Preparation of complex microbial inoculant for degrading enteromorpha polysaccharide and culture condition optimization method of complex microbial inoculant

Abstract

The invention discloses a method for preparing a complex microbial inoculant for degrading enteromorpha polysaccharide and optimizing culture conditions of the complex microbial inoculant, the complex microbial inoculant for degrading enteromorpha polysaccharide is prepared through antagonism detection and strain combination screening, and the complex microbial inoculant for degrading enteromorpha polysaccharide is obtained through methods such as a single factor test, a pramipt-Berman test, a steepest climbing test and response surface analysis. According to the method, the enteromorpha polysaccharide degradation strain is used as a raw material to prepare the complex microbial inoculant, and compared with a single strain, the complex microbial inoculant can combine the functions of various strains in one preparation, so that the specificity of a degradation product can be improved; the complex microbial inoculant does not generate harmful by-products in the degradation process, the pollution of degradation products to the environment is reduced, theoretical support and practical basis are provided for enteromorpha degradation and research and development of biological fertilizers, and a new thought is provided for degradation and efficient utilization of enteromorpha resources.

Description

Technical Field

[0001] The present invention relates to a preparation method of a composite bacterial agent and a method for optimizing its culture conditions, and more specifically, to a preparation method of a composite bacterial agent for degrading enteromorpha polysaccharide and a method for optimizing its culture conditions, and belongs to the field of biotechnology. Background Art

[0002] Enteromorpha prolifera belongs to the genus Enteromorpha of the family Ulvae of the Chlorophyta. It is common in estuaries and mid-tidal zones, attached to rocks, seawalls, ships, and other places on the shore. It can be seen in the waters of Taiwan, Shandong, Fujian, Liaoning, and Zhejiang in my country. There was once a large-scale Enteromorpha green tide crisis in the Yellow Sea, covering an area of ​​nearly 1,746 km 2 , producing more than 24 million tons of biomass. The dense network structure of the Ulva increases the difficulty of cleaning. If the large-scale outbreak of Ulva is not cleaned up in time, it will cause serious damage to the marine ecology, leading to a decrease in dissolved oxygen in the water, a decrease in biodiversity, the production of harmful and toxic substances, aggravated eutrophication of water bodies, obstruction of ship navigation and many other problems.

[0003] At present, the large-scale outbreaks of Enteromorpha are treated as waste. After being salvaged, they are piled up in a centralized manner and shipped to the open sea after being decomposed. This has failed to achieve the high-value utilization of Enteromorpha resources. Enteromorpha contains extremely rich trace minerals, vitamins, carbohydrates, proteins and other biologically active compounds. Among them, Enteromorpha polysaccharides, as the main component of Enteromorpha biomass, play an important role. After biodegradation to produce algae oligosaccharides, compared with general sugars, Enteromorpha polysaccharides have multiple physiologically active functions such as immunomodulation, anti-tumor, anti-viral, antioxidant, and hypolipidemic. However, the structure of Enteromorpha polysaccharides is extremely complex, and the problem of low efficiency is often encountered during the utilization process. The currently commonly used physical and chemical degradation methods have problems such as poor product specificity, difficult reaction control, and high degradation costs. These shortcomings hinder the realization of efficient degradation treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide and optimizing its culture conditions, which has the technical characteristics of being able to improve the utilization efficiency of Enteromorpha resources and provide new ideas for the high-value application of Enteromorpha resources.

[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0006] The present invention provides a method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide, and the preparation method comprises the following steps:

[0007] Step 1: Conduct antagonistic tests on multiple strains that have the ability to degrade Enteromorpha polysaccharides to determine if there is no antagonistic effect between the strains;

[0008] Step 2: By gradually increasing the number of strains, different bacterial liquids are mixed in equal proportions to make different composite bacterial agents and find the composite bacterial agent with the best degradation effect.

[0009] Preferably, the strain antagonism detection in step 2 is specifically as follows: streak the bacterial liquid of marine strains D202, D223, D294, D442 and D447 on 2216 solid culture medium, wait for 1-2 minutes until there is no obvious bacterial liquid on the surface of the culture medium, streak another strain, repeat the above operation, so that each strain just intersects with each other, invert and culture in a 28°C incubator for 2-3 days, take out and observe whether an inhibition zone appears at the intersection of each strain. If so, it indicates that there is antagonism, otherwise there is no antagonism.

[0010] The marine strains D202, D223, D294, D442 and D447 used are Halopseudomonasgallaeciensis D202, Microbacterium aquimarisD223, ExiguobacteriumqingdaonenseD294, Rossellomorea arthrocnemi D442, and Limimaricola soesokkakensisD447 respectively.

[0011] Preferably, the strain combination screening in step 2 is specifically as follows:

[0012] The screened Enteromorpha polysaccharide-degrading strains with no antagonistic effect on each other were taken, diluted to the same concentration using 2216 liquid culture medium, and then mixed in equal proportions by gradually increasing the number of strains to form different composite bacterial agents. The different mixed bacterial agents were inoculated into the fermentation medium at a ratio of 5% (v / v). After culturing at 160 rpm and 28°C for 3 days, the Enteromorpha polysaccharide degradation abilities of the different mixed bacterial agents were measured, and the composite bacterial agent with the best degradation ability was selected for the experiment.

[0013] Preferably, the degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents is determined by DNS (3,5-dinitrosalicylic acid) method, and the specific steps are as follows:

[0014] 1) Add 1 mL of 1% (w / v) Enteromorpha polysaccharide solution to a 20 mL constant volume tube and preheat in a 30°C water bath for 5 min;

[0015] 2) Take 2 mL of bacterial solution, centrifuge at 10000 rpm for 10 min, take the supernatant to obtain crude enzyme solution, dilute the crude enzyme solution, add 2 mL of diluted crude enzyme solution to the preheated Enteromorpha polysaccharide solution, shake well and react at 30°C for 30 min, and perform the same operation with the crude enzyme solution inactivated at high temperature as a blank control group;

[0016] 3) After the reaction is completed, add 1 mL of DNS solution to the constant volume tube, mix well and place in a boiling water bath for 5 min. After cooling to room temperature, add deionized water to 20 mL and measure the OD 540nm ;

[0017] 4) Based on the measured OD 540nm Value, according to sugar concentration-OD 540nm The standard curve was used to calculate the reducing sugar concentration in the solution, and then the degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents was calculated based on the reaction time.

[0018] Preferably, the DNS solution is prepared as follows: take a 1000 mL beaker, quickly add 700 mL ddH2O, 182 g potassium sodium tartrate, 6.3 g 3,5-dinitrosalicylic acid, 21 g NaOH, 5 g phenol, and 5 g anhydrous Na2SO3 in sequence, stir well, and fill to 1000 mL after all ingredients are completely dissolved to obtain a DNS solution, which is stored in a brown bottle for later use.

[0019] Preferably, the sugar concentration-OD 540nm The specific steps for drawing the standard curve are:

[0020] 1) Dilute 1g / L glucose solution to 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L;

[0021] 2) Take a 20mL constant volume tube, add 1mL of diluent and 1mL of DNS solution, then place in a boiling water bath and react for 5 minutes;

[0022] 3) After cooling to room temperature, add deionized water to make up to 20 mL;

[0023] 4) Treat the sample with deionized water in the same way as the blank control and measure the OD of the reaction solution. 540nm Absorbance;

[0024] 5) Plot sugar concentration-OD 540nm Standard curve.

[0025] Preferably, the definition and calculation method of the degradation capacity of Enteromorpha polysaccharide is as follows: the amount of crude enzyme solution required to catalyze the substrate to produce 1 μmol of reducing sugar per minute under the reaction conditions of 30° C. is used to represent 1 enzyme activity unit (U).

[0026] The present invention provides a method for optimizing the culture conditions of a composite bacterial agent for degrading Enteromorpha polysaccharide, comprising the following steps:

[0027] Step A: Taking the degradation ability of Enteromorpha polysaccharide of the composite bacterial agent as the investigation index, the carbon source, nitrogen source, metal ions, carbon source concentration, nitrogen source concentration, culture temperature, culture pH and initial salinity factors were used as the investigation objects for single factor analysis to determine the optimal influence range of each factor on the investigation index;

[0028] Step B: Based on the optimal range of each influencing factor determined by single factor analysis, the degradation capacity of Enteromorpha polysaccharide of the composite bacterial agent was used as the investigation index, and the Praxter-Berman test scheme was designed to find out the factors that have the most significant impact on the investigation index;

[0029] Step C: Based on the three factors that have the most significant impact on the degradation of Enteromorpha polysaccharide by the composite bacterial agent screened by the Praxter-Berman test, a reasonable step length and steepest climbing test scheme were designed according to the partial positive regression coefficients corresponding to the significant factors in the regression equation. The degradation ability of Enteromorpha polysaccharide by the composite bacterial agent was used as the investigation index to obtain the appropriate step length of each factor for subsequent response surface analysis;

[0030] Step D: Based on the appropriate step length of each factor for response surface analysis, a 3-factor 3-level response surface analysis experiment was designed using Design-expert software. The degradation capacity of the composite bacterial agent for Enteromorpha polysaccharide was used as the response value to determine the optimal culture conditions of the composite bacterial agent.

[0031] Preferably, the carbon source is analyzed: different carbon sources are added to the fermentation medium, 5% of the composite bacterial agent is added, and the degradation ability of Enteromorpha polysaccharide is measured after culturing at 160rpm and 28°C for 3 days; the carbon source includes Enteromorpha polysaccharide, maltose, sucrose, glucose, starch, and Ulva polysaccharide;

[0032] Analysis of carbon source concentration: The carbon source was prepared into fermentation medium with a concentration of 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, and 1.4% w / v, and 5% of the composite bacterial agent was added. The degradation capacity of Enteromorpha polysaccharide was measured at 160 rpm and 28°C for 3 days.

[0033] Analysis of nitrogen sources: Fermentation medium containing different nitrogen sources was prepared, and 5% of the composite bacterial agent was added respectively. The degradation capacity of Enteromorpha polysaccharide was measured after culturing at 160rpm and 28℃ for 3 days. The nitrogen sources included urea, peptone, yeast extract, beef extract, and ammonium sulfate.

[0034] Analysis of nitrogen source concentration: Fermentation medium containing w / v concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% was prepared, and 5% of the composite bacterial agent was added respectively. The degradation capacity of Enteromorpha polysaccharide was determined after culturing at 160 rpm and 28°C for 3 days;

[0035] Analysis of metal ions: 1% Enteromorpha powder was added to the metal ion combination to prepare a culture medium, and 5% of the composite bacterial agent was added to each culture medium. After culturing at 160 rpm and 28°C for 3 days, the degradation capacity of Enteromorpha polysaccharide was determined.

[0036] Analysis of culture temperature: 5% of the composite bacterial agent was added to the pre-prepared fermentation medium, and the medium was placed in a shaker at 160 rpm at 25°C, 30°C, 35°C, 40°C and 45°C for 3 days to determine the degradation capacity of Enteromorpha polysaccharide;

[0037] Analysis of culture pH: The optimal culture pH was determined by adjusting the fermentation medium to different pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0, and 40 mmol of different buffers 2-(N-morpholino)ethane sulfonic acid (pH 5.0-6.0), 3-morpholinopropanesulfonic acid (pH 6.5-7.5), and tricine buffer (pH8.0-8.5) were added for pH buffering, and 5% of the composite bacterial agent was inoculated. After culturing at 160 rpm and 28°C for 3 days, the degradation capacity of Enteromorpha polysaccharide was determined.

[0038] Analysis of initial salinity: The salinity was determined by using a fermentation medium containing different w / v NaCl concentrations of 0.5%, 1%, 2%, 3%, and 4%, inoculated with 5% composite bacterial agent, and cultured at 160 rpm and 28°C for 3 days to determine the degradation capacity of Enteromorpha polysaccharide;

[0039] The fermentation medium includes 0.5 g of peptone, 0.1 g of yeast extract powder, 20 g of NaCl, 5.9 g of MgCl2, 3.24 g of MgSO4, 1.8 g of CaCl2, 0.55 g of KCl, 10 g of Enteromorpha powder, and ddH2O diluted to 1000 mL.

[0040] Preferably, in step D, Box-Behnken Design is used to perform response surface analysis.

[0041] Beneficial effects: The present invention uses Enteromorpha polysaccharide-degrading strains as raw materials to make a composite bacterial agent. Compared with a single strain, the composite bacterial agent can combine the functions of multiple strains in one preparation, which can improve the specificity of degradation products; the composite bacterial agent will not produce harmful by-products during the degradation process, and reduce the pollution of the degradation products to the environment, providing theoretical support and practical basis for the research and development of Enteromorpha degradation and biofertilizer, and providing new ideas for the degradation and efficient utilization of Enteromorpha resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the antagonism detection among five Enteromorpha polysaccharide-degrading strains.

[0043] Figure 2 It is a bar graph showing the degradation ability of Enteromorpha polysaccharide by the composite bacterial agent under different single factor conditions.

[0044] Figure 3 It is a bar graph showing the degradation ability of Enteromorpha polysaccharide by the composite bacterial agent under different single factor conditions.

[0045] Figure 4 It is a bar graph showing the degradation ability of Enteromorpha polysaccharide by the composite bacterial agent under different single factor conditions.

[0046] Figure 5 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0047] Figure 6 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0048] Figure 7 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0049] Figure 8 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0050] Fig. 9 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0051] Fig.10 It is a line graph of the degradation ability of the composite bacterial agent into Enteromorpha polysaccharide under different single factor conditions.

[0052] Fig.11 is a half-normal probability effects plot for the standardized effects.

[0053] Fig.12 Pareto chart of the standardized effects. DETAILED DESCRIPTION

[0054] The following is in conjunction with the instruction manual Figure 1-12 , the present invention is further illustrated, but the present invention is not limited to the following embodiments.

[0055] Aiming at the problems of waste of enteromorpha resources and low efficiency of existing degradation methods, the present invention provides a preparation method of an enteromorpha polysaccharide-degrading composite bacterial agent and a culture condition optimization scheme thereof, which can improve the utilization efficiency of enteromorpha resources.

[0056] Embodiment 1:

[0057] Preparation of composite bacterial agent for degradation of Enteromorpha polysaccharide:

[0058] (1) Strain antagonism detection

[0059] Take appropriate amount of bacterial liquid of marine strains D202, D223, D294, D442 and D447 and streak on 2216 solid culture medium. Wait for 1-2 minutes until there is no obvious bacterial liquid on the surface of the culture medium. Then take another strain and streak. Repeat the above operation so that each strain just intersects with each other. Invert and culture in a 28℃ incubator for 2-3 days. Take out and observe whether there is an inhibition zone at the intersection of each strain. If there is, it means there is antagonism. Otherwise, there is no antagonism.

[0060] The marine strains D202, D223, D294, D442 and D447 used are Halopseudomonasgallaeciensis D202, Microbacterium aquimarisD223, ExiguobacteriumqingdaonenseD294, Rossellomorea arthrocnemi D442, and Limimaricola soesokkakensisD447 respectively.

[0061] (2) Strain combination screening

[0062] The screened Enteromorpha polysaccharide-degrading strains that have no antagonistic effect on each other were diluted to the same concentration using 2216 liquid culture medium, and then mixed in equal proportions to form different composite bacterial agents by gradually increasing the number of strains. Different mixed bacterial agents were inoculated into the fermentation medium at a ratio of 5% (v / v), and after culturing at 160rpm and 28°C for 3 days, the Enteromorpha polysaccharide degradation ability of different mixed bacterial agents was determined, and the composite bacterial agent with the best degradation ability was selected for the next experiment.

[0063] The degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents was determined by DNS (3,5-dinitrosalicylic acid) method, and the specific steps were as follows:

[0064] 1) Add 1 mL of 1% (w / v) Enteromorpha polysaccharide solution into a 20 mL constant volume tube and preheat in a 30°C water bath for 5 min.

[0065] 2) Take 2 mL of bacterial solution, centrifuge it at 10000 rpm for 10 min, take the supernatant to obtain the crude enzyme solution, dilute it a certain number of times to obtain the diluted crude enzyme solution of appropriate concentration, add 2 mL of the diluted crude enzyme solution to the preheated Enteromorpha polysaccharide solution, shake well and react at a constant temperature of 30°C for 30 min, and perform the same operation with the crude enzyme solution inactivated at high temperature as the blank control group.

[0066] 3) After the reaction, add 1 mL of DNS solution to the constant volume tube, mix well and place in a boiling water bath for 5 min. After cooling to room temperature, add deionized water to 20 mL and measure OD 540nm .

[0067] 4) Based on the measured OD 540nm Value, according to sugar concentration-OD 540nm The standard curve was used to calculate the reducing sugar concentration in the solution, and then the degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents was calculated based on the reaction time.

[0068] The DNS solution was prepared by taking a 1000 mL beaker, and adding 700 mL ddH2O, 182 g potassium sodium tartrate, 6.3 g 3,5-dinitrosalicylic acid, 21 g NaOH, 5 g phenol, and 5 g anhydrous Na2SO3 in sequence, stirring thoroughly, and filling to 1000 mL after all ingredients were completely dissolved to obtain a DNS solution, which was stored in a brown bottle for later use.

[0069] Sugar concentration-OD 540nm The specific steps for drawing the standard curve are as follows:

[0070] 1) Dilute 1g / L glucose solution to 0.11g / L, 0.21g / L, 0.31g / L, 0.41g / L, 0.51g / L, 0.61g / L, 0.71g / L, 0.81g / L;

[0071] 2) Take a 20mL constant volume tube, add 1mL of diluent and 1mL of DNS solution, then place in a boiling water bath and react for 5 minutes;

[0072] 3) After cooling to room temperature, add deionized water to make up to 20 mL;

[0073] 4) Treat the sample with deionized water in the same way as the blank control and measure the OD of the reaction solution. 540nm Absorbance;

[0074] 5) Plot sugar concentration-OD 540nm Standard curve.

[0075] The definition and calculation method of the degradation capacity of Enteromorpha polysaccharide is as follows: the amount of crude enzyme solution required to catalyze the substrate to produce 1 μmol of reducing sugar per minute under the reaction condition of 30° C. is used to represent 1 enzyme activity unit (U).

[0076] (3) Results analysis

[0077] like Figure 1As shown in the figure, each strain grows well at the intersection without antagonism, so any two of the 5 strains selected can be used in combination. As shown in Table 1, it was found that with the increase in the number of strains in the combination, the enzyme activity gradually increased. When the 5 strains were combined to make composite bacterial agent 4, the degradation capacity of Enteromorpha polysaccharide reached the highest 1.22U, which was higher than the sum of the enzyme activities of each single strain. It may be that there was a synergistic effect between the strains. Therefore, it is considered to select composite bacterial agent 4 as the composite bacterial agent for subsequent experimental research.

[0078] Table 1 shows the degradation capacity of Enteromorpha polysaccharide by cumulative combination of different strains

[0079]

[0080] Embodiment 2:

[0081] Optimization method for the culture conditions of Enteromorpha polysaccharide degradation composite bacterial agent:

[0082] (1) Preparation of fermentation medium

[0083] Peptone 0.5g, yeast extract powder 0.1g, NaCl 20g, MgCl2 5.9g, MgSO4 3.24g, CaCl21.8g, KCl0.55g, Enteromorpha powder 10g, and dilute to 1000mL with ddH2O.

[0084] (2) Single-factor experimental analysis of culture conditions

[0085] 1) Carbon source

[0086] Different carbon sources (Enteromorpha polysaccharide, maltose, sucrose, glucose, starch, Ulva polysaccharide) were added to the fermentation medium, 5% of the composite bacterial agent was inoculated and cultured at 160 rpm and 28°C for 3 days, and then the degradation capacity of Enteromorpha polysaccharide was measured.

[0087] 2) Carbon source concentration

[0088] The optimal carbon source was prepared into fermentation medium with different concentrations (0.4, 0.6, 0.8, 1.0, 1.2, 1.4%, w / v), inoculated with 5% composite bacterial agent, and cultured at 160 rpm, 28°C for 3 days to measure the degradation capacity of Enteromorpha polysaccharide.

[0089] 3) Nitrogen source

[0090] Fermentation medium containing different nitrogen sources (urea, peptone, yeast extract, beef extract, ammonium sulfate) was prepared (other components remained unchanged, 0.07 mol of nitrogen source was added), 5% of the composite bacterial agent was inoculated respectively, and the degradation ability of Enteromorpha polysaccharide was measured after culturing at 160rpm and 28°C for 3 days.

[0091] 4) Nitrogen source concentration

[0092] Fermentation medium containing different concentrations (0.1, 0.3, 0.5, 0.7, 0.9%, w / v) of the optimal nitrogen source was prepared, 5% of the composite bacterial agent was inoculated respectively, and the degradation capacity of Enteromorpha polysaccharide was measured after culturing at 160rpm and 28°C for 3 days.

[0093] 5) Metal ions

[0094] A commonly used ion combination was selected and 1% Enteromorpha powder was added to prepare a culture medium, and 5% of the composite bacterial agent was added respectively. After culturing at 160 rpm and 28°C for 3 days, the degradation capacity of Enteromorpha polysaccharide was determined.

[0095] 6) Culture temperature

[0096] 5% of the composite bacterial agent was inoculated into the pre-prepared fermentation medium, and the medium was cultured in a shaker at 160 rpm at different temperatures (25, 30, 35, 40 and 45° C.) for 3 days, and then the degradation capacity of Enteromorpha polysaccharide was measured.

[0097] 7) Culture pH

[0098] The optimal culture pH was determined by adjusting the fermentation medium to different pH values ​​(6.0, 6.5, 7.0, 7.5, 8.0). 40 mmol of different buffers, 2-(N-morpholino)ethane sulfonic acid (pH 5.0-6.0), 3-morpholinopropanesulfonic acid (pH 6.5-7.5) and tricine buffer (pH 8.0-8.5), were added for pH buffering. 5% of the composite bacterial agent was inoculated and cultured at 160 rpm and 28°C for 3 days. Then, the degradation ability of Enteromorpha polysaccharide was determined.

[0099] 8) Initial salinity

[0100] The salinity was determined by using a fermentation medium containing different concentrations (0.5, 1, 2, 3, 4%, w / v) of NaCl, inoculated with 5% composite bacterial agent, and cultured at 160 rpm and 28°C for 3 days to determine the degradation capacity of Enteromorpha polysaccharide.

[0101] (3) Plackett-Burman test analysis of culture conditions

[0102] On the basis of single factor analysis, factors favorable to the degradation of Enteromorpha polysaccharide by composite bacterial agent were selected from the optimum carbon source concentration, optimum nitrogen source concentration, optimum metal ions, culture temperature, culture pH and initial salinity, and the degradation ability of Enteromorpha polysaccharide was used as the investigation index to design the Prakt-Berman experimental scheme. The results were input into Design-Expert software to obtain the results of the Prakt-Berman test.

[0103] (4) Analysis of reasonable step length and steepest climbing test of culture conditions

[0104] Based on the results of the Praxter-Berman test, three factors with the most significant impact on the degradation of Enteromorpha polysaccharides by the composite bacterial agent (initial concentration of Enteromorpha polysaccharides, culture temperature and culture pH) were screened out, and a reasonable step length and steepest climbing experimental plan were designed according to the regression equation.

[0105] (5) Response surface analysis of culture conditions

[0106] Based on the results of the steepest climbing experiment, the range of three influencing factors was designed. The degradation capacity of Enteromorpha polysaccharide was used as the response value, and 17 groups of experiments were set up for the Box-Benhnken experiment in the response surface analysis. The experiment was carried out according to the designed experimental plan, and the results obtained from the experiment were input into the Design-Expert software. The data fitting and variance analysis were performed according to the software to obtain a multivariate regression equation model. Then, the obtained model was solved and analyzed using the software to determine the optimal conditions and predict the optimal response value.

[0107] like Figure 2-10 As shown in the figure, single factor experiments were first conducted on carbon source, nitrogen source and metal ion, and the factor with the greatest influence on the degradation efficiency of Enteromorpha polysaccharide in the composite bacterial agent was selected. Figure 2 , 3 4 It can be seen that Enteromorpha polysaccharide, yeast extract powder and Ca 2+ It has a great influence on the degradation effect of the composite bacterial agent Enteromorpha polysaccharide. Further single-factor experiments were used to find the optimal concentration of Enteromorpha polysaccharide. Figure 5 It can be seen that the degradation effect of Enteromorpha polysaccharide is best when the concentration reaches 0.6%. Figure 6 It can be found that the optimal yeast extract concentration of the composite bacterial agent is 0.3%. During the experiment, when the concentration continued to rise, the strain concentration increased significantly, but the degradation of Enteromorpha polysaccharide decreased. It should be that the fast growth of the strain consumes more nutrients and capacity, resulting in a reduction in the resources used for enzyme production. Figure 7 , 8 , 9, 10, we can see that the optimum Ca +2The concentration, culture temperature, culture pH, and initial salinity are 0.3%, 30°C, 7.5, and 2%, respectively. The range of the most suitable influencing factor in the single-factor experiment will be used as the center point of the Praxter-Berman test.

[0108] As shown in Table 2, the data were subjected to multiple regression fitting, and the regression equation of the Praxter-Berman test was obtained as follows: Y = 1.14 + 0.0483A + 0.05B ​​- ​​0.015C + 0.015D + 0.0567E + 0.025F. Fig.11 As shown in the figure, the standardized effect points of factors A, B and E are far away from the fitting line, so they are determined to be significant influencing factors (P<0.05), that is, the significant factors affecting the degradation of Enteromorpha polysaccharide by the composite bacterial agent are the initial concentration of Enteromorpha polysaccharide, culture temperature and culture pH, and the standardized effect points of other factors are smaller. Fig.12 The magnitude and importance of the effect can be further determined. Factors A, B, and E all exceed the t value and are considered significant factors.

[0109] Table 2 Experimental design and results of the Praxter-Berman test

[0110]

[0111] As shown in Table 3, the P value of the experimental model is 0.0091<0.05, which is a significant result, indicating that the model has a good fit in the regression interval studied and the model is reliable; the multiple correlation coefficient R 2 =0.9303, indicating a good correlation; the corrected determination coefficient R 2 adj =0.8467, indicating that this regression model can explain 84.67% of the variability of the experimental data; the coefficient of variation (CV) =3.56%, and the precision is the ratio of the effective signal to the noise (Adeq Precisior) =12.6091, so the experiment has good accuracy.

[0112] Table 3 shows the influence of various factors on the degradation efficiency of Enteromorpha polysaccharides in the Praxter-Berman test and the results of variance analysis

[0113]

[0114] As shown in Table 4, the composite bacterial agent Enteromorpha polysaccharide degradation efficiency of experimental group c was the highest, and it was considered as the central point of the response surface analysis, that is, the initial concentration of Enteromorpha polysaccharide was set to 0.6%, the culture temperature was set to 29°C, and the culture pH was set to 7.5.

[0115] Table 4 shows the experimental design and results of the steepest climbing test

[0116]

[0117] As shown in Table 5, the data were fitted with a quadratic multivariate regression, and the quadratic polynomial equation was obtained as follows: Y = 1.27-0.0887A-0.0525B+0.0213C-0.0375AB-0.055AC+0.1025BC-0.2355A 2 -0.103B 2 -0.1155C 2 .

[0118] Table 5 shows the experimental design and results of the Box-Behnken response surface methodology experiment.

[0119]

[0120] As shown in Table 6, the P value of the experimental model is <0.0001, which is a significant result, indicating that the model has a good fit in the regression interval studied and the model is reliable; the multiple correlation coefficient R 2 =0.9928, indicating a good correlation; the corrected determination coefficient R 2 adj =0.9835, indicating that this regression model can explain 98.35% of the response value changes; coefficient of variation (CV) = 2.2%, indicating that the higher the reliability and accuracy of the experiment. By comparing the F value, it was found that the influence of each factor on the degradation efficiency of the composite bacterial agent Enteromorpha polysaccharide was as follows: initial concentration of Enteromorpha polysaccharide> culture temperature> culture pH. After correction according to the actual situation, the optimal culture conditions of the composite bacterial agent after response surface optimization were as follows: initial concentration of Enteromorpha polysaccharide was 0.56%, culture temperature was 28℃, and culture pH was 7.5.

[0121] Table 6 shows the effects of various factors on the degradation efficiency of Enteromorpha polysaccharide in the Box-Behnken response surface methodology experiment and the results of variance analysis

[0122]

[0123] Embodiment 3:

[0124] Effects of Compound Microbial Agents on the Growth of Rice Seedlings

[0125] (1) Preparation of Enteromorpha liquid treated with composite bacterial agent

[0126] According to the optimized composite bacterial agent culture conditions, the components of the fermentation medium were adjusted accordingly, and 5% of the fresh composite bacterial agent and 5% of the fresh bacterial solution of strain D294 were added respectively, and cultured at the corresponding optimal temperature for 3 days. After centrifugation at 8000rpm for 15min, the supernatant was retained, and part of the treated liquid was taken out and filtered using a filter membrane with a pore size of 0.45μm to obtain the composite bacterial agent treated Enteromorpha solution and the strain D294 treated Enteromorpha solution for subsequent experiments. Among them, because strain D294 has the highest single strain Enteromorpha polysaccharide degradation ability, this strain was selected as the research object to verify the degradation effect of single strain Enteromorpha.

[0127] (2) Experimental group design

[0128] The Enteromorpha treatment solutions obtained previously were divided into 10 treatment groups.

[0129] Treatment 1: The compound bacterial agent-treated Enteromorpha solution was diluted 5 times with water;

[0130] Treatment 2: The compound bacterial agent-treated Enteromorpha solution was diluted 50 times with water;

[0131] Treatment 3: The compound bacterial agent-treated Enteromorpha solution was diluted 100 times with water;

[0132] Treatment 4: The Enteromorpha solution treated with strain D294 was diluted 5 times with water;

[0133] Treatment 5: strain D294 treated Enteromorpha solution diluted 50 times with water;

[0134] Treatment 6: strain D294 treated Enteromorpha solution diluted 100 times with water;

[0135] Treatment 7: 5 times the amount of water in the adjusted fermentation medium;

[0136] Treatment 8: Adjust the fermentation medium to dilute 50 times with water;

[0137] Treatment 9: Adjust the fermentation medium to dilute 100 times with water;

[0138] Treatment 10: Clear water was used as a blank control.

[0139] (3) Pretreatment of rice seeds and recording of plant growth

[0140] In this experiment, indica rice (long grain) was used as the experimental object. Uniform and full seeds were selected and poured onto clean paper. Large particles of impurities were picked out, and then the seeds were poured into a 1.5mL EP tube sterilized by high temperature. 1mL 70% ethanol was added. After slight shaking for 3 minutes, the seeds were rinsed 6 times with sterile water and blown dry on a sterile workbench. After the seeds were placed in a refrigerator at 4°C for 24 hours, they were placed in a culture dish padded with sterilized gauze and sprayed with the treatment solution twice a day. When the rice leaves reached about 10cm in length, the rice was transplanted into the culture pot for liquid culture according to different treatment groups. 5mL of the corresponding treatment solution was added to the culture pot every 3 days and the water was changed regularly. After the rice seeds were treated for 15 days, the various growth parameters of the rice plants were measured. The data of plant height, taproot length and wet weight were recorded.

[0141] As shown in Table 7, the plant height, root length and wet weight of rice seedlings treated with the composite bacterial agent Enteromorpha solution were improved. The most significant effect was in Treatment Group 2, which was the Enteromorpha solution treated with the composite bacterial agent diluted 50 times with clean water. Compared with the corresponding single strain treatment group and culture medium treatment group, the plant height increased by 0.4 cm and 0.8 cm, respectively, and increased by 1.3 cm compared with the clean water group, indicating that the nutrients and plant growth-promoting active substances of Enteromorpha treated with the composite bacterial agent were released.

[0142] Table 7 is a table showing the effects of different treatment groups on the growth of rice seedlings

[0143]

[0144] In summary, the present invention prepares the Enteromorpha polysaccharide-degrading composite bacterial agent through antagonism detection and strain combination screening, and obtains the optimal culture conditions of the Enteromorpha polysaccharide-degrading composite bacterial agent through single factor experiment, Praxter-Berman experiment, steepest climbing experiment and response surface analysis, etc., provides a preparation scheme for the Enteromorpha polysaccharide-efficient degradation composite bacterial agent and a method for optimizing its culture conditions, and provides new ideas for the high-value application of Enteromorpha resources.

[0145] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide, characterized in that The preparation method comprises the following steps: Step 1: Conduct antagonistic tests on multiple strains that have the ability to degrade Enteromorpha polysaccharides to determine if there is no antagonistic effect between the strains; Step 2: By gradually increasing the number of strains, different bacterial liquids are mixed in equal proportions to make different composite bacterial agents and find the composite bacterial agent with the best degradation effect.

2. The method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 1, characterized in that: The strain antagonism test in step 2 is specifically as follows: take the bacterial liquid of marine strains D202, D223, D294, D442 and D447 and streak it on 2216 solid culture medium, wait for 1-2 minutes until there is no obvious bacterial liquid on the surface of the culture medium, take another strain and streak it, repeat the above operation, so that each strain just intersects with each other, invert it in a 28°C incubator and culture it for 2-3 days, then take it out and observe whether an inhibition zone appears at the intersection of each strain. If it appears, it means that there is antagonism, otherwise there is no antagonism.

3. A method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 1 or 2, characterized in that: The strain combination screening in step 2 is specifically as follows: The screened Enteromorpha polysaccharide-degrading strains with no antagonistic effect on each other were taken, diluted to the same concentration using 2216 liquid culture medium, and then mixed in equal proportions by gradually increasing the number of strains to form different composite bacterial agents. The different mixed bacterial agents were inoculated into the fermentation medium at a ratio of 5% (v / v). After culturing at 160 rpm and 28°C for 3 days, the Enteromorpha polysaccharide degradation abilities of the different mixed bacterial agents were measured, and the composite bacterial agent with the best degradation ability was selected for the experiment.

4. The method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 3, characterized in that: The degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents was determined by DNS (3,5-dinitrosalicylic acid) method, and the specific steps were as follows: 1) Add 1 mL of 1% (w / v) Enteromorpha polysaccharide solution to a 20 mL constant volume tube and preheat in a 30°C water bath for 5 min; 2) Take 2 mL of bacterial solution, centrifuge at 10000 rpm for 10 min, take the supernatant to obtain crude enzyme solution, dilute the crude enzyme solution, add 2 mL of diluted crude enzyme solution to the preheated Enteromorpha polysaccharide solution, shake well and react at 30°C for 30 min, and perform the same operation with the crude enzyme solution inactivated at high temperature as a blank control group; 3) After the reaction is completed, add 1 mL of DNS solution to the constant volume tube, mix well and place in a boiling water bath for 5 min. After cooling to room temperature, add deionized water to 20 mL and measure the OD 540nm ; 4) Based on the measured OD 540nm Value, according to sugar concentration-OD 540nm The standard curve was used to calculate the reducing sugar concentration in the solution, and then the degradation capacity of Enteromorpha polysaccharide of different mixed bacterial agents was calculated based on the reaction time.

5. The method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 4, characterized in that: Preparation of the DNS solution: Take a 1000mL beaker, quickly add 700mL ddH2O, 182g potassium sodium tartrate, 6.3g 3,5-dinitrosalicylic acid, 21g NaOH, 5g phenol, and 5g anhydrous Na2SO3 in sequence, stir well, and fill to 1000mL after all ingredients are completely dissolved to obtain a DNS solution, which is stored in a brown bottle for later use.

6. A method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 4 or 5, characterized in that: The sugar concentration-OD 540nm The specific steps for drawing the standard curve are: 1) Dilute 1g / L glucose solution to 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L; 2) Take a 20mL constant volume tube, add 1mL of diluent and 1mL of DNS solution, then place in a boiling water bath and react for 5 minutes; 3) After cooling to room temperature, add deionized water to make up to 20 mL; 4) Treat the sample with deionized water in the same way as the blank control and measure the OD of the reaction solution. 540nm Absorbance; 5) Plot sugar concentration-OD 540nm Standard curve.

7. The method for preparing a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 4, characterized in that: The definition and calculation method of the degradation capacity of Enteromorpha polysaccharide is as follows: the amount of crude enzyme solution required to catalyze the substrate to produce 1 μmol of reducing sugar per minute under the reaction condition of 30° C. is used to represent 1 enzyme activity unit (U).

8. A method for optimizing the culture conditions of the composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 1, characterized in that The following steps are involved: Step A: Taking the degradation ability of Enteromorpha polysaccharide of the composite bacterial agent as the investigation index, the carbon source, nitrogen source, metal ions, carbon source concentration, nitrogen source concentration, culture temperature, culture pH and initial salinity factors were used as the investigation objects for single factor analysis to determine the optimal influence range of each factor on the investigation index; Step B: Based on the optimal range of each influencing factor determined by single factor analysis, the degradation capacity of Enteromorpha polysaccharide of the composite bacterial agent was used as the investigation index, and the Praxter-Berman test scheme was designed to find out the factors that have the most significant impact on the investigation index; Step C: Based on the three factors that have the most significant impact on the degradation of Enteromorpha polysaccharide by the composite bacterial agent screened by the Praxter-Berman test, a reasonable step length and steepest climbing test scheme were designed according to the partial positive regression coefficients corresponding to the significant factors in the regression equation. The degradation ability of Enteromorpha polysaccharide by the composite bacterial agent was used as the investigation index to obtain the appropriate step length of each factor for subsequent response surface analysis; Step D: Based on the appropriate step length of each factor for response surface analysis, a 3-factor 3-level response surface analysis experiment was designed using Design-expert software. The degradation capacity of the composite bacterial agent for Enteromorpha polysaccharide was used as the response value to determine the optimal culture conditions of the composite bacterial agent.

9. The method for optimizing the culture conditions of a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 8, characterized in that: Analysis of carbon sources: different carbon sources were added to the fermentation medium, 5% of the composite bacterial agent was added, and the degradation capacity of Enteromorpha polysaccharide was measured after culturing at 160rpm and 28°C for 3 days; the carbon sources included Enteromorpha polysaccharide, maltose, sucrose, glucose, starch, and Ulva polysaccharide; Analysis of carbon source concentration: The carbon source was prepared into fermentation medium with a concentration of 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, and 1.4% w / v, and 5% of the composite bacterial agent was added. The degradation capacity of Enteromorpha polysaccharide was measured at 160 rpm and 28°C for 3 days. Analysis of nitrogen sources: Fermentation medium containing different nitrogen sources was prepared, and 5% of the composite bacterial agent was added respectively. The degradation capacity of Enteromorpha polysaccharide was measured after culturing at 160rpm and 28℃ for 3 days. The nitrogen sources included urea, peptone, yeast extract, beef extract, and ammonium sulfate. Analysis of nitrogen source concentration: Fermentation medium containing w / v concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% was prepared, and 5% of the composite bacterial agent was added respectively. The degradation capacity of Enteromorpha polysaccharide was determined after culturing at 160 rpm and 28°C for 3 days; Analysis of metal ions: 1% Enteromorpha powder was added to the metal ion combination to prepare a culture medium, and 5% of the composite bacterial agent was added to each culture medium. After culturing at 160 rpm and 28°C for 3 days, the degradation capacity of Enteromorpha polysaccharide was determined. Analysis of culture temperature: 5% of the composite bacterial agent was added to the pre-prepared fermentation medium, and the medium was placed in a shaker at 160 rpm at 25°C, 30°C, 35°C, 40°C and 45°C for 3 days to determine the degradation capacity of Enteromorpha polysaccharide; Analysis of culture pH: The optimal culture pH was determined by adjusting the fermentation medium to different pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0, and 40 mmol of different buffers, 2-(N-morpholino)ethane sulfonic acid (pH 5.0-6.0), 3-morpholinopropanesulfonic acid (pH 6.5-7.5), and tricine buffer (pH 8.0-8.5) were added for pH buffering, and 5% of the composite bacterial agent was inoculated. After culturing at 160 rpm and 28°C for 3 days, the degradation capacity of Enteromorpha polysaccharide was determined. Analysis of initial salinity: The salinity was determined by using a fermentation medium containing different w / v NaCl concentrations of 0.5%, 1%, 2%, 3%, and 4%, inoculated with 5% composite bacterial agent, and cultured at 160 rpm and 28°C for 3 days to determine the degradation capacity of Enteromorpha polysaccharide; The fermentation medium includes 0.5 g of peptone, 0.1 g of yeast extract powder, 20 g of NaCl, 5.9 g of MgCl2, 3.24 g of MgSO4, 1.8 g of CaCl2, 0.55 g of KCl, 10 g of Enteromorpha powder, and ddH2O fixed to 1000 mL.

10. The method for optimizing the culture conditions of a composite bacterial agent for degrading Enteromorpha polysaccharide according to claim 8, characterized in that: In step D, Box-Behnken Design was used for response surface analysis.

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