Alcobacter PT7-4 and application thereof

Abstract

The invention belongs to the field of microorganisms, and provides an algibacter sp PT7-4 and application, the algibacter sp PT7-4 is preserved in Guangdong Province Microbial Culture Collection Center located in No.59 building of No.100 Courtyard, Xianlie Middle Road, Guangzhou City, Guangdong Province on July 18, 2025, and the preservation number is GDMCC NO: 66710. The phycobacterium PT7-4 has glucoside hydrolase capable of cutting off glucosidic bonds of ulva, sulfation and structural characteristics of a main chain of the ulva can be reserved, the ulva can be mildly and efficiently degraded into ulva oligosaccharide with biological activity, and the obtained ulva oligosaccharide is high in scavenging capacity for DPPH free radicals or superoxide free radicals and can be used for preparing the ulva oligosaccharide with biological activity. Good medicinal prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and specifically provides an Algibacter ulvanivorans PT7-4 and application thereof. BACKGROUND

[0002] Ulva is a kind of green algae with extremely strong reproductive capacity. Ulvan has multiple biological activities such as antioxidant, antitumor and immunomodulatory activity. However, the molecular weight of ulvan affects its biological activity, and the greater the molecular weight of ulvan, the more difficult it is to exert its biological activity. At present, the degradation of ulvan is mainly chemical-physical combined degradation, but this method can generate a large amount of hydroxyl radicals, which non-selectively attack the sugar ring, resulting in the loss of active groups and reducing the antioxidant activity. In the microbial enzymatic method, Pseudoalteromonas is mainly used as the research object, and the research on other bacteria is less. Therefore, it is urgent to mine ulvan-degrading bacteria. In addition, the degraded ulvan oligosaccharide can retain the multiple biological activities of ulvan, effectively solving the problems of low solubility and poor bioavailability. SUMMARY

[0003] Algibacter is the main branch of Flavobacteriaceae in marine environment, is a heterotrophic bacteria targeting complex organic matter, and is specialized in degrading organic polymers. Algibacter can break the glycosidic bond of seaweed polysaccharide by degrading enzyme to decompose complex organic matter, so as to maximize the degradation of seaweed polysaccharide with complex structure.

[0004] In one aspect, the present application provides an Algibacter ulvanivorans PT7-4, which was preserved in Guangdong Microbial Culture Collection Center, located at No. 59, Building 100, Martyrs' Park, Guangzhou, Guangdong Province, China on July 18, 2025, and the preservation number is GDMCC NO: 66710.

[0005] In another aspect, the present application provides a microbial agent, which comprises the above-mentioned Algibacter PT7-4.

[0006] In another aspect, the present application provides a crude enzyme solution produced by the above-mentioned Algibacter PT7-4.

[0007] Further, the preparation method of the crude enzyme solution comprises: inoculating the Algibacter PT7-4 into a liquid with ulvan as the only carbon source; culturing for 8 hours; centrifuging the culture medium to obtain supernatant, filtering impurities, and concentrating with an ultrafiltration tube with a molecular weight of 10 kDa.

[0008] In another aspect, the present application provides the application of the above-mentioned Algibacter PT7-4 or crude enzyme solution in degrading ulvan.

[0009] On the other hand, this application provides ulva oligosaccharides, which are obtained by degrading ulva polysaccharides with the above-mentioned Bacillus algae PT7-4 or crude enzyme solution.

[0010] Further, the preparation method of the Ulva polysaccharide includes: pulverizing dried Ulva powder, sieving it, soaking it in 75% ethanol overnight to remove pigments, and filtering it; drying the filtered powder, adding it to distilled water at a solid-liquid mass ratio of 1:50, and extracting it at 90°C for 4 hours; filtering to remove residues, and collecting the supernatant by centrifugation; concentrating the supernatant to 3 / 4 volume, then adding 3 times the volume of 95% ethanol after concentration, and precipitating it at 4°C; after precipitation, centrifuging to remove the supernatant, washing and drying the precipitate to obtain Ulva polysaccharide.

[0011] Furthermore, the preparation method of the Ulva oligosaccharide includes:

[0012] (1) The Bacillus algae PT7-4 was inoculated into a liquid culture medium with Ulva polysaccharide as the sole carbon source; cultured for 8 hours; the culture medium was centrifuged to collect the supernatant, filtered to remove impurities, and concentrated to one-third volume by ultrafiltration using an ultrafiltration tube with a molecular weight of 10 kDa to obtain crude enzyme solution.

[0013] (2) Take crude enzyme solution and ursolic acid in a ratio of 1:2 mL:g, add water to prepare reaction mixture, the concentration of ursolic acid in reaction mixture is 5mg / ml;

[0014] (3) After the reaction mixture is reacted at 40℃ for 5 hours, the temperature is raised to 100℃ and the mixture is inactivated for 5 minutes.

[0015] (4) Place the inactivated mixture into a centrifuge, centrifuge at 12000 rpm / min for 3 min, and collect the supernatant to obtain Ulva oligosaccharide.

[0016] On the other hand, this application provides the application of the above-mentioned Ulva oligosaccharide in the preparation of health products with auxiliary antioxidant effects.

[0017] On the other hand, this application provides the use of the above-mentioned Ulva oligosaccharides in the preparation of drugs with antioxidant effects.

[0018] In existing technologies, the control of Ulva protosan often utilizes *Pseudomonas aeruginosa* to degrade it. However, this invention uniquely isolates a strain of *Bacillus algae*, PT7-4, capable of efficiently degrading Ulva protosan from the surface of *Ulva*. *Bacillus algae* PT7-4 possesses glycosidic hydrolases capable of cleaving the glycosidic bonds of Ulva protosan, preserving the sulfation and structural characteristics of the protosan backbone, thus gently and efficiently degrading Ulva protosan into bioactive Ulva oligosaccharides. The Ulva oligosaccharides obtained by degradation by strain PT7-4 exhibit strong scavenging ability against DPPH and superoxide radicals, indicating that the Ulva oligosaccharides obtained by degradation by strain PT7-4 possess strong antioxidant bioactivity. Attached Figure Description

[0019] Figure 1 Transmission electron micrograph of Algibacter sp. PT7-4;

[0020] Figure 2 Phylogenetic tree of Algibacter sp. PT7-4;

[0021] Figure 3 The growth curve of Algibacter sp. PT7-4 in Ulva polysaccharide-only carbon source medium;

[0022] Figure 4 The change in the degree of oligosaccharide polymerization of Algibacter sp. PT7-4;

[0023] Figure 5 This is a graph showing the composition of monosaccharides in uranium.

[0024] Figure 6 The molecular weight peak diagram of uranyl peroxide is shown.

[0025] Figure 7 FACE degree of polymerization analysis of the enzymatic hydrolysis products of Ulva polysaccharide from concentrated crude enzyme solution of Algibacter sp. PT7-4;

[0026] Figure 8 The study demonstrated the DPPH free radical scavenging activity and superoxide anion free radical scavenging activity of Ulva oligosaccharides. Detailed Implementation

[0027] Example 1: Obtaining strain PT7-4

[0028] The green algae *Ulva fasciata* obtained from nearshore waters was used as a sample. The algae were rinsed with sterile saline to remove loosely attached bacteria. Bacteria were scraped from the algae surface, and the resulting bacterial suspension was serially diluted and plated on marine agar 2216E plates. After incubation at 25°C for 3 days, single colonies were picked and subjected to two consecutive streak purification processes to finally obtain strain PT7-4.

[0029] After culturing Bacillus PT7-4 on 2216E solid medium for 3 days, single colonies were picked and observed using transmission electron microscopy. Figure 1 As shown, the colonies on the plate are round and yellow, appearing as long rods under a microscope. They are non-flagellated. PT7-4 cells are 1.5-3.1 × 0.3-0.7 μm in size. Single colonies were picked and subjected to colony PCR amplification.

[0030] Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1);

[0031] Downstream primer 1492R: 5'-GGTTACCTTGTTACGACT-3' (SEQ ID NO.2);

[0032] The PCR reaction conditions were: 94℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for 30 cycles; 72℃ for 10 min.

[0033] The obtained 16S rRNA gene sequence was compared with highly similar sequences in the EzBioCloud database, and a phylogenetic tree was constructed. The results are as follows: Figure 2 As shown. PT7-4 was identified as *Algibacter sp.*, which was deposited on July 18, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCCNO: 66710). The address of the depository is Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0034] 16S rRNA gene sequence (SEQ ID NO.3):

[0035] TAGGCAAATCGGCAGCTACACATGCAGTCGAGGGGTAACATAGAGAAGCTTGCTTTTCTGATGACGACC

[0036] GGCGCACGGGTGCGTAACGCGTATAGAATCTGCCTTTTGCTGAGGAATAGCCCAGAGAAATTTGGATTA

[0037] ATGCCTCATAGTATACAGATTTCACATGAATTTTGTATTAAAGGTTACGGCAAAAGATGACTATGCGTC

[0038] CTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCGACGATAGGTAGGGGCCCTGAGAGGGGGATCC

[0039] CCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGACAATGGA

[0040] GGCAACTCTGATCCAGCCATGCCGCGTGCAGGAAGACTGCCCTATGGGTTGTAAACTGCTTTTATACAG

[0041] GAAAAACAGTTCTACGTGTAGAGCCTTGACGGTACTGTAAGAATAAGGATCGGCTAACTCCGTGCCAG

[0042] CAGCCGCGGTAATACGGAGGATCCAAGCGTTATCCGGAATCATTGGGTTTAAAGGGTCCGTAGGTGGAT

[0043] AATTAAGTCAGAGGTGAAAGTTTGCGGCTCAACCGTAAATGCCTTTGATACTGGTTATCTTGAATCA

[0044] TTATGAAGTAGTTAGAATGTAGTGTAGCGGTGAAATGCATAGATATTACATAGAATACCAATTGCGA

[0045] AGGCAGATTACTAATAATGTATTGACACTGATGGACGAAAGCGTAGGTAGCGAACGGGATTAGATACCC

[0046] CGGTAGTCTACGCCGTAAACGATGGATACTAGCTGTTGGGTTTCGGCTCAGTGGCTAAGCCGAAAGTGAT

[0047] AAGTATCCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC

[0048] GGTGGAGCATGTGGTTTAATTCGATCGCGAGGAACCTTACCAGGGCTTAAATGTAGATTGACAGG

[0049] TTTAGAGATAGACTTTTCTTCGGACAATTTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAG

[0050] GTGTCAGGTTAAGTCCTATAACGAGCGCAACCCCTGTTGTTAGTTGCCAGCATGTAAAGATGGGAACTC

[0051] TAACAAGACTGCCGGTGCAAACCGTGAGGAAGGTGGGGATGACGTCAAATCATCACGGCCCTTACGTCC

[0052] TGGGCTACACACGTGCTACAATGGTAGGGACAGAGAGCAGCCACTTCGCGAGAAGGAGCGAATCTATAA

[0053] ACCCTATCACAGTTCGGATCGGAGTCTGCAACTCGACTCCGTGAAGCTGGAATCGCTAGTAATCGCATA

[0054] TCAGCCATGATGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGCTGGGAGTGCCTGAAGTCCGTCACCGTAAGGAGCGGCATAGGTAATTTACGTT.

[0055] Example 2 Extraction of Ulva protopanax

[0056] After pulverizing the dried Ulva perfoliate powder and passing it through a 40-mesh sieve, soak it in 75% ethanol overnight to remove pigments, then filter it with gauze. Dry the filtered powder, take 20g and add it to 1L of distilled water, and extract it in a 90℃ water bath for 4 hours. First filter it with gauze to remove the residue, then collect the supernatant by centrifugation. Evaporate 3 / 4 of the volume of the supernatant at 80℃, then add 3 times the volume of 95% ethanol and place it in a 4℃ refrigerator for ethanol precipitation. After precipitation, centrifuge to remove the supernatant, wash it twice with 95% ethanol and once with acetone, and finally dry the precipitate to obtain crude Ulva perfoliate.

[0057] Example 3: Verification of the ability of strain PT7-4 to degrade uranium.

[0058] The culture medium with Ulva as the sole carbon source consists of: 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, 0.008 g / L disodium hydrogen phosphate, and 2 g / L Ulva.

[0059] Strain PT7-4 was inoculated into a single carbon source medium and cultured at 28℃ and 150 rpm in a constant temperature shaker. Samples were taken every 4 hours. 2 mL of bacterial culture was taken from each bottle each time, and the absorbance at 600 nm was measured and recorded. Figure 3 Through observation Figure 3 The growth curve of strain PT7-4 showed that it grew well. Bacterial culture samples were collected at different time points and centrifuged at 12000 rpm for 2 min; the supernatant was used for subsequent analysis. Changes in the oligosaccharide bands of *Ulva lactuca* in the fermentation broth at different time points were observed using fluorescence-assisted glycan electrophoresis (FACE). Figure 4 It was found that the low degree of polymerization oligosaccharides gradually increased within 8-32 hours, and strain PT7-4 produced abundant oligosaccharides in the only carbon source medium of Ulva protosan.

[0060] Example 4: Structural Composition Analysis of Ulva Propane

[0061] Prepare 2 mol / L trifluoroacetic acid and 0.5 mol / L PMP methanol solution, etc. Mix the polysaccharide with an equal volume of TFA and the sample solution at a 1:1 ratio, and hydrolyze in an oven at 110℃. Repeat the addition of methanol and drying with N2 twice to remove TFA. Finally, add NaOH solution to fully dissolve the residue. Then, perform monosaccharide derivatization. Mix the mixed monosaccharide standard solution with methanol solution, react in a 70℃ water bath, cool to room temperature, and extract with chloroform. Analyze using an Agilent 1100 high-performance liquid chromatograph. Dissolve the Ulva polysaccharide sample in 0.1M NaNO3 solution, filter at 0.45μm, and separate using Ohpak SB-805HQ and SB-803HQ tandem columns to determine the molecular weight of Ulva polysaccharide.

[0062] Obtained by HPLC (high performance liquid chromatography) Figure 5 It was found that Ulva polysaccharide is composed of 9 different monosaccharides, mainly including L-rhamnose, D-glucuronic acid, and D-glucose. In addition, it also contains L-guluronic acid, D-mannose, D-glucosamine, D-galactose, D-xylose, and L-arabinose.

[0063] The molecular weight distribution of ursolic acid was analyzed by GPC gel permeation chromatography and differential detector to obtain...Figure 6 The study found that the main peak value (Mw) was 580 kDa, accounting for 71.5% of the peak area; the second largest peak value (Mw) was 18 kDa, accounting for 22% of the peak area; and the last largest peak value (Mw) was 1088 Da, accounting for 6.5%.

[0064] Example 5: Structural Analysis of Ulva Oligosaccharides

[0065] Oligosaccharide polymerization degree analysis and oligosaccharide structure detection by FACE and ESI-MS were used, such as Figure 7 The study identified multiple degradation product components, such as △Rha3S-Xy1, Rha-Rha, Rha-Rha-Rha, and Rha-Xy12S-Rha-Glc, indicating that the crude enzyme solution of strain PT7-4 has the ability to cleave polysulfated polysaccharides and generate oligosaccharides with diverse structures. Furthermore, the crude enzyme solution of strain PT7-4 retains the core sulfation and structural features of the Ulva polysaccharide backbone during the cleavage process.

[0066] Example 6: Antioxidant activity of Ulva oligosaccharides

[0067] The *Bacillus algae* PT7-4 was inoculated into a liquid culture medium with Ulva as the sole carbon source; cultured for 8 hours; the culture medium was centrifuged, the supernatant was collected, filtered to remove impurities, and concentrated to one-third volume using an ultrafiltration tube with a molecular weight of 10 kDa to obtain a crude enzyme solution; the crude enzyme solution and Ulva were mixed at a ratio of 1:2 mL:g, and water was added to prepare a reaction mixture with a Ulva concentration of 5 mg / mL; the reaction mixture was reacted at 40℃ for 5 hours, then heated to 100℃ and inactivated for 5 minutes; the inactivated mixture was placed in a centrifuge and centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected to obtain Ulva oligosaccharide.

[0068] Its antioxidant activity was determined by its ability to scavenge DPPH free radicals and superoxide anions.

[0069] (1) Determination of DPPH radical scavenging rate: Sample solutions were prepared in deionized water to form three gradient sample solutions (0.5, 1.5, and 2.5 mg / mL). 40, 120, and 200 μL of oligosaccharide solution (excluding the blank tube) and 200 μL of DPPH solution (excluding the background tube) were added sequentially to 1.5 mL EP tubes. Each tube was then brought to a final volume of 400 μL with distilled water. The mixture was shaken and allowed to stand at room temperature for 30 min. The absorbance was measured at 517 nm using distilled water as the zeroing point. The DPPH radical scavenging capacity was calculated using Formula 1.

[0070] Formula 1:

[0071] In Formula 1: A DPPH A represents the absorbance of the DPPH solution without the sample added. 实验Add the absorbance of the sample to the DPPH solution; A 本底 The absorbance is the sample solution without DPPH added.

[0072] As determined by DPPH free radical scavenging ability, such as Figure 8 The study found that the concentration of 1.5 mg / mL of Ulva oligosaccharide had the highest average scavenging rate of 9.67%, with small fluctuations and good stability, making it the optimal concentration for scavenging effect. Its average DPPH free radical scavenging efficiency was 9.67%.

[0073] (2) Superoxide anion scavenging activity assay: 125 μL of 50 mM Tris-HCl buffer (pH 8.2) was added to a 1.5 mL EP tube and incubated at 25 °C for 20 min. Then, 200 μL of polysaccharide solutions of different concentrations (0.5, 1.5, 2.5 mg / mL) and 30 μL of 25 mM pyrogallol solution were added. The mixtures were incubated at 25 °C for 5 min, and then 50 μL of 8.0 mM HCl solution was added to each mixture to terminate the reaction. Finally, 200 μL of each reaction solution was pipetted into a 96-well plate, and the absorbance at 299 nm was measured using a microplate reader. The superoxide anion radical scavenging activity of Ulva protopanax senticosus was calculated according to Formula 2.

[0074] Formula 2:

[0075] In Formula 2: A is the absorbance of the sample; A0 is the absorbance after replacing pyrogallol with Tris-HCl buffer; A1 is the absorbance after replacing the sample with Tris-HCl buffer.

[0076] The superoxide anion radical scavenging ability of different concentrations of Ulva protoplasm degradation products was measured, such as... Figure 8 The study found that the scavenging rate of ulmoides oligosaccharides at a concentration of 1.5 mg / mL was the highest (51.22%), with a maximum of 60.91%. The data showed moderate fluctuations and good stability (variance of 14.3294), making it the optimal concentration for scavenging.

[0077] In summary, by measuring the DPPH and superoxide anion free radical scavenging abilities of Ulva oligosaccharides, it was found that the Ulva oligosaccharides obtained by degradation by strain PT7-4 showed good DPPH and superoxide anion free radical scavenging effects at a concentration of 1.5 mg / mL.

Claims

1. A strain of Algibacter sp. PT7-4, characterized in that, The Bacillus PT7-4 was deposited on July 18, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 66710.

2. A microbial agent, characterized in that, The bacterial agent comprises Bacillus algae PT7-4 according to claim 1.

3. Crude enzyme solution, characterized in that, The crude enzyme solution is produced by Bacillus algae PT7-4 according to claim 1.

4. The crude enzyme solution according to claim 3, wherein the preparation method of the crude enzyme solution comprises: The Bacillus PT7-4 was inoculated into a liquid with Ulva protease as the sole carbon source; 8 hours of training; Centrifuge the culture medium to obtain the supernatant, filter to remove impurities, and concentrate it using an ultrafiltration tube with a molecular weight of 10 kDa.

5. The application of Bacillus PT7-4 according to claim 1 or the crude enzyme solution according to claim 3 or 4 in the degradation of Ulva protosan.

6. Ulva oligosaccharides, characterized in that, The ulva oligosaccharide is obtained by degrading ulva polysaccharide with Bacillus PT7-4 according to claim 1 or with crude enzyme solution according to claim 3 or 4.

7. The ulva oligosaccharide according to claim 6, wherein the preparation method of the ulva polysaccharide comprises: After pulverizing the dried Ulva lactuca powder, sieve it, soak it in 75% ethanol overnight to remove pigments, and then filter it. The filtered powder was dried and added to distilled water at a solid-liquid mass ratio of 1:

50. It was then extracted at 90°C for 4 hours. The residue was removed by filtration, and the supernatant was collected by centrifugation. The supernatant was concentrated to 3 / 4 of its volume, and then 3 times the volume of 95% ethanol was added. The mixture was then placed at 4°C for alcohol precipitation. After precipitation, the supernatant was removed by centrifugation, and the precipitate was washed and dried to obtain uranyl peroxide.

8. The *Ulva* oligosaccharide according to claim 6 or 7, wherein the preparation method of the *Ulva* oligosaccharide comprises: (1) The Bacillus algae PT7-4 was inoculated into a liquid culture medium with Ulva polysaccharide as the sole carbon source; 8 hours of training; Centrifuge the culture medium to collect the supernatant, filter to remove impurities, and concentrate it to one-third of its volume using an ultrafiltration tube with a molecular weight of 10 kDa to obtain crude enzyme solution. (2) Take crude enzyme solution and ursolic acid in a ratio of 1:2 mL:g, add water to prepare reaction mixture, the concentration of ursolic acid in reaction mixture is 5mg / ml; (3) After the reaction mixture is reacted at 40℃ for 5 hours, the temperature is raised to 100℃ and the mixture is inactivated for 5 minutes. (4) Place the inactivated mixture into a centrifuge, centrifuge at 12000 rpm / min for 3 min, and collect the supernatant to obtain Ulva oligosaccharide.

9. The use of the oligosaccharide of Ulva as described in any one of claims 6-8 in the preparation of health products with auxiliary antioxidant effects.

10. The use of the oligosaccharide of Ulva as described in any one of claims 6-8 in the preparation of a drug with antioxidant activity.

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