New application of sargassum enzymolysis fermentation product

Abstract

The invention discloses a novel application of a sargassum enzymolysis fermentation product, which comprises the application of the sargassum enzymolysis fermentation product in preparation of a product for improving the immune activity of grass carp or improving the bacterial infection resistance of the grass carp. The preparation method of the sargassum enzymolysis fermentation product comprises the following steps: cleaning fresh sargassum, crushing, curing, soaking and adjusting the pH value to obtain a sargassum solution, adding a compound enzyme into the sargassum solution for enzymolysis, then carrying out enzyme deactivation treatment, adding a culture solution into the sargassum enzymolysis product, inoculating a compound strain into the sargassum enzymolysis product, and carrying out fermentation treatment to obtain the sargassum enzymolysis fermentation product. And sterilizing and drying to obtain the sargassum enzymolysis fermentation product. According to the sargassum enzymolysis fermentation product, the immunoregulation capability of a grass carp kidney tissue cell line and the immunoregulation capability of grass carp are improved, and the survival rate of the grass carp infected by bacteria, especially aeromonas hydrophila, can be improved by the sargassum enzymolysis fermentation product.

Description

Technical Field

[0001] The present invention belongs to the technical field of Sargassum, and particularly relates to a new use of an enzymolysis fermentation product of Sargassum. Background Art

[0002] Grass carp is one of the varieties with the highest yield in aquaculture in China. However, in a highly intensive aquaculture environment, grass carp is vulnerable to infections by pathogens such as bacteria, viruses, and parasites. Among them, bacterial inflammation caused by Aeromonas hydrophila is one of the most common diseases in grass carp aquaculture. Due to the insufficient research on the pathogenesis of fish diseases at present, there are great challenges in developing new drugs for aquaculture. Therefore, more and more research has begun to focus on the application of immunostimulants to improve the immunity of fish, thereby reducing the disease outbreaks caused by Aeromonas hydrophila.

[0003] In aquaculture, common polysaccharide immunostimulants mainly include polysaccharides from microbial sources (such as β-glucan, lipopolysaccharide, and peptidoglycan), polysaccharides from animal sources (such as chitosan), and seaweed polysaccharides. Seaweed polysaccharides can effectively stimulate the differentiation, maturation, and proliferation of various immune cells (such as macrophages, T lymphocytes, and B lymphocytes), thereby enhancing the immune response of the body. However, the extraction process of seaweed polysaccharides is relatively complex, the technology is not yet mature, and the production cost is relatively high. Therefore, using the enzymolysis fermentation product of seaweed as an immunostimulant has become an effective strategy to reduce costs and improve the immunity of fish.

[0004] Sargassum spp. belongs to the Phaeophyta and is widely distributed along the coast of China. Common species include Sargassum pallidum and Sargassum kjellmanianum, etc. Sargassum is rich in various bioactive components, but its macromolecular polysaccharides usually have a dense structure and poor water solubility, which limits the release of its active components. Therefore, using enzymolysis technology to hydrolyze the macromolecular polysaccharides in Sargassum that are difficult to utilize can significantly improve the utilization rate of active substances, which is a key step in exerting the biological activity function of Sargassum. In addition, the enzymes released during the solid-state fermentation of microorganisms can effectively degrade complex organic components such as alginate, cellulose, protein, and starch in Sargassum.

[0005] In the prior art, different enzymolysis or fermentation methods for treating Sargassum have been disclosed. However, due to different methods, there are significant differences in the degradation effect of Sargassum and the quality of the final product, and the uses are also different. There is currently no report on using the enzymolysis fermentation product of Sargassum to improve the anti-immunity of grass carp. Summary of the Invention

[0006] The purpose of the present invention is to provide a new use of an enzymolysis fermentation product of Sargassum.

[0007] The above object of the present invention can be achieved by the following technical solutions: a new use of the enzymatically hydrolyzed fermented product of Sargassum, and the new use includes the application of the enzymatically hydrolyzed fermented product of Sargassum in the preparation of products for improving the immune activity of grass carp or enhancing the ability of grass carp to resist bacterial infection.

[0008] Preferably, the improvement of the immune activity of grass carp includes that the enzymatically hydrolyzed fermented product of Sargassum improves the immunomodulatory ability of grass carp kidney tissue cell line and the enzymatically hydrolyzed fermented product of Sargassum improves the immunomodulatory ability of grass carp; wherein, the enzymatically hydrolyzed fermented product of Sargassum improving the immunomodulatory ability of grass carp kidney tissue cell line includes reducing the expression of inflammatory factors, tumor necrosis factor and inflammatory signaling pathway in grass carp kidney tissue cells, and the enzymatically hydrolyzed fermented product of Sargassum improving the immunomodulatory ability of grass carp includes reducing the expression of inflammatory factors, tumor necrosis factor and inflammatory signaling pathway in the head kidney tissue of grass carp.

[0009] Preferably, the bacterium is Aeromonas hydrophila.

[0010] As a preferred technical solution of the present invention, the present invention also provides a preparation method of the above-mentioned enzymatically hydrolyzed fermented product of Sargassum, including the following steps:

[0011] (1) Fresh Sargassum is washed, dried and ground into powder, and then sieved.

[0012] (2) The Sargassum powder is cooked in a high-temperature environment.

[0013] (3) The cooked Sargassum powder is added to phosphate buffer solution and soaked.

[0014] (4) Acid or alkali is added to the soaked Sargassum powder to adjust the pH value to obtain a Sargassum solution.

[0015] (5) A complex enzyme is added to the Sargassum solution for enzymatic hydrolysis, and the complex enzyme is cellulase, pectinase, papain and alginate lyase.

[0016] (6) After enzymatic hydrolysis, enzyme inactivation and sterilization are carried out to obtain an enzymatic hydrolysate, and the enzymatic hydrolysate is freeze-dried to obtain a Sargassum enzymatically hydrolyzed product.

[0017] (7) A culture medium is added to the Sargassum enzymatically hydrolyzed product, the ratio of material to liquid is adjusted, and the mixture is fully mixed and allowed to stand.

[0018] (8) A composite bacterial seed solution of Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus rhamnosus and recombinant yeast DH5α producing alginate lyase is inoculated for solid-state aerobic fermentation, and turning treatment is carried out during the fermentation process to obtain a fermentation product of the Sargassum enzymatically hydrolyzed product.

[0019] (9) The fermentation product of the Sargassum enzymatically hydrolyzed product is subjected to high-pressure sterilization, freeze-dried and stored frozen to obtain the enzymatically hydrolyzed fermented product of Sargassum.

[0020] In the above method for preparing the enzymatically hydrolyzed and fermented sargassum:

[0021] Preferably, when sieving in step (1), the mesh number of the sieve is 60 - 100.

[0022] Preferably, in step (2), the high - temperature environment is 90 - 100 °C, and the ripening time is 1 - 3 h.

[0023] Preferably, in step (3), the ripened sargassum powder is added to the phosphate - buffer solution and soaked for 3 - 6 h, where the amount of the phosphate - buffer solution is 5 - 10 times the total mass of the ripened sargassum powder.

[0024] Preferably, the phosphate - buffer solution PBS is prepared according to the conventional standard method. For example, for the PBS 1L formula, 0.24 g of potassium dihydrogen phosphate (KH2PO4), 1.44 g of disodium hydrogen phosphate (Na2HPO4), 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCI) are added. About 800 mL of deionized water is added and stirred well to dissolve. Then concentrated hydrochloric acid is added to adjust the pH to 7.4, and finally the volume is made up to 1L.

[0025] Preferably, in step (4), an acid or a base is used to adjust the pH value to 4.5 - 6.5.

[0026] Preferably, the acid is an aqueous hydrochloric acid solution or an aqueous acetic acid solution, and its mass percentage content is 8 - 12%. The base is an aqueous sodium hydroxide solution, and its mass percentage content is 1 - 3%.

[0027] Preferably, in step (5), the addition amount of the cellulase is 1 - 3% of the total mass of the sargassum solution, the addition amount of the pectinase is 1 - 3% of the total mass of the sargassum solution, the addition amount of the papain is 1 - 3% of the total mass of the sargassum solution, and the addition amount of the alginate lyase is 1 - 3% of the total mass of the sargassum solution.

[0028] Preferably, the activity unit of the cellulase is 100,000 U / g, the activity unit of the pectinase is 30,000 U / g, the activity unit of the papain is 100,000 U / g, and the activity unit of the alginate lyase is 100,000 U / g.

[0029] Preferably, in step (5), the enzymatic hydrolysis temperature is 40 - 60 °C, and the enzymatic hydrolysis time is 3 - 7 h.

[0030] Preferably, the cellulase, pectinase, papain, and alginate lyase in step (5) are commercially available products.

[0031] Preferably, in step (6), it is heated to 105 °C for enzyme inactivation and sterilization to obtain the enzymolysis solution.

[0032] Preferably, the culture medium in step (7) is a mixed culture medium of YPD culture medium, LB culture medium and MRS culture medium, and the volume ratio of the YPD culture medium, LB culture medium and MRS culture medium is 1:2:1.

[0033] Preferably, the formula of 1 L of the YPD culture medium is: 10 g / L yeast extract powder, 20 g / L glucose, 20 g / L sodium chloride, 20 g / L soy peptone.

[0034] Preferably, the formula of 1 L of the LB culture medium is: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0035] Preferably, the formula of 1 L of the MRS culture medium is: 10 g / L beef extract, 10 g / L tryptone, 20 g / L glucose, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate hydrogen, 5 g / L sodium acetate, 0.1% Tween 80, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate.

[0036] Preferably, the dosage relationship between the sargassum enzymolysate and the culture medium in step (7) is 1 g: 0.3 - 0.8 L.

[0037] Preferably, the mixing time in step (7) is 10 - 20 min, and the standing time is 1 - 3 h.

[0038] Preferably, the inoculation ratio of Bacillus subtilis in step (7) is 1% - 3%, v / v, the inoculation ratio of Saccharomyces cerevisiae is 1% - 4%, v / v, the inoculation ratio of Lactobacillus rhamnosus GG is 1% - 3%, v / v, and the inoculation ratio of the recombinant yeast DH5α producing alginate lyase is 2% - 4%, v / v.

[0039] Preferably, the inoculation density of Bacillus subtilis in step (8) is 2×10 7 -6×10 7 CFU / mL, the inoculation density of Saccharomyces cerevisiae is 2×10 7 -5×10 7 CFU / mL, the inoculation density of Lactobacillus rhamnosus GG is 4×10 7 -8×10 7 CFU / mL, and the inoculation density of the recombinant yeast DH5α producing alginate lyase is 1×10 8 -3×10 8 CFU / mL.

[0040] Preferably, in step (8), the temperature of the solid-state aerobic fermentation is 30-42°C, the fermentation period is 60-96 h, and the turning period is 20-26 h.

[0041] By using the method of the present invention to prepare the enzymolysis ferment of Sargassum, the contents of cellulose and hemicellulose in Sargassum can be reduced, and the contents of polysaccharides, alginic acid and algooligosaccharides in Sargassum calculated as glucose can be increased. Therefore, by using the method of the present invention, the degradation efficiency of Sargassum can be improved.

[0042] Therefore, the present invention also provides the application of the above method in improving the degradation efficiency of Sargassum.

[0043] The enzymolysis ferment of Sargassum obtained in the present invention is dissolved in double-distilled water, fully soaked and stirred to obtain an aqueous solution of the enzymolysis ferment of Sargassum. The aqueous solution is filtered and sterilized and added to the culture solution of grass carp kidney tissue cell line, and at the same time, the aqueous solution of the enzymolysis ferment of Sargassum is intraperitoneally injected into the grass carp.

[0044] The test results show that: the enzymolysis ferment of Sargassum improves the immunomodulatory ability of the grass carp kidney tissue cell line and the immunomodulatory ability of the grass carp; among them, improving the immunomodulatory ability of the grass carp kidney tissue cell line includes reducing the expression of inflammatory cytokines interleukin-1β (IL1β), tumor necrosis factor-α (TNFα) and the NF-kappa B signaling pathway (including p65, ikbα, ikkα1, ikkα1, kkβ) of the inflammatory signaling pathway, and improving the immunomodulatory ability of the grass carp includes reducing the expression of inflammatory cytokines interleukin-1β (IL1β), tumor necrosis factor-α (TNFα) and the NF-kappa B signaling pathway (including p65, ikbα, ikkα1, ikkα1, kkβ) of the head kidney tissue.

[0045] The present invention also provides the application of the above enzymolysis ferment of Sargassum in improving the resistance of grass carp to Aeromonas hydrophila infection.

[0046] The present invention conducts a challenge experiment on grass carp treated with the aqueous solution of the enzymolysis ferment of Sargassum, that is, observes the survival of grass carp after intraperitoneal injection of Aeromonas hydrophila. The test results show that: the enzymolysis ferment of Sargassum can improve the survival rate of grass carp under the infection of bacteria, especially Aeromonas hydrophila.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] (1) The enzymolysis ferment of Sargassum obtained in the present invention can enhance the immune activity of grass carp and improve the survival rate of grass carp under bacterial infection;

[0049] (2) The present invention also provides a method for preparing the enzymatically hydrolyzed and fermented product of Sargassum, which significantly reduces the content of macromolecular polysaccharides and improves the degradation efficiency of Sargassum. Description of the Drawings

[0050] Figure 1 It is for the effect of the enzymatically hydrolyzed and fermented product of Sargassum on the immune activity of grass carp kidney cell line in Test Example 2;

[0051] Figure 2 It is for the effect of the enzymatically hydrolyzed and fermented product of Sargassum on the immune activity of grass carp in Test Example 3;

[0052] Figure 3 It is for the effect of the enzymatically hydrolyzed and fermented product of Sargassum on the resistance of grass carp to Aeromonas hydrophila infection in Test Example 4. Detailed Embodiments

[0053] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments and drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; all other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0054] In the following embodiments, unless otherwise specified, they are all conventional methods. Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products.

[0055] The following embodiments recommend but are not limited to:

[0056] The recommended 1L formula of YPD culture medium is: 10 g / L yeast extract powder, 20 g / L glucose, 20 g / L sodium chloride, 20 g / L soy peptone.

[0057] The recommended 1L formula of LB culture medium is: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0058] The recommended 1L formula of MRS culture medium is: 10 g / L beef extract, 10 g / L tryptone, 20 g / L glucose, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate hydrogen, 5 g / L sodium acetate, 0.1% Tween 80, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate.

[0059] Bacillus subtilis was purchased from the official website of CGMCC with the item number CGMCC 1.1086, Saccharomyces cerevisiae was purchased from the China Industrial Microbiological Culture Collection Center with the item number CICC1251, and Lactobacillus rhamnosus was purchased from the official website of CGMCC with the item number CGMCC 1.577. The recombinant yeast DH5α producing alginate lyase was prepared by mutating yeast DH5α, namely H71K. The preparation method was based on previous research doi: 10.3389 / fbioe.2022.1011273.

[0060] Example 1

[0061] The method for preparing the enzymatic fermentation product of Sargassum provided in this embodiment comprises the following steps:

[0062] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0063] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0064] (3) adding phosphate buffered saline (PBS) at a mass of 8 times that of the base material and soaking for 4 h to allow the Sargassum powder to be soaked and softened;

[0065] Phosphate buffer PBS is prepared according to conventional standard methods. For example, the formula of PBS 1L is 0.24g of potassium dihydrogen phosphate (KH2PO4), 1.44g of disodium hydrogen phosphate (Na2HPO4), 8g of sodium chloride (NaCl), and 0.2g of potassium chloride (KCI). Add about 800mL of deionized water and stir thoroughly to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally adjust the volume to 1L.

[0066] (4) Add 2% NaOH or 10% acetic acid to adjust the pH to 5.0;

[0067] (5) adding a composite enzyme, including 1 wt% of cellulase, 1 wt% of pectinase, 1 wt% of papain and 1 wt% of alginate lyase, and performing enzymolysis at 50°C for 4 h, wherein the activity unit of the cellulase is 100,000 U / g, the activity unit of the pectinase is 30,000 U / g, the activity unit of the papain is 100,000 U / g, and the activity unit of the alginate lyase is 100,000 U / g;

[0068] (6) heating to 105° C. to sterilize the enzyme and obtain an enzymatic hydrolyzate. After freeze-drying, the enzymatic hydrolyzate of Sargassum is obtained;

[0069] (7) Prepare a mixed culture medium of YPD culture medium, LB culture medium and MRS culture medium in a volume ratio of 1:2:1, adjust the material-liquid ratio of Sargassum powder to the mixed culture medium to 1 g:0.5 L, mix thoroughly for 20 min, and let stand for 2 h;

[0070] (8) The inoculation volume is 1% and the density is 4×10 7 Bacillus subtilis / mL, volume is 1% and density is 3×10 7 / mL of Saccharomyces cerevisiae, with a volume of 1% and a density of 6×10 7 Lactobacillus rhamnosus, 2% by volume, and a density of 2×10 8 The recombinant yeast DH5α seed solution producing alginate lyase was prepared by solid-state aerobic fermentation at 30°C for 60 hours, with stirring once every 24 hours.

[0071] (9) sterilizing by high pressure, freeze-drying and then storing in a frozen state to obtain the enzymatic hydrolysis and fermentation product of Sargassum.

[0072] Example 2

[0073] The method for preparing the enzymatic fermentation product of Sargassum provided in this embodiment comprises the following steps:

[0074] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0075] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0076] (3) Add PBS 8 times the mass of the base material and soak for 4 h to soften the Sargassum powder;

[0077] (4) adding 2 wt % NaOH or 10 wt % acetic acid to adjust the pH to 5.0;

[0078] (5) adding a composite enzyme, including 2 wt% of cellulase, 2 wt% of pectinase, 2 wt% of papain and 2 wt% of alginate lyase, and performing enzymolysis at 50° C. for 4 h, wherein the activity unit of the cellulase is 100,000 U / g, the activity unit of the pectinase is 30,000 U / g, the activity unit of the papain is 100,000 U / g, and the activity unit of the alginate lyase is 100,000 U / g;

[0079] (6) heating to 105° C. to sterilize the enzyme and obtain an enzymatic hydrolyzate, which is freeze-dried to obtain a Sargassum enzymatic hydrolyzate;

[0080] (7) Prepare a mixed culture medium of YPD culture medium, LB culture medium and MRS culture medium in a volume ratio of 1:2:1, adjust the material-liquid ratio of Sargassum powder to the mixed culture medium to 1 g:0.5 L, mix thoroughly for 20 min, and let stand for 2 h;

[0081] (8) The inoculation volume is 2% and the density is 4×10 7 Bacillus subtilis / mL, volume is 2% and density is 3×10 7Saccharomyces cerevisiae at a density of 6×10 per mL, with a volume of 2% 7 Lactobacillus rhamnosus at a density of 2×10 per mL, with a volume of 3% 8 The seed solution of recombinant yeast DH5α producing alginate lyase at a density of 4×10 per mL, solid-state aerobic fermentation at 37°C for 72 h, turning once every 24 h;

[0082] (9) Sterilize by high pressure, freeze-dry and store frozen to obtain the enzymolysis fermentation product of Sargassum.

[0083] Example 3

[0084] The preparation method of the enzymolysis fermentation product of Sargassum provided in this example includes the following steps:

[0085] (1) Wash fresh Sargassum, remove impurities and surface salts, dry and grind into powder, and pass through a 60-mesh sieve;

[0086] (2) Ripen the Sargassum powder in a high-temperature environment of 95°C for 2 h;

[0087] (3) Add PBS with a mass 8 times that of the bottom material, soak for 4 h to fully soak and soften the Sargassum powder;

[0088] (4) Add 2 wt% NaOH or 10 wt% acetic acid to adjust the pH to 5.0;

[0089] (5) Add a composite enzyme, including 3 wt% cellulase, 3 wt% pectinase, 3 wt% papain, and 3 wt% alginate lyase, enzymolyze at 50°C for 4 h, the activity unit of the cellulase is 100,000 U / g, the activity unit of the pectinase is 30,000 U / g, the activity unit of the papain is 100,000 U / g, and the activity unit of the alginate lyase is 100,000 U / g;

[0090] (6) Heat to 105°C for enzyme inactivation and sterilization to obtain an enzymolysis solution, freeze-dry to obtain the enzymolysis product of Sargassum;

[0091] (7) Prepare a mixed culture solution of YPD culture solution, LB culture solution and MRS culture solution in a volume ratio of 1:2:1, adjust the material-liquid ratio of Sargassum powder to the mixed culture solution to 1 g:0.5 L, mix well for 20 min, and let stand for 2 h;

[0092] (8) Inoculate Bacillus subtilis at a density of 4×10 per mL with a volume of 3%, Saccharomyces cerevisiae at a density of 6×10 per mL with a volume of 3%, Lactobacillus rhamnosus at a density of 2×10 per mL with a volume of 4% 7 per mL, Saccharomyces cerevisiae at a density of 3×10 per mL with a volume of 3% 7 per mL, Lactobacillus rhamnosus at a density of 6×10 per mL with a volume of 3% 7 per mL, Lactobacillus rhamnosus at a density of 2×10 per mL with a volume of 4% 8The recombinant yeast DH5α seed solution producing alginate lyase was prepared by solid-state aerobic fermentation at 42°C for 72 hours, with stirring once every 24 hours.

[0093] Comparative Example 1

[0094] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0095] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0096] (3) Add PBS 8 times the mass of the base material and soak for 4 h to soften the Sargassum powder;

[0097] (4) adding 2 wt % NaOH or 10 wt % acetic acid to adjust the pH to 5.0;

[0098] (5) adding a composite enzyme inactivated at 105° C., including 1 wt % of cellulase, 1 wt % of pectinase, 1 wt % of papain, and 1 wt % of alginate lyase, and performing enzymolysis at 50° C. for 4 h;

[0099] (6) Heating to 105°C to sterilize the enzyme and obtain an enzymatic hydrolyzate. After freeze-drying, the enzymatic hydrolyzate of Sargassum is obtained.

[0100] (7) Prepare a mixed culture medium of YPD culture medium, LB culture medium and MRS culture medium in a volume ratio of 1:2:1, adjust the material-liquid ratio of Sargassum powder to the mixed culture medium to 1 g:0.5 L, mix thoroughly for 20 min, and let stand for 2 h;

[0101] (8) The inoculation volume is 2% and the density is 2×10 8 The recombinant yeast DH5α seed solution producing alginate lyase was prepared by solid-state aerobic fermentation at 30°C for 60 hours, with stirring once every 24 hours.

[0102] (9) sterilizing by high pressure, freeze-drying and then storing in a frozen state to obtain the enzymatic hydrolysis and fermentation product of Sargassum.

[0103] Blank example 1

[0104] Sterile phosphate buffered saline (PBS) was used as blank example 1.

[0105] Test Example 1

[0106] In Test Example 1, a series of specific detection methods were implemented to determine the contents of lignin, hemicellulose, and cellulose. These methods followed the detailed procedures in FUT-SOP(L)-37.1. In addition, to determine the polysaccharide content (calculated as glucose), a method compliant with the standard of SN / T 4260-2015 was adopted. The content of alginic acid was detected by a method following the standard of HG / T 5050-2016. Meanwhile, the content of alginate oligosaccharide was detected according to the method of the standard Q / 371083WX002-2016. The obtained test results were summarized in Table 1 for further analysis and discussion.

[0107] Table 1 Carbohydrate distribution of enzymatically hydrolyzed and fermented Sargassum

[0108]

[0109] Sargassum contains a large amount of dense and poorly water-soluble components such as lignin, hemicellulose, and cellulose. The high viscosity of these components hinders the effective utilization of nutrients and active ingredients. Through the enzymatic hydrolysis and fermentation treatment in the present invention, it is degraded into seaweed polysaccharides, alginic acid, alginate oligosaccharides, etc. with better water solubility.

[0110] As can be seen from Table 1, after the enzymatic hydrolysis and fermentation treatment, the contents of lignin, cellulose, and hemicellulose in Sargassum decreased significantly. At the same time, the contents of seaweed polysaccharides, alginic acid, and alginate oligosaccharides increased significantly. These results indicate that the degradation efficiency of Sargassum has been greatly improved after the enzymatic hydrolysis and fermentation treatment.

[0111] Test Example 2

[0112] Dissolve the enzymatically hydrolyzed and fermented Sargassum in Examples 1-3 and Comparative Example 1 in double-distilled water. The mass of double-distilled water is 5 times the total mass of the enzymatically hydrolyzed and fermented Sargassum. Soak and stir well. The magnetic stirring speed is 500 rpm, and the soaking time is 4 h to obtain an aqueous solution of enzymatically hydrolyzed and fermented Sargassum. Filter and sterilize the aqueous solution with a 0.22 μm filter membrane. Add the sterilized aqueous solution of Sargassum and the buffer solution in Blank Example 1 to the culture medium of grass carp kidney cell line (GIC, purchased from Shenyang Yike Biology), culture for 48 h, and determine the expression of inflammatory factors and inflammatory signaling pathways in grass carp kidney cells.

[0113] Use an ELISA commercial kit to determine the contents of inflammatory factors interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) in the kidney cell line; use qPCR technology to determine the expression of the NF-kappa B signaling pathway in the kidney cell line, including p65, ikbα, ikkα1, ikkα1, ikkβ. The results are as Figure 1 shown.

[0114] From Figure 1It can be seen that, compared with Blank Example 1 and Comparative Example 1, the contents of IL1β and TNFα in Comparative Example 1 were significantly reduced, and at the same time, the expression levels of p65, ikbα, ikkα1, ikkα1, and ikkβ were also significantly reduced. Examples 1-3 further enhanced this trend. These results indicate that the enzymatically hydrolyzed and fermented products of Sargassum significantly enhanced the immune activity of grass carp kidney cells.

[0115] Test Example 3

[0116] Take 100 μL of the aqueous solutions of the sterilized enzymatically hydrolyzed and fermented Sargassum in Examples 1-3 and Comparative Example 1, as well as the buffer solution in Comparative Example 1, and intraperitoneally inject grass carp, with the grass carp weighing about 100 g. The action time is 48 h. After 48 h, measure the expression of inflammatory factors and inflammatory signaling pathways in the head kidney tissues of grass carp.

[0117] It can be seen from Figure 2 that, compared with Comparative Example 1, the contents of IL1β and TNFα in Example 1, Example 2, and Example 3 were significantly reduced, and at the same time, the expression levels of p65, ikbα, ikkα1, ikkα1, and ikkβ were significantly reduced. Similar to the findings at the cellular level, the enzymatically hydrolyzed and fermented products of Sargassum also significantly enhanced the immune activity of grass carp.

[0118] Test Example 4

[0119] Conduct a challenge experiment on grass carp intraperitoneally injected with the aqueous solutions of the enzymatically hydrolyzed and fermented Sargassum in Examples 1-3, Comparative Example 1, and the buffer solution in Comparative Example 1. That is, intraperitoneally inject 100 μL of Aeromonas hydrophila at a concentration of 1×10 6 CFU / mL. Observe the death of grass carp every 24 h.

[0120] The results are as Figure 3 shown. Whether at each observation time point or after the challenge experiment, the treatment with the enzymatically hydrolyzed and fermented Sargassum greatly improved the survival rate of grass carp. Especially in the treatment group of Example 2, the anti-bacterial infection ability of grass carp reached the highest level.

[0121] It should be noted that the above examples are only further descriptions of the present invention, rather than limitations. Any adjustment or change by those skilled in the art within the equivalent meaning and scope of the technical solution of the present invention should be considered as included within the protection scope of the present invention.

Claims

1. A new use of Sargassum enzymatic fermentation product, characterized in that: The new use includes the use of the Sargassum enzymatic hydrolysis and fermentation product in the preparation of products that improve the immune activity of grass carp or improve the ability of grass carp to resist bacterial infection.

2. The new use according to claim 1, characterized in that: The method of improving the immune activity of grass carp includes that the enzymatic hydrolysis and fermentation product of Sargassum improves the immunoregulatory ability of grass carp kidney tissue cell line and that the enzymatic hydrolysis and fermentation product of Sargassum improves the immunoregulatory ability of grass carp; wherein, the enzymatic hydrolysis and fermentation product of Sargassum improves the immunoregulatory ability of grass carp kidney tissue cell line, including reducing the expression of inflammatory factors, tumor necrosis factor and inflammatory signaling pathways in grass carp kidney tissue cells, and the enzymatic hydrolysis and fermentation product of Sargassum improves the immunoregulatory ability of grass carp, including reducing the expression of inflammatory factors, tumor necrosis factor and inflammatory signaling pathways in grass carp head kidney tissue.

3. The new use according to claim 1, characterized in that: The bacterium is Aeromonas hydrophila.

4. The new use according to any one of claims 1 to 3, characterized in that: The Sargassum enzymatic fermentation product is prepared by the following method: (1) Wash fresh Sargassum, dry it, grind it into powder, and sieve it; (2) curing the Sargassum powder in a high temperature environment; (3) adding the matured Sargassum powder into phosphate buffer and soaking it; (4) adding an acid or a base to the soaked Sargassum powder to adjust the pH value to obtain a Sargassum solution; (5) adding a composite enzyme to the Sargassum solution for enzymatic hydrolysis, wherein the composite enzyme comprises cellulase, pectinase, papain and alginate lyase; (6) after enzymolysis, the enzyme is inactivated and sterilized to obtain an enzymolysis solution, and the enzymolysis solution is freeze-dried to obtain an enzymolysis product of Sargassum; (7) adding culture solution to the Sargassum hydrolysate, adjusting the material-liquid ratio, mixing thoroughly and allowing to stand; (8) inoculating a composite bacterial seed liquid of Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus rhamnosus and a recombinant yeast DH5α producing alginate lyase, performing solid-state aerobic fermentation, and performing a stirring treatment during the fermentation process to obtain a fermentation product of Sargassum enzymatic hydrolysate; (9) The fermentation product of the Sargassum enzymatic hydrolysate is sterilized by high pressure, freeze-dried and then frozen to obtain the Sargassum enzymatic hydrolysate fermentation product.

5. The new use according to claim 4, characterized in that: In step (2), the high temperature environment is 90-100° C. and the aging time is 1-3 hours.

6. The new use according to claim 4, characterized in that: In step (4), an acid or a base is used to adjust the pH value to 4.5 to 6.

5.

7. The new use according to claim 4, characterized in that: In step (5), the amount of cellulase added is 1-3% of the total mass of the Sargassum solution, the amount of pectinase added is 1-3% of the total mass of the Sargassum solution, the amount of papain added is 1-3% of the total mass of the Sargassum solution, the amount of alginate lyase added is 1-3% of the total mass of the Sargassum solution, the activity unit of the cellulase is 100,000 U / g, the activity unit of the pectinase is 30,000 U / g, the activity unit of the papain is 100,000 U / g, and the activity unit of the alginate lyase is 100,000 U / g; the enzymolysis temperature in step (5) is 40-60°C, and the enzymolysis time is 3-7h.

8. The new use according to claim 4, characterized in that: The culture medium in step (7) is a mixed culture medium of YPD culture medium, LB culture medium and MRS culture medium, and the volume ratio of the YPD culture medium, LB culture medium and MRS culture medium is 1:2:1; the dosage relationship of the Sargassum hydrolysate and the culture medium in step (7) is 1g:0.3-0.8L, the mixing time is 10-20min, and the standing time is 1-3h.

9. The new use according to claim 4, characterized in that: The inoculation ratio of Bacillus subtilis in step (8) is 1%-3%, v / v, the inoculation ratio of Saccharomyces cerevisiae is 1%-4%, v / v, the inoculation ratio of Lactobacillus rhamnosus is 1%-3%, v / v, and the inoculation ratio of the recombinant yeast DH5α producing alginate lyase is 2%-4%, v / v; the inoculation density of Bacillus subtilis in step (8) is 2×10 7 -6×10 7 The inoculation density of the brewer's yeast was 2×10 7 -5×10 7 / mL, and the inoculation density of Lactobacillus rhamnosus is 4×10 7 -8×10 7 The inoculation density of the recombinant yeast DH5α producing alginate lyase was 1×10 8 -3×10 8 Pieces / mL.

10. The new use according to claim 4, characterized in that: In step (8), the temperature of the solid aerobic fermentation is 30-42° C., the fermentation period is 60-96 h, and the stirring period is 20-26 h.

Images