A method for preparing bark algae liquid fertilizer using bark algae and its application

Abstract

The present invention relates to the technical field of seaweed liquid fertilizer, and specifically to a method for preparing bark algae liquid fertilizer using bark algae and its application. The present invention adopts an enzymatic hydrolysis and fermentation combined process to extract bark algae active substances, and a combination of multiple enzymes is used to degrade the bark algae, which can remove the fiber structure cell wall of the bark algae, so that the effective components of the bark algae are quickly released, and the bark algae enzymatic hydrolyzate is fermented by a composite bacterial agent to fully release the bark algae cell contents, and the macromolecular substances are decomposed into small molecular substances that are easily absorbed by plants. The bark algae liquid fertilizer of the present invention can enhance the drought resistance of Chinese cabbage and increase the yield of Chinese cabbage. The bark algae liquid fertilizer of the present invention effectively prevents and treats cucumber blight, and can promote the growth of cucumber seedlings and increase the yield.

Description

Technical Field

[0001] The invention relates to the technical field of seaweed liquid fertilizer, and in particular to a method for preparing seaweed liquid fertilizer by utilizing seaweed and application thereof. Background Art

[0002] Bark algae belongs to the genus Laminariaceae of the order Laminariaceae of the order Phaeophyceae. Bark algae originally grows on the coast of South Africa and Chile. China imports this algae species as a raw material for alginate production. Its outer layer is hard and gray-brown like bark, which makes it difficult to produce, process and extract active substances. Bark algae has high economic value and has great development potential in agriculture and industry. It is an important fertilizer, feed and industrial raw material. In addition to brown algae starch, the carbohydrates of bark algae mainly contain alginate, fucoidan, hemicellulose and cellulose.

[0003] Chinese patent CN118374377A, entitled "A marine bacterium for degrading South African bark algae and its application", discloses a marine bacterium for degrading bark algae and its application. The Klebsiella S401 has the activity of degrading alginate and can effectively degrade bark algae. After its degradation product was applied to plant rooting experiments, it was found that it can significantly promote the development of plant roots.

[0004] Seaweed fertilizer is a kind of fertilizer that is directly produced and processed from natural seaweed as raw material or mixed with a certain amount of nitrogen, phosphorus, potassium and trace elements, and refined by physical or biochemical methods. Its core substance is seaweed extract. The common types of seaweed fertilizer products on the market can be divided into flushing fertilizer and matrix fertilizer. Flush fertilizers are available in two types: liquid and water-soluble powder. They are concentrated from seaweed extract and are suitable for most crops. Flush fertilizers usually need to be diluted when used, and the dilution multiple is determined by foliar spraying, root flushing, drip irrigation and other methods or different growth periods of the plants. Seaweed matrix fertilizer refers to a fertilizer that uses seaweed as the main source of organic matter and is compounded with inorganic nutrients, other types of organic fertilizers or microbial fertilizers.

[0005] At present, there are three common methods for seaweed extraction: physical extraction, chemical extraction and biological extraction. Physical and chemical extraction methods have disadvantages such as low yield, destruction of product activity and poor controllability of the reaction process. Biological extraction has the characteristics of high efficiency, environmental protection and high product activity, and has become a preparation method with great application potential in the current seaweed extraction process. Biological extraction methods include microbial fermentation and enzymatic degradation.

[0006] The surface of the seaweed cell wall is usually composed of cellulose and alginate arranged alternately. The biological extraction method uses enzymes or microorganisms to hydrolyze specific glycosidic bonds at room temperature, evenly disintegrate the seaweed cell wall, and release the cell contents. The biological extraction method does not involve the use of strong acids and alkalis, high temperatures or high pressures. It can preserve the biological activity and nutrients in the seaweed to the greatest extent, and produce excellent fertilizer effects. The biological extraction method is technically difficult. The production process of seaweed fertilizer has a great influence on the type and content of active substances in the product. The key to the quality of seaweed fertilizer lies in the choice of production process. Summary of the invention

[0007] In view of the above-mentioned prior art, the present invention provides a method for preparing bark algae liquid fertilizer by utilizing bark algae and application thereof.

[0008] The present invention provides a method for preparing bark algae liquid fertilizer, comprising the following steps:

[0009] (1) Raw material processing: Wash the fresh bark algae, drain the surface water, grind, homogenize, and concentrate under reduced pressure until the volume is reduced by half to prepare a concentrated homogenate;

[0010] (2) First enzymatic hydrolysis: Take 35-50 portions of the concentrated homogenate, add 1000U / g cellulase, and perform enzymatic hydrolysis at 40-45℃ for 6-8h. During the enzymatic hydrolysis, the pH of the homogenate is adjusted to 5.0-5.5 to obtain the first enzymatic hydrolyzate;

[0011] (3) Second enzymatic hydrolysis: The first enzymatic hydrolysis solution was cooled to 33-38°C, and the compound enzyme was added for enzymatic hydrolysis for 20-25 hours. During the enzymatic hydrolysis, the pH was adjusted to 5.8-6.2, and the solution was centrifuged at 4000 rpm for 20-25 minutes to obtain the second enzymatic hydrolysis solution;

[0012] (4) Fermentation: After the enzymatic hydrolysis is completed, the pH is adjusted to 6.5-6.8, and the activated composite bacterial agent is added to the second enzymatic hydrolysis solution and fermented for 28-32 hours. During the fermentation period, the fermentation liquid is continuously stirred at a speed of 300-400 rpm; (5) The fermentation liquid is centrifuged to separate the supernatant, which is the bark algae liquid fertilizer.

[0013] Furthermore, the complex enzyme consists of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the added amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively.

[0014] Furthermore, the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium breve in a weight ratio of 2:3:2:4, and the addition amount of the composite bacterial agent is 6-10×10 8 CFU / L.

[0015] Furthermore, the bark algae is South African bark algae.

[0016] Since the cell wall surface structure of each seaweed is different, the biological extraction method has high requirements for the preparation of bacteria and enzymes. The same microorganism or enzyme has different affinities for different seaweed cell walls, and different enzymes will greatly affect the degradation efficiency. The seaweed extraction process has a great influence on the types and content of active substances in seaweed fertilizers.

[0017] The present invention adopts a method of combining enzymatic hydrolysis with microbial fermentation to prepare bark algae liquid fertilizer using bark algae as raw material. The algae cell wall is degraded by enzymatic hydrolysis to dissolve the intracellular active components, and the bark algae is enzymatically hydrolyzed by the composite enzyme, which has the advantages of short time, high extraction efficiency, mild reaction conditions, etc. Microbial fermentation decomposes macromolecules into small molecules that are easily absorbed by plants.

[0018] The bark algae liquid fertilizer treatment of the present invention can significantly enhance the activity of antioxidant enzymes in leaves, effectively remove oxygen free radicals in leaves, reduce oxidative damage to cell membranes, and improve the drought resistance of Chinese cabbage. The bark algae liquid fertilizer of the present invention can enhance the drought resistance of Chinese cabbage and increase the yield of Chinese cabbage. The bark algae liquid fertilizer of the present invention can effectively prevent and treat cucumber blight, and can promote the growth of cucumber seedlings and increase the yield.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention adopts an enzymatic hydrolysis and fermentation combined process to extract the active substances of the bark algae. A combination of multiple enzymes degrades the bark algae, which can remove the fiber structure cell wall of the bark algae, quickly release the effective components of the bark algae, well retain the effective active components in the bark algae, and the synergistic enhancement between different enzymes significantly improves the extraction rate of the active components. The compound bacterial agent ferments the bark algae enzymatic hydrolyzate, fully releases the bark algae cell contents, fully retains the effective active components in the bark algae, and decomposes the macromolecular substances into small molecular substances that are easily absorbed by plants.

[0021] The bark algae liquid fertilizer provided by the invention can directly act on plants, enhance the drought resistance of Chinese cabbage, and increase the yield of Chinese cabbage. The bark algae liquid fertilizer of the invention effectively prevents and treats cucumber blight, and can promote the growth of cucumber seedlings and increase the yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for the specific implementation methods are briefly introduced below.

[0023] Figure 1 Schematic diagram of the effect of different bark algae liquid fertilizer treatments on the yield increase of Chinese cabbage. Compared with the blank control group, # p<0.05, ## p<0.01; compared with the group in Example 1, *p<0.05, **p<0.01.

[0024] Figure 2 Schematic diagram of the effect of different bark algae liquid fertilizer treatments on the malondialdehyde content in Chinese cabbage. Compared with the blank control group, # p<0.05, ## p<0.01; compared with the group in Example 1, *p<0.05, **p<0.01.

[0025] Figure 3 Schematic diagram of the effect of different bark algae liquid fertilizer treatments on the SOD activity of Chinese cabbage. Compared with the blank control group, # p<0.05, ## p<0.01; compared with the group in Example 1, *p<0.05, **p<0.01.

[0026] Figure 4 Schematic diagram of the effect of different bark algae liquid fertilizer treatments on the POD activity of Chinese cabbage. Compared with the blank control group, # p<0.05, ## p<0.01; compared with the group in Example 1, *p<0.05, **p<0.01.

[0027] Figure 5 Schematic diagram of the effect of different bark algae liquid fertilizer treatments on the incidence of cucumber. Compared with the blank control group, # p<0.05, ## p<0.01; compared with the group in Example 1, *p<0.05, **p<0.01.

[0028] Figure 6 Schematic diagram of the control effect of different bark algae liquid fertilizer treatments on cucumber.

[0029] Figure 7 Schematic diagram of the increase in cucumber yield due to different bark algae liquid fertilizer treatments. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified; the materials described are commercially available unless otherwise specified.

[0032] Example 1

[0033] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate were taken, 1000U / g cellulase was added, and enzymolysis was performed at 42°C for 7 hours, and the pH of the homogenate was adjusted to 5.2 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 35°C, and a composite enzyme was added for enzymolysis for 22 hours, and the pH was adjusted to 6.0 during the enzymolysis process, and centrifuged at 4000rpm for 22 minutes to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.6, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 30 hours, and the fermentation solution was continuously stirred at a speed of 350rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0034] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 8×10 8 CFU / L.

[0035] Example 2

[0036] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 35 portions of the concentrated homogenate were taken, 1000U / g of cellulase was added, and enzymolysis was performed at 45°C for 8 hours, and the pH of the homogenate was adjusted to 5.0 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 38°C, and a composite enzyme was added for enzymolysis for 20 hours, and the pH was adjusted to 6.2 during the enzymolysis process, and centrifuged at 4000rpm for 20 minutes to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.5, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 32 hours, and the fermentation solution was continuously stirred at a speed of 300rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0037] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 10×10 8 CFU / L.

[0038] Example 3

[0039] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 50 portions of the concentrated homogenate were taken, 1000U / g of cellulase was added, and enzymolysis was performed at 45°C for 6 hours, and the pH of the homogenate was adjusted to 5.5 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 33°C, and a composite enzyme was added for enzymolysis for 20 hours, and the pH was adjusted to 5.8 during the enzymolysis process, and the second enzymolysis solution was obtained by centrifugation at 4000rpm for 25min; after the enzymolysis was completed, the pH was adjusted to 6.8, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 28 hours, and the fermentation solution was continuously stirred at a speed of 400rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0040] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 6×10 8 CFU / L.

[0041] Example 4

[0042] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 40 portions of the concentrated homogenate were taken, 1000U / g of cellulase was added, and enzymolysis was performed at 45°C for 6 hours, and the pH of the homogenate was adjusted to 5.5 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 38°C, and a composite enzyme was added for enzymolysis for 20 hours, and the pH was adjusted to 6.2 during the enzymolysis process, and centrifuged at 4000rpm for 25min to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.5, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 28 hours, and the fermentation solution was continuously stirred at a speed of 400rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0043] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 9×10 8 CFU / L.

[0044] Example 5

[0045] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 50 portions of the concentrated homogenate were taken, 1000U / g of cellulase was added, and enzymolysis was performed at 40°C for 6 hours, and the pH of the homogenate was adjusted to 5.5 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 33°C, and a composite enzyme was added for enzymolysis for 25 hours, and the pH was adjusted to 5.8 during the enzymolysis process, and centrifuged at 4000rpm for 25 minutes to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.5, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 28 hours, and the fermentation solution was continuously stirred at a speed of 400rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0046] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is -7×10 8 CFU / L.

[0047] Comparative Example 1

[0048] Compared with Example 1, the difference is that the compound bacterial agent is different.

[0049] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate were taken, 1000U / g cellulase was added, and enzymolysis was performed at 42°C for 7 hours, and the pH of the homogenate was adjusted to 5.2 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 35°C, and a composite enzyme was added for enzymolysis for 22 hours, and the pH was adjusted to 6.0 during the enzymolysis process, and centrifuged at 4000rpm for 22 minutes to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.6, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 30 hours, and the fermentation solution was continuously stirred at a speed of 350rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0050] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum and Lactobacillus rhamnosus, and the weight ratio is 2:4:2, and the addition amount of the composite bacterial agent is 8×108 CFU / L.

[0051] Comparative Example 2

[0052] Compared with Example 1, the difference is that the first enzymatic hydrolysis is not performed.

[0053] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate were taken, and a composite enzyme was added at 35°C for enzymatic hydrolysis for 22 hours. During the enzymatic hydrolysis, the pH was adjusted to 6.0, and the solution was centrifuged at 4000 rpm for 22 minutes to obtain an enzymatic solution; after the enzymatic hydrolysis was completed, the pH was adjusted to 6.6, and the activated composite bacterial agent was added to the enzymatic solution, and fermented for 30 hours. During the fermentation, the fermentation solution was continuously stirred at a speed of 350 rpm; the fermentation solution was centrifuged to separate the fermentation solution, and the supernatant obtained was the bark algae liquid fertilizer.

[0054] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the addition amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 8×10 8 CFU / L.

[0055] Comparative Example 3

[0056] Compared with Example 1, the difference is that the complex enzyme is different.

[0057] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate were taken, 1000U / g cellulase was added, and enzymolysis was performed at 42°C for 7 hours, and the pH of the homogenate was adjusted to 5.2 during the enzymolysis process to obtain a first enzymolysis solution; the first enzymolysis solution was cooled to 35°C, and a composite enzyme was added for enzymolysis for 22 hours, and the pH was adjusted to 6.0 during the enzymolysis process, and centrifuged at 4000rpm for 22 minutes to obtain a second enzymolysis solution; after the enzymolysis was completed, the pH was adjusted to 6.6, and the activated composite bacterial agent was added to the second enzymolysis solution, and fermented for 30 hours, and the fermentation solution was continuously stirred at a speed of 350rpm during the fermentation; the fermentation solution was centrifuged to separate the fermentation solution, and the obtained supernatant was the bark algae liquid fertilizer.

[0058] The composite enzyme is composed of cellobiohydrolase, saccharifying enzyme, α-maltase, alginate lyase, subtilisin and plant protease, and the addition amounts are 1000U / g, 1000U / g, 600U / g, 800U / g, 800U / g and 600U / g respectively; the composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve, and the weight ratio is 2:3:2:4, and the addition amount of the composite bacterial agent is 8×10 8 CFU / L.

[0059] Comparative Example 4

[0060] Compared with Example 1, the difference is that the second enzymolysis is not performed.

[0061] Fresh South African bark algae was washed, and after the surface water was drained, it was ground, homogenized, and concentrated under reduced pressure until the volume was reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate were added with 1000U / g cellulase, and enzymolysis was performed at 42°C for 7 hours. During the enzymolysis process, the pH of the homogenate was adjusted to 5.2 to obtain an enzymolysis solution. After the enzymolysis was completed, the pH was adjusted to 6.6, and the activated composite bacterial agent was added to the enzymolysis solution, and fermented for 30 hours. During the fermentation period, the fermentation solution was continuously stirred at a speed of 350rpm; the fermentation solution was centrifuged to separate the fermentation solution, and the supernatant obtained was the bark algae liquid fertilizer.

[0062] The composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium breve in a weight ratio of 2:3:2:4. The addition amount of the composite bacterial agent is 8×10 8 CFU / L.

[0063] Comparative Example 5

[0064] Compared with Example 1, the difference is that no fermentation is performed.

[0065] Fresh South African bark algae is washed, drained of surface water, ground, homogenized, and concentrated under reduced pressure until the volume is reduced by half to prepare a concentrated homogenate; 45 portions of the concentrated homogenate are added with 1000U / g cellulase, and enzymolyzed at 42°C for 7h, during which the pH of the homogenate is adjusted to 5.2 to obtain a first enzymolysis solution; the first enzymolysis solution is cooled to 35°C, and a composite enzyme is added for enzymolysis for 22h, during which the pH is adjusted to 6.0, and the solution is centrifuged at 4000rpm for 22min to obtain a second enzymolysis solution; the second enzymolysis solution is centrifuged to separate the supernatant, which is the bark algae liquid fertilizer.

[0066] The complex enzyme consists of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the added amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively.

[0067] Test Example 1

[0068] The seeds of Biqingcaixin were used as the test variety. After the surface of Biqingcaixin seeds was disinfected with 70% anhydrous ethanol for 15 minutes, they were rinsed with distilled water and then sown in the seedling tray. When the seedlings grew to four leaves and one heart, they were transplanted into pots filled with mixed soil (the mass ratio of surface paddy soil and fine sand was 3:1), with 2 plants transplanted per pot.

[0069] On the 7th and 17th days after transplanting the seedlings, dilute the liquid fertilizer 300 times and irrigate the roots, with 100 mL of fertilizer per seedling.

[0070] Example 1 group, Example 2 group, Control Example 1 group, Control Example 2 group, Control Example 3 group, Control Example 4 group, and Control Example 5 group were root-fertilized with Example 1, Example 2, Control Example 1, Control Example 2, Control Example 3, Control Example 4, and Control Example 5 bark algae liquid fertilizer, respectively.

[0071] The blank control group was operated the same as in Example 1 except that the bark algae liquid fertilizer was not sprayed.

[0072] During this period, the seedlings grew under normal water conditions (soil moisture content was 75%-85%). After the last root irrigation, water was cut off. The soil moisture content was 55%-68%, 45%-54%, 36%-42%, 28%-34%, and 23%-27% at 1, 2, 3, 4, and 5 days of water cutoff, respectively. The seedlings were harvested at 5 days of water cutoff to determine the fresh weight of the green cabbage heart seedlings, and the third unfolded leaf of the cabbage heart was taken to determine the relevant physiological indicators (malondialdehyde content, antioxidant enzyme activity). Randomized block arrangement was used during the treatment process, and each treatment was repeated 3 times.

[0073] The malondialdehyde content was determined by thiobarbituric acid colorimetry. Take 0.5g of Chinese cabbage leaves, add 5mL of 10% trichloroacetic acid, grind into slurry, and centrifuge for 10 minutes. Take 2mL of supernatant, add 2mL of 0.6% thiobarbituric acid, mix well, and heat in a boiling water bath for 15 minutes. After cooling, centrifuge for 10 minutes, and measure its absorbance at 600, 532, and 450nm respectively.

[0074] Determination of antioxidant enzyme activity: Weigh 0.5 g of Chinese cabbage sample, add 2 mL of 50 mmol / L phosphate buffer (PH7.8), grind into slurry under ice bath conditions, dilute to 5 mL with buffer, centrifuge at 10000 r / min, 4 ° C for 15 min, and the supernatant is used to determine the antioxidant enzyme activity. Superoxide dismutase (SOD) is determined by nitroblue tetrazolium photoreduction method; peroxidase (POD) is determined by guaiacol colorimetry.

[0075] like Figure 1As shown, under drought conditions, the growth of Chinese cabbage is inhibited. Compared with the blank control group, Example 1, Example 2, Control Example 1, Control Example 2, Control Example 3, Control Example 4, and Control Example 5 can improve the fresh quality of Chinese cabbage, and each group increases the yield by 81.70%, 72.48%, 45.39%, 36.60%, 23.83%, 29.65%, and 22.13%, respectively. The effect of bark algae liquid fertilizer in Example 1 and Example 2 on increasing the yield of Chinese cabbage is significantly better than that in the control group.

[0076] like Figure 2 As shown, after water cut-off, the cell membrane of the mesophyll cells was damaged and the content of malondialdehyde increased significantly. When water was cut off for 5 days, compared with the blank control group, the treatment with the bark algae liquid fertilizer of the present invention can significantly inhibit the production of malondialdehyde. The malondialdehyde content of the leaves treated in Example 1 and Example 2 was reduced by 44.19% and 40.17% respectively compared with the blank control group. The effect of the bark algae liquid fertilizer in Example 1 and Example 2 on inhibiting the production of malondialdehyde is significantly better than that in the control group.

[0077] like Figure 3 As shown, after 5 days of water cut-off, the SOD activities of the bark algae liquid fertilizer treatment in Example 1 and Example 2 were 24.86 and 23.17 U, respectively (based on the catalytic capacity of the enzyme per milligram of protein), which were 3.26 and 3.04 times that of the blank control group. The effect of the bark algae liquid fertilizer in Example 1 and Example 2 on improving the activity of the antioxidant enzyme SOD was significantly better than that in the control group.

[0078] like Figure 4 As shown, after 5 days of water cut-off, the POD activities of the bark algae liquid fertilizer treatment in Example 1 and Example 2 were 456.39 and 442.85 U, respectively (calculated as the catalytic capacity of the enzyme contained per milligram of protein), which were 1.36 and 1.32 times that of the blank control group. The effect of the bark algae liquid fertilizer in Example 1 and Example 2 on improving the antioxidant enzyme POD activity was significantly better than that in the control group.

[0079] The bark algae liquid fertilizer treatment of the present invention can significantly enhance the activity of antioxidant enzymes in leaves, effectively remove oxygen free radicals in leaves, reduce oxidative damage to cell membranes, and improve the drought resistance of Chinese cabbage. The bark algae liquid fertilizer of the present invention can enhance the drought resistance of Chinese cabbage and increase the yield of Chinese cabbage. The effects of the bark algae liquid fertilizer of Example 1 and Example 2 on enhancing the drought resistance of Chinese cabbage and increasing the yield of Chinese cabbage are significantly better than those of the control group.

[0080] Test Example 2

[0081] Cucumber blight, commonly known as blast and dead vine, is a disease of cucumber caused by infection with Phytophthora dermestii. Cucumber blight can occur throughout the entire growth period of cucumbers and can infect the leaves, stems and fruits of cucumbers, with more cases occurring at the base of the vines and tender stem nodes.

[0082] Cucumber blight is common and has a great impact on cucumber production. In greenhouses where it is serious, it is the cucumber harvesting season, and cucumber blight suddenly breaks out on a large scale, causing large areas of cucumber to die and severely reducing production.

[0083] The field disease prevention test of the bark algae liquid fertilizer of the present invention was carried out in a plastic greenhouse. The selected greenhouse had been continuously planted with cucumbers for more than 5 years, and the incidence of cucumber blight was high; the soil texture was sandy loam; the soil organic matter content was 14.6 g / kg, and the pH was 6.9. The test was divided into Example 1, Example 3, Control Example 1, Control Example 2, Control Example 3, Control Example 4, Control Example 5 and a blank control group. Each treatment was set to 3 replicates / plot, and the area of ​​each plot was 50 m 2 , each plot is randomly arranged.

[0084] Cucumbers were sown and transplanted into the greenhouse 22-25 days later. When transplanted, the cucumber seedlings were 12-16cm tall, with 3 true leaves and strong growth. When transplanting, the soil of the cucumber seedlings was inoculated with a sufficient amount of soil-borne pathogenic bacteria, cucumber blight. The bark algae liquid fertilizer of each group was diluted 300 times and then irrigated with roots. Each seedling was irrigated with 100mL of fertilizer. The blank control group was treated with clean water instead of bark algae liquid fertilizer.

[0085] Use it again at 7d and 20d respectively, and count the disease occurrence of cucumbers at 45d.

[0086] like Figure 5-Figure 7 As shown, the incidence of cucumber blight after treatment with the bark algae liquid fertilizer of the present invention is significantly lower than that of the blank control group. In the field test, the bark algae liquid fertilizer of Example 1 and Example 3 has a certain control effect and yield increase rate on cucumber blight, and the control effect is 63.74% and 61.40% respectively, and the yield increase rate is 83.61% and 75.34% respectively. The bark algae liquid fertilizer of the present invention effectively prevents and controls cucumber blight, and can promote the growth of cucumber seedlings and increase the yield.

[0087] 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 method for preparing bark algae liquid fertilizer, characterized in that: The following steps are involved: (1) Raw material processing: Wash the fresh bark algae, drain the surface water, grind, homogenize, and concentrate under reduced pressure until the volume is reduced by half to prepare a concentrated homogenate; (2) First enzymatic hydrolysis: Take 35-50 portions of the concentrated homogenate and add 1000 U / g cellulase for enzymatic hydrolysis to obtain the first enzymatic hydrolyzate; (3) Second enzymatic hydrolysis: Cool the first enzymatic hydrolysis solution, add compound enzyme for enzymatic hydrolysis, and centrifuge to obtain the second enzymatic hydrolysis solution; (4) Fermentation: After the enzymatic hydrolysis is completed, the pH is adjusted and the activated composite bacterial agent is added to the second enzymatic hydrolysis solution for fermentation. During the fermentation, the fermentation solution is continuously stirred at a speed of 300-400 rpm; (5) centrifuging the fermentation liquid to obtain the supernatant, which is the bark algae liquid fertilizer; The complex enzyme consists of cellobiohydrolase, saccharifying enzyme, α-amylase, pectinase, subtilisin and plant protease, and the added amounts are 800U / g, 800U / g, 600U / g, 1000U / g, 1000U / g and 600U / g respectively; The composite bacterial agent is composed of Bacillus subtilis, Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium breve in a weight ratio of 2:3:2:

4. The addition amount of the composite bacterial agent is 6-10×10 8 CFU / L; The temperature of the first enzymolysis is 40-45°C, the enzymolysis time is 6-8h, and the enzymolysis pH is 5.0-5.5; In the second enzymolysis, the first enzymolysis solution is cooled to 33-38° C., the enzymolysis time is 20-25 h, and the enzymolysis pH is 5.8-6.

2.

2. The method for preparing bark algae liquid fertilizer according to claim 1, characterized in that: The bark algae is South African bark algae.

3. The method for preparing bark algae liquid fertilizer according to claim 1, characterized in that: In the second enzymatic hydrolysis, the centrifugation condition is 4000 rpm for 20-25 min.

4. The method for preparing bark algae liquid fertilizer according to claim 1, characterized in that: The fermentation pH is 6.5-6.8 and the fermentation time is 28-32h.

5. Use of the bark algae liquid fertilizer prepared by the preparation method of the bark algae liquid fertilizer according to any one of claims 1 to 4 in enhancing the drought resistance of Chinese cabbage and increasing the yield of Chinese cabbage.

6. Use of the bark algae liquid fertilizer prepared by the preparation method of the bark algae liquid fertilizer according to any one of claims 1 to 4 in preventing and controlling cucumber blight and increasing cucumber yield.

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