A Vibrio strain that efficiently degrades alginate and its application

Abstract

This invention relates to the field of microbial technology, specifically to a Vibrio strain that efficiently degrades alginate and its applications. The Vibrio strain that efficiently degrades alginate is deposited at the China General Microbiological Culture Collection Center (CGMCC) and is named (…). Vibrio sp. MBP_DH9, accession number CGMCC No. 35925, accession date September 15, 2025, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The Vibrio strain provided by this invention produces an alginate lyase. The alginate lyase degrades alginate to produce only fucoidan, exhibiting high product specificity, which is beneficial for industrial application. Furthermore, this invention conducted further single-factor experiments on the fermentation culture conditions of the Vibrio strain, providing preliminary research for its subsequent large-scale industrial production. The Vibrio strain provided by this invention can efficiently degrade copper algae in a seawater environment, making it an advantageous strain for the resource utilization of brown algae. This strain also possesses environmental advantages.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and specifically to a Vibrio strain that efficiently degrades alginate and its applications. Background Technology

[0002] Alginate ( alginate Alginate is an acidic, hydrophilic heteropolysaccharide widely found in the cell walls of brown algae, polymerized from two uronic acid monomers: β-D-mannuronic acid (M) and α-L-guluronic acid (G). Due to its excellent thickening, gelling, and biocompatibility properties, it is commonly used as a food additive, drug carrier, and animal feed. However, the high molecular weight of alginate significantly limits its biological activity and application scope. Alginate oligosaccharides obtained from the degradation of alginate retain many of its physicochemical properties while, thanks to their low molecular weight, can more easily penetrate cell membranes and enter the cell interior, exerting anti-inflammatory, antibacterial, and immunomodulatory physiological activities. Therefore, they have broad application value in the food, pharmaceutical, and agricultural fields.

[0003] Compared to physical and chemical methods for preparing alginate oligosaccharides, the bio-enzymatic hydrolysis method with alginate lyase as the core has outstanding advantages such as high specificity, mild reaction conditions, high degradation efficiency, and environmental friendliness. It effectively avoids the problems of high energy consumption of physical methods and easy generation of pollutants by chemical methods. Therefore, it has become the preferred method for preparing alginate oligosaccharides by degrading alginate polysaccharides.

[0004] alginate lyase ( alginate lyase Alginate degradation is catalyzed by cleaving the 1,4-glycosidic bonds between alginate molecules via a β-elimination mechanism. Most reported alginate-lysin-producing strains originate from marine environments, including Vibrio species. Vibrio sp. Alternating Monoclonal bacteria () Pseudoalteromonas sp. ) and Bacillus spp. ( Bacillus sp. However, due to the diverse types of degrading enzymes produced by different strains, the degradation products of alginate by different strains exhibit diversity. Furthermore, the screened alginate-degrading strains generally suffer from low enzyme production and unstable enzyme activity, which restricts their application in actual production.

[0005] Zhao et al. screened a strain of Arctic pseudoalternomonas M9 from Sargassum fusiforme with an alginate lyase activity of 0.55 U / mL. Pseudoal teromonas sp. (Zhao et al., 2022). Wang et al. screened a strain with low-temperature-adapted alginate lyase activity from abalone viscera. Vibrio sp.W2 has an enzyme activity of 38.2 U / mL, and this enzyme can degrade alginate to produce oligosaccharides with a degree of polymerization of 2-6 (Wang et al., 2020). Therefore, screening a high-quality alginate lyase-producing strain to enhance the high-value and diversified application of algal biomass is crucial for the development and utilization of my country's marine resources.

[0006] Patent CN 114657085A discloses a high-yield alginate lyase strain and its applications. This strain exhibits strong alginate lyase activity, has a mature process, and is widely applicable. However, the degree of polymerization of the alginate-degrading products produced by the strain described in this patent ranges from 2 to 7, resulting in low product specificity, multiple product types, and difficulty in separation, thus increasing purification costs. Summary of the Invention

[0007] In view of the problems that existing technologies have limited types of strains that produce alginate lyase, low enzyme activity, and excessive algae in seawater that affect the marine environment, this invention provides a Vibrio strain that efficiently degrades alginate and its application.

[0008] The present invention is specifically implemented using the following technical solutions:

[0009] In a first aspect, the present invention provides a Vibrio strain that efficiently degrades alginate, the strain being deposited at the China General Microbiological Culture Collection Center (CGMCC) and named (…). Vibrio sp. MBP_DH9, accession number CGMCCNo.35925, accession date September 15, 2025, address: No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Secondly, the present invention provides an alginate lyase, which is prepared by fermentation using the aforementioned Vibrio strain.

[0011] Furthermore, the product of this alginate lyase that degrades alginate is fucoidan.

[0012] Thirdly, the present invention provides a method for fermenting and culturing the above-mentioned Vibrio strain, the method comprising the following steps: inoculating the seed liquid of activated Vibrio strain MBP_DH9 into alginate liquid culture medium and fermenting and culturing at a constant temperature for 15-17 hours.

[0013] Furthermore, the initial pH of the alginate liquid culture medium is 6-8, and the optimal initial pH of the present invention is 6.5.

[0014] Furthermore, the sodium alginate concentration in the alginate liquid culture medium is 0.2%-1.0%, and the optimal sodium alginate concentration in the alginate liquid culture medium of the present invention is 0.6%.

[0015] Furthermore, the culture temperature of the strain is 20℃-40℃, and the optimal culture temperature of the strain of the present invention is 30℃.

[0016] Fourthly, the present invention provides the above-mentioned Vibrio strain that efficiently degrades alginate and the application of the above-mentioned alginate lyase in the degradation of brown algae.

[0017] Fifthly, the present invention provides the above-mentioned Vibrio strain that efficiently degrades alginate and the application of the above-mentioned alginate lyase in the degradation of brown algae to prepare fucoidan.

[0018] The present invention has the following beneficial effects:

[0019] (1) The present invention provides a Vibrio strain that can produce alginate lyase. The alginate lyase degrades alginate to produce only fucoidan, which has high product specificity, high efficiency and single product, and has the function of efficiently degrading alginate. The Vibrio strain has efficient enzyme production and degradation capabilities, which is beneficial for its application in industrial production.

[0020] (2) The present invention conducted further single-factor experimental research on the fermentation culture conditions of the Vibrio strain, providing a prerequisite study for the subsequent large-scale industrial production of the strain.

[0021] (3) The Vibrio strain provided by the present invention can efficiently degrade copper algae in the seawater environment and is an advantageous strain for the resource utilization of brown algae. The strain has environmental advantages.

[0022] (4) The application of the strain described in this invention to degrade brown algae in seawater will not produce other pollutants, and the strain and enzyme themselves are degradable, which is beneficial to the protection of seawater resources. Attached Figure Description

[0023] Figure 1 This is a graph showing the results of plate screening for alginate lysin in the strain;

[0024] Figure 2 A graph showing the detection of reducing sugar content produced by the strain's degradation of alginate;

[0025] Figure 3 The figure shows the detection results of alginate lyase activity in the crude enzyme solution of the strain;

[0026] Figure 4 Thin-layer chromatography pattern of the strain degrading alginate to form alginate oligosaccharides;

[0027] Figure 5 The images show the colony morphology and Gram staining of MBP_DH9, where A is the colony morphology of MBP_DH9 and B is the Gram staining.

[0028] Figure 6To construct a phylogenetic tree of MBP_DH9 based on 16S rDNA;

[0029] Figure 7 Optimization diagram of fermentation conditions for producing alginate lyase from MBP_DH9;

[0030] Figure 8 Morphological images of copper algae thallus 48 h after inoculation with MBP_DH9 and before inoculation with MBP_DH9. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0032] Example 1: Vibrio ( Vibrio sp. Isolation, screening, and evaluation of alginate degradation ability of MBP_DH9

[0033] I. Experimental Materials

[0034] Alginate liquid culture medium: sodium alginate 5.0 g / L, (NH4)2SO4 5.0 g / L, NaCl 30.0 g / L, MgSO4·7H2O 1.0 g / L, K2HPO4·2H2O 2.0 g / L, FeSO4·7H2O 0.01 g / L, initial pH 7.5.

[0035] Alginate solid medium: 1.5% agar is added to the alginate liquid medium.

[0036] 0.3% sodium alginate solution: 0.3 g of sodium alginate was diluted to 100 mL with 0.01 mol / L PBS buffer, with an initial pH of 7.5.

[0037] II. Experimental Methods

[0038] (1) Isolation and screening of alginate-degrading bacteria

[0039] 100 μL of seawater sample from the Dongji Island area of ​​Zhoushan was added to 2 mL of alginate liquid medium and incubated at 37℃ with shaking at 250 rpm for 48 h. 1 mL of the culture was serially diluted with sterile physiological saline, and 100 μL of each diluted culture was spread onto alginate solid medium and incubated upside down at 37℃ for 2 days. Colonies with good growth and different morphologies were picked and streaked repeatedly to obtain the purified bacterial strain.

[0040] Purified single colonies were inoculated into alginate liquid medium and cultured at 37 ℃ with shaking at 250 rpm for 24 h to prepare glycerol-containing bacteria, which were then stored at -80 ℃. 1 μL of the activated bacterial culture was inoculated into alginate solid medium and incubated at 37 ℃ for 24 h. Lugol's iodine solution was then added for staining. The presence or absence of a clear zone around the colony indicated whether the strain possessed the ability to produce alginate lyase.

[0041] (2) Characterization of enzyme production activity of alginate-degrading bacteria

[0042] 10 μL of glycerol bacteria was added to the alginate seed culture medium and cultured at 37 ℃ with shaking at 250 rpm for 24 h. Then, 2% (v / v) of activated seed culture was inoculated into the alginate fermentation medium and cultured at 37 ℃ with shaking at 250 rpm. At different time points (4, 8, 12, 16, 20, 24 h), 1 mL of the fermentation broth was taken, centrifuged at 10000 rpm for 5 min, and the supernatant was collected as the sample to be tested. Using glucose as a standard, the reducing sugar content in the fermentation broth at each time point was determined by the 3,5-dinitrosalicylic acid (DNS) colorimetric method.

[0043] After activating the seeds as described above, the activated seed solution was inoculated into the alginate fermentation medium at a volume fraction of 2%, and cultured at 37 ℃ and 250 rpm for 16 h with constant temperature shaking. 1 mL of the fermentation broth was centrifuged at 10000 rpm for 5 min, and the supernatant was collected as the crude enzyme solution. The alginate lyase activity in the crude enzyme solution was determined using the DNS method. 50 μL of the crude enzyme solution was added to the substrate (0.3% alginate solution), and the reaction was terminated at 37 ℃ for 40 min, followed by a 10 min boiling water bath. 50 μL of the reaction product was added to 100 μL of DNS reagent and boiled for 10 min, then diluted with 450 μL of water. The absorbance was measured at 540 nm. The blank control sample was the inactivated crude enzyme solution. Enzyme activity unit (U) is defined as the amount of alginate lyase required to catalyze the release of 1 μg of reducing sugar from the substrate per minute under the test conditions. The enzyme activity calculation formula is:

[0044] Enzyme activity (U / L) = (m×N×1000) / (T×V)

[0045] In the formula: m is the reducing sugar content (mg); N is the dilution factor; T is the reaction time (10 min); V is the enzyme solution volume (mL).

[0046] (3) Qualitative analysis of the fermentation products of the strain

[0047] 10 μL of glycerol bacteria was added to the seed culture medium and cultured at 37 ℃ and 250 rpm for 24 h with constant temperature shaking. Then, 2% (v / v) activated seed culture was inoculated into the fermentation medium and cultured at 37 ℃ and 250 rpm with constant temperature shaking. At different time points (4, 8, 12, 16, 20 h), 1 mL of bacterial culture was taken, centrifuged at 10000 rpm for 5 min, and the supernatant was collected. Thin-layer chromatography (TLC) was used for preliminary characterization of the alginate oligosaccharides in the fermentation broth. A Qingdao marine silica gel chromatography plate (G plate) was used. Samples were spotted three times at each time point (samples were dried and then spotted again). The samples were developed with a developing solvent (butanol, formic acid, and water in a volume ratio of 4:5:1), air-dried at room temperature, sprayed with a colorimetric reagent (10% sulfuric acid ethanol), and developed in a 120 ℃ oven for 3-4 min. The M1-M3 standards are, in order, monosaccharides (glucose), disaccharides (L-guluronic acid disaccharide), and trisaccharides (L-guluronic acid trisaccharide).

[0048] III. Experimental Results

[0049] (1) Nine bacterial strains capable of degrading alginate were isolated from seawater in the Dongji Island area of ​​Zhoushan. The alginate lyase production capacity of these nine strains was tested using alginate solid plates, and the results are as follows: Figure 1 As shown, large clear zones appeared around the colonies of all nine strains, indicating that all nine strains were positive for alginate lyase.

[0050] (2) The fermentation results of the 9 strains in liquid culture medium with sodium alginate as the sole carbon source are as follows: Figure 2 As shown, the reducing sugar content in the fermentation broth of these nine strains all exhibited a trend of first increasing and then decreasing with the passage of fermentation time. Notably, at 16 h of fermentation, the reducing sugar content in the fermentation broth of strain MBP_DH9 was the highest, reaching 0.455 mg / mL, followed by MBP_Fz2, MBP_Fz1, and MBP_Fz4. Figure 2 Furthermore, the activity of alginate lyase in the crude enzyme solution of the strain was detected, and the results are as follows. Figure 3 As shown, the activity of alginate lyase in the crude enzyme solution of strain MBP_DH9 was significantly higher than that of the other eight strains, reaching 46.1 U / mL, followed by MBP_DH6, MBP_Fz1, and MBP_Fz2. In summary, among these nine strains, strain MBP_DH9 exhibited the strongest alginate degradation ability, as evidenced by higher reducing sugar content and stronger alginate lyase activity in the fermentation broth (Table 1).

[0051] Table 1. Results of reducing sugar content and alginate lyase activity in fermentation broths of different bacterial strains

[0052] .

[0053] (3) Qualitative results of the products of alginate degradation by the strain are as follows: Figure 4 As shown, MBP_Fz1, MBP_Fz2, MBP_DH6, and MBP_DH9 can all degrade macromolecular alginate into fucoidan oligosaccharides with different degrees of polymerization. Notably, the fucoidan oligosaccharides produced by strain MBP_DH9 have a lower degree of polymerization (degree of polymerization 2) and a relatively higher content. In summary, strain MBP_DH9 possesses the ability to efficiently degrade alginate and produce fucoidan oligosaccharides (disaccharides).

[0054] Example 2: Vibrio ( Vibrio sp. Identification of MBP_DH9 strains

[0055] I. Experimental Materials

[0056] Strains: MBP_DH9.

[0057] II. Experimental Methods

[0058] (1) Gram staining identification

[0059] Spread a single MBP_DH9 colony onto a glass slide, dilute it evenly with physiological saline, fix it, and then perform Gram staining.

[0060] (2) Physiological and biochemical identification

[0061] Referring to traditional methods of bacterial identification, the physiological and biochemical characteristics of MBP_DH9 were detected according to the "Handbook for Systematic Identification of Common Bacteria".

[0062] (3) Identification of 16S rDNA gene of strain

[0063] Single colonies were selected from alginate solid medium and amplified by PCR using universal primers 27F and 1492R. The PCR products were then sequenced. The sequence information was analyzed by BLAST against the NCBI database and compared with the 16S rDNA sequences of known microorganisms. A phylogenetic tree was constructed using MAGA12.

[0064] The nucleotide sequence of primer 27F is shown in SEQ ID NO.2, and the nucleotide sequence of primer 1492R is shown in SEQ ID NO.3.

[0065] III. Experimental Results

[0066] The colony morphology of strain MBP_DH9 is as follows: Figure 5As shown in Figure A, the colonies are white, nearly round, slightly convex, with a moist, smooth, and opaque surface and neat edges; the Gram staining results are as follows. Figure 5 As shown in Figure B, strain MBP_DH9 stains red with Gram stain, indicating that this strain is a Gram-negative bacterium. The cells are curved in an arc shape and are relatively active.

[0067] Physiological and biochemical tests on strain MBP_DH9 showed that strain MBP_DH9 can utilize glucose, mannitol, and inositol. It was positive for alkaline phosphatase, esterase (C4), esterase-lipase (C8), acid phosphatase, glucosidase, trypsin, and lipase (C14).

[0068] The 16S rDNA sequencing results of strain MBP_DH9 are shown in SEQ ID No. 1 of the sequence listing. The 16S rDNA sequence is 1489 bp, and the GenBank accession number is PX362803. Sequence homology was compared using the BLAST program in NCBI, and the results showed that strain MBP_DH9 is related to several Vibrio species (…). Vibrio sp. The strains showed a high degree of similarity (99.93%). For example... Figure 6 As shown in the phylogenetic tree, strain MBP_DH9 and Vibrio sp. The strain MY-2008-31d ​​is most closely related to Vibrio. Therefore, based on the morphological and biochemical characteristics of the strain, MBP_DH9 was determined to belong to the genus Vibrio. Strain MBP_DH9 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35925.

[0069] Example 3: Vibrio ( Vibrio sp. Optimization of enzyme production conditions for MBP_DH9

[0070] I. Experimental Materials

[0071] Strains: MBP_DH9.

[0072] Culture medium:

[0073] Alginate liquid culture medium: sodium alginate 5.0 g / L, (NH4)2SO4 5.0 g / L, NaCl 30.0 g / L, MgSO4·7H2O 1.0 g / L, K2HPO4·2H2O 2.0 g / L, FeSO4·7H2O 0.01 g / L, initial pH 7.5.

[0074] Copper algae culture medium: Take 1 g of copper algae, soak it in pure water for 4 h, clean it and cut it into pieces, add it to 250 mL of sterilized seawater containing 25 mL, and adjust the initial pH to 6.5.

[0075] II. Experimental Methods

[0076] The optimal parameters for three culture conditions (temperature, initial pH, and substrate concentration) affecting enzyme production were investigated using single-factor methods. The factors and their levels are shown in Table 2. Activated MBP_DH9 seed culture was inoculated into alginate liquid medium at a volume fraction of 2%, and cultured at 250 rpm with shaking for 16 h. One mL of the fermentation broth was then centrifuged at 10,000 rpm for 5 min, and the supernatant was collected as the crude enzyme solution. The alginate lyase activity in the crude enzyme solution was determined using the DNS method. The results of each optimization step were used in subsequent experiments.

[0077] Table 2. Distribution of univariate levels

[0078] .

[0079] III. Experimental Results

[0080] The optimization results of MBP_DH9 enzyme production conditions are as follows: Figure 7 As shown, the preferred fermentation conditions are a culture temperature of 30 °C, an initial pH of 6.5, and a sodium alginate concentration of 0.6%. Under these preferred enzyme-producing conditions, the alginate lyase activity of MBP_DH9 can reach 66.6 U / mL, which is 44.5% higher than that before optimization (46.1 U / mL).

[0081] Example 4: Vibrio ( Vibrio sp. Application of MBP_DH9 in fermenting copper algae thallus

[0082] I. Experimental Materials

[0083] Strains: MBP_DH9.

[0084] Culture medium:

[0085] Alginate liquid culture medium: sodium alginate 5.0 g / L, (NH4)2SO4 5.0 g / L, NaCl 30.0 g / L, MgSO4·7H2O 1.0 g / L, K2HPO4·2H2O 2.0 g / L, FeSO4·7H2O 0.01 g / L, initial pH 7.5.

[0086] Copper algae culture medium: Take 1 g of copper algae, soak it in pure water for 4 h, clean it and cut it into pieces, add it to 250 mL of sterilized seawater containing 25 mL, and adjust the initial pH to 6.5.

[0087] II. Experimental Methods

[0088] The activated MBP_DH9 seed culture was inoculated into the copper algae culture medium at a volume fraction of 2%, and cultured at 250 rpm and 30℃ for 48 h. The turbidity of the culture medium and the changes in the shape of the copper algae were observed.

[0089] III. Experimental Results

[0090] To further evaluate the degradation ability of MBP_DH9 on *Cotinus thunbergii*, optimized enzyme-producing conditions were used to ferment *Cotinus thunbergii*. Compared with the control group, after 48 h of fermentation with MBP_DH9, the strain showed good growth, the *Cotinus thunbergii* culture medium solution became turbid and darker in color, the *Cotinus thunbergii* clumps became smaller, and more debris was added. Figure 8 This indicates that MBP_DH9 can efficiently degrade brown algae in a seawater environment, making it a superior strain for the resource utilization of brown algae.

Claims

1. A Vibrio strain that efficiently degrades alginate ( Vibrio sp. MBP_DH9, accession number CGMCCNo.35925.

2. A method for fermenting and culturing the Vibrio strain according to claim 1, characterized in that, The method includes the following steps: Inoculating the seed culture of activated Vibrio strain MBP_DH9 into alginate liquid culture medium and fermenting at a constant temperature for 15-17 hours.

3. The method for fermentation culture of Vibrio strains as described in claim 2, characterized in that, The initial pH of the alginate liquid culture medium is 6-8.

4. The method for fermenting and culturing a Vibrio strain as described in claim 2, characterized in that, The sodium alginate concentration in the alginate liquid culture medium is 0.2%-1.0%.

5. The method for fermentation culture of Vibrio strains as described in claim 2, characterized in that, The culture temperature for the strain is 20℃-40℃.

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