Bacillus altitudinis, microbial inoculant and application thereof

Abstract

This invention discloses a strain of *Bacillus glabra*, a microbial inoculant, and its applications. *Bacillus glabra* (… Bacillus altitudinis Named Bacillus altitudinis The strain number is M1118, the accession number is GDMCC NO:67333, and the accession date is November 21, 2025. Using the *Bacillus hygroscopicus* strain of this invention to extract flavonoids from bamboo leaves significantly improves extraction efficiency: through microbial fermentation, the extraction rate of bamboo leaf flavonoids is increased by more than 50% compared to traditional methods. It is environmentally friendly: the fermentation conditions are mild, requiring no heating or electrical equipment. It is simple to operate and low in cost: the preparation of the inoculum and the fermentation process are easily scalable, reducing the cost of bamboo leaf processing. It allows for comprehensive resource utilization: the fermentation residue can be converted into organic fertilizer, achieving full resource utilization.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Bacillus subtilis, a microbial agent, and its application. Background Technology

[0002] Bamboo leaf flavonoids are a class of natural polyphenolic compounds widely found in bamboo leaves. They possess a wide range of biological activities, such as antioxidant, anti-inflammatory, antibacterial, and anticancer activities, and show great promise for applications in food, health products, pharmaceuticals, and cosmetics. For example, bamboo leaf flavonoids can be used as a natural preservative to replace synthetic additives, or for the prevention of cardiovascular diseases and metabolic syndrome.

[0003] Bamboo leaf flavonoids have huge market potential, but traditional extraction methods such as organic solvent extraction and hot water extraction have problems such as low extraction rate, high energy consumption and environmental pollution, which limit their industrial development.

[0004] Currently, the main extraction methods for bamboo leaf flavonoids include traditional processes such as organic solvent extraction, hot water extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. While these methods can extract bamboo leaf flavonoids, they still have certain limitations: organic solvent extraction uses reagents such as ethanol and acetone, resulting in generally low extraction rates, and solvent residues may pose food safety risks; hot water extraction is energy-intensive, has a long extraction cycle, and is prone to thermal degradation and inactivation of flavonoids; physical-assisted methods such as ultrasonic extraction can shorten the extraction time, but the equipment cost is high, large-scale application is difficult, and environmental pollution may occur. Existing technologies lack green and efficient methods, making it difficult to meet the requirements of sustainable development.

[0005] For example, the invention application with publication number CN115433085A discloses a production process for extracting bamboo leaf flavonoids and / or chlorogenic acid from bamboo leaves. The process involves filtering the aqueous extract of bamboo leaves through a surface-modified ceramic membrane to obtain a ceramic membrane filtrate containing bamboo leaf flavonoids and chlorogenic acid.

[0006] The invention application with publication number CN113527276A discloses a method for extracting high-purity bamboo leaf flavonoids, including the following steps: raw material processing: washing, sterilizing and drying the collected bamboo leaves; preparation of crude powder; alcohol extraction; reduced pressure alcohol collection; water dilution and centrifugation; chromatographic extraction; water-ethanol elution; alcohol collection and concentration; drying and detection; packaging and warehousing.

[0007] In recent years, microbial fermentation has attracted attention due to its environmental friendliness and mild conditions, such as the use of microbial enzymatic hydrolysis to enhance the dissolution of active ingredients in plants. Bacterial fermentation, in particular, exhibits unique advantages due to its strong adaptability and enzymatic hydrolysis capabilities. However, existing technologies lack bacterial strains specifically for the extraction of flavonoids from bamboo leaves; most strains focus on other plant matrices, resulting in insufficient extraction efficiency. Therefore, developing a highly efficient, specific, and safe microbial strain to disrupt the cell wall structure of bamboo leaves through bio-fermentation, thereby improving the extraction efficiency and purity of flavonoids, has become a crucial problem urgently needing to be solved in this field. Summary of the Invention

[0008] This application addresses the aforementioned deficiencies in the prior art by providing a strain of Bacillus subtilis, a microbial agent, and its application.

[0009] Most Bacillus hygroscopicus isolates are obtained from air or marine environments (Bacillus hygroscopicus isolates are...). Bacillus altitudinis HQ-51), mining area (the isolated strain of Bacillus hygroscopicus was...) Bacillus altitudinis RDK4), seeds (the isolated Bacillus hygroscopic strain is...) Bacillus altitudinis Isolated from sources such as KRS010, it generally exhibits good thermostability and extracellular enzyme secretion characteristics. This invention isolated a strain of Bacillus hygroscopicus M1118 (KRS010) from the surface of bamboo leaves. Bacillus altitudinis M1118, which naturally colonizes the surface environment of bamboo leaves, demonstrates good adaptability. Solid-state fermentation can significantly improve the flavonoid extraction rate. Compared with physical or chemical methods, the fermentation process of this strain is mild, easy to scale up, and the preparation of the inoculum is simple, making it suitable for continuous industrial production. This helps to reduce costs and energy consumption, providing a green technology path for the high-value utilization of bamboo leaf resources.

[0010] This invention first provides a strain of Bacillus hygroscopicus (B. hygroscopicus). Bacillus altitudinis ), named Bacillus altitudinis The strain number is M1118, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67333, and the deposit date is November 21, 2025.

[0011] The Bacillus subtilis provided by this invention is derived from moso bamboo (… Phyllostachys edulisThe strain was isolated and purified from leaves. Morphological observation and ITS sequence identification confirmed it as a new isolate of *Bacillus glaber*. The colonies of *Bacillus glaber* were irregularly shaped, milky white, with a rough surface and regular edges; under a scanning electron microscope, the cells were rod-shaped and lacked flagella. *Bacillus glaber* did not exhibit the ability to decarboxylate ornithine, arginine, or lysine. The strain did not form a hemolytic zone on blood agar plates, confirming that it does not express hemolysin and is a non-hemolytic strain. The 16S rRNA sequence of *Bacillus glaber*, as shown in SEQ ID NO: 1, showed over 99.9% homology with sequences of *Bacillus glaber* published in the GenBank database.

[0012] The present invention also provides a microbial agent containing the aforementioned Bacillus hygroscopicus, which can be directly used for solid-state fermentation extraction of bamboo leaf flavonoids.

[0013] Preferably, the microbial agent is prepared as a liquid agent or a solid agent. A liquid agent is prepared by preserving the bacterial cells in a liquid culture medium, while a solid agent can be a lyophilized formulation.

[0014] The preparation method of the microbial agent includes: inoculating the Bacillus hygroscopicus into a liquid culture medium, shaking and culturing at 25-35℃ for 48-72 hours, collecting the bacterial cells by centrifugation, and resuspending them in sterile water to the required concentration.

[0015] The present invention further provides the application of the aforementioned microbial agent in the fermentation and extraction of flavonoids from bamboo leaves.

[0016] The present invention also provides a fermentation extraction method for bamboo leaf flavonoids, wherein the Bacillus hygroscopicus is uniformly mixed with bamboo leaf raw material and then subjected to solid-state fermentation, and bamboo leaf flavonoids are separated and extracted from the fermentation product.

[0017] The raw material for bamboo leaves is bamboo leaf powder obtained by crushing bamboo leaves to 40-80 mesh.

[0018] Preferably, the fermentation time is no less than 2 days, such as 2-5 days. More preferably, the fermentation time is 2-3 days. When fermented for 3 days, the extraction rate of bamboo leaf flavonoids can reach a relatively high level. Increasing the fermentation time further, such as 4 or 5 days, does not significantly improve the extraction rate of bamboo leaf flavonoids.

[0019] Preferably, during fermentation, the Bacillus hygroscopicus is prepared into a fermentation suspension using a liquid culture medium; the ratio of the fermentation suspension to bamboo leaf raw material is 1 mL: 0.5-2 g, more preferably 1 mL: 0.5-1 g, and most preferably 3 mL: 2 g.

[0020] Preferably, the concentration of viable Bacillus hygroscopicus in the fermentation suspension is not less than 10. 6CFU / mL. More preferably, the concentration of viable Bacillus hygroscopicus in the fermentation suspension is 10. 6 -10 8 CFU / mL.

[0021] Preferably, the fermentation temperature is 25-35℃, and most preferably 30℃.

[0022] Compared with the prior art, the beneficial effects of the present invention include: (1) The extraction efficiency is significantly improved: through microbial fermentation, the extraction rate of bamboo leaf flavonoids is increased by more than 50% compared with traditional methods.

[0023] (2) Environmental friendliness: mild fermentation conditions and reduced energy consumption.

[0024] (3) Simple operation and low cost: the preparation and fermentation process of the microbial agent is easy to scale up, and the cost of bamboo leaf processing is reduced.

[0025] (4) Comprehensive utilization of resources: Fermentation residue can be converted into organic fertilizer, realizing full resource utilization. Attached Figure Description

[0026] Figure 1 The colony morphology of Bacillus hygroscopicus M1118.

[0027] Figure 2 This is a scanning electron microscope image of Bacillus hygroscopicus M1118.

[0028] Figure 3 Images of a hemolysis experiment using Bacillus hygroscopicus M1118.

[0029] Figure 4 This is the phylogenetic tree of Bacillus hygroscopicus M1118.

[0030] Figure 5 The growth curve of Bacillus hygroscopicus M1118 is shown.

[0031] Figure 6 Optimization of conditions for solid-state fermentation extraction of bamboo leaf flavonoids using Bacillus hygroscopicus M1118. Among them, Figure 6 (A) represents screening for different fermentation times. Figure 6 (B) in the figure represents screening for different liquid-to-solid ratios. Figure 6 (C) represents screening for different bacterial concentrations. Detailed Implementation

[0032] The bamboo leaves mentioned in this embodiment of the invention are those of the plant moso bamboo (Phyllostachys edulis). Phyllostachys edulisThe leaves of bamboo (from Anji County, Zhejiang Province) were harvested, naturally dried, and then pulverized through an 80-mesh sieve to obtain bamboo leaf powder. The bamboo leaf powder was stored in a desiccator for later use. The LB agar medium described in this embodiment is a finished beef extract peptone agar medium (Qingdao Haibo Biotechnology Co., Ltd.), prepared with distilled water at a concentration of 25 g / L, with a natural pH, sterilized by autoclaving at 121℃ for 20 minutes, and poured into sterile petri dishes with a diameter of 9 cm before solidification, 15-20 mL per dish.

[0033] The flavonoid content described in this embodiment of the invention was determined using the aluminum nitrate-sodium nitrite colorimetric method. Specifically, the bamboo leaf flavonoid extract was appropriately diluted with deionized water (ensuring the concentration was within the standard curve range). 1 mL of the sample solution was pipetted into a 10 mL stoppered tube, and 0.3 mL of 5% sodium nitrite solution, 0.3 mL of 10% aluminum nitrate solution, and 4 mL of 4% sodium hydroxide solution were added sequentially. The solution was then diluted to 10 mL with deionized water. After standing for 15 minutes, the absorbance was measured at a wavelength of 510 nm using a blank reagent as a reference. A standard curve was plotted using rutin as a standard, and the flavonoid content was calculated.

[0034] The yield of bamboo leaf flavonoids was calculated using the following formula: Flavonoid extraction yield = mass of extracted flavonoids / mass of bamboo leaf powder used for extraction.

[0035] Example 1: Isolation and screening of target bacterial strains This embodiment aims to isolate native strains from the microbial community on the surface of bamboo leaves.

[0036] (1) Sample processing: Take 5 g of dried bamboo leaves, put them in sterile water, shake for 5 min and then discard the bamboo leaves to obtain the microbial liquid on the surface of the bamboo leaves.

[0037] (2) Microbial culture and purification: 100 μL of microbial culture from the surface of bamboo leaves was spread on LB agar medium and cultured at 30℃ for 2 days. Single colonies with different morphologies were picked and purified to obtain 8 candidate strains (numbered M1 to M8).

[0038] Example 2: Screening of target strains for solid-state fermentation extraction of bamboo leaf flavonoids This embodiment details the process of extracting bamboo leaf flavonoids using solid-state fermentation assisted by candidate strains.

[0039] (1) Solid-state fermentation: Take 20 g of bamboo leaf powder and place it in a 250 mL Erlenmeyer flask, add 30 mL of the bacterial suspension prepared in Example 2 (the viable bacteria concentration is 10). 6 (CFU / mL), stir well. Seal the Erlenmeyer flask with breathable sealing film and incubate at 30℃ for 72 hours. After fermentation, a small amount of white mycelium can be seen on the surface of the bamboo leaf powder.

[0040] Control group: Bamboo leaf powder was directly extracted by hot water ultrasonic extraction.

[0041] Control fermentation group: Bamboo leaf powder was mixed with an equal amount of sterile physiological saline and extracted under the same fermentation conditions as the fermentation group.

[0042] Experimental group: Different strains were inoculated under the same fermentation conditions.

[0043] (2) Extraction: Add 500 mL of 70% ethanol solution (material-liquid ratio 1:25 g / mL) to the fermentation product, extract at room temperature for 30 min, filter with Buchner funnel after extraction, collect the filtrate and detect the total flavonoid content.

[0044] (3) Experimental results: The flavonoid extraction yield is shown in Table 1. The strain M8 group had the highest flavonoid extraction yield from bamboo leaves.

[0045] Table 1. Extraction yield of bamboo leaf flavonoids from different strains and the control group Therefore, strain M8 has a good ability to improve the extraction rate of bamboo leaf flavonoids. Thus, this strain was selected as the target strain for solid-state fermentation extraction of bamboo leaf flavonoids.

[0046] Example 3: Identification of target strains This embodiment aims to identify strains isolated from the microbial community on the surface of bamboo leaves that can help improve the extraction rate of bamboo leaf flavonoids.

[0047] (1) Morphological identification: such as Figure 1 As shown, strain M8 colonies on PDA plates are radial, milky white, with a smooth, raised surface and irregular edges. Figure 2 As shown, the cells appear rod-shaped under a scanning electron microscope and lack flagellar structures.

[0048] (2) Safety assessment: such as Figure 3 As shown, strain M8 did not produce a hemolytic zone when grown on defibrinated sheep blood agar plates. Furthermore, amino acid decarboxylation experiments revealed that strain M8 did not possess the ability to decarboxylate ornithine, arginine, or lysine, and did not produce highly toxic biogenic amines.

[0049] (3) Molecular identification: Genomic DNA was extracted from strain M8, and the 16S rDNA sequence was amplified. The sequencing results are as follows:

[0050] The sequencing results were compared with BLAST sequences on the NCBI website, confirming that strain M8 was *Bacillus hygroscopicus*. Bacillus altitudinis ), named Bacillus altitudinis Plant number M1118. Figure 4 This is the phylogenetic tree of Bacillus highlandii M1118. Furthermore, this strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 67333 and deposit date November 21, 2025.

[0051] (4) Growth viability of the strain: Bacillus hygroscopicus M1118 reached the logarithmic growth phase after 12 hours and the stationary phase after 24 hours in LB broth. This strain exhibited good growth viability, with a viable count of 10⁻⁶ cells upon reaching the logarithmic growth phase. 7 CFU. Figure 5 The growth curve of Bacillus hygroscopicus M1118 is shown.

[0052] Example 4: Preparation of Bacillus hygroscopicus inoculum This embodiment provides a highly efficient and stable method for preparing Bacillus alpineus inoculum.

[0053] (1) Preparation of fermentation culture: Bacillus hygroscopicus M1118 was inoculated into LB agar medium and cultured at 30°C for 24 hours. 10 mL of sterile physiological saline was added, and the mixture was stirred evenly to adjust the concentration of the culture to 10. 6 CFU / mL.

[0054] (2) Production of bacterial agent: The bacterial suspension was inoculated into LB broth liquid medium at an inoculation rate of 1%, and incubated at 30 °C and 150 °C. rpm Incubate with shaking for 24 hours. Collect bacterial cells by centrifugation, resuspend in sterile water, and adjust the viable cell concentration to 10⁻⁶. 9 The liquid bacterial agent was prepared at CFU / mL. Further, using 10% skim milk as a freeze-drying protectant, a powder was prepared, and the viable bacterial survival rate exceeded 80% when stored at -80℃.

[0055] The bacterial agent maintained a viable count of 10 after 15 days of storage at 4°C. 7 CFU or higher, meeting the needs of industrial applications.

[0056] Example 5: Optimization of fermentation conditions to improve the extraction rate of bamboo leaf flavonoids This embodiment explores the optimization of key fermentation parameters on flavonoid extraction yield.

[0057] (1) Fermentation time: fixed bacterial concentration 10 6The flavonoid extraction yield was compared after fermentation at CFU / mL, with a liquid-to-solid ratio of 3:2 for bacterial suspension and bamboo leaf powder, and a fermentation temperature of 30℃, after 1, 2, 3, 4, and 5 days of fermentation. The results are as follows: Figure 6 As shown in (A), the experimental results show that the yield of bamboo leaf flavonoids is close to the maximum after 3 days of solid-state fermentation. Fermentation for 4 days and 5 days has little effect on increasing the yield of bamboo leaf flavonoids. From an economic point of view, 3 days is the preferred fermentation time.

[0058] (2) Liquid-to-solid ratio: The fermentation time was fixed at 3 days, and the bacterial concentration was 10. 6 The flavonoid extraction yields were compared between bamboo leaf powder and bacterial suspension at CFU / mL and fermentation temperature of 30℃ for liquid-solid ratios (volume:mass = mL:g) of 1:2, 2:3, 1:1, 3:2, and 2:1. The results are as follows: Figure 6 As shown in (B), the experimental results show that the yield of bamboo leaf flavonoids is the highest when the liquid-solid ratio of bacterial suspension to bamboo leaf powder is 3:2. This ratio is preferred as the liquid-solid ratio of bamboo leaf powder to bacterial suspension during fermentation.

[0059] (3) Bacterial suspension concentration: Fermentation time was 3 days, the liquid-solid ratio of bacterial suspension to bamboo leaf powder was 3:2, the fermentation temperature was 30℃, and the bacterial suspension concentration was 10. 4 10 5 10 6 10 7 10 8 Extraction yield at CFU / mL. Results are as follows. Figure 6 As shown in (C), the experimental results indicate that the bacterial concentration is 10. 6 CFU / mL and 10 7 The yield of bamboo leaf flavonoids was high at CFU / mL. From an economic perspective, a bacterial concentration of 10 was chosen. 6 CFU / mL is the optimal inoculation concentration.

[0060] in conclusion: The above embodiments demonstrate that the *Bacillus hygroscopicus* and its fermentation-assisted extraction method provided by this invention can significantly improve the extraction yield of bamboo leaf flavonoids (more than 50% higher than traditional methods), and is simple to operate, environmentally friendly, and suitable for industrial production. Those skilled in the art can adjust the parameters according to actual needs, and all such adjustments fall within the protection scope of this invention.

Claims

1. A strain of Highland Bacillus ( Bacillus altitudinis ), characterized in that, Named Bacillus altitudinis The stock number is M1118, the accession number is GDMCC NO: 67333, and the accession date is November 21, 2025.

2. A microbial inoculant, characterized in that, It contains the Bacillus alpineus of claim 1, and the microbial agent is used for the extraction of flavonoids from bamboo leaves.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is prepared as a liquid or solid agent.

4. The application of Bacillus hygroscopicus as described in claim 1 in the fermentation and extraction of flavonoids from bamboo leaves.

5. A fermentation extraction method for bamboo leaf flavonoids, characterized in that, The Bacillus hygroscopicus of claim 1 is mixed with bamboo leaf raw material and fermented, and bamboo leaf flavonoids are separated and extracted from the fermentation product.

6. The fermentation extraction method for bamboo leaf flavonoids according to claim 5, characterized in that, Fermentation time should be no less than 2 days.

7. The fermentation extraction method for bamboo leaf flavonoids according to claim 5, characterized in that, During fermentation, the Bacillus hygroscopicus was prepared into a fermentation suspension using a liquid culture medium; the liquid-solid ratio of the fermentation suspension to the bamboo leaf raw material was 1 mL: 0.5-2 g.

8. The fermentation extraction method for bamboo leaf flavonoids according to claim 5, characterized in that, The concentration of Bacillus hygroscopicus in the fermentation suspension is not less than 10%. 6 CFU / mL.

9. The fermentation extraction method for bamboo leaf flavonoids according to claim 5, characterized in that, The concentration of Bacillus hygroscopicus in the fermentation suspension was 10. 6 -10 8 CFU / mL.

10. The fermentation extraction method for bamboo leaf flavonoids according to claim 7, characterized in that, The fermentation temperature is 25-35℃.

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