Bacillus velezensis KIBZ2 and application thereof
Through the synergy between Bacillus Bacillus Bacillus KIBZ2 and Bacillus composted Bacillus KIBZ1, the problem of incomplete degradation of Dendrobium polysaccharide is solved, and efficient and specific degradation is achieved, with a high degradation rate and short time.
Patent Information
- Application Number
- CN202510760902.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-08-22
AI Technical Summary
The existing polysaccharide degradation methods have problems such as incomplete degradation, high cost, pollution of the environment and poor enzyme stability, especially the insufficient utilization of Dendrobium polysaccharides and lack efficient and specific degradation methods.
Bacillus Bacillus Bacillus KIBZ2 and its microbial agent were used to degrade polysaccharides containing β-D-1,4 glucomanan structure through fermentation, and combined with compost Bacillus KIBZ1, it was used in a specific proportion to improve the degradation efficiency.
The molecular weight of polysaccharides was significantly reduced, the composition of mannose and glucose was maintained, the degradation rate reached more than 95%, and the degradation time was shortened to 10 hours, showing synergistic effects.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Bacillus velezensis KIBZ2 and an application thereof. Background Art
[0002] Polysaccharides are an important indicator for evaluating the quality of Dendrobium in the Chinese Pharmacopoeia (2020 edition). They are mainly composed of glucose and mannose connected by 1,4-glycosidic bonds, and some contain acetyl modifications. Studies have shown that Dendrobium polysaccharides have multiple biological activities such as antioxidant, immunomodulatory, and anti-tumor. However, their complex natural structure, large molecular weight, and poor solubility have severely limited their further development and utilization. Degrading large molecular weight polysaccharides into low molecular weight oligosaccharides is an effective way to improve their bioavailability and activity.
[0003] At present, the main methods for polysaccharide degradation include physical, chemical and enzymatic methods. Physical and chemical methods have problems such as incomplete degradation, high cost and environmental pollution. Although the enzymatic method has mild conditions, it has limitations such as poor enzyme stability and difficult storage. Microbial fermentation shows potential due to its simple reaction conditions and easy storage, but existing research has mostly focused on traditional fermentation bacteria, and the development of plant endophytes as natural polysaccharide degraders is insufficient. Plant endophytes have long-term symbiosis with the host and can efficiently utilize plant polysaccharides as a carbon source. The glycoside hydrolases they secrete have specific degradation capabilities for host polysaccharides. Therefore, there is an urgent need to develop efficient and specific degradation methods based on endophytes of Dendrobium to provide new strategies for the in-depth utilization of Dendrobium polysaccharides. Summary of the Invention
[0004] The present invention aims to provide a strain of Bacillus Velez KIBZ2 and its application. The Bacillus Velez KIBZ2 is extracted from Dendrobium officinale and is an endophyte of Dendrobium officinale, and can efficiently degrade Dendrobium polysaccharides containing β-D-1,4-glucomannan structure.
[0005] The invention provides a Bacillus velezensis KIBZ2 strain, with a deposit number of CGMCC No.33202.
[0006] The present invention also provides a microbial agent, the active ingredient of which includes the Bacillus Velezii KIBZ2 described in the above technical solution.
[0007] Preferably, the active ingredient of the microbial agent further includes Bacillus stercoris KIBZ1;
[0008] The deposit number of the composting Bacillus KIBZ1 is CGMCC No.33201.
[0009] Preferably, the ratio of the effective viable counts of KIBZ1 and KIBZ2 in the microbial agent is (1-9):(1-9).
[0010] The present invention also provides the use of the Bacillus Velezii KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution in degrading soluble starch.
[0011] The present invention also provides the use of the Bacillus Velez KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution in degrading polysaccharides; the polysaccharides contain glucose and mannose connected by β-D-1,4 glycosidic bonds.
[0012] Preferably, the polysaccharide comprises dendrobium polysaccharide;
[0013] The dendrobium polysaccharide is extracted from plants of the genus Dendrobium;
[0014] The Dendrobium plants include but are not limited to one or more of Dendrobium officinale, Dendrobium dentata, Dendrobium huoshanense, Dendrobium nobile and Dendrobium scutellariae.
[0015] The present invention also provides a method for degrading polysaccharides, comprising the following steps:
[0016] The Bacillus Velez KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution is fermented and cultured using a culture medium containing polysaccharides to obtain a fermentation liquid containing polysaccharide degradation products.
[0017] Preferably, when the Bacillus Velez subtilis KIBZ2 or microbial agent inoculated in the fermentation culture is a bacterial liquid, the OD of the bacterial liquid is 600 The value is 0.6~1.2.
[0018] Preferably, the temperature of the fermentation culture is 29° C. to 37° C.; and the shaking speed of the fermentation culture is 150 rpm to 250 rpm.
[0019] Beneficial effects:
[0020] The present invention provides a strain of Bacillus velezensis KIBZ2, with a deposit number of CGMCC No. 33202. The strain KIBZ2 exhibits excellent degradation of dendrobium polysaccharides, significantly reducing the molecular weight of the polysaccharide within 24 hours, while the degraded product retains its original mannose and glucose composition. The strain KIBZ2 has a specific degradation effect on polysaccharides containing a β-d-1,4-glucomannan structure, and can degrade dendrobium polysaccharides while also exhibiting good degradation activity against konjac polysaccharides and soluble starch. More notably, when the Bacillus Velez KIBZ2 described in the present invention is used in combination with the compost Bacillus (Bacillus stercoris) KIBZ1 in a specific ratio (such as 7:3), the degradation efficiency is greatly improved. The degradation rate of various Dendrobium polysaccharides (including Dendrobium dentata, Dendrobium Huoshanense, Dendrobium nobile and Dendrobium lip) can reach more than 95%, and the degradation time is shortened to 10 hours, showing a synergistic effect.
[0021] Biological deposit information
[0022] Bacillus velezensis KIBZ2, classified as Bacillus velezensis, was deposited on December 25, 2024 at the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33202.
[0023] Composting Bacillus KIBZ1, classified and named Bacillus stercoris, was deposited on December 25, 2024 at the General Microbiology Center of China Culture Collection Administration, abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33201. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0025] Figure 1 This is a flow chart for the isolation and screening of dendrobium polysaccharide-degrading bacteria;
[0026] Figure 2 is the growth curve of strains KIBZ1 and KIBZ2;
[0027] Figure 3 is the degradation curve of strains KIBZ1 and KIBZ2;
[0028] Figure 4The standard curves of different molecular weights are shown;
[0029] Figure 5 The monosaccharide composition analysis diagram of the degradation products of strains KIBZ1 and KIBZ2;
[0030] Figure 6 This is an analysis of the degradation of konjac polysaccharide and soluble starch by strains KIBZ1 and KIBZ2. DETAILED DESCRIPTION
[0031] The invention provides a Bacillus velezensis KIBZ2 strain, with a deposit number of CGMCC No.33202.
[0032] The Bacillus velezii KIBZ2 described in the present invention exhibits excellent degradation ability for dendrobium polysaccharides, can significantly reduce the molecular weight of polysaccharides within 24 hours, and the degradation product still maintains the original mannose and glucose composition. In addition, the Bacillus velezii KIBZ2 described in the present invention has a specific degradation effect on polysaccharides containing β-d-1,4 glucomannan structure, and can degrade dendrobium polysaccharides while also showing good degradation activity on konjac polysaccharides and soluble starch. The colonies of the Bacillus velezii KIBZ2 described in the present invention are round, with neat edges and a rough surface, and do not produce obvious pigments. The 16SrDNA sequencing assembly results of the Bacillus velezii KIBZ2 described in the present invention are shown in SEQ ID NO: 1:
[0033]
[0034] The present invention also provides a microbial agent, the active ingredient of which includes the Bacillus Velez KIBZ2 described in claim 1. As an embodiment, the active ingredient of the microbial agent of the present invention also includes composting Bacillus (Bacillus stercoris) KIBZ1; the deposit number of the composting Bacillus KIBZ1 is CGMCC No. 33201. The colony of the composting Bacillus KIBZ1 of the present invention is round, with neat edges and a rough surface, and does not produce obvious pigments. The 16S rDNA sequencing assembly result of the composting Bacillus KIBZ1 of the present invention is shown in SEQ ID NO: 2:
[0035]
[0036] As an embodiment, the ratio of the effective viable counts of KIBZ1 and KIBZ2 in the microbial agent of the present invention is (1-9): (1-9). As another embodiment, when KIBZ1 and KIBZ2 in the microbial agent of the present invention are bacterial solutions with the same OD value, the volume ratio of the bacterial solutions is (1-9): (1-9). As another embodiment, when KIBZ1 and KIBZ2 in the microbial agent of the present invention are bacterial solutions with the same OD value, the volume ratio of the bacterial solutions is 7:3. As an embodiment, the microbial agent of the present invention further comprises an excipient. As an embodiment, the excipient of the present invention comprises one or more of an adsorption material, a suspension matrix, and an embedding material. As an embodiment, the adsorption material of the present invention may be a solid culture medium. As an embodiment, the suspension matrix of the present invention may be a liquid culture medium.
[0037] The present invention also provides the use of the Bacillus Velezii KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution in degrading soluble starch. As an embodiment, the soluble starch described in the present invention is composed of α-1,4 linked glucose.
[0038] The present invention also provides the use of the Bacillus Velez subsp. KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution for degrading polysaccharides; the polysaccharides contain glucose and mannose linked by β-D-1,4 glycosidic bonds. In one embodiment, the polysaccharides of the present invention include Dendrobium polysaccharides; the source of the Dendrobium polysaccharides includes extraction from Dendrobium plants; the Dendrobium plants include, but are not limited to, one or more of Dendrobium officinale, Dendrobium dentata, Dendrobium huoshanense, Dendrobium nobile, and Dendrobium scutellariae. In one embodiment, the polysaccharides of the present invention include konjac polysaccharides. In one embodiment, the Bacillus Velez subsp. KIBZ2 or the microbial agent described in the present invention can depolymerize Dendrobium polysaccharides. In one embodiment, the degradation rate of Dendrobium dentata, Dendrobium huoshanense, Dendrobium nobile, and Dendrobium scutellariae polysaccharides by the Bacillus Velez subsp. KIBZ2 or the microbial agent described in the present invention can reach over 95% for each of these polysaccharides. In one embodiment, the polysaccharide containing a β-D-1,4-glucomannan structure of the present invention further comprises konjac polysaccharide. In another embodiment, when the ratio of the effective viable counts of KIBZ1 to KIBZ2 in the microbial agent of the present invention is 7:3, the degradation efficiency is significantly improved and the degradation time is significantly shortened, demonstrating a synergistic effect.
[0039] The present invention also provides a method for degrading polysaccharides, comprising the following steps:
[0040] The Bacillus Velez KIBZ2 described in the above technical solution or the microbial agent described in the above technical solution is fermented and cultured using a culture medium containing polysaccharides to obtain a fermentation liquid containing polysaccharide degradation products.
[0041] As an embodiment, when the Bacillus velez KIBZ2 or microbial agent inoculated in the fermentation culture of the present invention is a bacterial liquid, the OD600 value of the bacterial liquid is 0.6 to 1.2. As another embodiment, when the Bacillus velez KIBZ2 or microbial agent inoculated in the fermentation culture of the present invention is a bacterial liquid, the concentration of the bacterial liquid is OD600. 600 nm=0.6. As an embodiment, the temperature of the fermentation culture of the present invention is 29°C to 37°C; the shaking speed of the fermentation culture is 150rpm to 250rpm. As another embodiment, the temperature of the fermentation culture of the present invention is 29°C; the shaking speed of the fermentation culture is 200rpm. As an embodiment, the molecular weight of the completely degraded product in the fermentation broth of the present invention is significantly reduced compared with the dendrobium polysaccharide before degradation, and the yield is significantly improved. As an embodiment, the monosaccharide composition of the completely degraded product in the fermentation broth of the present invention is unchanged compared with the dendrobium polysaccharide before degradation. As an embodiment, the fermentation broth of the present invention can be applied to the fields of medicine, food and daily chemicals. As an embodiment, the mannose content of the completely degraded product in the fermentation broth of the present invention is significantly increased.
[0042] To further illustrate the present invention, a strain of Bacillus velezensis KIBZ2 and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0043] The reagents and consumables involved in the present invention can be purchased from commercial channels. If the specific experimental conditions are not specified, they are usually carried out according to conventional experimental conditions or the conditions recommended by the reagent company.
[0044] Example 1
[0045] Isolation and screening of strains KIBZ1 and KIBZ2
[0046] 1. Isolation and purification of endophytes from Dendrobium officinale:
[0047] Fresh stems of Dendrobium officinale were disinfected by soaking in 75% ethanol and then 3% sodium hypochlorite, followed by sterile washing three times. Sterile water was added and ground in a mortar. The volume was made up to 20 mL in a beaker, stirred with a magnetic stirrer for 1 hour, and allowed to stand. 100 μL of the supernatant was diluted in a series of dilutions. 100 μL of each dilution was spread onto ISP2, NA, and LB solid media and incubated at 29°C for 7 days. Based on the morphological characteristics of the strain, the strain was purified by plate streak method for three generations to obtain the endophytic bacteria.
[0048] 2. Extraction of crude polysaccharides from Dendrobium officinale:
[0049] The dried stems of Dendrobium officinale were crushed, distilled water was added at a solid-liquid ratio of 1:18, and the mixture was extracted with boiling water for 2 hours. The mixture was extracted twice, centrifuged, and the supernatants were combined. The protein in the supernatant was removed using the Sevag method (chloroform: n-butanol = 4:1) and extracted 2 to 3 times. The aqueous phase (i.e., the polysaccharide solution) was collected and rotary evaporated at 60°C until there was no reagent smell. Anhydrous ethanol was added to the Dendrobium officinale polysaccharide concentrate with stirring to adjust the final ethanol concentration to 60%. The mixture was placed in a refrigerator at 4°C for 4 hours or at room temperature overnight, then centrifuged (3600 rpm, 20 minutes) to collect the precipitate to obtain 60% alcohol-precipitated polysaccharide. The above alcohol-precipitated polysaccharide was re-dissolved in hot water, rotary evaporated at 60°C until there was no reagent smell, and named DOP-60%. It was freeze-dried and set aside.
[0050] 3. Screening of Dendrobium polysaccharide-degrading bacteria:
[0051] Prepare a polysaccharide culture solution containing only Dendrobium officinale polysaccharides as the sole carbon source and no other trace elements. The specific preparation process and formula are as follows: 5g crude Dendrobium officinale polysaccharide, 1L water, natural pH. Sterilize at 115°C and autoclave for 20 minutes before use.
[0052] Single endophytic bacterial colonies were selected, activated on plate culture media, and cultured at 29°C for 48 hours. An 8-mm-diameter endophytic bacterial cake was inoculated into a 50-mL Erlenmeyer flask containing 10 mL of polysaccharide culture medium and cultured at 200 rpm at 29°C for 5 days. Three parallel control groups were established, using uninoculated polysaccharide culture medium as a blank control. The fermented samples were sterilized by heating in boiling water for 30 minutes, centrifuged at 4000 rpm for 20 minutes, and the supernatant was sterilized by filtration through a 0.22-μm microporous filter. Changes in polysaccharide molecular weight were analyzed by HPLC.
[0053] The experimental results are as follows:
[0054] The isolation and screening flow chart of strains KIBZ1 and KIBZ2 is shown in the figure Figure 1 As shown in the figure, by analyzing the HPLC spectra of the fermentation products, two endophytic bacteria that can degrade Dendrobium polysaccharides were found and named KIBZ1 and KIBZ2.
[0055] Example 2
[0056] Identification of strains KIBZ1 and KIBZ2
[0057] Purified strains KIBZ1 and KIBZ2 were activated on plate culture media and sent to Paisono for 16S rDNA gene sequencing and identification. Endophytic bacterial DNA was extracted using a bacterial genomic DNA extraction kit, and PCR amplification primers (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3) and (1492R): 5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO: 4) were used. The PCR amplification system consisted of 1 μL genomic DNA, 5 μL 10× buffer, 1 μL Taq polymerase, 1 μL dNTPs, 1.5 μL each of the upstream and downstream primers, and 39 μL ddH2O. Reaction parameters included 35 cycles of initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min 30 s, and final extension at 72°C for 7 min. The reaction products were detected by 1% agarose gel electrophoresis.
[0058] DNA sequencing was performed using an ABI3730-XL sequencer. The spliced sequence files were compared with the data in the NCBI 16S database using the NCBI Blast program. The species with the greatest sequence similarity to the species under test was obtained, which was the preliminary identification result.
[0059] The sequencing and assembly results of strain KIBZ116S rDNA are shown in SEQ ID NO: 2 above;
[0060] The sequencing and assembly results of strain KIBZ216S rDNA are shown in SEQ ID NO: 1 above.
[0061] The results of 16S rDNA sequencing showed that strain KIBZ1 was Bacillus stercoris (NR_181952.1) (100%), and strain KIBZ2 was Bacillus velezensis (NR_075005.2) (99.86%).
[0062] Example 3
[0063] A microbial agent
[0064] 1. Composition of the microbial agent
[0065] Active ingredient: Bacillus Velezii KIBZ2 (deposit number: CGMCC No. 33202).
[0066] Culture medium: NA medium (3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 15 g agar and 1000 mL water) and NB medium (10 g peptone, 3 g beef extract powder, 5 g sodium chloride and 1000 mL water).
[0067] 2. Preparation Method
[0068] Bacillus velezensis KIBZ2 was activated using NA medium (cultured at 29°C for 48 hours). An 8 mm diameter bacterial mass was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of NB medium and cultured at 200 rpm at 29°C for 12 hours. A 2% inoculum of KIBZ2 was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of NB medium and cultured at 200 rpm at 29°C for 12 hours to prepare the seed solution.
[0069] Pipette 200 μL of KIBZ2 seed solution into a 50 mL Erlenmeyer flask containing 10 mL of NB medium and culture at 200 r / min and 29°C for 8 h until the strain is in the logarithmic growth phase. Add appropriate amount of sterile water to dilute the bacterial solution to OD 600 =0.6 working concentration. The microbial agent is obtained.
[0070] Example 4
[0071] A microbial mixture
[0072] 1. Composition of the microbial agent
[0073] Active ingredients: Bacillus velezensis KIBZ2 (deposit number: CGMCC No. 33202); Bacillus composting KIBZ1 (deposit number: CGMCC No. 33201).
[0074] Culture medium: NA medium (3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 15 g agar and 1000 mL water) and NB medium (10 g peptone, 3 g beef extract powder, 5 g sodium chloride and 1000 mL water).
[0075] 2. Preparation Method
[0076] Bacillus velezensis KIBZ2 and Bacillus composting KIBZ1 were activated using NA medium (cultured at 29°C for 48 hours). Bacterial clumps with a diameter of 8 mm were inoculated into 50 mL Erlenmeyer flasks containing 10 mL of NB medium and cultured at 200 rpm at 29°C for 12 hours. KIBZ1 and KIBZ2 bacterial solutions were inoculated into 50 mL Erlenmeyer flasks containing 10 mL of NB medium at a 2% inoculum rate and cultured at 200 rpm at 29°C for 12 hours to prepare seed solutions.
[0077] 200 μL of KIBZ1 and KIBZ2 seed solution were inoculated into 50 mL Erlenmeyer flasks containing 10 mL NB medium, and cultured at 200 r / min and 29°C for 8 h until the strains were in the logarithmic growth phase. Appropriate amount of sterile water was added to dilute the bacterial solution to OD 600 =0.6 working concentration, and then the diluted fermentation broth was mixed in a volume ratio of KIBZ1:KIBZ2=7:3 to obtain the microbial agent.
[0078] Example 5
[0079] Degradation of Dendrobium polysaccharides by strains KIBZ1 and KIBZ2
[0080] 1. Culture medium
[0081] NA medium: 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 15 g agar, and 1000 mL water;
[0082] NB medium: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g and water 1000 mL;
[0083] Polysaccharide culture solution: 5g crude polysaccharide from Dendrobium officinale and 1L water, natural pH.
[0084] 2. Growth curves of strains KIBZ1 and KIBZ2
[0085] The strains KIBZ1 and KIBZ2 were inoculated on NA plate medium and cultured at 29°C for 48 hours for activation. Bacterial blocks with a diameter of 8 mm were inoculated into a 50 mL triangular flask containing 10 mL NB medium and cultured at 200 r / min and 29°C for 12 hours. According to the inoculation amount of 2%, the KIBZ1 and KIBZ2 bacterial liquids were inoculated into a 50 mL triangular flask containing 10 mL NB medium and cultured at 200 r / min and 29°C for 12 hours to obtain the seed liquid. Centrifuge (3000 r / min, 1 min), take 200 μL of the supernatant and inoculate it into a 50 mL triangular flask containing 10 mL NB medium, set up 3 groups of parallel controls, cultured at 200 r / min and 29°C, and measure the OD600 nm values at 0 h, 1.5 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, and 24 h, with time as the horizontal axis and OD 600 The nm value is the vertical axis, and the growth curves of strains KIBZ1 and KIBZ2 are drawn.
[0086] 3. Degradation curves of strains KIBZ1 and KIBZ2
[0087] Based on the growth curves of strains KIBZ1 and KIBZ2, 200 μL of KIBZ1 or KIBZ2 culture was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of NB medium. Cultures were maintained at 200 rpm and 29°C for 8 h until the strains reached the logarithmic growth phase. Sterile water was added to dilute the culture to an OD600 nm concentration of 0.6. The culture was centrifuged at 3000 rpm for 1 min. KIBZ1 and KIBZ2 culture solutions were then added to a 50 mL Erlenmeyer flask containing 10 mL of polysaccharide culture medium at a 2% inoculum rate. Three parallel control groups were set up and incubated at 29°C and 200 rpm for 4, 8, 12, 16, 24, and 28 h, respectively. Uninoculated culture medium served as a blank control. After the cultured samples were sterilized (boiling water for 30 min, centrifugation at 4000 r / min for 20 min, and sterilization by 0.22 μm filtration), the changes in the molecular weight of the polysaccharide were analyzed by HPLC, and the degradation curve was drawn based on the HPLC spectrum.
[0088] The experimental results are as follows:
[0089] The growth curves of strains KIBZ1 and KIBZ2 are shown in Figure 2. Figure 2 As shown in the figure, strain KIBZ1 grew slowly from 0 to 4 hours, indicating a delayed growth phase. From 4 to 10 hours, the number of strain KIBZ1 increased rapidly, indicating a logarithmic growth phase. From 10 to 14 hours, the growth rate of strain KIBZ1 approached zero, while the total bacterial count remained constant, indicating a stable growth phase. From 14 to 20 hours, strain KIBZ1 began to die. Strain KIBZ2 was in a logarithmic growth phase from 6 to 10 hours.
[0090] The degradation curves of strains KIBZ1 and KIBZ2 are shown in Figure 2. Figure 3 As shown in the figure, the CK group is a blank control group. As can be seen from the figure, high-molecular-weight polysaccharides gradually accumulate towards low-molecular-weight polysaccharides as the culture time increases, and strains KIBZ1 and KIBZ2 completely degrade them after 16 hours of culture.
[0091] Example 6
[0092] Analysis of physicochemical properties of polysaccharides depolymerized by strains KIBZ1 and KIBZ2
[0093] The degradation products of strains KIBZ1 and KIBZ2 were named CDOP-1 and CDOP-2, respectively. The molecular weight and monosaccharide composition of Dendrobium officinale polysaccharide (DOP-60%) and CDOPs (CDOP-1 and CDOP-2) were determined.
[0094] 1. Molecular weight detection
[0095] The molecular weight of polysaccharides was determined using HPLC. Chromatographic conditions: Shodex KS 804 analytical column, column temperature of 30°C, mobile phase of ultrapure water, flow rate of 1 mL / min, sample concentration of 1 mg / mL. Standard dextran with different molecular weights (5000Da, 12000Da, 50000Da, 410000Da, 670000Da) and polysaccharide samples were prepared into 1 mg / mL solutions and filtered through a 0.45 μm microporous membrane. A molecular weight standard curve was drawn using the peak time of the standard dextran with different molecular weights as the horizontal axis and the logarithm of the molecular weight as the vertical axis. The peak time of the polysaccharide sample was substituted into the curve to obtain the corresponding molecular weight.
[0096] 2. Monosaccharide composition analysis
[0097] The monosaccharide composition of polysaccharide samples was detected by PMP pre-column derivatization method.
[0098] Hydrolysis of polysaccharides: Weigh 6 mg of polysaccharide sample into a round-bottom flask, add 1 mL of 4 MTFA to dissolve, add a stirrer, seal the flask, react in a 90°C oil bath for 8 h, cool to room temperature, add methanol several times and repeatedly evaporate to remove excess TFA to make the solution neutral, add 1 mL of pure water, and dissolve into 6 mg / mL polysaccharide hydrolyzate.
[0099] PMP derivatization: Take 50 μL of polysaccharide hydrolyzate, add an equal volume of 50 μL of 0.6 M NaOH solution, then add 100 μL of 0.5 M PMP-methanol solution, mix well, react at 70 ° C for 100 minutes, take out and cool to room temperature, add an equal volume of 50 μL of 0.6 M HCl to neutralize the solution to make it neutral, then add 1 mL of pure water and 1 mL of chloroform to extract, remove the organic phase, repeat several times until the upper aqueous phase is clear, collect the aqueous phase, add 1 mL of pure water to dissolve, filter with 0.22 μm microporous filter membrane, and use liquid chromatography analysis.
[0100] Derivatization of monosaccharide standards: Ten standards, including mannose (Man), glucose (Glc), rhamnose (Rha), galactose (Gal), xylose (Xyl), arabinose (Ara), fucose (Fuc), glucuronic acid (GlcA), galacturonic acid (GalA), and ribose (Rib), were weighed and dissolved in pure water to prepare a mixed standard solution with a concentration of 6 mg / mL. 50 μL of the mixed monosaccharide standard solution was taken and subjected to the PMP derivatization reaction according to the above procedure. The aqueous phase was filtered through a 0.22 μm microporous filter membrane and then injected into HPLC for analysis.
[0101] Chromatographic analysis conditions: chromatographic column: ZORBAX SB-C18 (5 μm, 4.6×250 mm); mobile phase A: acetonitrile, mobile phase B: phosphate buffered saline, pH 6.8; elution conditions: acetonitrile:phosphate buffered saline = 17:83 (v:v), isocratic elution, flow rate: 0.8 mL / min; detection wavelength: 245 nm; injection volume: 30 μL; column temperature: 30°C.
[0102] The experimental results are as follows:
[0103] 1. Molecular weight analysis
[0104] The standard curve is drawn with the peak time of different molecular weight dextran standards as the horizontal axis and the logarithm of molecular weight as the vertical axis. Figure 4 The HPLC spectra of DOP-60% and depolymerized polysaccharide CDOPs are shown in Figure 3 Substitute the peak time into the linear regression equation of the standard curve y = -0.6478x + 9.62 (R 2 =0.9966), and the relative molecular mass of the polysaccharide was calculated (Table 1).
[0105] Table 1 Molecular weight and yield analysis of DOP-60% and CDOPs
[0106] Peak time Molecular weight Yield strains DOP-60% 4.472～4.662 <![CDATA[3.78×10 6 ~5.01×10 6 ]]> 31.6 CDOP-1 9.210 <![CDATA[4.38×10 3 ]]> 92.2 KIBZ1 CDOP-2 9.417 <![CDATA[3.22×10 3 ]]> 79.7 KIBZ2
[0107] 2. Monosaccharide composition analysis
[0108] The monosaccharide compositions of DOP-60% and CDOPs are as follows Figure 5 As shown in Figure 2, the monosaccharide types of polysaccharides did not change after microbial degradation and were composed of mannose and glucose. However, the mannose content of CDOPs increased compared with DOP-60% (Table 2).
[0109] Table 2 Monosaccharide composition and ratio of DOP-60% and CDOPs
[0110] DOP-60% CDOP-1 CDOP-2 Mannose (Man) 4.24 4.30 6.65 Glucose (Glc) 1 1 1
[0111] Example 7
[0112] Depolymerization of different carbon sources by strains KIBZ1 and KIBZ2
[0113] Use ultrapure water to prepare konjac polysaccharide, soluble starch, chitin, pectin and sodium alginate into 5 mg / mL polysaccharide culture medium, and refer to the operating steps of "Screening of Dendrobium polysaccharide-degrading bacteria" in Example 1 to analyze the degradation effects of strains KIBZ1 and KIBZ2 on konjac polysaccharide, soluble starch, chitin, pectin and sodium alginate.
[0114] The experimental results are as follows:
[0115] Depend on Figure 6 It can be seen that strains KIBZ1 and KIBZ2 have a certain degradation effect on konjac polysaccharide and soluble starch, but have no degradation effect on chitin, pectin, and sodium alginate. The structure of konjac polysaccharide is composed of β-d-1,4 glucomannan, while the structure of soluble starch is composed of α-1,4-linked glucose. In addition, the previous analysis found that the strain can degrade Dendrobium officinale polysaccharide, and the structure of Dendrobium polysaccharide is mainly glucose and mannose linked by β-D-1,4 glycosidic bonds. Therefore, the strain has a certain depolymerization effect on polysaccharides containing glucose and mannose structures linked by β-D-1,4 glycosidic bonds.
[0116] Example 8
[0117] Degradation curves of Dendrobium polysaccharides by mixing strains KIBZ1 and KIBZ2 at different ratios
[0118] Strains KIBZ1 and KIBZ2 were inoculated onto NA plate media and incubated at 29°C for 48 hours for activation. Bacterial clumps with a diameter of 8 mm were inoculated into 50 mL Erlenmeyer flasks containing 10 mL NB culture medium and incubated at 200 rpm at 29°C for 12 hours. At a 2% inoculum rate, KIBZ1 and KIBZ2 bacterial solutions were inoculated into 50 mL Erlenmeyer flasks containing 10 mL NB culture medium and incubated at 200 rpm at 29°C for 12 hours to prepare seed solutions. After centrifugation (3000 rpm for 1 minute), different volumes (1-9):(1-9) of KIBZ1 or KIBZ2 supernatant were collected and mixed, and the polysaccharides were degraded according to the procedures described in Example 1, "Screening of Dendrobium Polysaccharide-Degrading Bacteria."
[0119] The experimental results are as follows:
[0120] Table 3 Degradation rate of Dendrobium officinale polysaccharide by mixed bacterial liquid with different proportions at different times
[0121] Mixed bacterial solution ratio (V) 2h 4h 6h 8h 10h 12h 14h 16h 18h 1：9 0 0 23.5 86.7 90.7 95.3 95.3 95.4 95.3 1：4 0 0 26.4 86.3 92.2 95.7 95.7 95.5 95.6 3：7 0 0 25.2 84.9 91.6 94.5 95.5 95.3 95.3 2：3 0 0 27.6 85.6 92.7 96.8 96.9 96.5 96.7 1：1 0 0 30.7 88.7 94.8 95.6 95.7 95.7 95.7 3：2 0 0 35.8 88.3 95.2 96.2 96.4 96.4 96.4 7：3 0 3.5 47.3 92.3 96.3 96.3 96.2 96.3 96.3 4：1 0 2.5 42.4 89.5 94.6 95.5 95.7 96.1 96.1 9；1 0 0 40.7 90.1 94.3 95.2 96.2 96.3 96.3
[0122] The degradation rates of Dendrobium officinale polysaccharides obtained by mixing strains KIBZ1 and KIBZ2 in different proportions at different times are shown in Table 3. It can be seen from Table 3 that the mixture of strains KIBZ1 and KIBZ2 can depolymerize Dendrobium officinale polysaccharides, and the depolymerization efficiency is improved. The optimal ratio is KIBZ1: KIBZ2 = 7:3.
[0123] Example 9
[0124] Degradation of different Dendrobium polysaccharides by strains KIBZ1 and KIBZ2
[0125] Strains KIBZ1 and KIBZ2 were inoculated onto NA plates and incubated at 29°C for 48 hours for activation. An 8 mm diameter bacterial mass was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of NB medium and incubated at 200 rpm at 29°C for 12 hours. At a 2% inoculum rate, KIBZ1 and KIBZ2 bacterial suspensions were inoculated into a 50 mL Erlenmeyer flask containing 10 mL of NB medium and incubated at 200 rpm at 29°C for 12 hours to prepare the seed solution. The mixture was centrifuged (3000 r / min, 1 min), and the supernatant of KIBZ1 and KIBZ2 was extracted in a volume ratio of KIBZ1:KIBZ2 = 7:3 to degrade Dendrobium officinale polysaccharides, Dendrobium huoshanense polysaccharides, Dendrobium nobile polysaccharides and Dendrobium lip polysaccharides. The preparation methods of the Dendrobium officinale polysaccharides, Dendrobium huoshanense polysaccharides, Dendrobium nobile polysaccharides and Dendrobium lip polysaccharides were the same as the operating steps of “Extraction of crude polysaccharides from Dendrobium officinale” in Example 1.
[0126] The experimental results are as follows:
[0127] Table 4 Degradation of different Dendrobium polysaccharides by KIBZ1 and KIBZ2 (10 h)
[0128] Sample name Degradation rate (%) Dendrobium candidum 95.6 Huoshan Dendrobium 96.2 Dendrobium nobile 95.5 Dendrobium officinale 95.9
[0129] The degradation results of different Dendrobium polysaccharides by KIBZ1 and KIBZ2 are shown in Table 4. As shown in Table 4, the degradation rates of polysaccharides from Dendrobium denticulate, Dendrobium huoshanense, Dendrobium nobile, and Dendrobium serratum were all above 95% when the strain KIBZ1:KIBZ2 was fermented for 10 h at a ratio of 7:3.
[0130] According to the above content, it can be seen that the Bacillus velezensis KIBZ2 provided by the present invention and its application can efficiently degrade Dendrobium polysaccharide containing β-D-1,4-glucomannan structure.
[0131] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A strain of Bacillus velezensis KIBZ2, deposited with CGMCC No. 33202.
2. A microbial agent, characterized in that: The active ingredient of the microbial agent includes the Bacillus Velezii KIBZ2 according to claim 1.
3. The microbial agent according to claim 2, characterized in that The active ingredients of the microbial agent also include Bacillus stercoris KIBZ1; The deposit number of the composting Bacillus KIBZ1 is CGMCC No.33201.
4. The microbial agent according to claim 2, characterized in that The ratio of the effective viable bacteria counts of KIBZ1 and KIBZ2 in the microbial agent is (1-9): (1-9).
5. Use of the Bacillus Velezii KIBZ2 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in degrading soluble starch.
6. Use of the Bacillus Velez KIBZ2 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in degrading polysaccharides; the polysaccharides contain glucose and mannose linked by β-D-1,4 glycosidic bonds.
7. The use according to claim 6, characterized in that The polysaccharide includes dendrobium polysaccharide; The dendrobium polysaccharide is extracted from plants of the genus Dendrobium; The Dendrobium plants include but are not limited to one or more of Dendrobium officinale, Dendrobium dentata, Dendrobium huoshanense, Dendrobium nobile and Dendrobium scutellariae.
8. A method for degrading polysaccharides, comprising the following steps: The Bacillus Velez subtilis KIBZ2 according to claim 1 or the microbial agent according to any one of claims 2 to 5 is fermented and cultured using a culture medium containing polysaccharides to obtain a fermentation liquid containing polysaccharide degradation products.
9. The method according to claim 8, characterized in that When the Bacillus Velez KIBZ2 or microbial agent inoculated in the fermentation culture is a bacterial liquid, the OD of the bacterial liquid is 600 The value is 0.6~1.
2.
10. The method according to claim 8, characterized in that The fermentation temperature is 29° C. to 37° C.; the shaker speed is 150 rpm to 250 rpm.