Sulfite bacillus algal inhibition exopolysaccharide as well as extraction method and application thereof

Abstract

The invention discloses algal-inhibiting exopolysaccharide of bacillus sulfite as well as an extraction method and application of the algal-inhibiting exopolysaccharide, and belongs to the field of extraction and application of algal-inhibiting active substances. The invention provides an application of algal inhibition extracellular polysaccharide of bacillus sulfite H46 in killing kagoda algae. The algal inhibition extracellular polysaccharide is obtained by extracting and purifying a metabolite with the molecular weight of 10-30kDa in fermentation filtrate of the bacillus sulfite H46. The preservation number of the bacillus sulfite H46 is CGMCC (China General Microbiological Culture Collection Center) No.27825. Experiments prove that the exopolysaccharide generated by the sulfite bacillus H46 can inhibit the growth of algal cells of the kagoda, and the algal inhibition rate in 24 hours is close to 96%. The algal-inhibiting exopolysaccharide provided by the invention can be used for preventing and treating the red tide of the kagoda algae, and has the characteristics of high efficiency and environment friendliness.

Description

Technical Field

[0001] The invention relates to the extraction and application of algae-inhibiting active substances, and in particular to a sulfite bacillus algae-inhibiting extracellular polysaccharide and an extraction method and application thereof. Background Art

[0002] Over-reproduction of marine microalgae in certain environments causes red tides. When a red tide breaks out, due to the high density of microalgae cells, they will cover a large area of ​​the sea surface and even attach to the gills of fish and shellfish, causing difficulty in breathing. In addition, during the growth of microalgae cells and the degradation of organic matter, a large amount of oxygen will be consumed, causing hypoxia in the marine environment and causing a large number of fish to die of hypoxia. At the same time, some red tide algae can also secrete algal toxins, such as the marine kadun algae red tide, which usually occurs in warm coastal waters, especially the red tide along the Gulf of Mexico coast of the United States. This phenomenon not only changes the color of the seawater, but also poses a major threat to the marine ecosystem, coastal economy and human health. The outbreak of the kadun algae red tide is usually accompanied by a rapid increase in the number of algae, causing the seawater to appear red or brown and releasing a neurotoxin called brevetoxins. This toxin is highly toxic to a variety of marine organisms, can kill a large number of fish, shellfish and marine mammals, and destroy marine biodiversity. In addition, the kadun algae red tide will be enriched through the food chain, threatening human health. If humans eat contaminated seafood, it may cause severe poisoning reactions and even damage the nervous system. It can directly kill marine life and even threaten human health through the food chain. According to statistics, the occurrence of red tides can cause the global economy to lose about US$8 billion each year. It has become a problem of widespread concern among marine ecologists around the world.

[0003] There are various methods for controlling red tides, including physical, chemical and biological means. Each method has its advantages and limitations in dealing with red tides. Compared with physical and chemical methods, biological control, especially microbial control, shows significant advantages in controlling red tides. Advantages: 1. Strong environmental protection: Biological control uses natural organisms or microorganisms. Compared with chemical agents, it has less pollution to the environment and will not cause secondary pollution. 2. Sustainability: By establishing a benign ecological balance, biological control can effectively control red tides in the long term and reduce the frequent occurrence of red tides. 3. Strong selectivity: Biological control methods can usually selectively target red tide algae and will not have a large-scale negative impact on other aquatic organisms, especially those that are beneficial to the ecosystem. 4. Diversified means: Biological control includes a variety of means, such as using specific predators, parasites or competitive algae to inhibit the growth of red tide algae, so that flexible response measures can be taken for different types of red tides.

[0004] At present, the anti-algae substances of microbial metabolism are basically small molecule compounds, including phenolic compounds, such as p-hydroxybenzoic acid, vanillic acid, etc.; fatty acids and their derivatives, such as unsaturated fatty acids and fatty acid ethyl esters, which are commonly found in a variety of microbial metabolites and can inhibit algae growth; alkaloids, such as pyrrole, indole and piperidine compounds, which play a role in inhibiting algae photosynthesis or destroying cell structure; polyphenolic compounds, such as ellagic acid and gallic acid, have antioxidant and anti-algae functions; organic acids, such as succinic acid and citric acid, can affect the metabolic process of algae and inhibit its growth. Although small molecule anti-algae substances have shown certain effects in inhibiting algae growth, their disadvantages are also obvious, including: (1) Poor selectivity: Most small molecule anti-algae substances show indiscriminate inhibitory effects on a variety of algae, lack selectivity for specific algae, and are likely to affect non-target algae and other aquatic organisms, destroying the ecological balance. (2) Easy to degrade: Small molecule compounds are usually easily degraded by microorganisms or light in the environment, resulting in a short effective time in the water body, a short-lasting algae inhibition effect, and the need for frequent replenishment. (3) Toxicity issues: Some small molecule compounds may be toxic to aquatic organisms or humans, such as phenols or organic acids. The dosage needs to be controlled when used to avoid environmental and health risks. These disadvantages make small molecule algae inhibitors have certain limitations in practical applications, especially in terms of environmental friendliness and long-term effectiveness.

[0005] Based on this, the present invention proposes an extracellular polysaccharide with an algae-inhibiting effect, which provides a new strategy for the prevention and control of red tides of Algae. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-algae exopolysaccharide of Bacillus sulfitans and an extraction method and application thereof to solve the problems existing in the above-mentioned prior art. The exopolysaccharide produced by Bacillus sulfitans H46 can inhibit the growth of algae cells of Cardunia schrenkiana, and the 24h algae inhibition rate is close to 96%. The anti-algae exopolysaccharide provided by the present invention can be used in the prevention and treatment of red tide of Cardunia schrenkiana, and has the characteristics of high efficiency and green environmental protection.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an application of sulfit bacillus H46 algae-inhibiting exopolysaccharide in killing cardun algae, wherein the algae-inhibiting exopolysaccharide is extracted and purified from a metabolite with a molecular weight of 10-30 kDa in the fermentation filtrate of sulfit bacillus H46;

[0009] The preservation number of the Bacillus sulfitans H46 is CGMCC No.27825, the preservation date is July 7, 2023, the preservation unit is the General Microbiology Center of the China Culture Collection Administration, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Furthermore, the algae-inhibiting extracellular polysaccharide destroys the external structure of the Chadronella cells and interferes with their normal metabolic activities, thereby killing Chadronella.

[0011] The present invention also provides a method for extracting and preparing the algae-inhibiting extracellular polysaccharide, comprising the following steps:

[0012] (1) fermenting and culturing Bacillus sulfitans H46, and centrifuging to obtain a fermentation filtrate;

[0013] (2) ultrafiltration of the fermentation filtrate to obtain metabolites with a molecular weight of 10-30 kDa;

[0014] (3) adding anhydrous ethanol to the metabolites for precipitation, collecting the liquid, centrifuging, and freeze-drying to obtain a crude extract;

[0015] (4) The crude extract is redissolved to obtain a reconstituted solution, a chloroform-n-butanol mixture is added to perform multiple purifications, the supernatant is collected, and freeze-dried to obtain a solid powder, which is the algae-inhibiting extracellular polysaccharide.

[0016] Furthermore, in step (1), the fermentation culture time is 24 hours, and the centrifugation conditions are 8000 rpm and 15 min.

[0017] Furthermore, in the step (3), the volume ratio of the metabolite to anhydrous ethanol is 1:3, and the alcohol precipitation condition is 4° C. for 12 hours.

[0018] Furthermore, in the step (3), the centrifugation conditions are 8000 rpm and 10 min.

[0019] Furthermore, in the step (4), the crude extract is redissolved with pure water, and the volume ratio of the redissolved solution to the chloroform-n-butanol mixed solution is 3:1;

[0020] The chloroform-n-butanol mixed liquid is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1.

[0021] The present invention also provides the algae-inhibiting extracellular polysaccharide prepared according to the extraction and preparation method.

[0022] The invention also provides a preparation for killing Carinthium chaena, comprising the algae-inhibiting extracellular polysaccharide.

[0023] The present invention discloses the following technical effects:

[0024] The inventors screened a bacterium Sulfitobacter pseudonitzschiae H46 in the algae environment that specifically kills marine red tides of Cargill algae. Experimental verification found that metabolites with a molecular weight in the range of 10-30kDa have strong algae inhibition activity, and the algae inhibition rate against Cargill algae can reach 96.6% in 24 hours, and the algae inhibition substance is a macromolecular substance with thermal stability. Further studies have firmly found that the algae inhibition substance is a polysaccharide.

[0025] The use of algae-inhibiting extracellular polysaccharides to control red tides of Cardun algae has the following advantages: 1. Environmentally friendly, polysaccharides are a natural product and are harmless to the environment. Unlike chemical agents, the extracellular polysaccharides secreted by H46 bacteria will not cause secondary pollution, nor will they cause significant harm to non-target organisms. Since polysaccharides can be naturally degraded in the ocean, they will not remain or pose a long-term threat to the environment. This environmental friendliness makes polysaccharides have a higher application potential as red tide control agents. 2. Strong selectivity can avoid the indiscriminate damage of traditional chemical agents to the entire ecosystem when inhibiting harmful algae. 3. Low cost and sustainable, another major advantage of biological control methods is that they are relatively low cost and sustainable. H46 bacteria can grow and reproduce naturally. Compared with expensive chemical agents, the use of microbial products for red tide control is more economically feasible.

[0026] In summary, the exopolysaccharide produced by Sulfatium sulfitum H46 inhibits the growth of Algae cells, and the 24h algae inhibition rate is close to 96%. The algae-inhibiting exopolysaccharide produced by H46 provided by the present invention can be used in the prevention and treatment of Algae red tide, and has the characteristics of high efficiency and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 is the inhibition rate of H46 cell-free supernatant on Algae.

[0029] Figure 2 It is the inhibition rate of H46 cell-free supernatant on Algae after heating at 100℃ for 30min;

[0030] Figure 3 is the inhibition rate of H46 metabolites with a molecular weight range of <3 kDa against Algae;

[0031] Figure 4 is the inhibition rate of H46 metabolites with a molecular weight range of 3-10 kDa against Cardun Algae;

[0032] Figure 5 is the inhibition rate of H46 metabolites with a molecular weight range of 10-30 kDa against Cardun Algae;

[0033] Figure 6 Fourier transform infrared spectrum of purified algae-inhibiting exopolysaccharide;

[0034] Figure 7 To purify the inhibition rate of extracellular polysaccharide on Algae.

[0035] Figure 8 The cell morphology of Algae before (A) and after (B) the addition of H46 exopolysaccharide. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 Identification of Sulfatbacillus H46 strain

[0042] The strain of Bacillus sulfitii H46 was screened from a sample of Prorocentrum donghaiense (CCMM5003) isolated from the surface seawater of the Pacific Ocean. The morphological observation, physiological and biochemical reactions of the H46 strain were carried out. The results showed that H46 was a Gram-negative bacterium with dense colonies, yellow throughout, opaque, and smooth edges. This morphology is the standard colony morphology of common marine Bacillus sulfitii.

[0043] The size and morphology of the bacterial cells were determined by scanning electron microscopy: the bacteria were rod-shaped, no flagella were observed, and the size was (1.31-2.19)×(0.42-0.44)μm (length×width).

[0044] The 16S rRNA gene sequence of H46 was BLAST-aligned on NCBI. The final alignment results showed that Sulfitobacter pseudonitzschiae H46 was most closely related to Sulfitobacter pseudonitzschiae H3, reaching 99.77%. Therefore, it was identified as Sulfitobacter pseudonitzschiae and named Sulfitobacter pseudonitzschiae H46. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on July 7, 2023, with the deposit number: CGMCC No.27825. The complete genome sequence of S.pseudonitzschiae H46 is deposited in GenBank, with the chromosome accession number: CP054599 and the accession numbers of 9 plasmids: CP054600-CP054608.

[0045] Example 2: The inhibitory effect of the fermentation liquid of Bacillus sulfitans H46 on Algae

[0046] (1) The red tide alga C. marina was selected as the target algae strain for algae inhibition. The selected C. marina was cultured with F / 2 (formula see Table 1) and placed in a light incubator at 20°C, 4000 lux for 12h light / 12h dark for 24h until the logarithmic growth phase; Sulfite Bacillus H46 was cultured with 2216E medium (formula see Table 2) at 28°C and 180rpm for 24h.

[0047] (2) Take the fermentation broth of Bacillus sulfitans H46, place it in a high-speed centrifuge at 8000 rpm and 4°C for 15 min to obtain the H46 cell-free supernatant for later use.

[0048] H46 cell-free supernatant and H46 cell-free supernatant heated at 100°C for 30 min were used as the experimental group, and 2216E culture medium treated with the same conditions and methods was used as the control group. They were added to the culture medium of Cardun Algae to maintain a final concentration of 10% (V / V). Each group of experiments was repeated 3 times, and the algae inhibition rate was calculated.

[0049] Algae inhibition rate (%) = (1-algae cell concentration of experimental group / algae cell concentration of control group) × 100%, wherein the algae cell concentration is obtained by counting under a microscope using a 0.1 mL plankton counting plate.

[0050] The experimental results showed that the metabolites of the growth process of Bacillus sulfitans H46 had a significant inhibitory effect on Algae, and the inhibition rate of Algae could reach 96% in 24 hours. Figure 1 ); At the same time, the anti-algae substances produced by its metabolism have strong thermal stability, and the inhibition rate of Cardun Algae reached 95% in 24 hours ( Figure 2 ).

[0051] Table 1F / 2 culture medium formula

[0052]

[0053] Table 2 2216E medium formula

[0054]

[0055] Example 3 Identification of Molecular Weight of Anti-algae Substances from Sulfatite Bacillus H46

[0056] (1) H46 strain was inoculated into liquid 2216E medium for activation. The activated bacterial solution was inoculated into liquid 2216E medium at 1% (v / v) for fermentation for 24 hours, and centrifuged at 8000 rpm for 15 minutes to obtain the fermentation filtrate.

[0057] (2) The fermentation filtrate was filtered through ultrafiltration tubes with different pore sizes (30 kDa, 10 kDa, and 3 kDa) to obtain metabolites with molecular weight ranges of <3 kDa, 3-10 kDa, and 10-30 kDa, respectively;

[0058] (3) Metabolites of different molecular weight ranges were added to the liquid of Algae in the exponential growth phase, and the inhibition rate of metabolites of different molecular weights on Algae was calculated. The filtrates of different molecular weights obtained by the above operation using bacteria-free 2216E culture medium were used as controls to calculate the inhibition rate.

[0059] The results showed that metabolites with molecular weight less than 3 kDa and metabolites with molecular weight range of 3-10 kDa had almost no anti-algae activity compared with the control group ( Figure 3 , Figure 4), while the metabolites with molecular weights in the range of 10-30 kDa have strong anti-algae activity, with an anti-algae rate of up to 96.6% against Cardun Algae in 24 hours ( Figure 5 ), thus it can be determined that the anti-algae substance is a macromolecular substance with thermal stability.

[0060] Example 4 Purification and structural identification of the anti-algae substance of Bacillus sulfitans H46

[0061] (1) Collect metabolites with a molecular weight range of 10-30 kDa after purification by ultrafiltration tube, add pre-cooled anhydrous ethanol and place at 4°C for 12 h for alcohol precipitation, metabolite (v): anhydrous ethanol (v) = 1:3;

[0062] (2) The alcohol precipitated liquid is placed in a high-speed centrifuge at 8000 rpm, centrifuged for 10 min, and freeze-dried to obtain a solid powder which is a crude extract of the algae-inhibiting substance;

[0063] (3) Use pure water to redissolve the crude extract to 100 mL to obtain a reconstituted solution, and add a chloroform-n-butanol mixture at a volume ratio of 3:1 for purification (the volume ratio of chloroform and n-butanol is 4:1). Vigorously shake to mix, then let stand to separate the layers (the sample solution is in the upper layer, and the protein forms a precipitate at the interface). Use a pipette to take the supernatant, remove the lower layer and the interface layer, and repeat the purification of the collected supernatant 1-2 times to ensure that the protein is removed; freeze-dry the supernatant obtained by the final purification, and the obtained solid powder is the purified anti-algae substance;

[0064] (4) Grind the purified solid powder and dry potassium chloride (KBr) evenly to prepare KBr tablets.

[0065] A small amount of solid sample and KBr powder were mixed evenly according to the ratio (1-2 mg sample: 200 mg KBr); a tablet press was used to press into a transparent sheet under vacuum conditions, and then placed in an FTIR instrument for testing to obtain a Fourier transform infrared spectrum ( Figure 6 ).

[0066] Depend on Figure 6 It can be seen that the substance is at 1625.5cm -1 、1072.3cm -1 and 645.1cm -1 There is an absorption peak at 1072.3 cm, which is consistent with the characteristics of bacterial extracellular polysaccharides. The COC bonds in extracellular polysaccharides usually connect monosaccharide units to form long-chain polysaccharides, which is consistent with the absorption peak at 1072.3 cm in the spectrum. -1 The strong peaks appearing are consistent; 1625.5cm -1The wave number region corresponds to the OH bending vibration of water and the vibration of the residual carboxyl group (C=O) in polysaccharides. This type of characteristic peak is very common in hyaluronic acid or similar carboxyl-containing sugars. Hyaluronic acid, as a typical extracellular polysaccharide, has this characteristic; 654.1cm -1 It is a peak in the low wave number region, at 600-700 cm -1 The absorption peak in this range is related to the deformation vibration of the sugar ring. Many extracellular polysaccharides, such as dextran, glucan and other polysaccharides produced by microorganisms, may show low-frequency vibration peaks in this region.

[0067] In summary, the anti-algae substance was identified as an extracellular polysaccharide having a hydroxyl group (-OH), an ether bond (COC), a carboxyl group (C=O) and a sugar ring structure, and a molecular weight of 10-30 kDa.

[0068] Example 5 Algicidal effect of purified polysaccharide on Algae

[0069] (1) The purified polysaccharide was re-dissolved to the original volume (100 mL) and added to the exponential growth phase of the cardun algae solution at 10% (v / v) for co-cultivation. A blank control was set up: the polysaccharide extracted from the sterile 2216E medium with the same volume. Three replicates were set for both the experimental group and the control group. Samples were taken at 4h, 12h and 24h, the algae cell concentration was counted, and the algae inhibition rate was calculated. Figure 7 The results showed that the polysaccharides produced by H46 inhibited the growth of algal cells, and the 24-hour algae inhibition rate was close to 96%. At the same time, the cell morphology of Cardun Algae was observed before and after the addition of H46 extracellular polysaccharides. Figure 8 It can be seen that after adding H46 exopolysaccharide, the cell membrane of the algae is lysed and the cell contents flow out. The H46 exopolysaccharide can destroy the external structure of the algae cells, thereby interfering with its normal metabolic activities and killing the algae. The above results show that the exopolysaccharide of Bacillus sulfitans H46 has an algae-dissolving effect on the algae, and can be used to inhibit the algae and control the red tide of the marine algae.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of the algae-inhibiting exopolysaccharide of Bacillus sulfitans H46 in killing Cardun Algae is characterized in that: The algae-inhibiting exopolysaccharide is extracted and purified from a metabolite with a molecular weight of 10-30 kDa in the fermentation filtrate of Bacillus sulfitans H46; The deposit number of the bacterium Sulfite Bacillus H46 is CGMCC No.27825.

2. The use according to claim 1, characterized in that The algae-inhibiting extracellular polysaccharide destroys the external structure of the cells of the Chadronella and interferes with its normal metabolic activities, thereby killing the Chadronella.

3. The method for extracting and preparing the algae-inhibiting extracellular polysaccharide as claimed in claim 1, characterized in that: The following steps are involved: (1) fermenting and culturing Bacillus sulfitans H46, and centrifuging to obtain a fermentation filtrate; (2) ultrafiltration of the fermentation filtrate to obtain metabolites with a molecular weight of 10-30 kDa; (3) adding anhydrous ethanol to the metabolites for precipitation, collecting the liquid, centrifuging, and freeze-drying to obtain a crude extract; (4) The crude extract is redissolved to obtain a reconstituted solution, a chloroform-n-butanol mixture is added to perform multiple purifications, the supernatant is collected, and freeze-dried to obtain a solid powder, which is the algae-inhibiting extracellular polysaccharide.

4. The extraction and preparation method according to claim 3, characterized in that: In the step (1), the fermentation culture time is 24 hours, and the centrifugation conditions are 8000 rpm and 15 min.

5. The extraction and preparation method according to claim 3, characterized in that: In the step (3), the volume ratio of the metabolite to anhydrous ethanol is 1:3, and the alcohol precipitation condition is 4° C. for 12 hours.

6. The extraction and preparation method according to claim 3, characterized in that: In the step (3), the centrifugal conditions are 8000 rpm and 10 min.

7. The extraction and preparation method according to claim 3, characterized in that: In the step (4), the crude extract is redissolved with pure water, and the volume ratio of the redissolved solution to the chloroform-n-butanol mixed solution is 3:1; The chloroform-n-butanol mixed liquid is obtained by mixing chloroform and n-butanol in a volume ratio of 4:

1.

8. The algae-inhibiting extracellular polysaccharide prepared by the extraction and preparation method according to any one of claims 3 to 7.

9. A preparation for killing Caruncle algae, characterized in that: The invention comprises the algae-inhibiting extracellular polysaccharide according to claim 8.

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