrifuging the culture to obtain a supernatant as a crude enzyme liquid.

[0020] The present invention also provides a use of the feruloyl esterase-producing Streptococcus bovis Q6, the bacterial suspension or the fermentation liquid in preparing feruloyl esterase.

[0021] The present invention also provides a use of the ferulic acid esterase-producing bovine Streptococcus Q6, the bacterial suspension or the fermentation broth in inhibiting pathogenic bacteria, wherein the pathogenic bacteria is any one or more combinations of Escherichia coli E. coli, Salmonella pullorum S. pullorum and Staphylococcus aureus S. aureus.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention isolates and screens a bovine Streptococcus Q6, with a deposit number of CCTCC NO: M2024254. When cultured on a solid plate, a transparent zone appears with a diameter of 18.09±1.01 mm. Testing shows that the transparent zone represents feruloyl esterase. A crude enzyme solution is obtained by fermenting the bovine Streptococcus Q6, and testing shows that the feruloyl esterase activity is 1.53±0.06 mU / mL.

[0024] The bovine Streptococcus Q6 isolated and obtained in the present invention is isolated from the rumen, so the possibility of colonization in the rumen is much higher than that of other exogenous strains. The ferulic acid esterase produced by the streptococcus Q6 has an effect on the ingested feed, and can regulate the intestine and promote digestion.

[0025] 2. Streptococcus bovis Q6 has the ability to produce ferulic acid esterase. Therefore, the ferulic acid esterase produced by this bacterium can be used to ferment agricultural waste, such as straw, to obtain ferulic acid in agricultural waste. Ferulic acid has antioxidant properties and can be used in the manufacture of cosmetics, health products and other products.

[0026] 3. The bovine Streptococcus Q6 can also effectively inhibit the growth of Escherichia coli E. coli, Salmonella pullorum S. pullorum and Staphylococcus aureus S. aureus, and has the potential to prepare an antibacterial agent.

[0027] 4. Based on the structural characteristics of lignocellulose in plant cell walls, ferulic acid esterase can break the ferulic acid ester bonds therein, thereby destroying the cell wall, thereby improving the digestibility of ruminants and achieving the goal of efficient utilization of roughage. Therefore, the bovine Streptococcus Q6 of the present invention can be applied in the following two ways:

[0028] ① Spray the strain on roughage (such as straw), create an anaerobic environment through sealing and other means, and ferment for a certain period of time so that the strain can improve the feed quality and increase the utilization rate of roughage by animals through the dual characteristics of lactobacillus and ferulic acid esterase.

[0029] ② Feed the strain directly to animals to take advantage of its isolation from the rumen, allowing it to colonize in the rumen and produce effects in the rumen to increase the animal's digestibility and improve rumen health.

[0030] Information on the deposit of biological materials

[0031] Q6, referred to as Streptococcus bovis Q6 in this application, was deposited in the China Center for Type Culture Collection on January 29, 2024, with the deposit number: CCTCC NO: M 2024253. The depository address is Wuhan University, Wuhan, China, Postal Code: 430072, and the classification name is: Streptococcus boviae Q6 Streptococcus boviae Q6. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a streak plate image of strain Q6 in the present invention.

[0034] Figure 2It is the Oxford cup transparent circle of the strain Q6 in the present invention in the culture dish.

[0035] Figure 3 This is the growth curve of strain Q6 in the present invention.

[0036] Figure 4 The inhibitory effect of strain Q6 of the present invention on different pathogens;

[0037] In the figure, a, b, and c are the antibacterial effects of Q6 strain on E. coli, and a, b, and c are three parallel experiments;

[0038] d, e, and f are the antibacterial effects of strain Q6 on Salmonella pullorum S. pullorum, and d, e, and f are three parallel experiments;

[0039] g, h, and i are the antibacterial effects of Q6 strain on Staphylococcus aureus S. aureus, and g, h, and i are three parallel experiments. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0041] Example 1: Isolation, screening and identification of a ferulic acid esterase-producing Lactobacillus mucosae strain.

[0042] 1. Experimental Materials and Methods

[0043] 1. Samples and culture medium

[0044] (1) MRS liquid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, anhydrous sodium acetate 5 g, diammonium hydrogen citrate 2 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.17 g, Tween-80 1 mL, distilled water 1 L.

[0045] MRS solid medium: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, anhydrous sodium acetate 5 g, diammonium hydrogen citrate 2 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.17 g, Tween-80 1 mL, agar 14 g, distilled water 1 L.

[0046] (2) Screening medium: Sterilize MRS solid medium without adding glucose, cool to about 60°C, add 15 mL / L of filter-sterilized ethyl ferulate solution, and shake well immediately.

[0047] Liquid fermentation enzyme production medium: MRS medium without glucose, sterilized and cooled to about 60℃, and added with 50mL / L methyl ferulate solution;

[0048] The methyl ferulate solution is prepared by dissolving methyl ferulate in dimethylformamide at a mass volume ratio of 1%.

[0049] Preparation of ethyl ferulate solution: dissolve ethyl ferulate in dimethylformamide at a mass volume ratio of 10%, and filter and sterilize.

[0050] (3) Rumen fluid was collected from adult Kazakh rams, frozen at -20°C, and brought back to the laboratory.

[0051] 2. Initial screening of ferulic acid esterase-producing strains

[0052] The rumen fluid thawed at room temperature was inoculated into MRS liquid culture medium at an inoculum rate of 1%, and cultured anaerobically at 37°C for 12 hours. Then, the screening culture medium was inoculated by the cross-streaking method and cultured at 37°C for 72 hours. The streaking inoculation was repeated until a transparent phenomenon, such as a transparent zone or a transparent circle, appeared. It can be preliminarily considered that a strain producing ferulic acid esterase was obtained.

[0053] 3. Rescreening of ferulic acid esterase-producing strains

[0054] Plate clearing zone: After streaking the strains obtained in the initial screening onto MRS solid medium, use an inoculating needle to pick up the clear-looking colony and place it on the screening medium. Incubate at 37°C for 72 hours and observe whether a clearing zone appears on the plate. Strains that produce a clearing zone can be preliminarily identified as producing feruloyl esterase (FAE).

[0055] Oxford cup clearing zone: For strains producing clearing zones, the relationship between enzyme activity and clearing zone size was further determined. After overnight culture in MRS liquid medium, the cells were centrifuged at 8000 rpm for 5 minutes and harvested. The cells were washed three times with sterile saline and resuspended in deionized water. An Oxford cup was placed on the screening medium, and 200 μL of the suspension was resuspended in deionized water. The suspension was then incubated anaerobically for 3 days. The size of the clearing zone was observed.

[0056] 4. Save

[0057] Select a single colony that can produce a transparent circle on the screening medium, purify it for more than three generations, pick a single colony with stable characteristics and put it into MRS liquid medium, culture it anaerobically at 37℃, and OD 600Preserve the bacteria at 0.6-0.8°C with 25% glycerol and store at -80°C.

[0058] 5. Main biochemical reactions

[0059] Inoculate a loopful of the resuspension into a bacterial microbiochemical reaction tube, culture at 37°C for 2-3 days, and then observe the color change.

[0060] 6. Determination of strain growth curve

[0061] The growth curve of the strains was determined using a Ningbo Xinzhi MGC-200 microbial growth curve analyzer. A 96-well plate was sealed in an anaerobic environment, 200 μL of culture medium was added to each well, and 2.5 μL of bacterial solution was inoculated and cultured at 37°C for 48 h.

[0062] 7. Ferulic acid esterase activity assay

[0063] (1) Preparation of crude enzyme solution

[0064] Culture the strain overnight (pick a single colony and inoculate it into MRS liquid medium, 37°C, 12 hours, anaerobic culture), centrifuge at 8000 rpm for 5 minutes, and collect the bacteria. Wash the bacteria three times with sterile saline and resuspend in deionized water to obtain a bacterial suspension. Inoculate the bacterial suspension (2% inoculum) into liquid fermentation and enzyme production medium, and ferment anaerobically at 37°C for 2 days to obtain a fermentation broth. Centrifuge the fermentation broth at 8000 rpm for 5 minutes, and remove the supernatant to obtain the crude enzyme solution.

[0065] (2) Calculation of enzyme activity

[0066] The invention patent of Yang Hongjian, "A method for detecting ferulic acid esterase activity," was used with slight modifications. 100 μL of crude enzyme solution diluted 6.25 times was added to a 96-well plate and preheated at 39°C for 15 min. 200 μL of preheated methyl ferulate solution was added to the blank group, and 200 μL of MOPS (100 mM) was added to the reaction group. The reaction was carried out at 39°C for 30 min. OD values ​​were measured using a microplate reader before and after the reaction. 340 .

[0067] Calculated using the following formula:

[0068]

[0069] The meanings of the parameters are:

[0070] OD 反应起始 : OD value at the start of the reaction;

[0071] OD 反应终止 : OD value at which the reaction was terminated;

[0072] OD 空白起始 : OD value at the start of the reaction without enzyme addition;

[0073] OD 空白终止 : OD value at which the reaction was terminated without enzyme addition;

[0074] V 体系 =300μL; l ε阿魏酸甲酯 =9.467L / mM; l ε阿魏酸 =2.049L / mM; V 样品 =100 μL;

[0075] Sample dilution factor = 6.25; reaction time = 30 min.

[0076] 8. Identification

[0077] The bacteria stored at -80°C were thawed at room temperature and inoculated into MRS liquid culture medium at a 1% inoculum volume and cultured anaerobically at 37°C for 12 h. The culture was then collected and stored in 1.5 mL centrifuge tubes and sent to Shanghai Sangon Biotechnology Co., Ltd. for 16S rDNA full sequence analysis.

[0078] 9. Oxford cup antibacterial test

[0079] (1) Preparation of indicator bacteria suspension:

[0080] Three pathogenic bacteria, E. coli, Salmonella pullorum, and Staphylococcus aureus, were used as indicator bacteria. The strains were preserved in the Feed Biotechnology Laboratory, College of Animal Science, Xinjiang Agricultural University. The pathogenic bacteria were activated and cultured twice using LB medium. A single colony was picked and incubated in LB liquid medium at 37°C, 170 rpm, and a concentration of 10 8 CFU / mL to obtain the indicator bacteria suspension.

[0081] (2) Determination of antibacterial activity

[0082] A single colony of Streptococcus bovis Q6 that produced a transparent circle was inoculated into MRS liquid medium and cultured until 10 8 CFU / mL. Place 200 μL of bacterial solution in an Oxford cup and measure the diameter of the inhibition zone using ProtoCOL3P3HD / 1129 manufactured by SYNBIOSIS Integrated Technology Co., Ltd., UK. Record the diameter of the Oxford cup inhibition zone and subtract 8 mm from the Oxford cup to obtain the diameter of the inhibition zone.

[0083] 2. Experimental Results

[0084] 1. Flat plate streaking screening results

[0085] The MRS liquid medium cultured overnight was streaked onto the screening medium. One strain that could produce transparency was found on the screening medium and was named Q6. The streaked culture plate was as follows: Figure 1shown.

[0086] 2. Oxford Cup Transparent Ring Screening Results

[0087] The size of the transparent zone produced by strain Q6 was further determined by the Oxford cup transparent zone test. The transparent zone produced after dark culture was as follows: Figure 2 shown.

[0088] Record the diameter of the transparent ring of the Oxford cup (minus the diameter of the Oxford cup). The results are shown in Table 1.

[0089] Table 1 Size of Oxford cup transparent zone produced by strains

[0090]

[0091] 3. Main biochemical reactions of strains

[0092] Table 2 Main biochemical reactions

[0093]

[0094] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0095] As shown in Table 2, referring to GB4789.35-2023, strain Q6 showed positive reactions to esculin, maltose, salicin, sorbitol, sucrose, raffinose inulin, and lactose, and negative reactions to mannitol and sodium hippurate. Among them, the positive test for esculin indicated that the strain belonged to group D streptococcus, which was consistent with the results of species identification.

[0096] 4. Strain growth curve determination

[0097] like Figure 3 As shown, during the 0-2h cultivation, strain Q6 grew slowly and was in a stagnant period; during the 2-13h cultivation, it entered a logarithmic growth period with the fastest growth rate; after 13h of cultivation, the growth of the strain entered a plateau period and gradually stabilized.

[0098] 5. Determination of ferulic acid esterase crude enzyme activity

[0099] The feruloyl esterase activity of the feruloyl esterase-producing strain Q6 was calculated using the formula according to the microplate reader assay, and the results are shown in Table 3. The feruloyl esterase activity of the strain Q6 was 1.53±0.06 mU / mL.

[0100] Table 3 Ferulic acid esterase activity of strains

[0101] strain number Enzyme activity (mU / mL) Q6 1.53±0.06

[0102] 6. Strain sequence comparison results

[0103] Sequence comparison results show that strain Q6 has a 100% similarity to Streptococcus bovis PP916666 in Genbank. Therefore, strain Q6 was identified as Streptococcus bovis.

[0104] 7. Inhibition of bovine Streptococcus Q6 on pathogens

[0105] Table 4 Size of inhibition zone produced by Q6 strain

[0106] Indicator strain Diameter of inhibition zone (mm) E. coli 7.37±0.66 Salmonella pullorum 6.48±0.97 Staphylococcus aureus 9.91±0.64

[0107] The results are shown in Table 4 and Figure 4 As shown in the figure, in terms of antibacterial effect, Streptococcus bovis Q6 exhibited inhibitory effects on Escherichia coli, Salmonella pullorum and Staphylococcus aureus, with the most significant antibacterial effect on Staphylococcus aureus.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0109] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A ferulic acid esterase-producing Streptococcus bovis Q6, characterized in that: The bovine Streptococcus Q6 was deposited in the China Center for Type Culture Collection on January 29, 2024, with the deposit number: CCTCC NO: M2024253.

2. A method for preparing a bacterial suspension, characterized in that: Pick a single colony of the bovine Streptococcus Q6 described in claim 1 into MRS liquid culture medium, culture anaerobically at 37° C. to 38° C. for 12 h to 16 h, centrifuge at 7000 rpm to 8000 rpm for 5 min to 10 min, collect the bacteria, wash them with sterile saline, and resuspend them in deionized water to obtain a bacterial suspension.

3. The method for preparing the bacterial suspension according to claim 2, wherein The centrifugal conditions were: 8000 r / min for 5 min.

4. A bacterial suspension, characterized in that The bacterial suspension is prepared by the method for preparing the bacterial suspension according to claim 2 or 3.

5. A fermentation broth, characterized in that: Obtained by fermentation of the bacterial suspension according to claim 4.

6. A method for preparing the fermentation broth according to claim 5, characterized in that: Inoculate the bacterial suspension into a liquid fermentation enzyme production medium at an inoculum volume of 2% to 3%, and perform anaerobically fermentation at 37°C to 38°C for 2 days to 2.5 days to obtain a fermentation liquid; The liquid fermentation enzyme production medium is as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 5 g of anhydrous sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.17 g of manganese sulfate, 1 mL of Tween-80, 14 g of agar, 50 mL of methyl ferulate solution, and 1 L of distilled water; The methyl ferulate solution is prepared by dissolving methyl ferulate in dimethylformamide at a mass volume ratio of 1%.

7. The method for preparing a fermentation broth according to claim 6, wherein: The inoculation amount of the bacterial suspension is 2% to 3%, and the fermentation condition is anaerobic fermentation at 37° C. to 38° C. for 2 days.

8. A method for producing ferulic acid esterase, characterized in that: The bovine Streptococcus Q6 culture of claim 1 is centrifuged and then resuspended with deionized water to obtain a bacterial suspension, which is then inoculated into a liquid fermentation enzyme production medium for anaerobic fermentation to obtain a fermentation liquid, which is centrifuged and the supernatant is collected as a crude enzyme liquid.

9. Use of the feruloyl esterase-producing Streptococcus bovis Q6 according to claim 1, the bacterial suspension according to claim 4 or the fermentation broth according to claim 5 in the preparation of feruloyl esterase.

10. Use of the ferulic acid esterase-producing Streptococcus bovis Q6 according to claim 1, the bacterial suspension according to claim 4, or the fermentation broth according to claim 5 in inhibiting pathogenic bacteria, characterized in that: The pathogenic bacteria are any one or more of Escherichia coli, Salmonella pullorum and Staphylococcus aureus.

Images