CN108409877A - Sea grass polysaccharide, enteromorpha oligosaccharide, medical composition and its use - Google Patents

Abstract

The invention discloses sea grass polysaccharide and its catabolite enteromorpha oligosaccharide antibiosis purposes.The sea grass polysaccharide and its catabolite enteromorpha oligosaccharide of the present invention or its there is stronger destruction to the bacterial biof iotalm formed with antibiotic physical mixture, bactericidal range is wide, has good inhibiting effect to the formation of biomembrane.Meanwhile the sea grass polysaccharide and its catabolite enteromorpha oligosaccharide or its drug resistance that can effectively reduce bacterium with antibiotic physical mixture to be generated in the presence of biological form membrane, bacterium is improved to the sensibility of conventional antibiotic, reduces the usage amount of conventional antibiotic.

Description

Enteromorpha polysaccharide, Enteromorpha oligosaccharide, pharmaceutical composition and use thereof

technical field

The invention relates to the field of bacterial film prevention and control, in particular to Enteromorpha polysaccharide and its degradation product Enteromorpha oligosaccharide, a pharmaceutical composition and its antibacterial application.

Background technique

The causes of clinical persistent infection and hospital-acquired infection are mostly related to the formation of biofilm (Biofilm) by pathogenic bacteria on the surface of the host or medical materials. Biofilm, also known as biofilm, is a bacterial community and its extracellular matrix complex with a special microenvironment. Bacterial or fungal colonies attach to the surface of objects or the gas-liquid boundary, and secrete a large amount of exopolysaccharides, proteins and DNA, thereby forming biofilm colonies. The resistance of pathogenic bacteria in pellicle state to antibiotics and other drugs is often hundreds to thousands of times higher than that of planktonic bacteria. Traditional drugs are not effective in treating bacterial film-related infections. Therefore, there is an urgent need for drugs that can effectively target pathogenic bacteria in the bacterial film state.

Enteromorpha (Enteromorpha prolifera) is an algal plant of the genus Chlorophyta, Chlorophyceae, Ulva, and the genus Enteromorpha, commonly known as Enteromorpha, sea greens, mosses, and moss strips. Enteromorpha polysaccharide is the main active substance of Enteromorpha. Studies have shown that Enteromorpha polysaccharide has a significant effect on lowering blood sugar and blood cholesterol, and has significant inhibitory activity on Ehrlich cancer and skin cancer.

Enteromorpha polysaccharide is a polyanionic sugar chain composed of rhamnose, xylose, glucuronic acid and rich in sulfate groups. Tang et al. (2013) found that the monosaccharide composition of Enteromorpha polysaccharides was rhamnose, xylose, and glucuronic acid, with mass percentages of 64.2%, 18.2%, and 12.6%, respectively, and a sulfate group content of 19.6%. is 103.51 kDa. Ray et al. (2006) showed that rhamnose in Enteromorpha is connected by (1→2,4) bonds, glucuronic acid is connected by (1→4) bonds, xylose is connected by (1→4) bonds, and sulfate group Rhamnose sulfate is formed at the C-3 end of rhamnose.

Studies have found that Enteromorpha polysaccharides have anti-tumor, anti-coagulation, lowering blood sugar and blood lipids, improving immunity, and antioxidant activity. Enteromorpha polysaccharides contain two potential functional sugar components: glucuronic acid and rhamnose sulfate. Among them, glucuronic acid has the function of protecting the liver and detoxifying, and is the main additive of functional drinks, food and cosmetics. Rhamnose can participate in the synthesis of spices, cardiac glycosides and lectins, etc., and has obvious immune activity. Fucose sulfate, which is similar in structure to rhamnose sulfate, has been proved to have important physiological functions such as nerve conduction and immune regulation, so rhamnose sulfate is a potential functional component.

However, the higher molecular weight and viscosity of polysaccharides reduce their physiological functions during application.

Chinese patent application CN105695537A discloses a method for preparing glycosylated Enteromorpha oligosaccharides for lowering blood sugar and regulating intestinal flora. It prepares a glycosylated Enteromorpha oligosaccharides, which have a strong inhibitory effect on α-glucosidase activity, can significantly promote the proliferation of intestinal probiotics, and has the effect of improving intestinal flora, and can significantly promote the proliferation of intestinal probiotics Bifidobacteria.

Chinese patent application CN106305721A discloses a green algae oligosaccharide plant vaccine and its preparation method. The plant vaccine is composed of Enteromorpha oligosaccharides and laminin which are prepared by precise extraction of natural green algae Enteromorpha as raw materials. The reaction conditions produce oligosaccharides of different sizes, which can induce the expression of some genes related to disease resistance and stress resistance in plants, thereby improving the stress resistance of plants. Enteromorpha oligosaccharide vaccine can significantly stimulate the immune system response of plants, and has a remarkable effect in the application of resistance to wheat whole rust and wheat sheath blight.

Chinese patent application CN104962490A discloses a method for preparing Enteromorpha microbiota, comprising the following steps:

S1, taking fresh Enteromorpha to make slurry to obtain Enteromorpha slurry;

S2, aseptically culture the preserved Streptomyces flavinus, Lactobacillus casei, Bacillus subtilis, Rhodopseudomonas palustris and Pichia membranoidis to obtain biological strains;

S3, get 30-35 parts by weight of Enteromorpha slurry obtained in step S1, 3-5 parts by weight of biological strains obtained in step S2, and 3-5 parts by weight of molasses, 8-12 parts by weight of auxiliary agents and 40-60 parts by weight of water Mix the parts by weight, and control the temperature below 35°C for aerobic fermentation;

S4, when more than 80% of the propagules of the bacillus class in the bacterial liquid transform into empty spores, stop aeration and stirring, and continue anaerobic fermentation;

S5, inspecting and packaging the obtained fermentation product to obtain the liquid microbial agent.

The bacterial agent disclosed in this application can be applied to the microbial bacterial agent required by crop planting, livestock and poultry and aquaculture. Soil and water quality.

However, the use of Enteromorpha polysaccharides in the prior art is mostly focused on microbial agents, but its bactericidal effect has not been reported.

The inventors were surprised to find that Enteromorpha polysaccharides and their degradation products Enteromorpha oligosaccharides can effectively remove bacteria in the pellicle state. In addition, the effect of Enteromorpha polysaccharides and its degradation products Enteromorpha oligosaccharides and antibiotic physical mixtures is significantly better than that of antibiotics at the same concentration .

Contents of the invention

The invention provides an antibacterial application of Enteromorpha polysaccharide and its degradation product Enteromorpha oligosaccharide.

The invention provides an Enteromorpha polysaccharide, which is characterized in that the raw material for preparing the Enteromorpha polysaccharide is Enteromorpha sea area.

As a better choice of the above polysaccharide of Enteromorpha, the molecular weight range of the polysaccharide of Enteromorpha is 5000Da-1000000Da.

The present invention also provides an Enteromorpha oligosaccharide, the molecular weight of the Enteromorpha polysaccharide ranges from 200 Da to 3000 Da, and the Enteromorpha oligosaccharide can be obtained by degrading the Enteromorpha polysaccharide.

The present invention also provides a kind of Enteromorpha oligosaccharide, comprising xylose, glucose, rhamnose, galactose and glucuronic acid in the Enteromorpha oligosaccharide, the molar ratio range of these components is 0.5-2:1- 3:2-6:0.1-1:1-5.

The present invention also provides a kind of Enteromorpha oligosaccharide, described Enteromorpha oligosaccharide is made up of xylose, glucose, rhamnose, galactose and glucuronic acid, and the molar ratio scope of these components is 0.5-2:1- 3:2-6:0.1-1:1-5.

Enteromorpha polysaccharide provided by the invention can be prepared according to the following method:

(1) Provide a mixture of Enteromorpha and water, the mass ratio of Enteromorpha and water in the mixture is 1:15-30, then add 0.5%-2% degrading enzyme, and react at 40-70°C for 4-6h , to obtain the reaction solution;

(2) The reaction solution is filtered to obtain an extract, and the extract is spray-dried at 60-80° C. to obtain a crude dry powder;

(3) The crude extracted dry powder is washed with a solvent, and after standing overnight, a precipitate is obtained, and the dry powder of Enteromorpha polysaccharide is obtained after spray drying.

Further, the degrading enzyme is papain, cellulase or pectinase.

The reaction temperature can also be adjusted according to actual conditions, such as being adjusted to 20-40 degrees Celsius.

As a better choice of the above method, the enteromorpha is a powder obtained by drying the raw material enteromorpha and grinding it into powder.

Further, the solvent in step 3) may be 75% ethanol.

Further, the Enteromorpha polysaccharide provided by the invention can be prepared according to the following method:

(1) Provide a mixture of Enteromorpha and water, the mass ratio of Enteromorpha and water in the mixture is 1:15-30, then add 0.5%-2% papain, and react at 40-70°C for 4-6 hours, get the reaction solution;

(2) The reaction solution is filtered to obtain an extract, and the extract is spray-dried at 60-80° C. to obtain a crude dry powder;

(3) The rough extracted dry powder is washed with 75% ethanol, and after standing overnight, a precipitate is obtained, and the dry powder of Enteromorpha polysaccharide is obtained after spray drying.

As a better choice of the above method, the enteromorpha is a powder obtained by drying the raw material enteromorpha and grinding it into powder.

Preferably, the Enteromorpha polysaccharide is prepared according to the following method:

(1) Provide a mixture of Enteromorpha and water, the mass ratio of Enteromorpha and water in the mixture is 1:20, then add 1% papain, and react at 65°C for 4 hours to obtain a reaction solution;

(2) The reaction solution described in step (1) is filtered to obtain an extract, and the extract is spray-dried at 60° C. to obtain a crude dry powder;

(3) The rough extracted dry powder in step (2) is washed with 75% ethanol, and after standing overnight, a precipitate is obtained, and the dry powder of Enteromorpha polysaccharide is obtained after spray drying.

Further, the present invention also provides a method for preparing Enteromorpha oligosaccharides, comprising:

A degradation agent is added to the dry powder of the enteromorpha polysaccharide to degrade the enteromorpha polysaccharide into enteromorpha oligosaccharides.

The degradation agent can be a biological degradation agent, such as an enzyme degradation agent; it can also be a chemical degradation agent, such as degradation by free radicals.

Preferably, the Enteromorpha polysaccharide is hydrolyzed and degraded by mixing trifluoroacetic acid, and the obtained reaction solution is freeze-dried to obtain Enteromorpha oligosaccharides. Here trifluoroacetic acid acts as a degradant, other chemical degradants that can be used here include but are not limited to hydrochloric acid, sulfuric acid, hydrogen peroxide and acetic acid.

Preferably, the Enteromorpha polysaccharide is degraded by an enzyme degradation agent, and the enzyme degradation agent that can be used includes cellulase, dextranase, carding glycosidase, galactosidase or xylanase.

In one embodiment of the present invention, a certain quality of Enteromorpha polysaccharide dry powder is added with 10mg/ml 1M trifluoroacetic acid, and hydrolyzed at 100°C for 60-120min to obtain a reaction solution; the reaction solution is freeze-dried to obtain Enteromorpha oligosaccharides. dry powdered sugar.

The present invention also provides the use of the above-mentioned Enteromorpha polysaccharides and Enteromorpha oligosaccharides as microorganism inhibitors, and the microorganisms are Gram-positive bacteria, Gram-negative bacteria and fungi. After the composition of Enteromorpha oligosaccharides or polysaccharides is defined, it can be used to inhibit microorganisms, rather than play a role in the proliferation of microorganisms.

Preferably, wherein said Gram-positive bacteria are Staphylococcus aureus, Streptococcus, Listeria monocytogenes.

Preferably, wherein said Gram-negative bacteria are Pseudomonas aeruginosa, Escherichia coli, Salmonella.

Preferably, said fungus is Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus.

The present invention also provides a pharmaceutical composition for the inhibitor of microorganisms deep in the biofilm, which comprises:

(1) Enteromorpha polysaccharide and/or Enteromorpha oligosaccharide;

(2) One or more antibacterial drugs.

The pharmaceutical composition can be provided in the form of powder. In the pharmaceutical composition, the content of Enteromorpha polysaccharides and/or Enteromorpha oligosaccharides is 5-99 wt%, and the proportion of antibacterial drugs can be adjusted according to actual needs.

When necessary, the pharmaceutical composition may also include an adjuvant, and the adjuvant may be a pharmaceutically acceptable substance used to assist shaping, flavoring agent and the like.

The present invention further provides a pharmaceutical composition for an inhibitor of microorganisms deep in a biofilm, which comprises:

(1) Enteromorpha polysaccharide and/or Enteromorpha oligosaccharide;

(2) One or more antibacterial drugs;

(3) solvent;

Wherein, the weight concentration of Enteromorpha polysaccharide in the composition is 10 μg/mL-5000 μg/mL

Preferably, the weight concentration of Enteromorpha polysaccharide in the composition is 100-500 μg/mL.

The pharmaceutical compositions of the present invention may also include water.

Preferably, the solvent is water.

Preferably, the mass ratio of Enteromorpha polysaccharide and antibiotic in the composition is 5:1˜1:5.

Preferably, the mass ratio of Enteromorpha polysaccharide and antibiotic in the composition is 1:1.

Preferably, the antibacterial drugs are aminoglycoside antibiotics.

Preferably, the antibacterial drug is streptomycin, kanamycin or gentamicin, more preferably streptomycin.

Preferably, the pharmaceutical composition also contains excipients.

The invention also provides the use of Enteromorpha polysaccharides and Enteromorpha oligosaccharides for preparing pharmaceutical compositions.

The invention relates to enteromorpha polysaccharide and its degradation product, enteromorpha oligosaccharide, which are used as active substances and prepared into antibacterial film drugs in various dosage forms with pharmaceutically acceptable auxiliary materials. Wherein the pharmaceutically acceptable adjuvant is an adjuvant commonly used in this field. The pharmaceutical excipients refer to the excipients and additives used in the production of medicines and formulation of prescriptions. In a specific implementation process, the present invention can be implemented and applied through various methods known in the art.

Wherein the antibacterial film drug is more preferably an antibacterial film drug. The bacteria are conventional pathogenic bacteria in this field. The bacteria are preferably Gram-positive and Gram-negative bacteria. The Gram-positive bacteria are preferably Staphylococcus aureus, and the Gram-negative bacteria are preferably Pseudomonas aeruginosa.

The present invention relates to a composition with antibacterial film effect, which comprises Enteromorpha polysaccharide or Enteromorpha oligosaccharide, solvent and antibacterial drug, wherein the weight concentration of Enteromorpha polysaccharide or Enteromorpha oligosaccharide in the composition is 10-5000 μg /mL, preferably 100-500 μg/mL.

As a better choice of the pharmaceutical composition with antibacterial film effect, the solvent is water.

As a better choice of the pharmaceutical composition with antibacterial film effect, the antibacterial drug includes one or more of antibiotics and antifungal drugs. The antibacterial drugs can be conventional antibacterial drugs in the art. The antibiotics are preferably aminoglycoside antibiotics. The aminoglycoside antibiotic is a glycoside antibiotic formed by linking aminosugar and aminocyclic alcohol through an oxygen bridge. The antibiotic is preferably streptomycin, kanamycin or gentamicin. The antibiotic of the present invention is preferably streptomycin.

The mass ratio of Enteromorpha polysaccharide and antibiotic in the composition is 5:1˜1:5, preferably 1:1.

The positive progress effect of the present invention is: experiment proves, described Enteromorpha polysaccharide, Enteromorpha oligosaccharide, Enteromorpha polysaccharide and antibiotic physical mixture, Enteromorpha oligosaccharide antibiotic physical mixture have stronger destruction to the bacterial biofilm that has formed It has a wide range of sterilization and has a good inhibitory effect on the formation of biofilm. At the same time, the Enteromorpha polysaccharide, Enteromorpha oligosaccharide, Enteromorpha polysaccharide and antibiotic physical mixture, and Enteromorpha oligosaccharide antibiotic physical mixture can effectively reduce the drug resistance produced when bacteria exist in the form of biofilm, and improve the resistance of bacteria to traditional antibiotics. Sensitivity, reduce the use of traditional antibiotics.

Description of drawings

Fig. 1, the molecular weight distribution of Enteromorpha polysaccharide identified by HPLC;

Fig. 2. The molecular weight distribution of Enteromorpha polysaccharide identified by HPLC-GPC;

Figure 3. The monosaccharide composition of Enteromorpha polysaccharide identified by capillary electrophoresis;

Figure 4. Identification of Enteromorpha oligosaccharides by MALDI-TOF mass spectrometry.

Detailed ways

The following are specific embodiments of the present invention, which are only used to explain the present invention rather than limit it.

Example 1 Preparation and structural characterization of Enteromorpha polysaccharides and Enteromorpha oligosaccharides

In this example, the preparation and structural characterization of Enteromorpha polysaccharides and Enteromorpha oligosaccharides were carried out. The specific implementation is as follows:

A certain quality of Enteromorpha enteromorpha is dried and ground into powder, mixed with 15 to 30 times the weight of water, then added with 0.5% to 2% papain, and reacted at 40 to 70°C for 4 to 6 hours to obtain a reaction solution; the reaction solution The supernatant is collected by centrifugation, and the supernatant is spray-dried at 60-80° C. to obtain a crude dry powder. The crude dry powder is washed with 75% ethanol, centrifuged to obtain the precipitate, and spray-dried to obtain the enteromorpha polysaccharide dry powder. Take a certain quality of Enteromorpha polysaccharide dry powder, add 10mg/ml 1M trifluoroacetic acid, hydrolyze at 100°C for 60-120min to obtain a reaction solution; freeze-dry the reaction solution to obtain Enteromorpha oligosaccharide dry powder.

Those skilled in the art can also refer to other technical solutions in the summary of the invention to optimize the reaction conditions, such as selecting cellulase, dextranase, carding glycosidase, galactosidase or xylanase to degrade it, which The amount added can be the same as that of papain, or can be adjusted according to actual needs.

The present invention characterizes the molecular weight distribution of Enteromorpha polysaccharide and Enteromorpha oligosaccharide by HPLC method, determines that the molecular weight range of Enteromorpha polysaccharide is 5000Da～1000000Da (see accompanying drawing 1), and Enteromorpha oligosaccharide molecular weight range is 200Da～3000Da (see Accompanying drawing 2), corresponding molecular weight distribution sees the following table:

time Peak area Peak height peak width Symmetry factor Peak area% Area% logMw Molecular weightDa 1 13.965 1846988.4 14351.4 1.778 0.707 51.6 51.6 3.4569865 2864.088939 2 17.001 1642169.5 80677.7 0.3044 1.176 45.877 45.877 2.6861461 485.4517824 3 17.556 83834.8 3574.6 0.3816 0.531 2.342 2.342 2.5452316 350.9389723 4 18.148 6475.9 346.8 0.2891 0.205 0.181 0.181 2.3949228 248.2691744

The monosaccharide composition of Enteromorpha polysaccharides was determined by capillary electrophoresis, and its components were determined to include xylose, glucose, rhamnose, galactose, and glucuronic acid, among which rhamnose and glucuronic acid were Enteromorpha polysaccharides The main constituents (see accompanying drawing 3) and following table.

The structure of Enteromorpha oligosaccharides was analyzed by mass spectrometry using MALDI-TOF mass spectrometry, and it was determined that it is mainly the core unit formed by rhamnose and sulfated rhamnose, and forms the main structure of its oligosaccharides with xylose and glucuronic acid (see Figure 4).

Applicant also provides different enteromorpha oligosaccharide compositions, and its composition is as follows:

Example 2 Enteromorpha polysaccharides and Enteromorpha oligosaccharides destroy the effect of Staphylococcus aureus film

In this example, the destruction effect of Enteromorpha polysaccharides and Enteromorpha oligosaccharides on the biofilm of Staphylococcus aureus was studied. The specific implementation is as follows:

The wild-type Staphylococcus aureus used was from the China General Microorganism Culture Collection and Management Center. Staphylococcus aureus was shaken and cultivated in TSB liquid medium at 37°C overnight, and 100 μL ~ 2×10 ⁷ CFU bacterial liquid was added to a 96-well plate for static culture at 37°C for 24 hours to form a mature biofilm.

Enteromorpha polysaccharide, Enteromorpha oligosaccharide, Enteromorpha polysaccharide+streptomycin mixture (mass ratio 1:1), Enteromorpha oligosaccharide+streptomycin mixture (mass ratio 1:1) were prepared for this study. After the bacterial film matured, remove the supernatant, and add 100 μL of TSB liquid medium containing 250 μg/mL of the above samples respectively. Add 100 μL TSB liquid medium to the untreated group. To the positive control group, 100 μL of TSB liquid medium containing 250 μg/mL streptomycin was added. After acting at 37°C for 24 hours, the effect of bacterial film destruction was detected by crystal violet staining.

The experimental results showed that Enteromorpha polysaccharide, Enteromorpha oligosaccharide group, Enteromorpha polysaccharide+streptomycin mixture group, and Enteromorpha oligosaccharide+streptomycin mixture group all had better bacterial film removal effects, among which Enteromorpha polysaccharide+streptomycin The mycin mixture group was the best, which significantly improved the bacterial film removal effect compared with the streptomycin group. See Table 1 below for specific results.

Table 1 Destruction effect of Staphylococcus aureus bacterial film

Group Bacteria removal rate (%) untreated group 0 Enteromorpha polysaccharide 17.1 Enteromorpha oligosaccharides 16.4 Enteromorpha polysaccharide + streptomycin mixture 37.8 Enteromorpha oligosaccharide + streptomycin mixture 14.3 streptomycin 0

The applicant also provides other treatment effects on Gram-positive bacterial film:

Group Types of biofilm Bacteria removal rate (%) Enteromorpha polysaccharide Streptococcus 12.3 Enteromorpha oligosaccharides Streptococcus 16.0 Enteromorpha polysaccharide single augmented lister 15.1 Enteromorpha oligosaccharides single augmented lister 18.5 untreated group Streptococcus 0 untreated group single augmented lister 0

Example 3 Enteromorpha polysaccharides and Enteromorpha oligosaccharides destroy effect experiment on Pseudomonas aeruginosa bacterial film

In this example, the destruction effect of Enteromorpha polysaccharides and Enteromorpha oligosaccharides on the bacterial film of Pseudomonas aeruginosa was studied. The specific implementation is as follows:

The wild-type Pseudomonas aeruginosa PAO1 used was from the China General Microorganism Culture Collection and Management Center. After Pseudomonas aeruginosa was shaken and cultivated in LB liquid medium at 37°C overnight, 100 μL ~ 2×10 ⁷ CFU bacterial solution was added to a 96-well plate and cultured statically at 30°C for 24 hours to form a mature bacterial film.

Enteromorpha polysaccharides and Enteromorpha oligosaccharides were prepared separately for this study. After the bacterial film matured, the supernatant liquid was removed, and 100 μL of LB liquid medium containing 250 μg/mL of the above sample was added respectively. Add 100 μL LB liquid medium to the untreated group. Add 100 μL LB liquid medium containing 250 μg/mL streptomycin to the positive control group. After acting at 30°C for 24 hours, the effect of bacterial film destruction was detected by crystal violet staining.

The experimental results show that both Enteromorpha polysaccharide and Enteromorpha oligosaccharide group have good effect on removing bacterial film, among which Enteromorpha polysaccharide has the best effect, which significantly improves the effect of removing bacterial film compared with streptomycin group. The specific results are shown in the table below 2

Table 2 Destruction effect of Pseudomonas aeruginosa biofilm

Group Bacteria removal rate (%) untreated group 0 Enteromorpha polysaccharide 35.0 Enteromorpha oligosaccharides 26.9 streptomycin 30.1

Applicants also provide other treatment effects on Gram-negative bacterial pellicles:

Group Types of biofilm Bacteria removal rate (%) untreated group Escherichia coli 0 Enteromorpha polysaccharide Escherichia coli 27.6 Enteromorpha oligosaccharides Escherichia coli 23.3 untreated group salmonella 0 Enteromorpha polysaccharide salmonella 28.9 Enteromorpha oligosaccharides salmonella 29.1

Example 4 Enteromorpha polysaccharides and Enteromorpha oligosaccharides inhibit the effect of Candida albicans film

In this example, the inhibitory effect of Enteromorpha polysaccharide on the bacterial film of Candida albicans was studied. The specific implementation is as follows:

The wild-type Candida albicans used was from the China General Microorganism Culture Collection and Management Center. After Candida albicans was shaken and cultured overnight at 30°C in YPD liquid medium, 100 μL ~ 2×10 ⁷ CFU bacterial solution was added to a 96-well plate. Add 100 μL of YPD liquid medium containing 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, and 5 mg/mL Enteromorpha polysaccharide, respectively. Add 100 μL YPD liquid medium to the untreated group. After static culture at 30°C for 24 hours, the effect of bacterial film inhibition was detected by crystal violet staining.

The experimental results show that Enteromorpha polysaccharides have good fungal film inhibitory effect, among which 2mg/mL Enteromorpha polysaccharides have the best effect, see the following table 3 for specific results

Table 3 Candida albicans film inhibitory effect

Group Bacteria removal rate (%) untreated group 0 1mg/mL Enteromorpha polysaccharide 5.2 2mg/mL Enteromorpha polysaccharide 30.6 3mg/mL Enteromorpha polysaccharide 15.5 4mg/mL Enteromorpha polysaccharide 13.3 5mg/mL Enteromorpha polysaccharide 28.8

The applicant also provides other treatment effects on fungal pellicles:

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit them. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications or equivalent replacements to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all of them should be included in the scope of the present invention. within the scope of the claims.

Claims (16)

1. a kind of sea grass polysaccharide, which is characterized in that the raw material for preparing sea grass polysaccharide is the Enteromorpha of Qingdao sea area.

2. sea grass polysaccharide according to claim 1, which is characterized in that the molecular weight ranges of the sea grass polysaccharide are 5000Da ~1000000Da.

3. kind of enteromorpha oligosaccharide, which is characterized in that the molecular weight ranges of the sea grass polysaccharide are 200Da~3000Da, and the Enteromorpha is few Sugar is obtained by any sea grass polysaccharide degradations of claim 1-2.

4. a kind of enteromorpha oligosaccharide, the enteromorpha oligosaccharide includes xylose, glucose, rhamnose, galactolipin and glucuronic acid.

5. sea grass polysaccharide or enteromorpha oligosaccharide are used as the purposes of microbial inhibitor, it is characterised in that：The microorganism is gram Positive bacteria, Gram-negative bacteria and fungi.

6. purposes according to claim 5, wherein the gram-positive bacteria is staphylococcus aureus.

7. purposes according to claim 5, wherein the Gram-negative bacteria is pseudomonas aeruginosa.

8. purposes according to claim 5, wherein the fungi is Candida albicans.

9. a kind of pharmaceutical composition of inhibitor for biofilm depths microorganism, it includes：

(1) sea grass polysaccharide and/or enteromorpha oligosaccharide；

(2) one or more antibacterials；

(3) solvent；

Wherein, sea grass polysaccharide weight concentration is 10 μ g/mL-5000 μ g/mL in the composition.

10. pharmaceutical composition according to claim 9, it is characterised in that：Sea grass polysaccharide weight concentration in the composition For 100-500 μ g/mL.

11. pharmaceutical composition according to claim 9, it is characterised in that：The solvent is water.

12. pharmaceutical composition according to claim 9, it is characterised in that：Sea grass polysaccharide and antibiotic in the composition Mass ratio be 5:1~1:5.

13. pharmaceutical composition according to claim 12, it is characterised in that：Sea grass polysaccharide and antibiotic in the composition Mass ratio be 1:1.

14. pharmaceutical composition according to claim 9, which is characterized in that the wherein described antibacterials are aminoglycoside Antibiotic.

15. the pharmaceutical composition according to claim 9 or 14, which is characterized in that the antibacterials are streptomysin, block that Mycin or gentamicin.

16. a kind of pharmaceutical composition of inhibitor for biofilm depths microorganism, it includes：

(1) sea grass polysaccharide and/or enteromorpha oligosaccharide；

(2) one or more antibacterials.