Bacillus altitudinis, microbial inoculum, preparation method and application

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a Bacillus altitudinis, a microbial agent, a preparation method and application. The present application provides a Bacillus altitudinis ICBR-C01 with a preservation number of CGMCC No. 29900. The present application utilizes the Bacillus altitudinis ICBR-C01 to ferment bamboo shoots. The results of the examples show that the strain has a good fermentation capacity for bamboo shoots, can convert and form more active polypeptide substances, can increase the polypeptide content in the bamboo shoots from 0.34 g / L before fermentation to 1.69 g / L after fermentation, and the fermentation liquor has a good antioxidant activity, thereby endowing the bamboo shoots with better health care efficacy.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of Bacillus hygroscopicus, a bacterial agent, a preparation method, and its application. Background Technology

[0002] Bamboo shoots are the swollen buds and tender stems that sprout and differentiate from the buds at the base of the bamboo rhizome or culm. Bamboo shoots are delicious, crisp, and tender, with a unique flavor; they have a long history of consumption in my country. Modern research shows that bamboo shoots contain various essential trace minerals, vitamins, dietary fiber, and protein, while also being low in energy and fat. Bamboo shoots are rich in protein, with a high content and variety of amino acids, making them a highly nutritious natural food. Fermenting bamboo shoot protein through microorganisms to prepare more easily absorbed and utilized active peptides may have a series of beneficial physiological activities. Fermented bamboo shoot products are a traditional Chinese delicacy. After microbial transformation, bamboo shoots acquire a more unique flavor and crisp texture, and their nutritional value is also enhanced. For example, many fermented bamboo shoot products exhibit good antioxidant, blood pressure-lowering, and blood sugar-lowering biological activities, making them very popular among consumers.

[0003] The giant panda (Ailuropoda melanoleuca) is a precious protected animal in my country and is considered a "flagship species" for global biodiversity conservation. Research has found that the gut microbiota plays a crucial role in the giant panda's immune, digestive, and metabolic functions. In both wild and captive conditions, giant pandas primarily feed on bamboo, with bamboo shoots being their favorite food. However, they still retain digestive system characteristics of carnivores. Numerous studies have clearly demonstrated that giant pandas mainly rely on their gut microbiota to obtain nutrients from bamboo. Laccase produced by Pseudomonas in the giant panda's gut oxidizes lignin in bamboo fiber, exposing cellulose. Cellulase produced by Clostridium and Bacillus amyloliquefaciens further degrades this cellulose into sugars that the giant panda can utilize. Therefore, the giant panda's gut microbiota may possess a specific advantage in the transformation and utilization of bamboo shoot nutrients, making it an excellent source for finding and obtaining bamboo shoot fermentation strains with unique fermentation characteristics.

[0004] Currently, the main methods for preparing peptides from natural protein sources include chemical hydrolysis, enzymatic hydrolysis, and microbial fermentation. Different methods yield peptides with varying purity, physicochemical properties, and functional activities. Microbial fermentation utilizes proteases produced during microbial fermentation to break down proteins into peptides or free amino acids. Simultaneously, enzymes other than proteases are used to degrade complex carbohydrates and lipids. Furthermore, mixed enzymes produced by microbial metabolic activities release peptides through hydrolysis. In addition, microorganisms can enhance their growth and enzyme production capacity with the help of peptide hydrolysates, resulting in a cyclical and efficient process. Microbial fermentation offers advantages over the other two methods, including lower cost and higher protease yield. The biological applications of peptides largely depend on their size and structure, and their absorption and bioavailability in target tissues play a crucial role. Peptides possess various active functions, such as immunomodulation, antioxidation, antitumor activity, inhibition of angiotensin-converting enzyme (ACE), and hypoglycemic effects, which have been extensively studied. Food-derived plants are numerous and, compared to animal proteins, are cheaper, more readily available, and have advantages such as good dispersibility and easy digestibility. Bioactive peptides prepared from plant proteins have broad application prospects in the fields of food, pharmaceuticals, and cosmetics due to their wide availability, low cost, and general non-toxicity.

[0005] With the increasing demand for functional foods, bamboo shoots, as a forest vegetable with both edible and medicinal value, have shown their unique value. Through microbial fermentation, bamboo shoot peptides can be prepared to expand the source of plant protein, and it is possible to develop high-value plant-derived bioactive peptides and carry out commercial production.

[0006] Bacillus is an important microbial resource that produces stress-resistant spores, generating various functional metabolites used in biopesticides, biofertilizers, biofeeds, probiotics, and food fermentation. For example, Bacillus subtilis and Bacillus amyloliquefaciens are widely used in the production of fermented foods. These functional microorganisms can regulate the synthesis of flavor- and quality-related substances, as well as carcinogenic, sensitizing, or adverse substances during the fermentation process through their own metabolism, thereby improving the safety or flavor and quality of fermented foods. Studies have shown that Bacillus hygrophila, due to its growth-promoting, protease-producing, and stress-resistant functions, has the potential to be used as a microbial fertilizer and fermentation strain. However, currently, bamboo shoot fermentation in my country mainly relies on mixed-culture natural fermentation, utilizing microorganisms from the natural environment. While natural fermentation is simple and low-cost, it suffers from problems such as excessively long fermentation times, incomplete fermentation, susceptibility to contamination, and uncontrollable fermentation quality. Therefore, pure-culture fermentation has begun to be developed, involving the deliberate addition of selected fermenting agents for inoculation. However, there are no reports of Bacillus hygrophila fermenting bamboo shoots in existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Bacillus altitudinis ICBR-C01, which can ferment bamboo shoots and increase the polypeptide content of the bamboo shoot fermentation liquid.

[0008] To address the aforementioned technical problems, the following technical solutions are proposed:

[0009] This invention provides a strain of Bacillus altitudinis ICBR-C01, with accession number CGMCC No.29900.

[0010] The present invention provides a microbial agent, the effective components of which include the Bacillus hygroscopicus ICBR-C01 or the fermentation broth of Bacillus hygroscopicus ICBR-C01 as described in the above technical solution.

[0011] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating the Bacillus hygroscopicus ICBR-C01 into a culture medium for activation to obtain an activated solution; inoculating the activated solution into a culture medium for propagation to obtain the bacterial agent.

[0012] Preferably, the propagation temperature is 26–40°C, the rotation speed is 150–200 rpm, and the time is 6–18 hours.

[0013] This invention provides the application of the above-described Bacillus hygroscopicus ICBR-C01 or the above-described bacterial agent or the bacterial agent prepared by the above-described preparation method in one or more of the following 1) to 3).

[0014] 1) Fermented bamboo shoots and / or fruits and vegetables;

[0015] 2) Increase the polypeptide content in bamboo shoot fermentation liquid;

[0016] 3) Improve the antioxidant activity of bamboo shoot fermentation liquid.

[0017] This invention provides a method for preparing fermented bamboo shoots, comprising the following steps:

[0018] Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 described in the above technical solution, or the inoculum prepared by the above technical solution or the preparation method described in the above technical solution.

[0019] Preferably, the fermentation temperature is 20–37°C; the time is 10–24 h; and the rotation speed is 150–250 rpm.

[0020] Preferably, before fermentation, the bamboo shoots are homogenized.

[0021] Preferably, during homogenization, the ratio of bamboo shoots to water is 1 kg:(3-10) L.

[0022] This invention provides a method for increasing the polypeptide content in fermented bamboo shoots and / or improving the antioxidant activity of bamboo shoot fermentation broth, comprising the following steps:

[0023] Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 described in the above technical solution, or the inoculum prepared by the above technical solution or the preparation method described in the above technical solution.

[0024] The beneficial effects of this invention: This invention provides a strain of Bacillus altitudinis ICBR-C01, with the preservation number CGMCC No. 29900. This strain of Bacillus altitudinis ICBR-C01 is the first to be isolated from the feces of captive giant pandas. During its growth, it produces proteases, and this strain exhibits excellent fermentation ability in bamboo shoots, converting them into more active substances. The results of the examples show that the fermented bamboo shoots prepared have a high polypeptide content. Therefore, the Bacillus altitudinis ICBR-C01 of this invention can increase the polypeptide content of bamboo shoots through proteolytic hydrolysis, and it can also disrupt the cell walls of bamboo shoots, promoting the release and digestion of nutrients, and improving their antioxidant activity. This provides technical support for the development of bamboo shoot fermentation products and has broad application prospects.

[0025] Biological Preservation Instructions

[0026] Bacillus altitudinis ICBR-C01 was deposited on February 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29900. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 The image shows the morphological observation of Bacillus altissima ICBR-C01 in this invention; where A represents the basic morphology of Bacillus altissima, B represents the Gram staining result of Bacillus altissima, and C represents the spore staining result of Bacillus altissima.

[0029] Figure 2 This is the growth curve of Bacillus hygroscopicus ICBR-C01 in this invention;

[0030] Figure 3The Bacillus hygroscopicus ICBR-C01 in this invention possesses proteolytic enzyme activity;

[0031] Figure 4 This invention illustrates the effect of different microbial fermentation processes on the polypeptide content in bamboo shoot fermentation broth.

[0032] Figure 5 This is a graph showing the effect of Bacillus hygroscopicus fermentation on DPPH free radical scavenging rate before and after the fermentation process in this invention. Detailed Implementation

[0033] This invention provides a strain of Bacillus altitudinis ICBR-C01, with accession number CGMCC No.29900.

[0034] The *Bacillus hygroscopicus* ICBR-C01 strain described in this invention was screened from feces of captive giant pandas. The colony morphology on TSB agar plates was as follows: colonies were off-white, viscous, wrinkled, and regularly shaped; the strain was Gram-positive; and the vegetative cells were regular, uniform, and neatly arranged short rods. The main fatty acids of *Bacillus hygroscopicus* ICBR-C01 were trans-isomerized pentadecane saturated fatty acid C15: 0anteiso, isomerized pentadecane saturated fatty acid C15: 0iso, palmitoleic acid (unsaturated fatty acid) C16: 1ω5c, trans-isomerized heptadecanane saturated fatty acid C17: 0anteiso, and isomerized heptadecanane saturated fatty acid C17: 0iso, with contents of 24.04%, 28.61%, 12.20%, 7.82%, and 6.70%, respectively. The API 50CH assay results for *Bacillus hygroscopicus* ICBR-C01 were as follows: positive reactions included ribose, glucose, fructose, mannose, mannitol, arbutin, aesculin, salinomyol, cellobiose, sucrose, geraniol, and D-tagatose; weak positive reactions included glycerol, L-arabinose, N-acetylglucosamine, amygdalin, and trehalose. *Bacillus hygroscopicus* ICBR-C01 possesses proteolytic enzyme activity, which can increase the polypeptide content in fermentation products.

[0035] The present invention provides a microbial agent, the effective components of which include the Bacillus hygroscopicus ICBR-C01 or the fermentation broth of Bacillus hygroscopicus ICBR-C01 as described in the above technical solution.

[0036] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating the Bacillus hygroscopicus ICBR-C01 into a culture medium for activation to obtain an activated solution; inoculating the activated solution into a culture medium for propagation to obtain the bacterial agent.

[0037] Before activating the *Bacillus hygroscopicus* ICBR-C01, it is preferable to purify the *Bacillus hygroscopicus* ICBR-C01. The preferred purification method is to spread the *Bacillus hygroscopicus* ICBR-C01 onto a TSB solid medium plate for purification culture to obtain single colonies of *Bacillus hygroscopicus* ICBR-C01. The preferred purification culture temperature is 26–37°C, more preferably 30–33°C, and the preferred time is 20–26 h, more preferably 24 h. The purification culture is preferably a static culture.

[0038] The TSB solid culture medium of this invention preferably uses distilled water as a solvent and comprises 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L glucose, 5 g / L sodium chloride, 2.5 g dipotassium hydrogen phosphate, and 18–20 g / L agar, with a pH of 7.3 ± 0.2. After preparation, it is autoclaved at 121°C for 15 min.

[0039] Single colonies of *Bacillus hygroscopicus* ICBR-C01 were obtained. Preferably, these single colonies were inoculated into a culture medium for activation to obtain an activated solution. The activation culture temperature was preferably 26–40°C, more preferably 30–37°C; the activation time was preferably 22–26 h, more preferably 24 h; and the activation culture rotation speed was preferably 180–200 rpm, more preferably 180 rpm. The culture medium preferably included TSB liquid medium. The TSB liquid medium preferably consisted of 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L glucose, 5 g / L sodium chloride, and 2.5 g dipotassium hydrogen phosphate, with a pH of 7.3 ± 0.2, using distilled water as a solvent. After preparation, the solution was autoclaved at 121°C for 15 min.

[0040] The activated solution is then inoculated into a culture medium for propagation to obtain a bacterial agent. The inoculation amount is preferably 1%–5% of the culture medium volume, more preferably 2%–3%. The propagation temperature is preferably 26–40℃, more preferably 30–37℃; the propagation time is preferably 22–26 h, more preferably 24 h; the propagation speed is preferably 180–200 rpm, more preferably 180 rpm; and the propagation time is preferably 6–18 h, more preferably 12 h. The culture medium preferably includes TSB liquid culture medium; the TSB liquid culture medium has been discussed above and will not be repeated here. The bacterial solution obtained after propagation is the bacterial agent, and the viable count of the bacterial agent is preferably ≥1×10⁻⁶. 7 CFU / mL.

[0041] This invention provides the application of the above-described Bacillus hygroscopicus ICBR-C01 or the above-described bacterial agent or the bacterial agent prepared by the above-described preparation method in one or more of the following 1) to 3).

[0042] 1) Fermented bamboo shoots and / or fruits and vegetables;

[0043] 2) Increase the polypeptide content in bamboo shoot fermentation liquid;

[0044] 3) Improve the antioxidant activity of bamboo shoot fermentation liquid.

[0045] In this embodiment of the invention, bamboo shoots are fermented using Bacillus subtilis ICBR-C01, and the resulting bamboo shoot fermentation liquid has a high polypeptide content.

[0046] This invention provides a method for preparing fermented bamboo shoots, comprising the following steps:

[0047] Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 described in the above technical solution, or the inoculum prepared by the above technical solution or the preparation method described in the above technical solution.

[0048] Before fermentation, the present invention preferably homogenizes and sterilizes the bamboo shoots sequentially. Before homogenization, the bamboo shoots are preferably peeled, washed, and chopped into pieces and crushed. The parameters for peeling, washing, chopping, and crushing are conventional and not specifically limited. After crushing, homogenization is performed. During homogenization, the material-to-liquid ratio of bamboo shoots to water is preferably 1 kg:(3-10) L, more preferably 1 kg:(8-10) L. The homogenization time is preferably 8-15 min, more preferably 10 min; the temperature is preferably 20-30℃. After homogenization, the resulting bamboo shoot homogenate is sterilized and then inoculated with Bacillus hygroscopicus ICBR-C01. The sterilization parameters are preferably 121℃ for 20 min. The inoculation method of Bacillus hygroscopicus ICBR-C01 preferably includes a bacterial agent. The preparation method of the bacterial agent of the present invention has been discussed above and will not be repeated here. The fermentation temperature is preferably 20–37°C, more preferably 37°C; the fermentation time is preferably 10–24 h, more preferably 12 h; and the fermentation speed is preferably 150–250 rpm, more preferably 180–200 rpm. Fermented bamboo shoots prepared using the technical solution of this invention can increase the polypeptide content from 0.34 g / L to 1.69 g / L, thereby giving the bamboo shoots better health benefits.

[0049] This invention provides a method for increasing the polypeptide content in fermented bamboo shoots, comprising the following steps:

[0050] Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 described in the above technical solution, or the inoculum prepared by the above technical solution or the preparation method described in the above technical solution.

[0051] The preparation method of the bacterial agent described in this invention has been discussed above and will not be repeated here.

[0052] The parameters for peeling, cleaning, chopping, crushing, and homogenizing the bamboo shoots have been discussed above and will not be repeated here. The fermentation parameters have also been discussed above and will not be repeated here. The prepared bamboo shoot fermentation broth has a high peptide content.

[0053] This invention provides a method for improving the antioxidant activity of bamboo shoot fermentation broth, comprising the following steps: fermenting bamboo shoots using the Bacillus subtilis ICBR-C01 described in the above technical solution, or the bacterial agent described in the above technical solution, or the bacterial agent prepared by the preparation method described in the above technical solution.

[0054] The preparation method of the microbial agent described in this invention has been discussed above and will not be repeated here. The parameters of the bamboo shoots have been discussed above and will not be repeated here. The fermentation parameters have been discussed above and will not be repeated here. The prepared bamboo shoot fermentation broth has a high DPPH free radical scavenging rate.

[0055] This invention screens panda gut microbiota, uses bamboo shoots as raw material, ferments and develops polypeptide products with excellent bioactivity, explores the physiological functions of fermentation broth, realizes the efficient utilization of active ingredients in bamboo shoots, and conducts in-depth development in multiple aspects, angles, and directions, which is of great significance to the sustainable development of the bamboo shoot industry.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Unless otherwise specified, all raw materials, reagents and instruments used in this invention are well known in the art.

[0058] Example 1: Microbial Isolation, Purification, and Identification

[0059] 1. Isolation, purification, and protease production screening of microorganisms from captive giant panda feces

[0060] Fresh panda feces were collected and separated using the dilution-spread plate method. The specific method is as follows: Under aseptic conditions, 5g of fecal sample (from the center of the fecal bolus) was weighed and placed in 45mL of 0.9% physiological saline to prepare 10... -1 Diluent. 0.9% physiological saline is physiological saline with a mass concentration of 0.9%. 10 -1 Centrifuge the diluent (4000 rpm, 1 min), and take 1 mL of 10 mL. -1 The supernatant of the diluent was prepared by a 10-fold dilution method to obtain 10... -5 10 -6 10-7 A diluted fecal solution.

[0061] 200 μL of fecal samples at different dilutions were spread onto LB agar plates. The plates were then inverted and incubated at 37°C for 18 h.

[0062] Select colonies that are distinguishable to the naked eye and have different characteristics from the plate, streak them again on a new LB medium plate, and continue to incubate for 24 hours to observe the colony morphology.

[0063] Colonies were picked and diluted with 0.9% physiological saline at dilution gradients of 10⁻⁶. -1 10 -2 10 -3 10 -4 Take 10 -4 200 μL of colony dilution was evenly spread on the surface of skim milk solid medium plates and incubated at 37°C for 24 h. The ratio of the diameter of the transparent hydrolysis zone to the colony diameter is denoted as DH. The composition of the skim milk solid medium is: 62% skim milk (v:v), 36% distilled water (v:v), and 2% agar (w:v).

[0064] Colonies with high DH values ​​were selected from the skim milk solid medium (i.e., isolated plate) and purified using the three-zone streak method. After incubation in an incubator, single colonies were obtained, which was recorded as the first purification. The obtained single colonies were then purified again using the three-zone streak method, which was recorded as the second purification. The three-zone streak method was used for the third purification to obtain single colonies.

[0065] The purified single colonies were inoculated onto skim milk solid medium plates and stored at 37°C for 24 hours. Based on the DH value, the strain with the highest DH value, i.e., the high-protease-producing strain, was preliminarily screened and named ICBR-C01. (See below) Figure 3 The preservation solution was prepared by mixing TSB liquid medium and glycerol at a volume ratio of 1:1 and a glycerol mass concentration of 40%.

[0066] The strain ICBR-C01 was cultured in TSB liquid medium to obtain the bacterial suspension of strain ICBR-C01. The bacterial suspension was then stored in a preservation solution at a temperature of -80℃.

[0067] 2. Strain identification

[0068] 2.1 Observation of colony morphology

[0069] A small amount of bacterial culture was taken from the cryopreservation tube and placed in TSB liquid medium. The culture was incubated at 37°C and 120 rpm for 12 hours to obtain a seed culture. The seed culture was then transferred to fresh TSB liquid medium at a ratio of 10% of the medium volume and incubated again under the same conditions for 12 hours to obtain the culture medium. The culture medium was diluted and spread onto TSB solid agar plates, labeled, and incubated at 37°C for at least 12 hours. The morphological characteristics of the colonies were observed. The results are as follows: Figure 1 As shown.

[0070] from Figure 1 As can be seen from Figure A, the colonies of strain ICBR-C01 are off-white, with a viscous surface, wrinkles, and regular edges.

[0071] 2.2 Observation of bacterial cell morphology

[0072] Will Figure 1 A small amount of bacterial growth was picked from the A plate and placed on a clean glass slide. 30 μL of distilled water was added and spread on the slide. The slide was then air-dried and fixed. Gram staining was then performed. The prepared slide was placed under a microscope. The morphology of the bacteria was observed first under low power, then under high power, and finally under oil immersion. The Gram staining reaction of the bacteria was recorded.

[0073] from Figure 1 China B and Figure 1 Based on the staining results of C, strain ICBR-C01 is a Gram-positive bacterium. The vegetative cells of strain ICBR-C01 are regular, uniform, and neatly arranged short rods.

[0074] It can be seen that the colony morphology, cell morphology and Gram staining characteristics of strain ICBR-C01 are similar to those of the genus Bacillus.

[0075] 2.3 Physiological and biochemical characteristics of the strain

[0076] The fatty acid composition of strain ICBR-C01 was analyzed using the Microbial Fatty Acid Rapid Identification System (MIDI). The results showed that the main fatty acids in strain ICBR-C01 were trans-isomerized pentadecane saturated fatty acid C15:0anteiso, isomerized pentadecane saturated fatty acid C15:0iso, palmitoleic acid (unsaturated fatty acid) C16:1ω5c, trans-isomerized heptadecanane saturated fatty acid C17:0anteiso, and isomerized heptadecanane saturated fatty acid C17:0iso, with contents of 24.04%, 28.61%, 12.20%, 7.82%, and 6.70%, respectively. This is consistent with the main cellular fatty acid characteristics of the genus Bacillus.

[0077] The carbohydrate metabolism of strain ICBR-C01 was studied using the API 50CH identification system. The API 50CH detection results were as follows: positive reactions included ribose, glucose, fructose, mannose, mannitol, arbutin, aesculin, salinomyol, cellobiose, sucrose, geraniol, and D-tagatose; weakly positive reactions included glycerol, L-arabinose, N-acetylglucosamine, amygdalin, and trehalose; negative reactions included the control, erythrose, D-arabinose, D-xylose, L-xylose, azirmonol, β-methyl-D-xylosinoside, and galactoside. Sugars, sorbitol, rhamnose, eugenol, inositol, sorbitol, α-methyl-D-mannoside, α-methyl-D-glucoside, maltose, lactose, melibiose, inulin, pinosyl, raffinose, starch, glycogen, xylitol, D-pinobiose, D-lythose, D-rockose, L-rockose, D-arabinitol, L-arabinitol, gluconate, 2-keto-gluconate, and 5-keto-gluconate. Consistent with the biochemical metabolic characteristics of the genus *Bacillus*.

[0078] 2.416S rDNA Molecular Biological Identification

[0079] The 16S rRNA gene sequence was determined using universal primers 27F and 1492R (SEQ ID NO.2: 27f5'-GTTTGATCMTGGCTCAG-3'; SEQ ID NO.3: 1492r5'-TACGGYTACCTTGTTACGACTT-3'), yielding a 1447 bp gene fragment, as shown in SEQ ID NO.1. Comparison with known type bacteria using the EzbioCloud website (http: / / www.ezbiocloud.net / eztaxon / ) showed that the strain exhibited the highest similarity (100%) to the known type strain *Bacillus altitudinis* 41KF2bT.

[0080] SEQ ID NO.1:

[0081]

[0082] 2.5 gyrB gene sequence analysis of strain ICBR-C01

[0083] The conserved gyrB gene was determined using universal primers up-1 and up-2r (SEQ ID NO. 5: UP-1-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA; SEQ ID NO. 6: UP-2r-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT), yielding a 1187 bp gene fragment, as shown in SEQ ID NO. 4. NCBI alignment revealed that strain ICBR-C01 showed the highest similarity (99.01%) to the known model bacterium Bacillus altitudinis 41KF2bT. The strain clustered with Bacillus altitudinis 41KF2bT into the same clade.

[0084]

[0085] In summary, based on morphological and molecular identification, strain ICBR-C01 is identified as Bacillus saltitudinis ICBR-C01.

[0086] 3. Growth curve of Bacillus hygroscopicus ICBR-C01

[0087] Bacillus subtilis ICBR-C01 was inoculated onto TSB solid agar plates and cultured at 37°C for 24 h. Single colonies in good growth condition (i.e., purified single colonies) were picked and inoculated into TSB liquid agar for activation, yielding an activated solution. This activated solution was then inoculated into TSB liquid agar at 1% (v / v) and incubated statically for 24 h. The OD of the bacterial suspension was measured every 1 h. 600 And plot the growth curve of Bacillus hygroscopicus. Figure 2 As shown, Bacillus hygroscopicus ICBR-C01 enters a stable growth phase after 13 hours of culture.

[0088] Example 2: Application of microorganisms in fermented bamboo shoots

[0089] 1. Microbial culture

[0090] The *Bacillus hygroscopicus* ICBR-C01 strain isolated and purified above, as well as *Bacillus amyloliquefaciens* and *Bacillus subtilis* preserved in the laboratory, were cultured separately using the following methods:

[0091] (1) Bacillus hygroscopicus ICBR-C01: Two loops of Bacillus hygroscopicus ICBR-C01 were picked from the TSB solid medium plate obtained during the growth curve determination and inoculated into TSB liquid medium. The volume of TSB liquid medium in a 250mL Erlenmeyer flask was 30mL. The culture was then incubated at 37℃ and 200rpm for 12h with shaking to obtain the seed culture. The seed culture was then inoculated into new TSB liquid medium at an inoculation rate of 10% of the medium volume. The volume of TSB liquid medium in a 250mL Erlenmeyer flask was 30mL. The culture was then incubated at 37℃ and 200rpm for 12h with shaking to obtain the seed fermentation broth ICBR-C01. The viable count of the seed fermentation broth ICBR-C01 was 1×10⁻⁶. 7 CFU / mL.

[0092] (2) Bacillus amyloliquefaciens: Pick two loops of Bacillus amyloliquefaciens from the LB solid medium plate and inoculate them into LB liquid medium. The volume of LB liquid medium in a 250mL Erlenmeyer flask is 30mL. Incubate at 37℃ and shake at 120rpm for 12h to obtain seed culture. Then, inoculate the seed culture into a new LB liquid medium at an inoculation amount of 10% of the volume of the medium. The volume of LB liquid medium in a 250mL Erlenmeyer flask is 30mL. Incubate at 37℃ and shake at 120rpm for 10h to obtain Bacillus amyloliquefaciens seed fermentation broth, abbreviated as Bacillus amyloliquefaciens-seed fermentation broth.

[0093] (3) Bacillus subtilis: Two loops of Bacillus subtilis were picked from the LB solid plate and inoculated into LB liquid medium. The volume of LB liquid medium in a 250mL Erlenmeyer flask was 30mL. The culture was then incubated at 37℃ and 120rpm for 12h with shaking. Then, the seed culture was inoculated into a new LB liquid medium at an inoculation volume of 10% of the culture medium volume. The volume of LB liquid medium in a 250mL Erlenmeyer flask was 30mL. The culture was then incubated at 37℃ and 120rpm for 10h with shaking to obtain Bacillus subtilis seed fermentation broth, abbreviated as Bacillus-seed fermentation broth.

[0094] 2. Fermentation of bamboo shoots

[0095] Experimental Group: Bamboo Shoot Fermentation with Bacillus subtilis ICBR-C01: Bamboo shoots were peeled, washed, chopped, and crushed to obtain bamboo shoot flesh. The bamboo shoot flesh and distilled water were homogenized at a ratio of 1 kg:10 L for 10 min at room temperature, followed by sterilization at 121℃ for 20 min to prepare bamboo shoot homogenate. 8% of the seed fermentation broth ICBR-C01 was inoculated into the bamboo shoot homogenate for fermentation at 37℃ under aerobic conditions. The fermentation was carried out in a temperature-controlled shaker at 200 rpm. After 12 h of fermentation, the fermentation broth was transferred to centrifuge tubes and centrifuged at 6000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter to terminate the reaction, yielding the supernatant ICBR-C01.

[0096] Comparison 1: Fermentation of bamboo shoots with Bacillus amyloliquefaciens B: The method was the same as the experimental group, except that the seed fermentation broth ICBR-C01 was replaced with Bacillus amyloliquefaciens seed fermentation broth, and Bacillus amyloliquefaciens supernatant was obtained.

[0097] Comparison 2: Bacillus subtilis C fermentation of bamboo shoots: The method was the same as the experimental group, except that the seed fermentation broth ICBR-C01 was replaced with Bacillus subtilis-seed fermentation broth to obtain Bacillus subtilis-supernatant.

[0098] Unfermented bamboo shoot group (CK): The method was the same as the experimental group, except that the seed fermentation liquid ICBR-C01 was replaced with distilled water to obtain unfermented supernatant.

[0099] 3. Determination of bioactive components

[0100] (1) Recovery of low molecular weight peptides

[0101] The polypeptides with a molecular weight less than 3 kDa in the supernatant ICBR-C01 were recovered by centrifugal ultrafiltration. The specific method was as follows: 0.5 mL of supernatant ICBR-C01 was placed in a 2 mL ultrafiltration centrifuge tube and centrifuged at 10,000 rpm for 20 min to obtain the ultrafiltration centrifuge filtrate ICBR-C01.

[0102] Polypeptides with a molecular weight less than 3 kDa in the decomposition supernatant were recovered by centrifugation ultrafiltration. The centrifugation ultrafiltration method was the same as described above, and the decomposition ultrafiltration centrifugation filtrate was obtained.

[0103] Polypeptides with a molecular weight less than 3 kDa in the supernatant were recovered by centrifugal ultrafiltration. The centrifugal ultrafiltration method was the same as described above, and the supernatant was obtained by centrifugal ultrafiltration.

[0104] Polypeptides with a molecular weight less than 3 kDa in the unfermented supernatant were recovered by centrifugal ultrafiltration. The centrifugal ultrafiltration method was the same as described above, and the ultrafiltration centrifugal filtrate was obtained as the unfermented supernatant, referred to as the unfermented-ultrafiltration centrifugal filtrate.

[0105] (2) Preparation of peptide standard curve: Take 6 stoppered test tubes and prepare 0.0 g / L (tube 1), 1.0 g / L (tube 2), 2.0 g / L (tube 3), 3.0 g / L (tube 4), 4.0 g / L (tube 5), and 5.0 g / L (tube 6) of Gly-Gly-Tyr-Arg (SEQ ID NO. 7) tetrapeptide standard solution sequentially with distilled water. Then, take 0.3 mL of the standard solution from tubes 1 to 6, add 0.3 mL of biuret reagent A, mix well, and then add 15 μL of biuret reagent B. Measure the OD value of each tube at 540 nm (using tube 1 as a blank control). Plot a standard curve with peptide concentration as the abscissa (x) (g / L) and absorbance as the ordinate (y), y = 0.1012x - 0.0014, R0 2 =0.9973.

[0106] (3) Determination of peptide content: Take 0.3 mL of ultrafiltration centrifuged filtrate ICBR-C01, add 0.3 mL of biuret reagent A, mix well, and then add 15 μL of biuret reagent B. Measure the absorbance value at 540 nm, and calculate the peptide content (g / L) in the ultrafiltration centrifuged filtrate according to the obtained standard curve;

[0107] The method for determining the peptide content in ultrafiltration centrifuged filtrate is the same as that for ultrafiltration centrifuged filtrate ICBR-C01;

[0108] The method for determining the peptide content in the ultrafiltration centrifuge filtrate is the same as that for the ultrafiltration centrifuge filtrate ICBR-C01;

[0109] The method for determining the peptide content of unfermented ultrafiltration centrifuged filtrate is the same as that for ultrafiltration centrifuged filtrate ICBR-C01.

[0110] After determining the polypeptide content in the ultrafiltration centrifuged filtrate, the polypeptide content of the bamboo shoot fermentation broth was calculated according to formula (1).

[0111] The polypeptide content of bamboo shoot fermentation broth = (M1×0.6) / 0.3(1)

[0112] In formula (1), M1 is the polypeptide content obtained from the standard curve equation, in g / L.

[0113] The results are as follows Figure 4 As shown, the polypeptide content of unfermented bamboo shoots was 0.34 g / L; the polypeptide content of bamboo shoot fermentation broth obtained from Bacillus hygroscopicus ICBR-C01 was 1.69 g / L; and the polypeptide content of bamboo shoot fermentation broth obtained from Bacillus amyloliquefaciens and Bacillus subtilis was 0.41 g / L and 0.69 g / L, respectively.

[0114] 4. Determination of antioxidant activity of fermentation broth

[0115] The DPPH free radical scavenging method was used. 0.004 g of DPPH was weighed and dissolved in 100 mL of methanol, and sonicated until completely dissolved to obtain the DPPH working solution.

[0116] (1) Determination of supernatant ICBR-CO1: 100 μL LPPH working solution and 200 μL supernatant ICBR-CO1 from step 2 were mixed evenly and reacted in the dark for 30 min. The absorbance value A1 was measured at a wavelength of 517 nm.

[0117] Mix 100 μL of distilled water and 200 μL of supernatant ICBR-C01 thoroughly, react in the dark for 30 min, and measure the absorbance A2 at a wavelength of 517 nm.

[0118] Mix 100 μL of DPPH working solution and 200 μL of distilled water thoroughly, react in the dark for 30 min, and measure the absorbance A0 at a wavelength of 517 nm.

[0119] (2) Determination of the supernatant: The determination method is the same as step (1), except that the supernatant ICBR-C01 is replaced with the supernatant, and A1 and A2 are determined.

[0120] (3) Determination of supernatant: The determination method is the same as step (1), except that the supernatant ICBR-C01 is replaced with supernatant, and A1 and A2 are determined.

[0121] (4) Determination of unfermented supernatant (i.e. CK): The determination method is the same as step (1), except that the supernatant ICBR-C01 is replaced with unfermented supernatant, and A1 and A2 are determined.

[0122] (5) Determination of Vitamin C (Vc): The determination method is the same as step (1), except that the supernatant ICBR-C01 is replaced with vitamin C at a concentration of 20 mg / L, and A1 and A2 are determined.

[0123] The DPPH scavenging rate in steps (1) to (5) was calculated using the formula SR = [1 - (A1 - A2) / A0] × 100%. Three parallel experiments were set up for each step (1) to (5). The effects of bamboo shoot fermentation broth from the three bacteria on the DPPH free radical scavenging rate are as follows: Figure 5 As shown, the DPPH free radical scavenging rates of the fermentation broths of bamboo shoots from the three fungi were all higher than those of the unfermented group. Among them, the DPPH free radical scavenging rate of CK was 19.07%, the DPPH free radical scavenging rate of the fermentation broth after Bacillus subtilis fermentation was the highest at 40.95%, the DPPH free radical scavenging rate of the fermentation broth after Bacillus amyloliquefaciens fermentation was the highest at 12.25%, the DPPH free radical scavenging rate of the fermentation broth after Bacillus glabra fermentation was the highest at 55.73%, and the DPPH free radical scavenging rate of the positive control Vc was 79.84%.

[0124] In summary, the Bacillus subtilis ICBR-C01 strain provided by this invention is the first strain isolated from the feces of captive giant pandas. This strain ICBR-C01 exhibits excellent fermentation ability in bamboo shoots, capable of converting them into more polypeptide active substances. The ICBR-C01 strain of this invention can secrete proteases, which, through enzymatic hydrolysis, not only increase the polypeptide content of bamboo shoots but also disrupt the cell walls of bamboo shoots, promoting the release and digestion and absorption of nutrients, and enhancing their antioxidant activity. This provides technical support for the development of bamboo shoot fermentation products and has broad application prospects.

[0125] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Highland Bacillus ( Bacillus altitudinis ICBR-C01, accession number CGMCCNo.29900.

2. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes the Bacillus hygroscopicus ICBR-C01 as described in claim 1.

3. The method for preparing the microbial agent according to claim 2, characterized in that, The method includes the following steps: inoculating the Bacillus cereus ICBR-C01 of claim 1 into a culture medium for activation to obtain an activated solution; and inoculating the activated solution into a culture medium for propagation to obtain a bacterial agent.

4. The preparation method according to claim 3, characterized in that, The propagation temperature is 26~40℃, the rotation speed is 150~200rpm, and the time is 6~18h.

5. The application of the Bacillus cereus ICBR-CO1 of claim 1, the inoculum of claim 2, or the inoculum prepared by the preparation method of claim 3 or 4 in one or more of the following 1) to 3): 1) Fermented bamboo shoots; 2) Increase the polypeptide content in bamboo shoot fermentation liquid; 3) Improve the antioxidant activity of bamboo shoot fermentation liquid.

6. A method for preparing fermented bamboo shoots, characterized in that, Includes the following steps: Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 of claim 1, the inoculum of claim 2, or the preparation method of claim 3 or 4.

7. The preparation method according to claim 6, characterized in that, The fermentation temperature is 20~37℃; the time is 10~24h; and the rotation speed is 150~250rpm.

8. The preparation method according to claim 6, characterized in that, Before fermentation, the bamboo shoots are homogenized.

9. The preparation method according to claim 8, characterized in that, During homogenization, the ratio of bamboo shoots to water is 1 kg: (3~10) L.

10. A method for increasing the polypeptide content and / or antioxidant activity of bamboo shoot fermentation broth, characterized in that, Includes the following steps: Bamboo shoots are fermented using the Bacillus subtilis ICBR-C01 of claim 1, the inoculum of claim 2, or the preparation method of claim 3 or 4.

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