Bacillus and application thereof in production of beta-glucosidase

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus and application thereof in production of beta-glucosidase. The invention relates to bacillus LP-1, the Latin literature name of the bacillus LP-1 is bacillus sp.LP-1, and the preservation number of the bacillus LP-1 is CCTCC (China Center For Type Culture Collection) M 20232261. The invention further provides application of the bacillus LP-1 in the aspect of producing beta-glucosidase. The bacillus LP-1 has the advantages that the bacillus LP-1 has good capacity of producing the beta-glucosidase, the heat stability of the produced beta-glucosidase is relatively high, performance results show that the enzyme activity is 75.33 U / mL, the optimum reaction temperature is 80 DEG C, the optimum pH is 7.0, and the bacillus LP-1 has a great application prospect. The method has important significance for reasonably developing microbial resources in the Liupao tea and promoting the industrial development of the Liupao tea.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus species and its application in the production of β-glucosidase. Background Technology

[0002] Liubao tea is a famous historical tea from Guangxi, belonging to the post-fermented dark tea category along with Pu'er tea and Fuzhuan tea, boasting a history of over 1500 years. Unlike the other five major tea categories, bacteria and fungi continuously engage in physiological activity during the production, aging, and storage of dark tea. It is generally believed that the combined action of microorganisms and enzymes—namely, polymerization or degradation—generates macromolecules and small molecules, thus influencing and shaping the quality characteristics of Liubao tea. In recent years, Liubao tea has been exported overseas, continuously increasing its market share, and scientific research has received greater attention. Regarding microbial research, compared to other dark teas, there are fewer reports on the microorganisms in Liubao tea. The diversity of microorganisms in Liubao tea has not yet been fully explored and utilized, leaving considerable room for further research.

[0003] β-glucosidase has promising development prospects and is an important class of enzyme preparations. It can be used to improve food flavor and generate bioactive monomeric components, and has wide applications in food processing, daily chemical industry, agriculture, and medicine. Food raw materials such as tea and fruits contain various flavor substances and some secondary metabolites. These substances themselves have no aroma and are non-volatile. β-glucosidase can be used to enzymatically hydrolyze them, producing natural flavor substances and thus achieving a natural aroma-enhancing effect. Studies have shown that adding β-glucosidase can promote the conversion of important flavor substances in fruit juices and wines, enhancing their taste. In the debittering of plums, β-glucosidase plays an important role, breaking down amygdalin, thereby significantly reducing bitterness. β-glucosidase can also significantly promote the hydrolysis of lignocellulose, greatly improving the efficiency of cellulose conversion to fuel ethanol. In dairy processing, β-glucosidase is often used to break down lactose, working with other enzymes to produce glucose and single-cell proteins. The application of β-glucosidase activity also plays an indispensable role in the diagnosis and treatment of certain cancers. In recent years, research on the sources of β-glucosidase has mainly focused on fungi such as Aspergillus, Penicillium, and yeast. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a Bacillus strain and its application in the production of β-glucosidase.

[0005] The primary objective of this invention is to provide a strain of Bacillus LP-1, whose Latin scientific name is Bacillus sp .LP-1, accession number CCTCC M 20232261.

[0006] The second objective of this invention is to provide the application of a Bacillus LP-1 strain in the production of β-glucosidase.

[0007] Preferably, the method for producing β-glucosidase specifically includes the following steps: S1. Strain activation: Activate Bacillus LP-1 in basal medium, pick the activated strain and inoculate it into basal medium, and culture in a shaker at 20-40℃ for 20-30 hours; centrifuge, wash, and resuspend to adjust the bacterial suspension concentration to 10. 7 cfu / mL is the seed solution; S2. Inoculation: Inoculate the seed culture into the fermentation medium at an inoculation rate of 4-6%; culture in a shaker at a temperature of 35-37℃ and a rotation speed of 130-180 r / min for 3-5 days. S3. Take the fermentation broth after fermentation, centrifuge at 7000~10000r / min for 8~15min, collect the supernatant, and obtain crude β-glucosidase enzyme solution. S4. Purify the crude enzyme solution and freeze-dry it under vacuum to obtain β-glucosidase powder.

[0008] Preferably, the fermentation medium comprises the following weight components: 4-6g yeast extract, 8-12g peptone, 8-12g glucose, 0.2-0.4g NH4Cl, 0.3-0.5g KH2PO4, and 0.1-1g p-nitrophenyl glucoside.

[0009] Preferably, the fermentation medium is prepared as follows: weigh the yeast extract, peptone, glucose, NH4Cl and KH2PO4 0.4g in sequence, sterilize at 100~121℃ for 15~25min, cool to 65~75℃ and add p-nitrophenyl glucoside.

[0010] Preferably, in step S2, the pH needs to be adjusted to 6.5-7.5 before shaking culture.

[0011] Preferably, the inoculation amount of seed liquid in step S2 is 5%; the temperature of the shaking culture is 37°C and the rotation speed is 150 r / min.

[0012] Preferably, the temperature in step S1 is 30°C.

[0013] Preferably, the centrifugation speed is 8000 r / min and the time is 10 min.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) Bacillus LP-1 has a good ability to produce β-glucosidase and the β-glucosidase has high thermal stability. Performance results show that the enzyme activity is 75.33 U / mL and the optimal reaction temperature is 80℃ and the optimal pH is 7.0, which has great application prospects.

[0015] (2) This invention studies the diversity of microorganisms in Liubao tea from a new perspective. It is of great significance for the rational development of microbial resources in Liubao tea and further promotes the development of the Liubao tea industry. It also provides inspiration for expanding the application research of β-glucosidase to a certain extent. Attached Figure Description

[0016] Figure 1 This describes the colony characteristics of the LP-1 strain on the culture medium provided in the embodiments of the present invention.

[0017] Figure 2 The images provided in this embodiment of the invention show the colony characteristics (A) and a magnified view (B) of the strain observed under a microscope on a culture medium.

[0018] Figure 3 This is a phylogenetic tree of the strain based on 16S rDNA provided in the embodiments of the present invention.

[0019] Figure 4 This is the growth curve of Bacillus LP-1 provided according to an embodiment of the present invention.

[0020] Figure 5 The results show the effect of pH on the growth of Bacillus LP-1 according to embodiments of the present invention.

[0021] Figure 6 The results show the effect of temperature on the growth of Bacillus LP-1 according to embodiments of the present invention.

[0022] Figure 7 This is a standard curve for p-nitrophenol provided according to an embodiment of the present invention.

[0023] Figure 8 The results show the effect of temperature on β-glucosidase activity according to embodiments of the present invention.

[0024] Figure 9 The results show the effect of pH on β-glucosidase activity according to embodiments of the present invention. Detailed Implementation

[0025] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0027] Example 1 I. Preparation of Culture Medium LB medium: 10g peptone, 10g sodium chloride, 5g yeast extract, 1000mL water, pH adjusted to 7.0, sterilized for 20 min.

[0028] Aescin medium: 1g aescin, 0.5g ferric citrate, 10g peptone, 10g sodium chloride, 15g agar, 5g yeast extract, 1000mL water, pH adjusted to 7.0, sterilized for 20 min.

[0029] The yeast extract, peptone, agar, sodium chloride, aescin, ferric citrate, and other reagents used above were all domestically produced analytical grade reagents.

[0030] According to the culture medium formula, weigh all reagents on a balance, heat them to dissolve, add the required amount of water, adjust the pH to neutral, sterilize for 20 minutes, and set aside.

[0031] II. Isolation and purification of β-glucosidase-producing microorganisms Raw material: Liubao tea leaves, i.e., fresh leaves of the tea tree, whose Latin name is *Camellia sinensis* (L.) O. Ktze. Take an appropriate amount of Liubao tea leaves (purchased from Guangxi Wuzhou Huaxiang Liubao Tea Industry Co., Ltd.), add them to LB liquid medium, and incubate at 37℃ for 24 hours; dilute the culture medium, diluting 1 mL of culture medium to 10 mL in sequence. -6 Using a pipette, 1 mL of the bacterial suspension for each dilution was evenly spread onto aescin medium, with three plates for each dilution. The plates were then incubated upside down at 37°C. In this medium, Fe... 3+ The colonies that react with esculin to produce a brownish-black color are β-glucosidase-producing microorganisms. Brownish-black colonies are picked up with an inoculation needle and streaked onto a plate to isolate and purify single β-glucosidase-producing colonies. These colonies are then incubated upside down at 37°C. Finally, the single colonies are inoculated onto solid LB agar slant and incubated at 37°C for further culture and identification.

[0032] Results: One strain that produced a deep brownish-black color on esculin-containing medium was selected from β-glucosidase-producing strains. This strain grew rapidly on esculin-containing medium. Figure 1As shown, the colonies are round, with neat edges, moist surfaces, and slightly raised, and are milky white and opaque in color. After 7 days of cultivation, the colonies turn into flattened mycelial moss, with irregular shapes, rough surfaces, and a grayish-white, dull color.

[0033] II. Identification of β-glucosidase-producing microorganisms 1. Morphological identification Sterilize clean glass slides in 75% alcohol, using them only as needed, and burning off excess alcohol before each use. Pick a small amount of bacterial growth from the culture medium and spread it onto a slide dipped in sterile water. Heat-fix the slide over an alcohol lamp flame. Once the slide is dry, stain it according to the Gram staining procedure. After the slide is dry, observe the morphological characteristics of the microorganisms under a microscope.

[0034] like Figure 2 As shown, under a microscope, the bacterial strain is rod-shaped, arranged in a chain, and stains purple with Gram stain. It is a Gram-positive bacterium with blunt, rounded ends and is capable of forming spores, which are oval in shape.

[0035] 2. Molecular biological identification LB solid slant culture medium was sent to Shanghai Sangon Biotech Co., Ltd. to determine its 16S rDNA gene sequence. The BLAST program in the NCBI database was used to search for standard sequences with high similarity. Then, MEGA 7.0 software was used to perform cluster analysis and construct a phylogenetic tree.

[0036] Results: Sequencing results showed that the amplified sequence was 1051 bp in length, and the strains with high similarity to it were all Bacillus. The top 10 strains with the highest sequence similarity were Bacillus toyosum BCT-7112 (… Bacillus toyonensis BCT-7112), Bacillus kinosinophila 0711P9-1 ( Bacillus mobilis 0711P9-1), Bacillus paclitaxel EB422 ( Bacillus pacificus EB422), Bacillus weizmannii FSL W8-0169 ( Bacillus wiedmannii FSL W8-0169), Bacillus TD42 ( Bacillus proteolyticus TD42), Bacillus albus N35-10-2 ( Bacillus albus N35-10-2), Bacillus TD41 ( Bacillus luti TD41), Bacillus subtilis 17-SMS-01 ( Bacillus fungorum 17-SMS-01), Bacillus cereus ATCC 14579 ( Bacillus cereu ATCC 14579) and Bacillus paramyxoides NH24A2 ( Bacillus paramycoidesNH24A2), which is related to Bacillus thymosus BCT-7112 ( Bacillus toyonensis The homology of BCT-7112 reached 100%, and a phylogenetic tree was constructed from typical strains with high similarity ( Figure 3 The strain was preliminarily identified as belonging to the genus Bacillus and named Bacillus LP-1, with its sequence shown in SEQ ID NO.1 of the sequence listing. Bacillus LP-1 Latin scientific name Bacillus sp .LP-1 was deposited at the China Center for Type Culture Collection on November 17, 2023, with accession number CCTCC M 20232261; the address of the depository is Wuhan University, Wuhan, China.

[0037] 3. Determination of the growth curve of the strain Seed culture cultured for 24 hours with shaking was inoculated into LB liquid medium at an inoculation rate of 2% (volume fraction). The culture was then incubated at 37℃ and 150 rpm for 24 hours with shaking. Samples were taken every two hours for analysis, using sterile blank LB medium as a control. The OD values ​​of each bacterial culture were measured. 600 The value is the average of three measurements. Plot pH on the X-axis, and OD... 600 Using Origin 7.5 software as the Y-axis, a scatter plot of bacterial count in the bacterial culture was drawn.

[0038] Depend on Figure 4 It can be seen that Bacillus LP-1 grows rapidly, with exponential growth in the period from 2 to 16 hours. During this period, the reproduction rate is relatively fast, and the OD value reaches 1.938, which is the logarithmic growth phase. After 16 hours, it enters the stationary phase. After 24 hours, the OD value of the bacterial solution does not show a significant decrease.

[0039] 4. Determination of suitable pH for strain growth The culture time was 24 hours, and the pH of the culture medium was set to 4, 5, 6, 7, 8, and 9. Other culture conditions, data measurement, and processing methods were the same as those for the growth curve determination of the strain.

[0040] Depend on Figure 5 It is known that the optimal pH for the growth of Bacillus LP-1 is 7.0. Between pH 5.0 and 7.0, the OD value increases with increasing pH; between pH 7.0 and 9.0, the OD value decreases with increasing pH. At a culture medium pH of 4.0, the OD value of the bacterial solution is below 0.1, which is unsuitable for the growth of the strain.

[0041] 5. Determination of the optimal growth temperature for the strain The culture time was 24 hours, and the ambient temperature was set at 20℃, 25℃, 30℃, 35℃, and 40℃. Other culture conditions, data measurement, and processing methods were the same as those for the growth curve determination of the strain.

[0042] Depend on Figure 6 It is known that Bacillus LP-1 can grow normally in an environment temperature range of 20–40℃, belonging to the mesophilic type. When the temperature is between 15 and 30℃, the OD value increases with increasing temperature, and the OD value reaches its highest value at 30℃; when the temperature exceeds 30℃, the OD value decreases with increasing temperature, indicating that the optimal growth temperature is 30℃.

[0043] Example 2 This example verifies the β-glucosidase production performance of Bacillus LP-1, and the specific operation is as follows: I. Preparation of Culture Medium Fermentation medium: 5g yeast extract, 10g peptone, 10g glucose, 0.3g NH4Cl, 0.4g KH2PO4, sterilized at 121℃ for 20min, and 0.1% p-NPG added when the medium temperature drops to about 70℃.

[0044] The reagents used above, including p-nitrophenol (p-NP), anhydrous sodium carbonate, citric acid, disodium hydrogen phosphate, p-nitrophenyl glucoside (p-NPG), peptone, yeast extract, sodium chloride, and disodium hydrogen phosphate, were all domestically produced analytical grade reagents.

[0045] II. Preparation of crude enzyme solution Seed culture cultured for 24 hours was inoculated into fermentation medium at a 5% inoculation rate. The temperature-controlled shaker was set to 150 rpm and 37°C, and cultured for 4 days. The fermentation broth was then transferred to centrifuge tubes and centrifuged at 8000 rpm for 10 minutes. After centrifugation, the bacterial precipitate at the bottom of the centrifuge tube was removed, and the resulting supernatant was the crude enzyme solution. This solution was stored at 4°C for later use. The experimental results were the average of three measurements.

[0046] III. Standard Curve Plotting The determination method of p-nitrophenol standard curve by Zhang Jiameng was referenced and modified. 0.0139 g of p-nitrophenol (p-NP) was weighed on a balance, dissolved in distilled water, and diluted to 100 mL. 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of the prepared p-nitrophenol solution were taken and diluted to 100 mL with 1 mol / L Na₂CO₃ solution, then shaken well. Nitrophenol standard solutions of different concentrations (10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, and 60 μmol / L) were prepared. Each solution was diluted to 100 mL with 1 mL of deionized water using 1 mol / L Na₂CO₃ solution as a blank control group, and the absorbance was measured at 400 nm. The results were taken as the average of three measurements. A standard curve was plotted using Origin 7.5 software, with p-NP concentration (μmol / L) as the X-axis and OD400 as the Y-axis. Figure 7 ).

[0047] The regression equation for p-nitrophenol is Y = 0.0257x + 0.0062, and the correlation coefficient R0 is [missing value]. 2 =0.9998, which indicates that the absorbance is linearly related to the p-nitrophenol content.

[0048] IV. Determination of β-glucosidase activity 1. Measurement Method Mix 100 μL of crude enzyme solution with 200 μL of 35 mmol / L p-NPG solution (prepared with citrate-phosphate buffer at pH 5.0) and shake well. Incubate in a 50°C water bath for 10 min, then add 2 mL of 1 mol / L Na₂CO₃ to terminate the reaction. Measure OD₄₀ and take the average of three measurements. The blank control group is a solution without enzyme. Calculate the enzyme activity according to the standard curve equation.

[0049] The enzyme activity unit (U) of β-glucosidase is defined as the amount of p-NP produced by hydrolyzing p-NPG in 1 mL of enzyme solution in 1 min under reaction conditions of pH 5.0 and 50℃.

[0050] 2. Enzymatic properties of β-glucosidase (1) Effect of temperature on β-glucosidase activity The water bath temperatures were set to 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃. The highest enzyme activity was defined as 100%, and the relative enzyme activity at each temperature was calculated.

[0051] Result: As Figure 8 As shown, the relative enzyme activity is above 65% between 30℃ and 80℃. The activity of β-glucosidase increases with increasing temperature. The enzyme activity reaches its maximum at 80℃, with a relative activity of 100%, indicating that the optimal reaction temperature for β-glucosidase is 80℃.

[0052] (2) Effect of pH on β-glucosidase activity With pH values ​​set to 3, 4, 5, 6, 7, and 8, and the highest enzyme activity defined as 100%, the relative enzyme activity at each pH value was calculated.

[0053] Result: As Figure 9 As shown, the relative enzyme activity is above 85% between pH 3 and 7. With increasing pH, the activity of β-glucosidase also increases, reaching its maximum at pH 7 with a relative activity of 100%. Therefore, the optimal pH for β-glucosidase is 7. At pH 8, the activity of β-glucosidase decreases.

[0054] In summary, the biological characteristics test results of Bacillus LP-1 show that the growth curve of Bacillus LP-1 cultured at 37℃ with shaking at 150 r / min exhibits a logarithmic growth phase from 2 h to 16 h and a stationary phase from 16 h to 24 h. During the culture period, the growth rate did not show a significant decrease, and the decline phase was not obvious. The optimal pH was 7.0, and the optimal growth temperature was 30℃. Bacillus LP-1 possesses strong growth capacity and belongs to the mesophilic bacteria.

[0055] The p-NPG method was used to evaluate the β-glucosidase production performance of Bacillus LP-1. The results showed an enzyme activity of 75.33 U / mL, an optimal reaction temperature of 80℃, and an optimal pH of 7.0. Bacillus LP-1 exhibits good β-glucosidase production capacity and high thermostability of the produced β-glucosidase, indicating promising application prospects.

[0056] Example 3 The production method of β-glucosidase specifically includes the following steps: S1. Strain activation: Bacillus LP-1 was activated in basal medium. The activated strain was then inoculated into the basal medium and cultured in a shaker at 30°C for 24 hours. The culture was then centrifuged, washed, and resuspended to adjust the bacterial suspension concentration to 10⁻⁶. 7 cfu / mL is the seed solution; S2. Inoculation: Inoculate the seed culture into the fermentation medium at an inoculation rate of 5%; culture in a shaker at a temperature of 35~37℃ and a rotation speed of 150r / min for 4 days; S3. Take the fermentation broth after fermentation, centrifuge at 8000 r / min for 10 min, collect the supernatant, and obtain crude β-glucosidase enzyme solution. S4. Purify the crude enzyme solution and freeze-dry it under vacuum to obtain β-glucosidase powder.

[0057] Specifically, the purification method is as follows: Preliminary purification was performed using ammonium sulfate separation and precipitation. 30 mL of crude enzyme solution was taken, and ammonium sulfate was added to achieve saturations of 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively. The solutions were then incubated at 4°C for 12 hours, centrifuged at 9000 r / min for 20 min, and the precipitates were reconstituted with buffer. The β-glucosidase activity and protein content in the supernatant and precipitates were measured to determine the saturation point of the ammonium sulfate fractionation. The crude enzyme solution after preliminary purification was dialyzed to remove salts. The enzyme solution was then loaded onto a Sephadex G-75 column (45.0 cm × Φ3.5 cm) with an elution buffer of pH 5.0 (acetic acid-sodium acetate buffer) and an elution gradient of 0.5 mL / min. The elution peaks containing β-glucosidase were collected and mixed, and then loaded onto a DEAE Cellulose 52 weak anion exchange column (28.0 cm × Φ3.0 cm) with an elution buffer of 0.5 mol / L NaCl and an elution gradient of 0.5 mL / min. The fraction containing β-glucosidase was collected for enzyme activity assay and SDS-PAGE. The purified enzyme was then desalted and freeze-dried under vacuum to prepare β-glucosidase powder.

[0058] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0059] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A strain of Bacillus LP-1, Latin name is Bacillus LP-1 Bacillus sp LP-1, the preservation number is CCTCC M20232261.

2. The application of Bacillus sp. LP-1 in the production of beta-glucosidase.

3. Use according to claim 2, characterized in that: The method for producing beta-glucosidase comprises the following steps: S1, strain activation: Bacillus LP-1 is activated in the basic medium, and the activated strain is inoculated into the basic medium and cultured in a shaker at a temperature of 20-40°C for 20-30 h; centrifuged, washed, resuspended, and adjusted to a concentration of 10 7 cfu / mL, i.e. seed liquid; S2, inoculation: inoculate the seed liquid into the fermentation medium at an inoculation amount of 4-6%; and perform shaking culture in a shaking table at a temperature of 35-37℃ and a rotating speed of 130-180 r / min for 3-5 days; S3, centrifuge the fermentation liquid at a rotating speed of 7000-10000 r / min for 8-15 min to collect the supernatant to obtain the crude enzyme liquid of beta-glucosidase; S4, purify the crude enzyme liquid, and freeze-dry it under vacuum to obtain the beta-glucosidase enzyme powder.

4. Use according to claim 3, characterized in that: The fermentation medium comprises the following components by weight: yeast extract 4-6 g, peptone 8-12 g, glucose 8-12 g, NH4Cl 0.2-0.4 g, KH2PO4 0.3-0.5 g, and p-nitrophenyl glucoside 0.1-1 g.

5. Use according to claim 4, characterized in that: The preparation method of the fermentation medium is as follows: weigh the yeast extract, peptone, glucose, NH4Cl and KH2PO4 0.4 g in sequence, sterilize them at 100-121℃ for 15-25 min, and then add the p-nitrophenyl glucoside after the temperature is reduced to 65-75℃.

6. Use according to claim 5, characterized in that: The pH needs to be adjusted to 6.5-7.5 before the step S2.

7. Use according to claim 6, characterized in that: The inoculation amount of the seed liquid in the step S2 is 5%; the temperature of the shaking culture is 37℃, and the rotating speed is 150 r / min.

8. Use according to claim 7, characterized in that: The temperature in the step S1 is 30℃.

9. Use according to claim 8, characterized in that: The centrifugal rotating speed is 8000 r / min, and the time is 10 min.

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