Microbial compound preparation for quickly decomposing enteromorpha as well as preparation method and application of microbial compound preparation

Abstract

The invention provides a microbial compound preparation for quickly decomposing enteromorpha as well as a preparation method and application of the microbial compound preparation. The microbial agent comprises bacillus YH-B7 and an enzyme preparation, the bacillus YH-B7 is classified and named as Bacillus sp.YH-B7, the decomposition temperature is 37-40 DEG C, the bacillus YH-B7 is preserved in the China Center for Type Culture Collection on May 26, 2022, and the preservation number is CCTCC M 2022728; the addition amount of the enzyme preparation is 0.5-3% of the mass of the bacillus YH-B7; the invention further provides a preparation method of the microbial compound preparation, and the microbial compound preparation is used for rapid decomposition of enteromorpha. The enteromorpha prolifera degrading agent is specially developed for enteromorpha prolifera, fermentation is rapid, and the degrading effect is good; according to the thoroughly decomposed enteromorpha, the suspension lengths of frond cellulose and cyst branches are greatly reduced, frond cells are shrunk, discolored and gradually broken, contents disappear and are in a dead state, the degradation period is short, and the thoroughly decomposed effect of the enteromorpha is greatly improved.

Description

[0001] This application is a divisional application with application number 202210892939.8, application date July 27, 2022, and invention name “A microbial composite preparation for rapid decomposition of Enteromorpha, its preparation method and application”. Technical Field

[0002] The invention relates to the technical field of microbial preparations, and in particular to a microbial composite preparation for rapidly decomposing enteromorpha, and a preparation method and application thereof. Background Art

[0003] Enteromorpha prolifera, also known as moss, is the main cause of green tides in the Yellow Sea of ​​my country. Enteromorpha grows rapidly, and the algae are dark green or bright green, with hollow tubular structures and high branches. Vegetative reproduction is a common way for Enteromorpha to reproduce. During the peak period of green tides, each gram of Enteromorpha can produce hundreds of millions of new algae. Enteromorpha algae tend to float on the sea surface, weakening the light intensity entering the seawater, destroying the growth environment of other organisms in the seawater, and causing economic losses to the aquaculture industry. It is urgent to control Enteromorpha.

[0004] Microbial preparations, commonly known as live bacteria, are currently mostly used in the market for the degradation of plant straw and its waste. Compared with plants, Enteromorpha contains more sulfated heteropolysaccharides, and common microorganisms and enzyme preparations have limited effects on the degradation of Enteromorpha. Moreover, the degradation cycle is relatively long, generally 5-10 days, which seriously affects the treatment efficiency of Enteromorpha. Summary of the invention

[0005] The purpose of the present invention is to provide a microbial composite preparation for rapidly decomposing Enteromorpha, and a preparation method and application thereof, in order to solve the problem that the degradation effect of microbial preparations in the prior art on Enteromorpha is limited and the degradation cycle is long, which seriously affects the treatment efficiency of Enteromorpha.

[0006] In order to solve the above technical problems, the technical solution of the present invention is achieved as follows:

[0007] In one aspect, the present invention provides a microbial composite preparation for quickly decomposing Enteromorpha, comprising Bacillus YH-B7 and an enzyme preparation; the Bacillus YH-B7 is classified and named Bacillus sp. YH-B7, and was deposited in the China Center for Type Culture Collection on May 26, 2022, with a deposit number of CCTCC M2022728; the added amount of the enzyme preparation is 0.5-3% of the mass of the Bacillus YH-B7, and the decomposition temperature of the Bacillus YH-B7 is 37-40°C.

[0008] The microbial composite preparation of the present invention is composed of Bacillus sp. YH-B7 and enzyme preparation. The composting temperature of sp.YH-B7 is 37-40°C, and the Enteromorpha can be fully composted within 0.5-2 days, and it has the characteristics of rapid fermentation and good degradation effect; the enzyme preparation can degrade the Enteromorpha algae in the early stage of fermentation, release a small amount of reducing sugar for the activation and proliferation of the strain, and accelerate the efficiency of bacterial growth and composting; this is a microbial composite preparation specially developed for Enteromorpha, and after the Enteromorpha is composted by the microbial composite preparation, the length of the algal cellulose and the hanging cyst branches is greatly reduced, the algal cells shrink, change color and gradually break up, the contents disappear, and are in a dead state, and the degradation effect is good. After composting, the total amount of chlorophyll of the Enteromorpha is reduced by more than 85%, and the content of substances with a molecular weight of less than 10kDa is significantly increased. The degradation cycle is short, and the single composting time is 1-2 days, which effectively improves the utilization rate of the microbial composite preparation and reduces the processing cost of Enteromorpha. It can also achieve multiple cumulative composting. In the case of 3-5 consecutive compostings, the composting of the newly added Enteromorpha can still be completed in 1 day, which greatly improves the composting effect of Enteromorpha.

[0009] As a preferred embodiment, the enzyme preparation is any one or more of cellulase, hemicellulase, pectinase, and saccharifying enzyme. The enzyme preparation of the present invention is cumbersome in variety, cheap and easy to obtain, the enzyme preparation pre-treats the Enteromorpha, softens the Enteromorpha fiber, shortens the decomposition time, and the enzyme preparation is scientifically matched with Bacillus sp. YH-B7, so as to achieve rapid and efficient decomposition of Enteromorpha.

[0010] In another aspect, a method for preparing a microbial composite preparation of a fast-decomposing Enteromorpha of the present invention comprises the following steps: 1) inoculating Bacillus YH-B7 into a broth culture medium, wherein the broth culture medium is composed of: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH value 7.0, and culturing at 35-37°C with shaking, activating, and increasing the bacteria to 5×10 6 1) adding corn syrup powder to the Enteromorpha hydrolyzate in an amount of 0.1% of the mass of the Enteromorpha hydrolyzate, adjusting the pH value to 6.5-7.0, sterilizing at 115°C for 20 min, and obtaining a fermentation medium; 2) taking the first-level seeds obtained in step 1), transferring them to the fermentation medium obtained in step 2) at an inoculum amount of 5-10%, and shaking and culturing them at 35-37°C for 48-60 h until the bacterial concentration reaches 10 8 / mL or more to obtain a fermentation broth; 4) adding a flocculant to the fermentation broth obtained in step 3), the amount of the flocculant added is 10-100ppm by weight of the fermentation broth, and performing precipitation and flocculation, removing the supernatant to make the bacterial weight 12-15%, spray drying, the inlet air temperature is 160-180°C, and the outlet temperature is 80-90°C to obtain a microbial agent; 5) adding an enzyme preparation to the microbial agent obtained in step 4), stirring, mixing evenly, to obtain a microbial composite preparation.

[0011] The preparation method of the microbial composite preparation for rapidly decomposing Enteromorpha of the present invention comprises the following steps: first culturing Bacillus YH-B7, then inoculating it into a fermentation medium, and finally flocculating, drying, and mixing an enzyme preparation to obtain the preparation; the preparation method is convenient to operate, has mild conditions, has no special requirements for equipment, has a high viable bacteria rate, and has low cost, thus achieving the rapid decomposition effect of Enteromorpha and being easy to realize industrialization.

[0012] As a preferred embodiment, in the step 1), Bacillus YH-B7 is first preserved in a glycerol tube under sterile conditions and inoculated on a broth slant culture medium, the composition of the broth slant culture medium is: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, cultured at 35-37°C for 2 days, and then inoculated into the broth culture medium. Usually, Bacillus YH-B7 is preserved in a glycerol tube under sterile conditions. In the present invention, the preserved Bacillus YH-B7 is first slant-activated in a broth slant culture medium, and then cultured.

[0013] As a preferred embodiment, in the step 2), the preparation method of the enteromorpha hydrolyzate is: take enteromorpha dry powder, add it to a sulfuric acid aqueous solution with a molar concentration of 0.1-0.3 mol / L, the mass ratio of enteromorpha dry powder to sulfuric acid is 20:3-5, at 110-120°C, high temperature acid hydrolysis for 30-50min, gauze filtering to remove residues, and obtain enteromorpha hydrolyzate. The enteromorpha hydrolyzate of the present invention adopts a high temperature acid hydrolysis method, which is simple to operate and easy to implement, and the obtained enteromorpha hydrolyzate has high active ingredients and few impurities.

[0014] As a preferred embodiment, in step 4), the flocculant is any one or more of ammonium sulfate, sodium alginate, alkali starch, gelatin, tannic acid, and chitosan. The function of the flocculant is to concentrate the fermentation broth to improve the efficiency of subsequent spray drying; there are many types of flocculants, and they have been commercialized. Usually, the flocculant is a composite flocculant.

[0015] In another aspect, the present invention provides an application of a microbial composite preparation for rapidly decomposing Enteromorpha, wherein the microbial composite preparation is used for rapidly decomposing Enteromorpha. This microbial composite preparation can, on the one hand, be used for salvaging Enteromorpha for decomposition and release during a large-scale outbreak of Enteromorpha green tide, shortening the release cycle and effectively improving the release efficiency; on the other hand, it can be used for rapid decomposition and degradation of Enteromorpha, increasing the content of soluble substances in the degradation products; furthermore, it can also be used for the extraction of sulfated polysaccharides and Enteromorpha polysaccharides.

[0016] As a preferred embodiment, the application of a microbial composite preparation for quickly decomposing Enteromorpha of the present invention comprises the following steps: 1) taking the microbial composite preparation, adding it to water, the amount of the microbial composite preparation added is 0.1-1% of the mass of the water, stirring, dissolving, and obtaining a decomposing liquid; 2) taking Enteromorpha, adding it to the decomposing liquid obtained in step 1), the mass ratio of Enteromorpha to the decomposing liquid is 3-5:1, standing for fermentation, turning over every 6-12 hours, fermenting for 1-2 days, and obtaining a decomposed Enteromorpha.

[0017] The microbial composite preparation of the present invention can achieve primary fermentation and composting of Enteromorpha. After static fermentation for 1-2 days, the Enteromorpha is inactivated, and the primary fermentation and composting of Enteromorpha is completed. The Enteromorpha after primary composting can be released into the sea, thereby achieving rapid growth and composting of Enteromorpha, effectively improving the utilization rate of the microbial composite preparation and reducing the processing cost of Enteromorpha.

[0018] As a preferred embodiment, after the step 2), the method further comprises: 3) adding the decomposition liquid and water obtained in step 1) to the once-mature Enteromorpha obtained in step 2), stirring evenly, wherein the amount of the decomposition liquid added is 0-4% of the mass of the once-mature Enteromorpha, so that the water content of the Enteromorpha is above 90%, continuing to add Enteromorpha, wherein the amount of Enteromorpha added is 3-5:5 of the mass of the once-mature Enteromorpha, stirring and standing for fermentation, fermenting for 1 day to obtain twice-mature Enteromorpha; 4) continuing to add the decomposition liquid, water and Enteromorpha to the twice-mature Enteromorpha obtained in step 3) according to the method of step 3), standing for fermentation to obtain thrice-mature Enteromorpha, and so on to obtain continuously-mature Enteromorpha.

[0019] The microbial composite preparation of the present invention can also realize continuous fermentation and maturation of Enteromorpha. A maturation liquid is first prepared, and continuous addition is performed to realize continuous maturation of Enteromorpha. The dosage of the microbial composite preparation is reduced, and the purpose of reducing costs is achieved. After one or more times of maturation, the total amount of chlorophyll in Enteromorpha is reduced by more than 85%, cells atrophy and die, cell wall structure is destroyed, intracellular substances are released in large quantities, and the content of substances with a molecular weight of less than 10 kDa is significantly increased.

[0020] As a preferred embodiment, the continuously decomposed Enteromorpha is Enteromorpha that has been continuously decomposed 3-5 times. After the Enteromorpha is decomposed once using the microbial composite preparation of the present invention, decomposition liquid, water and Enteromorpha are continuously added to continuously ferment the Enteromorpha; this continuous fermentation can be accumulated for multiple times, and in the case of 3-5 continuous fermentations, the microbial composite preparation can still complete the decomposition of the newly added Enteromorpha in 1 day.

[0021] Compared with the prior art, the invention has the following beneficial effects: the microbial composite preparation of the invention is developed for Enteromorpha, the composting temperature of Bacillus sp.YH-B7 strain is 37-40°C, Enteromorpha can be fully composted within 0.5-2 days, and it has the characteristics of rapid fermentation and good degradation effect. The enzyme preparation can degrade Enteromorpha algae in the early stage of fermentation, release a small amount of reducing sugar for the activation and proliferation of the strain, and accelerate the efficiency of bacterial growth and composting; after Enteromorpha is composted by the microbial composite preparation, the length of the algae cellulose and the hanging cyst branches is greatly reduced, the algae cells shrink, change color and gradually break apart, the contents disappear, and the Enteromorpha is in a dead state, the degradation effect is good, the total amount of chlorophyll of Enteromorpha is reduced by more than 85% after composting, the content of substances with a molecular weight of less than 10 kDa is significantly increased, the degradation cycle is short, the single composting time is 1-2 days, the utilization rate of the microbial composite preparation is effectively improved, the processing cost of Enteromorpha is reduced, and multiple cumulative composting can be achieved. In the case of 3-5 consecutive compostings, the composting of newly added Enteromorpha can still be completed in 1 day, and the composting effect of Enteromorpha is greatly improved.

[0022] Biological Deposit

[0023] The Bacillus YH-B7 provided by the present invention is classified and named Bacillus sp. YH-B7, and has been deposited in the China Center for Type Culture Collection on May 26, 2022. The abbreviation of the depository unit is CCTCC, the address is: Wuhan University, Wuhan, China, Postal Code: 430072, and the deposit number is CCTCC M 2022728. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A microbial colony diagram of Bacillus YH-B7 in the microbial composite preparation provided by the present invention;

[0025] Figure 2 for Figure 1 Cell morphology of Bacillus subtilis YH-B7;

[0026] Figure 3 This is a photo of the appearance of the control sample in the present invention;

[0027] Figure 4 This is a photo of the appearance of the Enteromorpha obtained by the present invention after once being decomposed;

[0028] Figure 5 This is a microstructure diagram of the control sample in the present invention under an electron microscope;

[0029] Figure 6 This is a microscopic structure diagram of the once-composted Enteromorpha obtained by the present invention under an electron microscope;

[0030] Figure 7 The high performance liquid chromatography analysis diagram of Enteromorpha before and after one-time decomposition using the microbial composite preparation of Example 1 of the present invention;

[0031] A-microorganism complex preparation of the present invention; B-control sample. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention discloses a microbial composite preparation for quickly decomposing Enteromorpha, comprising Bacillus YH-B7 and an enzyme preparation; the Bacillus YH-B7 is classified and named Bacillus sp. YH-B7, which was deposited in the China Center for Type Culture Collection on May 26, 2022, with a deposit number of CCTCC M 2022728; the added amount of the enzyme preparation is 0.5-3% of the mass of the Bacillus YH-B7, and the decomposition temperature of the Bacillus YH-B7 is 37-40°C.

[0034] Preferably, the enzyme preparation is any one or more of cellulase, hemicellulase, pectinase and saccharifying enzyme.

[0035] The method for preparing a microbial composite preparation for rapidly decomposing Enteromorpha of the present invention comprises the following steps:

[0036] 1) Inoculate Bacillus YH-B7 into broth medium, the composition of which is: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH 7.0, culture at 35-37°C with shaking, activate, and increase the bacteria to 5×10 6 pcs / mL or more, as first-class seeds;

[0037] 2) taking the Enteromorpha hydrolyzate, adding corn steep liquor powder, the amount of corn steep liquor powder added is 0.1% of the mass of the Enteromorpha hydrolyzate, adjusting the pH value to 6.5-7.0, sterilizing at 115° C. for 20 min, and obtaining a fermentation medium;

[0038] 3) Take the first-class seeds obtained in step 1) and transfer them to the fermentation medium obtained in step 2) at an inoculum rate of 5-10%, and culture them at 35-37°C with shaking for 48-60 hours until the bacterial concentration reaches 10 8 / mL or more, and the fermentation liquid is obtained;

[0039] 4) Adding a flocculant to the fermentation broth obtained in step 3) in an amount of 10-100 ppm based on the weight of the fermentation broth, performing precipitation and flocculation, removing the supernatant to make the bacterial weight 12-15%, spray drying, with an inlet air temperature of 160-180° C. and an outlet air temperature of 80-90° C., to obtain a bacterial agent;

[0040] 5) adding enzyme preparation to the bacterial agent obtained in step 4), stirring and mixing evenly to obtain a microbial composite preparation.

[0041] Preferably, in the step 1), Bacillus YH-B7 is first preserved in a glycerol tube under sterile conditions and inoculated into a broth slant culture medium, the composition of the broth slant culture medium is: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, cultured at 35-37°C for 2 days, and then inoculated into the broth culture medium.

[0042] Preferably, in the step 2), the preparation method of the enteromorpha hydrolyzate is: take enteromorpha dry powder, add it to a sulfuric acid aqueous solution with a molar concentration of 0.1-0.3 mol / L, the mass ratio of enteromorpha dry powder to sulfuric acid is 20:3-5, high temperature acid hydrolysis is performed at 110-120°C for 30-50min, and the residue is filtered through gauze to obtain the enteromorpha hydrolyzate.

[0043] Preferably, in step 4), the flocculant is any one or more of ammonium sulfate, sodium alginate, alkali starch, gelatin, tannic acid, and chitosan.

[0044] The invention discloses an application of a microbial composite preparation for rapidly decomposing Enteromorpha, wherein the microbial composite preparation is used for rapidly decomposing Enteromorpha.

[0045] Preferably, the steps include:

[0046] 1) Take a microbial composite preparation, add it to water, the amount of the microbial composite preparation added is 0.1-1% of the water mass, stir, dissolve, and obtain a decomposed liquid;

[0047] 2) Take Enteromorpha, add it to the decomposing liquid obtained in step 1), the mass ratio of Enteromorpha to the decomposing liquid is 3-5:1, let it stand for fermentation, stir it every 6-12 hours, ferment it for 1-2 days, and obtain decomposed Enteromorpha.

[0048] Furthermore, after the step 2), the method further comprises: 3) adding the decomposition liquid and water obtained in step 1) to the once-matured Enteromorpha obtained in step 2), stirring evenly, wherein the amount of the decomposition liquid added is 0-4% of the mass of the once-matured Enteromorpha, so that the water content of the Enteromorpha is above 90%, continuing to add Enteromorpha, wherein the amount of Enteromorpha added is 3-5:5 of the mass of the once-matured Enteromorpha, stirring and standing for fermentation, fermenting for 1 day, and obtaining twice-mature Enteromorpha; 4) continuing to add the decomposition liquid, water and Enteromorpha to the twice-mature Enteromorpha obtained in step 3) according to the method of step 3), standing for fermentation, and obtaining thrice-mature Enteromorpha, and so on, to obtain continuously-mature Enteromorpha.

[0049] Preferably, the continuously decomposed Enteromorpha is Enteromorpha that has been continuously decomposed 3-5 times.

[0050] Experiment 1

[0051] Screening and identification of Bacillus sp. YH-B7

[0052] The rotten seaweed samples were obtained from the Yellow Sea offshore, homogenized, resuspended in physiological saline, allowed to stand, 1 mL of the supernatant was drawn, and the mixture was washed with water for 10 min. -3 , 10 -4 , 10 -5 , 10 -6 Perform gradient dilution, spread on broth plates, and culture upside down at 35°C; pick single colonies and streak them multiple times to obtain pure culture; transfer to Enteromorpha hydrolyzate (with 0.5% yeast powder) for culture to obtain fermentation broth.

[0053] The fermentation liquid and Enteromorpha were stirred and mixed in a ratio of 1:1, cultured at natural temperature, stirred once every 12 hours, and decomposed for 48 hours. The decomposition ability of the strain was preliminarily determined by the release of soluble reducing sugars in the decomposed Enteromorpha (DNS method). Finally, a Bacillus sp. strain named YH-B7 was obtained, which had the best soluble reducing sugar release effect.

[0054] Gram staining of the strain showed that it was a Gram-positive bacterium. Figure 2 It can be seen that the strain has the morphology of Bacillus and the ability to produce spores. After culturing in broth medium for 24 hours, the attached Figure 1 It can be seen that the colonies are flat, rough and irregular, dry and easy to pick up, milky white, with uniform color on both sides, and the colonies are facultative anaerobic.

[0055] 16S rDNA (SEQ ID NO: 1) was extracted for molecular biological identification, and it was determined that the strain belonged to Bacillus sp., and was deposited in the China Type Culture Collection of Wuhan University, China on May 26, 2022, with the deposit number CCTCCM 2022728.

[0056] 16S rDNA (SEQ ID NO: 1):

[0057]

[0058] The method for preparing a microbial composite preparation for rapidly decomposing Enteromorpha of the present invention comprises the following steps:

[0059] 1) Slant activation of Bacillus sp. YH-B7

[0060] Under sterile conditions, the strain Bacillus sp. YH-B7 obtained in Experiment 1 was preserved in a glycerol tube and inoculated into a broth slant medium. The composition of the broth slant medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, and cultured at 37°C for 2 days.

[0061] 2) Seed cultivation

[0062] The cultured Bacillus YH-B7 obtained in step 1) was inoculated into a broth medium, wherein the composition of the broth medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH 7.0, and the culture was shaken at 37°C for activation and the bacteria were enriched to 5×10 6 / mL, as the first-level seeds;

[0063] 3) Fermentation medium preparation

[0064] Take the Enteromorpha hydrolyzate, add corn steep liquor powder, the addition amount of corn steep liquor powder is 0.1% of the mass of the Enteromorpha hydrolyzate, adjust the pH value to 6.5, sterilize at 115° C. for 20 min, and obtain a fermentation medium;

[0065] 4) Fermentation broth preparation

[0066] Take the first-class seeds obtained in step 2) and transfer them to the fermentation medium obtained in step 3) at a 10% inoculum amount, and culture them at 37°C with shaking for 48 hours until the bacterial concentration reaches 10 8 / mL, and the fermentation liquid was obtained;

[0067] 5) Concentration and drying

[0068] Adding flocculant, chitosan, to the fermentation liquid obtained in step 4) in an amount of 10 ppm based on the weight of the fermentation liquid, performing precipitation and flocculation, removing the supernatant to make the bacterial weight 12%, spray drying, with an inlet air temperature of 160°C and an outlet air temperature of 80°C, to obtain a bacterial agent;

[0069] 6) Preparation of microbial compound preparations

[0070] Add enzyme preparation to the bacterial agent obtained in step 5), the amount of enzyme preparation added is 0.5% of the mass of the bacterial agent, i.e., Bacillus YH-B7, and stir and mix evenly to obtain a microbial composite preparation.

[0071] Embodiment 2

[0072] The method for preparing a microbial composite preparation for rapidly decomposing Enteromorpha of the present invention comprises the following steps:

[0073] 1) Slant activation of Bacillus sp. YH-B7

[0074] Under sterile conditions, the strain Bacillus sp. YH-B7 obtained in Experiment 1 was preserved in a glycerol tube and inoculated into a broth slant medium. The composition of the broth slant medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, and cultured at 35°C for 2 days.

[0075] 2) Seed cultivation

[0076] The cultured Bacillus YH-B7 obtained in step 1) was inoculated into a broth medium, wherein the composition of the broth medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH 7.0, and the culture was shaken and activated at 35°C, and the bacteria were increased to 8×10 6 / mL, as the first-level seeds;

[0077] 3) Fermentation medium preparation

[0078] Take the Enteromorpha hydrolyzate, add corn steep liquor powder, the addition amount of corn steep liquor powder is 0.1% of the Enteromorpha hydrolyzate mass, adjust the pH value to 7.0, sterilize at 115° C. for 20 min, and obtain a fermentation medium;

[0079] 4) Fermentation broth preparation

[0080] Take the first-class seeds obtained in step 2) and transfer them to the fermentation medium obtained in step 3) at a 5% inoculum amount. Incubate at 35°C with shaking for 60 hours until the bacterial concentration reaches 10 8 / mL or more, and the fermentation liquid is obtained;

[0081] 5) Concentration and drying

[0082] Adding flocculant, alkali starch, to the fermentation liquid obtained in step 4) in an amount of 100 ppm based on the weight of the fermentation liquid, performing precipitation and flocculation, removing the supernatant to make the bacterial weight 15%, spray drying, with an inlet air temperature of 180° C. and an outlet air temperature of 90° C., to obtain a bacterial agent;

[0083] 6) Preparation of microbial compound preparations

[0084] Add enzyme preparation to the bacterial agent obtained in step 5), the amount of enzyme preparation added is 2.0% of the mass of the bacterial agent, i.e., Bacillus YH-B7, and stir and mix evenly to obtain a microbial composite preparation.

[0085] Embodiment 3

[0086] The method for preparing a microbial composite preparation for rapidly decomposing Enteromorpha of the present invention comprises the following steps:

[0087] 1) Slant activation of Bacillus sp. YH-B7

[0088] Under sterile conditions, the strain Bacillus sp. YH-B7 obtained in Experiment 1 was preserved in a glycerol tube and inoculated into a broth slant medium. The composition of the broth slant medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, and cultured at 36°C for 2 days.

[0089] 2) Seed cultivation

[0090] The cultured Bacillus YH-B7 obtained in step 1) was inoculated into a broth medium, wherein the composition of the broth medium was: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH 7.0, and the culture was shaken at 36°C for activation and the bacteria were enriched to 5×10 6 pcs / mL or more, as first-class seeds;

[0091] 3) Preparation of Enteromorpha Hydrolyzate

[0092] Take 10 g of Enteromorpha dry powder, add it to 100 mL of sulfuric acid aqueous solution with a molar concentration of 0.2 mol / L, acid hydrolyze it at 115°C for 40 min, filter it with gauze to remove the residue, and obtain Enteromorpha hydrolyzate;

[0093] 4) Fermentation medium preparation

[0094] Take the Enteromorpha hydrolyzate obtained in step 3), add corn steep liquor powder, the amount of corn steep liquor powder added is 0.1% of the mass of the Enteromorpha hydrolyzate, adjust the pH value to 6.8, sterilize at 115° C. for 20 min, and obtain a fermentation medium;

[0095] 5) Fermentation broth preparation

[0096] Take the first-class seeds obtained in step 2) and transfer them to the fermentation medium obtained in step 3) at an inoculum rate of 8%, and culture them at 36°C with shaking for 52 hours until the bacterial concentration reaches 10 8 / mL or more, and the fermentation liquid is obtained;

[0097] 6) Concentration and drying

[0098] Adding a flocculant, sodium alginate, to the fermentation liquid obtained in step 5) in an amount of 80 ppm based on the weight of the fermentation liquid, performing precipitation and flocculation, removing the supernatant, making the bacterial weight 13.5%, spray drying, with an inlet air temperature of 170° C. and an outlet air temperature of 90° C., to obtain a bacterial agent;

[0099] 7) Preparation of microbial compound preparations

[0100] Add enzyme preparation to the bacterial agent obtained in step 6), the amount of enzyme preparation added is 3.0% of the mass of the bacterial agent, i.e., Bacillus YH-B7, and stir and mix evenly to obtain a microbial composite preparation.

[0101] Experiment 2

[0102] The application of the microbial composite preparation in the primary fermentation and decomposition of Enteromorpha includes the following steps:

[0103] 1) Take 5 kg of each of the microbial composite preparations prepared in Example 1 to Example 3, add them to 1 ton of tap water, stir and mix thoroughly, dissolve, and obtain a decomposed liquid;

[0104] 2) Take 4 tons of newly salvaged Enteromorpha, add 1 ton of each to the decomposed liquid obtained in step 1), set at natural pH value, and ferment under natural conditions. During the fermentation process, stir once every 12 hours. Ferment for 1 day to obtain decomposed Enteromorpha.

[0105] Evaluation of composting effect

[0106] 1. Apparent changes

[0107] The appearance of the above-obtained once-composted Enteromorpha was compared with that of the newly-fished Yellow Sea green tide Enteromorpha (control sample). Figure 3 It can be seen that the newly salvaged Enteromorpha is bright green, with hanging branches, forming air sacs, floating in the water, with many slender branches and clear branches; however, Figure 4 It can be seen that after being once composted by the microbial composite preparation of the present invention, the color of the Enteromorpha becomes light yellow due to the release of substances, and the branches are no longer hanging, but are broken and sink to the bottom of the water. At this time, the Enteromorpha is inactivated and can be released into the sea.

[0108] 2. Chlorophyll content

[0109] Take 10 g of the above-obtained once-decomposed Enteromorpha and the newly salvaged Green Tide Enteromorpha from the Yellow Sea (control sample), resuspend them in 100 mL of water, filter and dry them using filter paper, dissolve the algae on the filter paper in 80% acetone and grind them, fix the volume in 100 mL of 80% acetone solution after grinding, measure the absorbance of the sample at wavelengths of 645 nm and 663 nm using a spectrophotometer, and calculate the chlorophyll content. The experimental results are listed in Table 1; the calculation process of the chlorophyll content is as follows:

[0110] Chlorophyll a=12.71OD 663 -2.59OD 645 ;

[0111] Chlorophyll b = 22.88 OD 645 -4.67OD 663 ;

[0112] Total chlorophyll = a + b = 8.04OD 663 +20.29OD 645 .

[0113] Table 1 Determination results of chlorophyll content in different Enteromorpha

[0114] Sample name Chlorophyll a(mg / g) Chlorophyll b(mg / g) Total chlorophyll content (mg / g) Chlorophyll reduction (%) Embodiment 1 0.128 0.045 0.173 85.99 Embodiment 2 0.100 0.049 0.149 87.94 Embodiment 3 0.063 0.075 0.138 88.83 Control 0.760 0.475 1.235 ---

[0115] It can be seen from Table 1 that the total amount of chlorophyll in the Enteromorpha after being once decomposed using the microbial composite preparation of the present invention is 0.138-0.173 mg / g, however, the total amount of chlorophyll in the newly salvaged Yellow Sea green tide Enteromorpha (control sample) is 1.235 mg / g; therefore, the chlorophyll content of the Enteromorpha after being once decomposed using the microbial composite preparation of the present invention is significantly reduced, and the reduction amount is more than 85%.

[0116] 3. Changes in algal fiber structure

[0117] The once-ripened Enteromorpha obtained by using the microbial composite preparation of Example 1 and the newly salvaged Enteromorpha spp. from the Yellow Sea (control sample) were placed under an OLYMPUS CX33 electron microscope for observation. Figure 5 It can be seen that the newly salvaged green tide Enteromorpha (control sample) in the Yellow Sea has formed air sacs, arranged neatly, green in color, with thick cell walls and full cytoplasm, showing a vegetative cell state; however, Figure 6 It can be seen that in the Enteromorpha that has been once decomposed using the microbial composite preparation of the present invention, the cytoplasm gradually shrinks, the air sacs disappear, the algae appear transparent and reticular, the cell walls become thinner and break in large quantities, the contents are released, and the cells are in a dead state.

[0118] 4. Release of small molecule soluble substances

[0119] Take 10 g each of the once-ripened Enteromorpha obtained by the microbial composite preparation of Example 1 and the newly salvaged Green Tide Enteromorpha from the Yellow Sea (control sample), resuspend them in 100 mL of water, filter them with filter paper, collect the filtrate, and place it in a high performance liquid chromatography (HPLC) for analysis. The analytical column is a high-resolution molecular sieve gel column SUPERDEX PEPTIDE 10 / 300GL (GE Healthcare, 100-7000Da), the flow rate is 1 mL / min, the detection wavelength is 214 nm, and the mobile phase is pure water.

[0120] By the attached Figure 7It can be seen that compared with the newly salvaged green tide Enteromorpha from the Yellow Sea (control sample), the content of small molecules with a molecular weight of less than 10 kDa in the Enteromorpha once decomposed using the microbial composite preparation of the present invention is greatly increased.

[0121] Experiment 3

[0122] The application of the microbial composite preparation in the continuous fermentation and ripening of Enteromorpha includes the following steps:

[0123] 1) Take 5 kg of each of the microbial composite preparations prepared in Examples 1 to 3, add them to 1 ton of seawater, stir and mix thoroughly, dissolve, and obtain a decomposed liquid;

[0124] 2) taking 4 tons of newly salvaged Enteromorpha, adding 1 ton of the decomposed liquid obtained in step 1) to the natural pH value, and letting it stand and ferment under natural conditions. During the fermentation process, the liquid was stirred once every 6 hours, and fermented for 1 day to obtain the decomposed Enteromorpha;

[0125] 3) Adding 100 kg of the decomposition liquid obtained in step 1) and an appropriate amount of seawater to the once-decomposed Enteromorpha obtained in step 2), and stirring evenly. The amount of water added depends on the actual situation of the Enteromorpha fishing and decomposition site, so that the water content of the Enteromorpha is above 90%. Newly-fished Enteromorpha basically does not need to be supplemented with water; continue to add 4 kg of newly-fished Enteromorpha, adjust the pH value to the natural value, stir and immerse it fully, and stand and ferment under natural conditions. During the fermentation process, stir once every 6 hours, and ferment for 1 day to obtain the twice-decomposed Enteromorpha;

[0126] 4) According to the method of step 3), the decomposition liquid, seawater and Enteromorpha are continuously added to the twice-decomposed Enteromorpha obtained in step 3), and the mixture is allowed to ferment to obtain thrice-decomposed Enteromorpha.

[0127] Evaluation of composting effect

[0128] 1. Apparent changes:

[0129] The appearance of the three-times-ripened Enteromorpha obtained above was compared with the newly-fished Yellow Sea green tide Enteromorpha (control sample) according to the method of Experiment 3. Similarly, the newly-fished Enteromorpha was bright green, showed a hanging branch phenomenon, formed air sacs, floated in the water, had many slender branches, and had clear branches; however, the Enteromorpha after three-times-ripened by the microbial composite preparation of the present invention showed a light yellow color due to the release of substances, and the branches were no longer hanging, but broke and sank to the bottom of the water. At this time, the Enteromorpha was inactivated and could be released into the sea.

[0130] 2. Chlorophyll content

[0131] The chlorophyll content of the three-times-ripened Enteromorpha obtained above and the newly-fished Yellow Sea green tide Enteromorpha (control sample) was determined according to the method of Experiment 3. The experimental results are listed in Table 3.

[0132] Table 3 Determination results of chlorophyll content in different Enteromorpha

[0133] Sample name Chlorophyll a(mg / g) Chlorophyll b(mg / g) Total chlorophyll content (mg / g) Chlorophyll reduction (%) Embodiment 1 0.130 0.050 0.180 85.43 Embodiment 2 0.118 0.053 0.171 86.15 Embodiment 3 0.076 0.080 0.156 87.37 Control 0.760 0.475 1.235 ---

[0134] It can be seen from Table 3 that the total amount of chlorophyll in the Enteromorpha after being decomposed three times using the microbial composite preparation of the present invention is 0.156-0.180 mg / g, however, the total amount of chlorophyll in the newly salvaged Yellow Sea green tide Enteromorpha (control sample) is 1.235 mg / g; therefore, the chlorophyll content of the Enteromorpha after being decomposed three times using the microbial composite preparation of the present invention is significantly reduced, and the reduction amount is more than 85%.

[0135] 3. Changes in algal fiber structure

[0136] The Enteromorpha obtained after thrice decomposition and the newly salvaged Green Tide Enteromorpha from the Yellow Sea (control sample) were placed under an electron microscope for observation in Experiment 3. The observation results showed that the algae of the newly salvaged Green Tide Enteromorpha from the Yellow Sea (control sample) formed air sacs, were arranged neatly, were green in color, had thick cell walls, and had full cytoplasm, showing a vegetative cell state; however, in the Enteromorpha after thrice decomposition using the microbial composite preparation of the present invention, the cytoplasm gradually shrank, the air sacs disappeared, the algae showed a transparent network, the cell walls became thin and broke in large quantities, the contents were released, and the cells were in a dead state.

[0137] 4. Release of small molecule soluble substances

[0138] The three-times-ripened Enteromorpha obtained above and the newly-fished Yellow Sea green tide Enteromorpha (control sample) were used to measure the content of small molecules with a molecular weight of less than 10 kDa according to the method of Experiment 3. The results of HPLC analysis showed that, compared with the newly-fished Yellow Sea green tide Enteromorpha (control sample), the content of small molecules with a molecular weight of less than 10 kDa in the Enteromorpha that was three-times-ripened using the microbial composite preparation of the present invention was greatly increased.

[0139] Therefore, compared with the prior art, the present invention has the following beneficial effects: the microbial composite preparation of the present invention is developed for Enteromorpha, the fermentation temperature of Bacillus sp.YH-B7 strain is 37-40°C, Enteromorpha can be fully fermented within 0.5-2 days, and it has the characteristics of rapid fermentation and good degradation effect. The enzyme preparation can degrade Enteromorpha algae in the early stage of fermentation, release a small amount of reducing sugar for the activation and proliferation of the strain, and accelerate the efficiency of bacterial growth and fermentation; after Enteromorpha is fermented by the microbial composite preparation, the length of the algal cellulose and the hanging cyst branches is greatly reduced, the algal cells shrink, change color and gradually break, the contents disappear, and are in a dead state, the degradation effect is good, the total amount of chlorophyll of Enteromorpha after fermentation is reduced by more than 85%, the content of substances with a molecular weight of less than 10 kDa is significantly increased, the degradation cycle is short, and the single fermentation time is 1-2 days, which effectively improves the utilization rate of the microbial composite preparation, reduces the processing cost of Enteromorpha, and can also achieve multiple cumulative fermentation. In the case of 3-5 consecutive fermentations, the fermentation of newly added Enteromorpha can still be completed in 1 day, which greatly improves the fermentation effect of Enteromorpha.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A microbial composite preparation for rapidly decomposing Enteromorpha, characterized in that: The microbial composite preparation comprises Bacillus YH-B7 and an enzyme preparation; The Bacillus sp. YH-B7 was classified and named Bacillus sp. YH-B7, and was deposited in the China Center for Type Culture Collection on May 26, 2022, with the deposit number CCTCC M 2022728; The added amount of the enzyme preparation is 0.5-3% of the mass of the Bacillus YH-B7, and the decomposition temperature of the Bacillus YH-B7 is 37-40°C.

2. The microbial composite preparation for rapid decomposition of Enteromorpha according to claim 1, characterized in that: The enzyme preparation is any one or more of cellulase, hemicellulase, pectinase and saccharifying enzyme.

3. The method for preparing the fast-decomposing enteromorpha microbial composite preparation according to claim 1 or 2, characterized in that: The following steps are involved: 1) Inoculate Bacillus YH-B7 into broth medium, the composition of which is: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, pH 7.0, culture at 35-37°C with shaking, activate, and increase the bacteria to 5×10 6 pcs / mL or more, as first-class seeds; 2) taking the Enteromorpha hydrolyzate, adding corn steep liquor powder, the amount of corn steep liquor powder added is 0.1% of the mass of the Enteromorpha hydrolyzate, adjusting the pH value to 6.5-7.0, sterilizing at 115° C. for 20 min, and obtaining a fermentation medium; 3) Take the first-class seeds obtained in step 1) and transfer them to the fermentation medium obtained in step 2) at an inoculum rate of 5-10%, and culture them at 35-37°C with shaking for 48-60 hours until the bacterial concentration reaches 10 8 / mL or more, and the fermentation liquid is obtained; 4) Adding a flocculant to the fermentation broth obtained in step 3) in an amount of 10-100 ppm based on the weight of the fermentation broth, performing precipitation and flocculation, removing the supernatant to make the bacterial weight 12-15%, spray drying, with an inlet air temperature of 160-180° C. and an outlet air temperature of 80-90° C., to obtain a bacterial agent; 5) adding enzyme preparation to the bacterial agent obtained in step 4), stirring and mixing evenly to obtain a microbial composite preparation.

4. The method for preparing the fast-decomposing enteromorpha microbial composite preparation according to claim 3, characterized in that: In the step 1), Bacillus YH-B7 is first preserved in a glycerol tube under sterile conditions and inoculated into a broth slant culture medium, the composition of which is: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, 2% agar, pH 7.0, and cultured at 35-37° C. for 2 days, and then inoculated into the broth culture medium.

5. The method for preparing the fast-decomposing enteromorpha microbial composite preparation according to claim 3, characterized in that: In the step 2), the preparation method of the enteromorpha hydrolyzate is: Take Enteromorpha dry powder, add it to a sulfuric acid aqueous solution with a molar concentration of 0.1-0.3 mol / L, the mass ratio of Enteromorpha dry powder to sulfuric acid is 20:3-5, high temperature acid hydrolysis is performed at 110-120°C for 30-50 minutes, and the residue is filtered through gauze to obtain Enteromorpha hydrolyzate.

6. The method for preparing the fast-decomposing enteromorpha microbial composite preparation according to claim 3, characterized in that: In the step 4), the flocculant is any one or more of ammonium sulfate, sodium alginate, alkali starch, gelatin, tannic acid, and chitosan.

7. An application of the fast-decomposing enteromorpha microbial composite preparation according to claim 1 or 2, characterized in that: The microbial composite preparation is used for rapidly decomposing Enteromorpha.

8. The use of the fast-decomposing enteromorpha microbial composite preparation according to claim 7, characterized in that: The following steps are involved: 1) Take a microbial composite preparation, add it to water, the amount of the microbial composite preparation added is 0.1-1% of the water mass, stir, dissolve, and obtain a decomposed liquid; 2) Take Enteromorpha, add it to the decomposing liquid obtained in step 1), the mass ratio of Enteromorpha to the decomposing liquid is 3-5:1, let it stand for fermentation, stir it every 6-12 hours, ferment it for 1-2 days, and obtain decomposed Enteromorpha.

9. The use of the fast-decomposing enteromorpha microbial composite preparation according to claim 8, characterized in that: After step 2), the method further comprises: 3) adding the decomposition liquid obtained in step 1) and water to the once-mature Enteromorpha obtained in step 2), stirring evenly, wherein the amount of the decomposition liquid added is 0-4% of the mass of the once-mature Enteromorpha, so that the water content of the Enteromorpha is above 90%, continuing to add Enteromorpha, wherein the amount of Enteromorpha added is 3-5:5 of the mass of the once-mature Enteromorpha, stirring, standing and fermenting, and fermenting for 1 day to obtain a second-mature Enteromorpha; 4) According to the method of step 3), the decomposition liquid, water and Enteromorpha are continuously added to the twice-decomposed Enteromorpha obtained in step 3), and the mixture is allowed to ferment to obtain thrice-decomposed Enteromorpha, and so on to obtain continuously-decomposed Enteromorpha.

10. The use of the fast-decomposing enteromorpha microbial composite preparation according to claim 9, characterized in that: The continuously decomposed Enteromorpha is Enteromorpha that has been continuously decomposed 3-5 times.

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