Polygonatum sibiricum endophytic bacillus cereus and application thereof
By screening the SK6 strain of Bacillus cereus from the Taishan area, we have achieved efficient production of extracellular polysaccharides, solving the problems of resource scarcity and high cost in the extraction of Polygonatum polysaccharides, and improving production efficiency and stability.
Patent Information
- Application Number
- CN202511943744.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-02-10
AI Technical Summary
Existing Polygonatum polysaccharide extraction technologies face challenges such as scarcity of wild resources, long cultivation cycles, high costs, low yields, and poor fermentation stability. Furthermore, the geographical origin and diversity of Polygonatum endophytic bacteria are insufficient, making it difficult to meet market demand.
A strain of Bacillus cereus SK6 from the Taishan area was isolated and screened. Its preservation number is CGMCC No.36060. The fermentation broth with an extracellular polysaccharide content as high as 44.22 g/L was obtained by culturing it in a fermentation medium at 35℃~38℃ for 24h~36h.
It significantly improves the production efficiency and stability of Polygonatum polysaccharides, reduces production costs, and decreases dependence on wild Polygonatum resources, which is conducive to ecological environmental protection and in-depth utilization of resources.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of endophytic Bacillus cereus from Polygonatum sibiricum and its application. Background Technology
[0002] Polygonatum ( Polygonatum sibiricum Polygonatum is a plant with important medicinal value. Its rhizome is rich in polysaccharides, and Polygonatum polysaccharide is the main active ingredient in Polygonatum. Modern research shows that Polygonatum polysaccharide has various biological activities such as immunomodulation, antioxidation, hypoglycemia, and antitumor effects, and has broad application prospects in the fields of medicine, food, and cosmetics.
[0003] Traditional methods for extracting Polygonatum polysaccharides face numerous challenges: wild Polygonatum resources are scarce, while artificially cultivated Polygonatum grows slowly, requiring 3-7 years from planting to harvest, resulting in high cultivation costs. Furthermore, the artificial extraction process for Polygonatum polysaccharides is complex, costly, and yields low rates (typically only 2%-5%), failing to meet consumer demand. Endophytic bacteria of Polygonatum are a type of microorganism that live in long-term symbiosis with Polygonatum. The extracellular polysaccharides produced by their metabolism have similar structures and activities to Polygonatum polysaccharides. Studies have shown that the antioxidant and antitumor activities of polysaccharides produced by some endophytic bacteria are even higher than those extracted using traditional methods. Endophytic bacteria of Polygonatum grow and reproduce rapidly, and the fermentation process is easily controlled, allowing for the rapid accumulation of large amounts of extracellular polysaccharides, significantly reducing energy consumption and production costs while improving production efficiency. For these reasons, utilizing the fermentation of Polygonatum endophytic bacteria to produce polysaccharides represents a highly promising alternative.
[0004] However, currently available endophytic bacteria fermentation technology for polysaccharide production has significant shortcomings: First, polysaccharide production efficiency is generally low, with existing publicly available strains typically having polysaccharide content in fermentation broths ranging from 10 g / L to 25 g / L. Some strains also rely on special culture media containing Polygonatum extract, further increasing costs. Second, fermentation adaptability is poor, resulting in unstable production; polysaccharide yield fluctuations between different batches can reach 20% to 30%. Third, the geographical origin and diversity of strains are insufficient; current research is mostly concentrated in a few areas of Huangshan and Yunnan, with limited exploration of other production areas with unique ecological advantages. With the continuous growth of the Polygonatum polysaccharide market, the need to discover more high-quality Polygonatum endophytic bacteria is becoming increasingly urgent. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a strain of *Bacillus cereus* endophytic in *Polygonatum* and its applications. This invention isolated a strain of *Bacillus cereus* SK6, an endophytic bacterium of *Polygonatum*, from wild *Polygonatum* in the Taishan region. This strain exhibits excellent extracellular polysaccharide synthesis capabilities; the fermentation broth obtained after fermentation at 37°C for 24 h contains an extracellular polysaccharide content as high as 44.22 g / L.
[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a strain of *Bacillus cereus* endophytic in *Polygonatum sibiricum*, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 36060.
[0007] A second aspect of the present invention provides the application of the above-described Bacillus cereus in the fermentation production of extracellular polysaccharides.
[0008] The third aspect of the present invention provides a method for producing extracellular polysaccharides: the seed culture of Bacillus cereus described above is inoculated into a fermentation medium and fermented at 35℃~38℃ for 24h~36h to obtain a fermentation broth containing extracellular polysaccharides.
[0009] Furthermore, the fermentation culture temperature is 37°C, and the fermentation culture time is 24 hours.
[0010] Furthermore, the content of extracellular polysaccharides in each liter of the fermentation broth is ≥44.22g.
[0011] Furthermore, the seed culture of Bacillus cereus is obtained by culturing Bacillus cereus in LB liquid medium at 35℃~38℃ with shaking for 10h~14h.
[0012] Furthermore, the rotation speed of the oscillation culture is 110 r / min to 130 r / min.
[0013] Furthermore, based on the volume of the fermentation medium, the inoculation volume of the Bacillus cereus seed solution is 3% to 8% of the fermentation medium volume.
[0014] Furthermore, the fermentation medium is LB medium, beef extract peptone medium, or corn flour and soybean meal medium.
[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolates and screens an endophytic bacterium, Bacillus cereus strain SK6, from wild Polygonatum sibiricum in the Taishan region. This strain is classified and named Bacillus cereus. Bacillus cereus The strain of *Bacillus cereus* provided in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 36060. It exhibits excellent extracellular polysaccharide synthesis capabilities, with an extracellular polysaccharide content as high as 44.22 g / L in the fermentation broth obtained after fermentation at 37°C for 24 hours.
[0016] The strain provided by this invention is suitable for large-scale fermentation production, thus providing a new approach for the industrial production of Polygonatum polysaccharides. It can significantly improve production efficiency, reduce production costs, and reduce dependence on wild Polygonatum resources, which is conducive to ecological environmental protection and the in-depth development and utilization of Polygonatum resources.
[0017] Instructions for the Preservation of Biological Materials The Bacillus cereus strain SK6 in this invention is classified and named as: Bacillus cereus. Bacillus cereus Its Latin name is: Bacillus cereus It was deposited on September 25, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36060. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 The colony morphology and spore morphology of strain RG1 are shown. Figure 1 In this context, A represents the colony morphology. Figure 1 B in the text represents the spore morphology.
[0020] Figure 2 The colony morphology and spore morphology of strain SK5 are shown. Figure 2 In this context, A represents the colony morphology. Figure 2 B in the text represents the spore morphology.
[0021] Figure 3 The colony morphology and spore morphology of strain SK6 are shown. Figure 3 In this context, A represents the colony morphology. Figure 3 B in the text represents the spore morphology.
[0022] Figure 4 The colony morphology and spore morphology of strain HQ1 are shown. Figure 4 In this context, A represents the colony morphology. Figure 4 B in the text represents the spore morphology.
[0023] Figure 5 The colony morphology and spore morphology of strain HQ2 are shown. Figure 5 In this context, A represents the colony morphology. Figure 5 B in the text represents the spore morphology.
[0024] Figure 6 The colony morphology and spore morphology of strain HQ3 are shown. Figure 6 In this context, A represents the colony morphology. Figure 6 B in the text represents the spore morphology.
[0025] Figure 7 The colony morphology and spore morphology of strain HQ5 are shown. Figure 7 In this context, A represents the colony morphology. Figure 7 B in the text represents the spore morphology.
[0026] Figure 8 The colony morphology and spore morphology of strain HQ7 are shown. Figure 8 In this context, A represents the colony morphology. Figure 8 B in the text represents the spore morphology.
[0027] Figure 9 The colony morphology and spore morphology of strain YQ1 are shown. Figure 9 In this context, A represents the colony morphology. Figure 9 B in the text represents the spore morphology.
[0028] Figure 10 The colony morphology and spore morphology of strain YQ8 are shown. Figure 10 In this context, A represents the colony morphology. Figure 10 B in the text represents the spore morphology.
[0029] Figure 11 The colony morphology and spore morphology of strain YQ10 are shown. Figure 11 In this context, A represents the colony morphology. Figure 11 B in the text represents the spore morphology.
[0030] Figure 12 The colony morphology and spore morphology of strain YQ11 are shown. Figure 12 In this context, A represents the colony morphology. Figure 12 B in the text represents the spore morphology.
[0031] Figure 13 This is the standard curve for glucose.
[0032] Figure 14 Phylogenetic tree of the constructed SK6 strain. Detailed Implementation
[0033] 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 to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0034] While Polygonatum polysaccharides have broad application prospects, traditional extraction methods face bottlenecks such as scarcity of wild resources, long cultivation cycles, low extraction rates, and high costs. Polygonatum endophytic bacteria are a type of microorganism that have long coexisted with Polygonatum, and their metabolites, extracellular polysaccharides, have similar structures and activities to Polygonatum polysaccharides. Utilizing Polygonatum endophytic bacteria for polysaccharide production through fermentation represents a highly promising alternative. However, currently available Polygonatum endophytic bacteria suffer from drawbacks such as low sugar production efficiency, poor fermentation stability, and insufficient geographical origin and strain diversity.
[0035] This invention isolates and screens an endophytic bacterium, Bacillus cereus strain SK6, from wild Polygonatum sibiricum in the Taishan region (CGMCC No. 36060). This strain exhibits excellent extracellular polysaccharide synthesis capabilities, with an extracellular polysaccharide content as high as 44.22 g / L in the fermentation broth obtained after fermentation at 37°C for 24 hours.
[0036] Example 1: Obtaining the SK6 strain of *Bacillus cereus* endophytic in *Polygonatum sibiricum* I. Experimental Methods 1. Source of Polygonatum Four different sources of Polygonatum materials were selected: wild Polygonatum near the entrance of Tianzhufeng Mountain in Taishan District, Tai'an City, Shandong Province; wild Polygonatum around the West Main Canal in Huangqian Town, Taishan District, Tai'an City, Shandong Province; wild Polygonatum in Yuquan Temple in Taishan District, Tai'an City, Shandong Province; and artificially cultivated Polygonatum in Shankou Town, Taishan District, Tai'an City, Shandong Province.
[0037] 2. Initial processing of raw materials Collect the rhizomes of Polygonatum and rinse them under running water to remove small roots, surface soil, dust, and impurities. For samples with more surface dirt, gently scrub them with a soft-bristled brush.
[0038] Disinfection: Immerse the cleaned sample in 75% ethanol solution for surface disinfection for 30 seconds. After disinfection, rinse the sample with sterile water, repeating the rinsing process 3 times (performed in a laminar flow hood).
[0039] 3. Prepare the culture medium (1) Culture medium: beef extract peptone medium, LB liquid medium.
[0040] (2) Culture medium formulation and preparation Beef extract peptone medium: beef extract 3 g / L, peptone 10 g / L, sodium chloride 5 g / L, yeast extract 3 g / L Add g / L of agar and 2% of distilled water to make up the difference; adjust the pH to 7.2.
[0041] LB liquid medium: 10 g / L peptone, 5 g / L beef extract, 5 g / L yeast extract, 5 g / L sodium chloride, and distilled water to make up the difference; adjust the pH to 7.0.
[0042] (3) Culture medium dispensing and sterilization: Aseptically dispense the culture medium, avoiding contact with the bottle mouth, seal with cotton plugs or sealing film, and wrap the bottle mouth with newspaper. Sterilize with high-pressure steam at 121℃ and 101 kPa for 20 min.
[0043] (4) Pouring plates: After sterilization, wait for the culture medium to cool to about 50°C, pour 15 mL to 20 mL of culture medium into each plate on a sterile table, let it cool and solidify, and then invert it for later use.
[0044] 4. Isolation and culture of endophytic bacteria After sterilization, the Polygonatum tissue was cut into pieces, and endophytic bacteria were extracted using grinding and leaching methods respectively (operations were performed in a clean bench).
[0045] (1) Grinding method: Take about 1g of Polygonatum rhizome, cut it into pieces and place it in a sterile mortar. Add sterile quartz sand and 2 mL of sterile water and grind it into a paste. Then add 8 mL of sterile water and stir to prepare a bacterial suspension. Transfer the suspension to a 15 mL sterile test tube. Heat treat in a 90℃ water bath for 15 min. After cooling, take 50 μL of the bacterial suspension and spread it on beef extract peptone medium. Incubate at 37℃ upside down for 48 h.
[0046] (2) Leaching method: Slice Polygonatum sibiricum, take about 1g of Polygonatum sibiricum slices and place them in a centrifuge tube containing 10 mL of sterile water. Shake at 150r / min for 3 h to prepare a bacterial suspension. Heat treat in a water bath at 90℃ for 15 min. After cooling, take 50μL of the bacterial suspension and spread it on beef extract peptone medium. Incubate upside down at 37℃ for 48 h.
[0047] 5. Selection and purification of endophytic bacteria (1) Picking single colonies: After 48 h of culture, use an inoculation loop to pick single colonies that are plump and conform to the typical characteristics of Bacillus.
[0048] (2) Purification of single colonies by streak plating: The selected single colonies were purified by the three-zone streak plating method. After streak plating, the strain information was marked and the colonies were incubated upside down at 37°C for 48 h.
[0049] (3) Repeated purification and pure culture verification: The single colony obtained in step (2) was streaked three times for purification. After each purification, the morphology and color of the colony were observed, and the morphology and size of the cells were observed by Gram staining until the colony and cell morphology were uniform and free of contaminants, thus confirming that a pure culture was obtained.
[0050] (4) Characteristics of target Bacillus strains: Target strains are selected based on the colony characteristics of Bacillus. The colonies are mostly round or nearly round with neat edges. Some species may be wavy or serrated. The colony diameter is 2 mm to 5 mm. The colony surface is generally dry and rough with a frosted texture. The texture is hard and opaque. The color is mainly milky white, grayish white or light yellow. The degree of elevation is mostly mild or moderate. Some species (such as Bacillus subtilis) may also show slight shrinkage in the late stage due to spore production.
[0051] 6. Preservation of bacterial strains (1) Glycerin tube preparation: Prepare a 50 v / v% glycerin solution, autoclave at 121℃ for 20 min, and take 0.75 mL of glycerin into a 2.0 mL cryovial for later use.
[0052] (2) Transfer and culture of strains: The purified strains were inoculated onto LB medium slant under aseptic conditions and cultured at 37°C for 48 h.
[0053] (3) Preservation of bacterial strains: Add 5 mL of physiological saline to the slant cultured with bacterial moss for 48 h to prepare a bacterial suspension. Take 0.75 mL of the bacterial suspension and mix it with glycerol in a glycerol tube. Seal the tube with sealing film and label it. After pre-cooling at 4℃ for 0.5 h and freezing at -20℃ for 2 h, transfer it to an ultra-low temperature freezer at -80℃ for long-term storage.
[0054] 7. Gram staining and microscopic observation (1) Slide preparation and Gram staining: Use an inoculation loop to pick up the bacterial growth of Bacillus spores, prepare a smear and fix it, and then perform Gram staining.
[0055] (2) Oil immersion observation and cleaning: Place the stained slide under a microscope and observe under 100× oil immersion. The target spores are Gram-positive. The vegetative body is straight rod-shaped with blunt or truncated ends. They are usually arranged singly, in pairs or in chains. The spores are round or oval in shape. Their size is smaller than the diameter of the bacterial body. They are mostly located in the mesophyll or near the mesophyll, and some are located at the end of the bacterial cell. They generally do not cause the mother cell to bulge obviously.
[0056] 8. Determination of the strain's ability to produce polysaccharides (1) Culture of strains: The strains activated by slant were inoculated into Erlenmeyer flasks containing LB liquid medium and cultured at 37°C and 120 r / min for 12 h with shaking.
[0057] (2) Reagent preparation: Prepare a 100 μg / mL glucose standard solution. Prepare an 80% phenol stock solution and store it in the dark and refrigerated. Dilute it to a 6% phenol solution before use.
[0058] (3) Standard curve preparation: Take 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose standard solution and place them in stoppered test tubes. Add distilled water to each tube to make up to 2.0 mL. Add 1.0 mL of 6% phenol solution and 5.0 mL of concentrated sulfuric acid in sequence. After standing for 20 min, measure the absorbance at 490 nm. Plot the standard curve with glucose concentration as the abscissa and absorbance as the ordinate.
[0059] (4) Determination of fermentation time of strain: In order to determine the appropriate fermentation time of strain, fermentation broths were taken at different time periods of 0 h, 24 h, 36 h and 48 h, and their OD values at 600 nm were measured by spectrophotometer.
[0060] (5) Determination of extracellular polysaccharide content: Centrifuge 1 mL of fermentation broth at 3000 r / min for 20 min and collect the supernatant. Take 0.2 mL of the supernatant, dilute it to 2.0 mL with 1.8 mL of distilled water, prepare the sample to be tested, add 1.0 mL of 6% phenol solution and 5.0 mL of concentrated sulfuric acid, let it stand for 20 min, and then measure the absorbance at 490 nm. Based on the absorbance of the sample, convert the corresponding glucose concentration through the glucose standard curve that has been plotted, and calculate the actual content of extracellular polysaccharides by combining the conversion factor.
[0061] II. Experimental Results 1. One artificially cultivated Polygonatum plant was selected: RG1. Two Polygonatum plants were selected near the entrance to Tianzhu Peak in Mount Tai: SK5 and SK6. Five Polygonatum plants were selected from the Huangqian area: HQ1, HQ2, HQ3, HQ5, and HQ7. Four Polygonatum plants were selected from the Yuquan Temple area: YQ1, YQ8, YQ10, and YQ11.
[0062] After streak plating and preliminary screening, a total of 12 candidate strains were obtained. The obtained strains underwent colony morphology observation and Gram staining analysis. The results are as follows: Figures 1-12 As shown.
[0063] The colony morphology of the artificially cultivated Polygonatum strain RG1 was initially screened as round with irregularly lobed edges, a rough surface, dense texture, and a light yellow, opaque color. The colonies were flat. Figure 1 A in the text). RG1 spores are oval and Gram-positive. Figure 1 (B in the middle).
[0064] Preliminary screening of Polygonatum strain SK5 in the Yamaguchi area showed that the colonies were round with irregular edges, rough surface, dense texture, light yellow and opaque color, and flat. Figure 2 A in the text). SK5 spores are short rod-shaped and Gram-positive. Figure 2 (B in the middle).
[0065] Preliminary screening of Polygonatum strain SK6 in the Yamaguchi area showed that the colonies were round, waxy droplets with irregular edges, dry surface, dense texture, milky white and opaque, and the colonies were flat and slightly raised. Figure 3 A in the text). SK6 spores are oval and Gram-positive. Figure 3 (B in the middle).
[0066] The preliminary screening of Polygonatum strain HQ1 in Huangqian area showed irregularly round colonies with uneven edges, rough surfaces, a viscous and dense texture, and a light yellow, opaque appearance. The colonies were flat. Figure 4 A in the middle). HQ1 spores are oval and Gram-positive ( Figure 4 (B in the middle).
[0067] The preliminary screening of Polygonatum strain HQ2 in Huangqian area showed a crater-like colony morphology with irregular edges, wrinkled surface, dry and rough texture, and white and opaque color. The colonies were raised. Figure 5 A in the middle). HQ2 spores are short and columnar, Gram-positive ( Figure 5 (B in the middle).
[0068] The preliminary screening of Polygonatum strain HQ3 in Huangqian area showed that the colonies were round with irregular edges, dry surface, dense texture, white and opaque color, and slightly raised. Figure 6 A in the middle). HQ3 spores are short and columnar, Gram-positive ( Figure 6 (B in the middle).
[0069] The preliminary screening of Polygonatum strain HQ5 in Huangqian area showed that the colonies were round with relatively neat edges, viscous and dense texture, light yellow color, and opaque. Figure 7 A in the middle). HQ5 spores are cylindrical and Gram-positive ( Figure 7 (B in the middle).
[0070] The preliminary screening strain HQ7 of Polygonatum sibiricum from the Huangqian area has a round colony morphology with irregular edges, a dry surface, and small wrinkles and bulges in the center. The colonies spread flat on the surface of the culture medium, and are white and opaque. Figure 8 A in the middle). HQ7 spores are short columnar, Gram-positive ( Figure 8 (B in the middle).
[0071] Preliminary screening of Polygonatum strain YQ1 in the Yuquansi area revealed nearly circular colonies with irregular edges, smooth surfaces, moist and dense texture, milky white color, and opaque appearance. The colonies were flat and slightly raised. Figure 9 A in the middle). YQ1 spores are short rod-shaped and Gram-positive ( Figure 9 (B in the middle).
[0072] Preliminary screening of Polygonatum strain YQ8 in the Yuquansi area showed that the colonies were round with rounded edges, smooth surface, moist and dense texture, bright yellow color, opaque, and slightly raised. Figure 10 A in the middle). YQ8 spores are oval, Gram-positive ( Figure 10 (B in the middle).
[0073] Preliminary screening of Polygonatum strain YQ10 in the Yuquansi area revealed nearly round colonies with rounded edges, dry surface, dense texture, white color, and opaque appearance; the colonies were flat. Figure 11 A in the middle). YQ10 spores are oval and Gram-positive. Figure 11 (B in the middle).
[0074] Preliminary screening of Polygonatum strain YQ11 in the Yuquansi area revealed nearly circular colonies with a dry surface, capable of forming crater-like ridges, irregular edges, dense texture, and a white, opaque color. Figure 12 A in the middle). YQ11 spores are oval and Gram-positive ( Figure 12 (B in the middle).
[0075] 2. Evaluation of the extracellular polysaccharide production capacity of different strains 2.1 Determination of the fermentation endpoint of the strain To determine the fermentation time of the strains, five Bacillus strains (SK6, SK5, HQ7, YQ8, and RG1) were selected for fermentation. The Bacillus strains were cultured in LB liquid medium at 37°C and 120 rpm for 12 hours to obtain a seed culture. This seed culture was then inoculated into LB medium at a 5% inoculum volume, and fermentation was carried out at 37°C. The OD values of the fermentation broth at 0 h, 24 h, 36 h, and 48 h were measured. 600 value.
[0076] Table 2. OD values of five Bacillus strains at different fermentation times 600 value As shown in Table 2, the growth performance of different strains varied, with strain SK6 exhibiting the strongest growth ability and OD. 600 The highest value was 2.384; strain RG1 had the weakest growth ability, with the lowest OD value. 600 The highest value was 1.889. The five strains exhibited the most vigorous growth activity from 0h to 24h, and their growth activity increased slowly and then stabilized from 24h to 48h. Therefore, this invention selected the 24h fermentation broth for determining the polysaccharide content.
[0077] 2.2 Results of Polysaccharide Production Performance of the Strains The glucose standard curve is shown below. Figure 13The linear regression equation between the absorbance and concentration of the glucose standard solution was y = 0.0109x + 0.2634, with R² = 0.999. The polysaccharide yields of the 12 bacterial strains are shown in Table 3.
[0078] Table 3 Polysaccharide yield of each strain Table 3 shows the endophytic strains of wild Polygonatum sibiricum from the Yamaguchi area: SK5, SK6; wild Polygonatum sibiricum from the Huangqian area: HQ1, HQ2, HQ3, HQ5, HQ7; wild strains from the Yuquansi area: YQ1, YQ8, YQ10, YQ11; and artificially cultivated strain: RG1.
[0079] The results showed that the extracellular polysaccharide production of strains SK6 and YQ8 was significantly higher than that of other strains. Specifically, the polysaccharide production in the fermentation broth of strain SK6 was 44.22 g / L, and that of strain YQ8 was 34.50 g / L. Strain RG1 had the lowest polysaccharide production among the 12 strains, at only 8.17 g / L. The average polysaccharide production of the preliminarily screened wild Polygonatum strains from the Yamaguchi area was 35.97 g / L, the average polysaccharide production of the preliminarily screened wild Polygonatum strains from the Yuquansi area was 27.20 g / L, and the average polysaccharide production of the preliminarily screened wild Polygonatum strains from the Huangqian area was 21.71 g / L.
[0080] 3. Strain identification DNA was extracted from strain SK6 using the Tiangen DNA Extraction Kit. PCR amplification was performed using universal primers 27F and 1492R. The amplified products were sequenced to obtain the 16S rDNA sequence of strain SK6, as shown in SEQ ID NO.1. The sequence in SEQ ID NO.1 was compared with known sequences using BLAST analysis in the NCBI database, and a phylogenetic tree was constructed using MEGA software. Figure 14 ).
[0081] SEQ ID NO.1:
[0082] from Figure 14 The phylogenetic tree of strain SK6 shows its evolutionary relationship with several Bacillus strains, among which SK6 is related to... Bacillus cereus With a high degree of similarity and 98% homology, combined with morphological identification and 16S rDNA sequence analysis, strain SK6 can be identified as Bacillus cereus. Bacillus cereus ).
[0083] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0084] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A strain of *Bacillus cereus* endophytic in *Polygonatum sibiricum*, characterized in that... The Bacillus cereus is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36060.
2. The use of Bacillus cereus as described in claim 1 in the production of extracellular polysaccharides.
3. A method for producing extracellular polysaccharides, characterized in that, The seed culture of Bacillus cereus as described in claim 1 is inoculated into a fermentation medium and fermented at 35℃~38℃ for 24h~36h to obtain a fermentation broth containing extracellular polysaccharides.
4. The method according to claim 3, characterized in that, The fermentation culture was conducted at a temperature of 37°C for 24 hours.
5. The method according to claim 4, characterized in that, The content of extracellular polysaccharides in each liter of the fermentation broth is ≥44.22g.
6. The method according to claim 5, characterized in that, The seed culture of Bacillus cereus is obtained by culturing Bacillus cereus in LB liquid medium at 35℃~38℃ with shaking for 10h~14h.
7. The method according to claim 6, characterized in that, The rotation speed of the oscillation culture is 110 r / min to 130 r / min.
8. The method according to claim 7, characterized in that, Based on the volume of the fermentation medium, the inoculation volume of the Bacillus cereus seed solution is 3% to 8% of the fermentation medium volume.
9. The method according to claim 8, characterized in that, The fermentation medium is LB medium, beef extract peptone medium, or corn flour and soybean meal medium.