Novel microbacterium strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a new strain of microbacterium and application thereof. The bacterial strain is separated from rapana venosa, is a new species of Microbacterium sp. M-28, is registered and preserved in the China Center for Type Culture Collection (CCTCC), and has a preservation number of CCTCC NO: M 20241861. The invention also discloses a method for preparing the bacterial strain. The microbacterium sp. M-28 strain disclosed by the invention has obvious bacteriostatic activity on bacillus subtilis, shewanella, staphylococcus aureus and proteusbacillus vulgaris, has obvious acetylcholin esterase inhibitory activity, has obvious antioxidant activity, has alpha-glucosidase inhibitory activity, can generate casein enzyme and amylase, and can be used for preparing a food additive. The strain can be applied to preparation of a bacteriostatic agent, an acetylcholin esterase inhibitor, an antioxidant, an alpha-glucosidase inhibitor, casein and amylase, can also replace the bacteriostatic agent, the antioxidant, the casein and the amylase in a viable form to be applied to industrial production, and has a very good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a new strain of *Microbacterium* isolated from *Rhodotorula pulveratum*. Microbacterium sp . M-28 and its applications. Background Technology

[0002] Red snail ( Rapana venosa *Rhodotorula pulvinata* belongs to the phylum Mollusca, subclass Prosobranchia, order Neogastropoda, family Nematopoda, and genus *Rhodotorula*. It is a species that grows rapidly, is easy to manage, and has high economic value. In recent years, its aquaculture area has been continuously expanding, making it a new type of marine aquaculture species in northern my country. *Rhodotorula pulvinata* can be eaten fresh, frozen, or processed into dried products or canned goods, and is very popular in domestic and international markets. It also has certain medicinal value; its meat can clear heat and improve eyesight, while its shell can be used raw or calcined to treat stomach ailments, lymph node tuberculosis, and spasms. Furthermore, the symbiotic bacteria of marine animals have various biological functions and can produce antibiotics, anticancer substances, and other bioactive substances, showing broad development prospects in the food and pharmaceutical fields. However, the symbiotic bacteria and metabolites of *Rhodotorula pulvinata* are currently not fully utilized. Summary of the Invention

[0003] This invention aims to overcome the shortcomings of existing technologies and provide a novel strain of microbacteria isolated from *Rhodotorula rubrum*. Microbacterium sp . M-28 strain exhibits significant antibacterial activity against Bacillus subtilis, Shewanella, Staphylococcus aureus, and Proteus vulgaris. It also demonstrates significant cholinesterase inhibitory activity, antioxidant activity, and α-glucosidase inhibitory activity. Furthermore, it produces casein and amylase, making it suitable for the preparation of antibacterial agents, acetylcholinesterase inhibitors, antioxidants, α-glucosidase inhibitors, casein, and amylase. It can also be used in live bacterial form to replace antibacterial agents, antioxidants, casein, and amylase in industrial production, demonstrating promising development and application prospects.

[0004] The first aspect of this invention is to provide a strain of Microbacterium isolated from *Rhodotorula pulveratum*, which is a new species of the genus *Microbacterium*, named as follows: Microbacterium sp . M-28 was deposited at the China Center for Type Culture Collection (CCTCC) on August 27, 2024, with accession number CCTCC NO: M 20241861.

[0005] A second aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in non-therapeutic antibacterial activity, characterized in that the antibacterial activity includes inhibition of Bacillus subtilis, Shewanella, Staphylococcus aureus and Proteus vulgaris.

[0006] A third aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of a drug for the prevention and treatment of diseases caused by Staphylococcus aureus.

[0007] A fourth aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of a medicament for the prevention and treatment of diseases caused by Sivali.

[0008] The fifth aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of acetylcholinesterase inhibitors.

[0009] The sixth aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of antioxidant agents.

[0010] The seventh aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of α-glucosidase inhibitors.

[0011] The eighth aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of casein.

[0012] The ninth aspect of the present invention is to provide the use of the Microbacterium M-28 strain described in the first aspect of the present invention in the preparation of amylase.

[0013] The microbacteria of the present invention Microbacterium sp . M-28 Gram staining was positive, and the cells were rod-shaped. After incubation on TSA medium at 30°C for 24 h, the colonies were pale yellow, round, smooth, moist, slightly convex, and opaque. PCR technology was used to detect the microbes. Microbacterium sp . The 16S rRNA gene sequence of strain M-28 was amplified using universal primers 27F (5'-GTTTGATCMTGGCTC AG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT -3'). Sequencing yielded a 1419 bp gene fragment sequence. Comparison with known type bacteria on the EzbioCloud website (http: / / www.ezbiocloud.net / eztaxon / ) confirmed that the strain... Microbacterium sp . M-28 and known type strains Microbacterium gallinarum Sa1CUA4 T , Microbacterium terricola KV-448 T and Microbacterium gorillaeG3 had the highest similarity, at 98.34%, 98.26%, and 98.19%, respectively.

[0014] right Microbacterium sp . Whole-genome sequencing analysis was performed on strain M-28, and ANI value analysis was conducted using the JSpeciesWS website. Microbacterium sp . The ANI values ​​of M-28 and known similar strains range from 75.02% to 75.75%, all below the threshold of 95%, indicating that the strain... Microbacterium sp . M-28 is a new species. DNA-DNA hybridization (DDH) analysis. Microbacterium sp . The DDH values ​​of strain M-28 compared with known similar strains ranged from 19.9% ​​to 21.1%, all below the 70% threshold, further indicating that... Microbacterium sp . M-28 is a new species with a genome size of 3,120,384 bp and a G+C content of 70.7%. Biological Preservation

[0015] This invention provides a new species of microbacterium, named as follows: Microbacterium sp . M-28 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241861, on August 27, 2024. The deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. This invention relates to the microbacterium... Microbacterium sp . M-28 was obtained by separating and screening *Rhododendron molle* from the nearshore waters of Changhai County, Dalian City, Liaoning Province, China. Attached Figure Description

[0016] Figure 1 yes Microbacterium sp . Colony morphology of M-28 cells cultured in TSA medium at 30°C for 24 h.

[0017] Figure 2 yes Microbacterium sp . M-28 Gram staining diagram.

[0018] Figure 3 yes Microbacterium sp . M-28 transmission electron microscope image.

[0019] Figure 4 It is based on the 16S rRNA gene sequence. Microbacterium sp . Phylogenetic tree diagram of M-28 and major type strains of Microbacterium.

[0020] Figure 5 It is based on genome sequence. Microbacterium sp . M-28 phylogenetic tree diagram.

[0021] Figure 6 yes Microbacterium sp . Image of casein activity of strain M-28.

[0022] Figure 7 yes Microbacterium sp . Image of amylase activity of strain M-28. Detailed Implementation

[0023] This invention provides a new species of microbacterium, named as follows: Microbacterium sp . M-28 was deposited at the China Center for Type Culture Collection (CCTCC) on August 27, 2024, with accession number CCTCC NO: M 20241861. The deposit address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. This invention relates to the microbacterium... Microbacterium sp . M-28 was isolated and screened from the nearshore waters of Changhai County, Dalian City, Liaoning Province, China.

[0024] The present invention will be further described below with reference to specific embodiments to better understand the present invention, but these embodiments should not be construed as limiting the scope of protection of the present invention.

[0025] 1. 16S rRNA gene analysis and morphological observation of the strain 1.1 Source of strains Samples of *Rhodotorula rubra* were collected from the nearshore waters of Changhai County, Dalian City, Liaoning Province, China. The collected *Rhodotorula rubra* were kept alive at low temperature and transported to the laboratory for immediate processing. Under aseptic conditions, the samples were minced and ground into a homogenate, which was then serially diluted to 10⁻⁶ with sterile physiological saline. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Seven dilutions; take 10 -3 ~10 -7Each dilution of 100 μL was spread evenly on tryptic soy agar (TSA) using the plate spread method. Three parallel petri dishes were prepared for each concentration. The dishes were incubated upside down in a 20°C incubator for 1-5 days. Single colonies were picked, streaked to isolate pure cultures, and numbered.

[0026] Preparation method of tryptic soy agar (TSA): 15.0 g tryptic, 5.0 g soybean papain hydrolysate, 5.0 g sodium chloride, 15.0 g agar, pH 7.3±0.2, 1000 mL water. Autoclave at 121℃ for 20 minutes, cool to approximately 50℃ and pour into plates.

[0027] 1.2. 16S rRNA gene analysis of the strain The 16S rRNA gene sequence of the purified strains was determined using universal primers 27F and 1492R (27F 5'-GTTTGATCMTGGCTC AG-3'; 1492R 5'-TACGGYTACCTTGTTACGACTT -3'). A 1419 bp gene fragment was obtained from strain M-28. Comparison with known type bacteria using the EzbioCloud website (http: / / www.ezbiocloud.net / eztaxon / ) confirmed that strain M-28 is comparable to known type strains. Microbacterium gallinarum Sa1CUA4 T The highest similarity was 98.34% (similarities to other model bacteria are shown in Table 1). The comparison results indicate that strain M-28 belongs to the genus Microbe, and it was named... Microbacterium sp. M-28, and constructed a phylogenetic tree of strain M-28 and related taxa based on 16S rRNA gene sequences (see instruction manual). Figure 4 ).

[0028] Table 1 Microbacterium Comparative analysis of the 16S rRNA gene sequence of sp. M-28

[0029] 1.3 Strains Microbacterium Morphological observation of sp. M-28 Separate and purify Microbacterium sp . When M-28 is inoculated onto TSA medium and incubated at 30°C for 24 hours, the resulting colonies are pale yellow, round, smooth, moist, slightly convex, and opaque (see instruction manual). Figure 1 Under an optical microscope, the bacterium showed Gram-positive staining and the cells were rod-shaped (see instruction manual). Figure 2Under an electron microscope, the bacterium appears as a rod-shaped bacterium, approximately 6.0–7.5 × 18.0–24.0 μm in size, without spores, flagella, or pili (see instruction manual). Figure 3 The above morphological and Gram staining characteristics are consistent with... Microbacterium The morphological characteristics of the genera are similar.

[0030] 2 strains Microbacterium sp. M-28 genomic analysis Microbacterium sp. The genome sequencing analysis of M-28 was performed by the Agricultural Microbiology Center of the China Culture Collection Committee. A5-MiSeq and SPAdes were used to assemble the sequencing data (with adapter sequences removed) de novo, constructing contigs and scaffolds. The assembled results were evaluated and compared, and base correction was performed using Pilon software. Prokka annotation of the genome draft sequence was performed using PGCGAP software.

[0031] 2.1 Strains Microbacterium sp. Basic characteristics of the M-28 genome After annotation, the strain was obtained. Microbacterium sp. The M-28 genome is 3,120,384 bp in size and contains 70.7% G+C. Other relevant information is shown in Table 2. Genome characteristics and Microbacterium The other species in the genus are basically the same.

[0032] Table 2 Strains Microbacterium sp.M-28 genome-wide information

[0033] 2.2 Strains Microbacterium sp. M-28 genome ANI value analysis Using the JSpeciesWS website (http: / / jspecies.ribohost.com / jspeciesws / #home) to analyze the ANI values ​​of the strains, the strains can be determined. Microbacterium sp. The ANI values ​​of M-28 and known closely related strains are all below 95%, as detailed in Table 3. Generally, the ANI value among strains of the same species is ≥95%. Therefore, it can be concluded that strain M-28... Microbacterium sp. M-28 is a new species.

[0034] Table 3 Strains MicrobacteriumAnalysis of ANI values ​​(%) of sp. M-28 and known similar strains

[0035] 2.3 Strains Microbacterium sp. M-28 genome DDH value analysis Analysis using GGDC (Genome-to-Genome Distance Calculator 2.1, http: / / ggdc.dsmz.de / ggdc.php#) revealed that the strain Microbacterium sp. The DDH (Dual Degree of DNA) of M-28 was less than 70% compared to the genome sequences of known closely related strains (Table 4). Typically, the DDH (DNA-DNA hybridization) value is ≥70% among strains of the same species. Therefore, this further confirms the presence of the strain. Microbacterium sp. M-28 is a new species.

[0036] Table 4 Strains Microbacterium DDH value analysis (%) of sp. M-28 and known similar strains

[0037] 2.4 Strains Microbacterium sp. M-28 Genome-based Phylogenetic Tree Build Microbacterium sp. Phylogenetic tree of strain M-28 and its closely related species (attached to instruction manual) Figure 5 ), Microbacterium sp. M-28 clustered individually on the same branch and was clustered close to the model bacteria, which also indicates that it is a new species.

[0038] 3. Physiological and biochemical characteristics analysis 3.1 Strains ​ sp. pH growth assay of M-28 Prepare TSA liquid culture media with different pH gradients of 4, 5, 6, 7, 8, 9, and 10, and dispense 5 mL portions into test tubes for later use. [The text then abruptly shifts to a seemingly unrelated topic:] ...the bacterial strains... ​ sp. M-28 was inoculated at a 1% inoculum into TSA liquid medium at pH 4, 5, 6, 7, 8, 9, and 10, with three replicates for each gradient. The culture was carried out with shaking at 180 rpm and 30°C for 24–48 h. 100 μL of each tested strain was aspirated into a 96-well plate, and OD was measured. 600Value and analyze, if OD 600 A value greater than 0.1 indicates that the strain can grow. Results of the strain... ​ sp. M-28 can grow at pH 6-10, but not at pH 4 and pH 5. The optimal growth pH is 6 and 7.

[0039] 3.2 Strains ​ sp. NaCl growth assay of M-28 Prepare LB liquid culture medium containing different concentrations of NaCl (0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12%), and dispense 5 mL into test tubes for later use. [The text then abruptly shifts to a seemingly unrelated topic:] ...the bacterial strains... ​ sp. M-28 was inoculated at a 1% inoculum into LB liquid medium containing NaCl concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12%, with three replicates for each concentration. The culture was carried out with shaking at 30°C and 180 rpm for 24–48 h. 100 μL of each tested strain was aspirated into a 96-well plate, and the OD was measured. 600 Value and analyze, if OD 600 A value greater than 0.1 indicates that the strain can grow. Results of the strain... ​ sp. M-28 can grow in NaCl concentrations of 0-9%, but does not grow in concentrations of 10-12%, with the optimal NaCl concentration for growth being 0-5%.

[0040] 3.3 ​ sp. M-28 Temperature Growth Measurement strain ​ sp. M-28 was transferred to TSA medium using an inoculation loop and cultured at different temperatures (4℃, 20℃, 30℃, 37℃, 41℃, 45℃) for 24-48 h, and its growth was observed. Results of the strain... ​ sp. M-28 can grow at temperatures of 20℃, 30℃, and 37℃, but cannot grow at 4℃, 41℃, and 45℃. The optimal growth temperature is 30℃.

[0041] 3.4 ​ sp. M-28 fatty acid composition analysis Detection of strains using the Microbial Fatty Acid Rapid Identification System (MIDI) ​ sp. The whole-cell fatty acid composition of M-28, with C as the main fatty acid.15:0 anteiso, C 15:0 iso, C 17:0 anteiso and C 16:0 ISO content was 49.48%, 20.65%, 15.87%, and 8.99%, respectively. The specific fatty acid results are shown in Table 5, which are consistent with... ​ The main cellular fatty acid characteristics of the genus.

[0042] Table 5 Strains ​ fatty acids of sp.M-28

[0043] 3.5 ​ sp. API 50CH Reaction Analysis of M-28 API 50CH test results: Positive reactions included fructose, mannose, mannitol, aesculin, and maltose; weakly positive reactions included glycerol, D-xylose, glucose, sucrose, trehalose, geraniol, D-minobiose, and gluconate; negative reactions included control, erythrose, D-arabinose, L-arabinose, ribose, L-xylose, azirone, β-methyl-D-xyloside, galactose, sorbitol, rhamnose, eugenol, and inositol. Sorbitol, α-methyl-D-mannoside, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicylic acid, cellobiose, lactose, melibiose, inulin, pinosyl arabinose, raffinose, starch, glycogen, xylitol, D-lysose, D-tagatose, D-rockose, L-rockose, D-arabinitol, L-arabinitol, 2-keto-gluconate and 5-keto-gluconate, conforming to ​ Biochemical metabolic characteristics of the genus.

[0044] 3.6 Identification by API ZYM biochemical reaction Use API ZYM test strips (follow the instructions for use) to determine the strains. ​ sp. M-28 ​ KV-448 T (JCM 14903) Microbacterium gorilla G3 T The enzyme activity of (DSM26203) was measured. The results of the measurements for the three are shown in Table 6. Microbacterium sp. The enzyme reaction characteristics of M-28 are consistent with Microbacterium It exhibits genus-like characteristics, differs from similar type strains, and is consistent with the results of genomic analysis.

[0045] Table 6 Strains Microbacterium Comparison of reaction results between sp. M-28 and its closely related microbes ZYM

[0046] Note: +, positive; -, negative; w, weak positive. Based on the above analysis of 16S rRNA sequencing and phylogenetic analysis, whole genome analysis, colony morphology, and physiological and biochemical characteristics, the strain... Microbacterium sp. M-28 was identified as Microbacterium A new species of the genus has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241861.

[0047] 4. Strains Microbacterium sp. M-28's biological activity 4.1 Antibacterial activity Preparation of beef extract peptone culture medium: Weigh 3 g of beef extract, 10 g of peptone, 5 g of NaCl, and 17 g of agar powder, pour them into a ceramic jar, add 1000 mL of distilled water, heat with an induction cooker and stir constantly with a glass rod until boiling, dispense into Erlenmeyer flasks, stopper and seal, sterilize at 121℃ for 20 min, and set aside for later use.

[0048] strain Microbacterium sp. Preparation of M-28 fermentation broth: Under aseptic conditions, pick up the broth with an inoculation loop. Microbacterium sp. Single colonies of strain M-28 were inoculated into 10 mL of beef extract peptone medium, citrate medium or Czapek's medium, and cultured in a shaker at 30°C at 150 r / min for 2-6 days. After centrifugation at 5000 rpm for 10 min, the supernatant was collected for later use.

[0049] Preparation of indicator bacteria fermentation broth: Under aseptic conditions, Bacillus subtilis (Bacillus subtilis) was picked up separately using an inoculation loop. Bacillus subtilis ATCC 11562), Shewanella ( Shewanella marisflavi MCCC 1B00304), Proteus vulgaris ( Proteus vulgaris ATCC 13315) and Staphylococcus aureus ( Staphylococcus aureus ATCC 6538 colonies were inoculated into 10 mL of beef extract peptone liquid medium and incubated in a shaker at 37°C at 150 r / min for 24–48 h for later use.

[0050] Oxford cup method determination Microbacterium sp. M-28 Antibacterial Activity: Under aseptic conditions, approximately 10 mL of sterile beef extract peptone agar medium was poured into sterile 90 mm petri dishes, and Oxford cups were placed on top after solidification. 500 μL of indicator bacterial fermentation broth (Bacillus subtilis, Shewanella, Proteus vulgaris, and Staphylococcus aureus) was pipetted into each dish and mixed with 100 mL of sterile beef extract peptone agar medium at approximately 55°C. This mixture was then poured into petri dishes containing Oxford cups, approximately 15 mL per dish. The mixture was allowed to solidify completely before removing the Oxford cups and pipetting the contents into each dish. Microbacterium sp. 20 μL each of fermentation broth of strain M-28, 75% ethanol, and uninoculated blank liquid culture medium were added to different wells. Three replicates were performed for each indicator bacterium. The culture dishes were placed in a 4°C refrigerator overnight, followed by incubation at 37°C for 1-2 days. The size of the inhibition zone was measured to determine the antibacterial activity. Results of the strain Microbacterium sp. M-28 fermentation broth showed significant inhibitory effects on four indicator bacteria (Table 7), and can be used to prepare therapeutic or non-therapeutic antibacterial agents to prevent and treat various diseases caused by bacteria or for preservation.

[0051] Table 7 Strains Microbacterium Inhibitory effect of sp. M-28 on four indicator bacteria

[0052] 4.2 Antioxidant activity 4.2.1 Determination of hydroxyl radical scavenging ability by salicylic acid method Principle: Salicylic acid reacts with hydroxyl radicals (·OH) to form 2,3-dihydroxybenzoic acid, which has a maximum absorption wavelength at 510 nm. By utilizing the reaction of hydrogen peroxide (H₂O₂) with ferrous sulfate heptahydrate (FeSO₄·7H₂O) to generate ·OH, and adding a substance with free radical scavenging ability, the formation of 2,3-dihydroxybenzoic acid can be inhibited, thus reducing the absorbance at 510 nm.

[0053] Specific steps: A1: Take respectively Microbacterium sp. Supernatant of fermentation broth from strain M-28 and 10 mg / mL V C Add 0.5 mL of 9 mmol / L FeSO4 solution, 0.5 mL of 9 mmol / L salicylic acid-ethanol solution, and 0.5 mL of 8.8 mmol / L H2O2 solution to each of the 0.5 mL solutions. Incubate in a water bath at 37 °C for 30 min. After shaking well, measure the absorbance at a wavelength of 510 nm. Perform three replicates for each sample.

[0054] A2: Take respectively Microbacterium sp. Supernatant of fermentation broth from strain M-28 and 10 mg / mL V C Add 0.5 mL of 9 mmol / L FeSO4 solution, 0.5 mL of 9 mmol / L salicylic acid-ethanol solution, and 0.5 mL of distilled water to each sample. Incubate in a water bath at 37℃ for 30 min. After shaking well, measure the absorbance at a wavelength of 510 nm. Perform three replicates for each sample.

[0055] A0: Take 0.5 mL of distilled water and add 0.5 mL of 9 mmol / L FeSO4 solution, 0.5 mL of 9 mmol / L salicylic acid-ethanol solution and 0.5 mL of 8.8 mmol / L H2O2 solution. React in a water bath at 37℃ for 30 min, shake well and measure the absorbance value at a wavelength of 510 nm. Perform three replicates for each sample.

[0056] OH removal rate calculation formula: ·OH removal rate = [(A0 - (A1 - A2)) / A0] × 100% result Microbacterium The supernatant clearance rate of sp. M-28 in Gao's medium was 93.64%, only slightly lower than that of the positive control at 10 mg / mL. C The solution clearance rate is slightly low at 99.7%, but it has a very good ability to remove ∙OH.

[0057] 4.2.2 Ability to reduce iron oxide Reducing agents can reduce the Fe in ferricyanide compounds. 3+ Reduced to Fe 2+ Fe 2+ During this process, there will be color changes. The absorbance value measured by a spectrophotometer at a wavelength of 700 nm is used to determine the level of the sample's original ability. The reducing ability is directly proportional to the absorbance value.

[0058] Specific experimental methods: Take respectively Microbacterium Supernatant of fermentation broth from sp. M-28 strain and 10 mg / mL V C Add 0.2 mL of the solution to 0.5 mL of PBS buffer (0.2 mol / L, pH=6.6) and 0.2 mL of 1% potassium ferricyanide solution, react in a water bath at 50℃ for 20 min, then add 1 mL of 10% trichloroacetic acid (TCA) solution and centrifuge at 3000 rpm for 10 min.

[0059] A1: Take 0.5 mL of the supernatant after centrifugation, add 0.5 mL of distilled water and 0.1 mL of 0.1% ferric chloride solution, shake well and let stand for 10 min, and measure the absorbance value at a wavelength of 700 nm. Perform three parallel tests for each sample.

[0060] A2: Take 0.5 mL of the supernatant after centrifugation, add 0.6 mL of distilled water, shake well and let stand for 10 min, and measure the absorbance value at a wavelength of 700 nm. Perform three parallel sets for each sample.

[0061] Calculation formula: Reducing power = A1 - A2 Calculated Microbacterium The reducing power of the supernatant from the fermentation broth of strain M-28 was 89%, slightly lower than that of V at 10 mg / mL. C The reducing power of the solution is 91.2%.

[0062] In conclusion, Microbacterium sp. M-28 exhibits significant antioxidant activity and can be used to prepare antioxidants.

[0063] 4.3 α-Glucosidase Inhibitory Activity The principle of determining α-glucosidase inhibitory activity by the PNPG (4-nitrophenyl-α-D-glucopyranoside) method is as follows: At 37℃, PNPG decomposes under the action of α-glucosidase to produce a colored substance PNP. PNP needs to be further developed in an alkaline environment. Then, the absorbance value of the reaction solution is detected at 405 nm using a UV-Vis spectrophotometer, and the bacterial inhibition rate is calculated based on the absorbance value.

[0064] Specific methods: The strains were prepared according to method 4.1. Microbacterium The supernatant from the M-28 fermentation was used as a positive control, with 0.2 mg / mL acarbose aqueous solution as the positive control blank. Sterile water was used as the positive control blank. Sterile beef extract peptone liquid medium was used as the negative control. The reaction systems for each group were prepared as shown in Table 8. Three parallel experiments were set up and the average value was taken.

[0065] Table 8 Reaction system for α-glucosidase inhibitory activity assay

[0066] Calculation formula: α-glucosidase inhibition rate = (A 对照 -A 样品 ) / A 对照 ×100% In the formula A 对照 As a negative control (blank fermentation broth) or a positive blank control (deionized water), A 样品 Acarbose was used as a bacterial culture or positive control. The strains were calculated... Microbacterium The supernatant of sp. M-28 fermentation showed an inhibition rate of 30%, which can be used to prepare α-glucosidase inhibitors.

[0067] 4.4 Casein activity Preparation of skim milk culture medium: Weigh 40 mL of skim milk, 7.2 g of agar powder, and 400 mL of distilled water. No pH adjustment is needed (the pH can be adjusted according to the amount used each time). Pour the mixture into a ceramic jar, heat it with an induction cooker, and stir constantly with a glass rod until it boils. Dispense the mixture into Erlenmeyer flasks, stopper them, and sterilize them at 121℃ for 20 min.

[0068] Assay procedure: Pour 20 mL of sterile skim milk culture medium into a sterile petri dish, let it stand for a period of time until the culture medium in the petri dish is completely solidified, and then use an inoculation loop to pick up the bacterial strain. Microbacterium Single colonies of sp. M-28 were inoculated onto the surface of skim milk culture medium, and the petri dishes were inverted and incubated at 30°C for 3 days. The activity of casein was determined by measuring the clear zone formed around the colony, and the data was recorded by taking a picture.

[0069] exist Microbacterium sp. M-28 colonies form a clear band approximately 2 mm wide, exhibiting significant casein activity, and can be used to prepare casein.

[0070] 4.5 Amylase activity Starch medium: Weigh 10 g peptone, 5 g NaCl, 5 g beef extract, 2 g soluble starch, 17 g agar powder, and 1000 mL distilled water. No pH adjustment is needed (adjustment can be made according to the amount used each time). Pour the mixture into a ceramic jar, heat it with an induction cooker and stir constantly with a glass rod to ensure the medium is heated evenly until it becomes transparent. Dispense the medium into Erlenmeyer flasks of the corresponding capacity.

[0071] Divide each petri dish into two sections using a marker, pour in 20 mL of sterilized starch culture medium, and let stand for a period of time until the medium in the petri dish has completely solidified. Then, use an inoculation loop to pick up the bacterial strain. Microbacterium Single colonies of sp. M-28 were inoculated into two zones on the surface of starch medium. The inverted petri dishes were incubated at 37°C for 3 days. Iodine solution was then added dropwise to the surface of the starch medium, covering the entire surface. The diameter of the clear zone around each colony was measured to determine amylase activity. Results Microbacterium The colonies of sp. M-28 form a 7 mm clear zone and exhibit very strong amylase activity, making them suitable for the preparation of amylase.

[0072] 4.6 Acetylcholinesterase inhibitory activity The strains were prepared according to method 4.1. Microbacterium sp. M-28 fermentation supernatant.

[0073] In the in vitro AChE inhibitory activity assay, iodinated thioacetylcholine solution (ATCI, 5 mg / mL), 5,5-dimercapto-2,2-dinitrobenzic acid solution (DTNB, 0.3 mg / mL), and AChE solution (1 U / mL) were prepared using phosphate-buffered saline (PBS) (0.1 mol / L, pH 8.0). All solutions were refrigerated and prepared fresh before use.

[0074] 120 μL of PBS solution, 20 μL of sample solutions of different concentrations, and 15 μL of AChE solution were added sequentially to a 96-well plate and incubated at 37°C for 20 min. Then, 20 μL of ATCI solution and 70 μL of DTNB solution were added, and the mixture was incubated at 37°C for another 20 min. The absorbance of the reaction mixture at 412 nm was measured using a microplate reader (B1). The sample control group (B2) used an equal volume of PBS solution instead of AChE solution. The negative control group (A1) used an equal volume of blank culture medium solution instead of sample solution, and the negative control group (A2) used an equal volume of blank culture medium solution instead of sample solution and an equal volume of PBS solution instead of AChE solution. Gatacrine was used as a positive control. The inhibition rate was calculated using the following formula:

[0075] The test results showed that the positive control tacrine inhibited acetylcholinesterase by 85.32%. Microbacterium The supernatant of sp. M-28 fermentation showed an inhibition rate of 35.4% against acetylcholinesterase, indicating its potential application in the preparation of acetylcholinesterase inhibitors.

[0076] The specific embodiments of the present invention have been described in detail above, but they are only examples of implementation of the present invention. The present invention is not limited to the specific embodiments described above. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A new strain of Microbacterium, isolated from *Rhodotorula pulveratum*, is named: Microbacterium sp . M-28 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241861.

2. The application of the Microbacterium M-28 strain as described in claim 1 in non-therapeutic bacteriostasis.

3. The application as described in claim 2, characterized in that, The antibacterial activity includes inhibition of Bacillus subtilis, Shewanella, Staphylococcus aureus, and Proteus vulgaris.

4. The use of the Microbacterium M-28 strain as described in claim 1 in the preparation of drugs for the prevention and treatment of diseases caused by Staphylococcus aureus.

5. The use of the Microbacterium M-28 strain as described in claim 1 in the preparation of drugs for the prevention and treatment of diseases caused by Shewanella.

6. The application of the Microbacterium M-28 strain as described in claim 1 in the preparation of preservatives that inhibit Staphylococcus aureus, Bacillus subtilis, Shewanella and Proteus vulgaris.

7. The use of the Microbacterium M-28 strain according to claim 1 in the preparation of acetylcholinesterase inhibitors.

8. The use of the Microbacterium M-28 strain according to claim 1 in the preparation of antioxidant agents.

9. The use of the Microbacterium M-28 strain according to claim 1 in the preparation of α-glucosidase inhibitors.

10. The use of the Microbacterium M-28 strain according to claim 1 in the preparation of casein.

11. The use of the Microbacterium M-28 strain according to claim 1 in the preparation of amylase.

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