Sargassum zymolyte as well as preparation method and application thereof

Abstract

The invention discloses a sargassum zymolyte and a preparation method thereof. The method comprises the following steps: cleaning, drying, crushing and curing sargassum; soaking in a PBS (Phosphate Buffer Solution), adjusting the pH, and adding a compound enzyme for enzymolysis; and after inactivating the enzyme, concentrating and freeze-drying to obtain the zymolyte. The compound enzyme component comprises cellulase, pectinase, papain and alginate lyase. According to the method, the content of macromolecular polysaccharide is remarkably reduced, the degradation efficiency of the sargassum is improved, the bioavailability of protein is improved, and the metabolite composition is optimized; the aqueous solution of the obtained sargassum zymolyte can significantly enhance the free radical scavenging ability, improve the antioxidant ability of liver cell lines and livers of the grass carps, improve the anti-stress ability of the grass carps under ammonia nitrogen stress, and improve the survival rate of the grass carps under ammonia nitrogen stress.

Description

Technical Field

[0001] The invention belongs to the technical field of Sargassum, and in particular relates to a Sargassum enzymolysate and a preparation method and application thereof. Background Art

[0002] Sargassum is a major algae in the Phaeophyta, with a wide distribution of resources in my country. Common species include Sargassum sargassum, Sorghum sphaerocephala, Salvia thunbergii and Sargassum creeping. Sargassum contains a variety of bioactive ingredients, so it has a wide range of application value in the fields of medicine, health care, food and feed.

[0003] In order to improve the utilization efficiency of active ingredients, methods such as thermal extraction, acid-base extraction, enzymatic extraction, ultrasonic extraction and fermentation are widely used. Among them, the enzymatic method is particularly in line with actual production needs due to its characteristics of specific hydrolysis site, high efficiency and mild reaction. This method degrades polysaccharides through specific or non-specific enzymes, can effectively control the degradation process and the molecular weight of the product, and has less pollution to the environment, showing good industrial application prospects. Therefore, the use of enzymatic technology to hydrolyze the macromolecular polysaccharides in Sargassum and improve the utilization rate of active substances is a key link in giving full play to the antioxidant potential of Sargassum.

[0004] Although various enzymatic hydrolysis methods for Sargassum are disclosed in the prior art, the enzymatic hydrolysis effects and the quality of the obtained products vary greatly due to the different methods, and the uses are also different. Summary of the invention

[0005] The object of the present invention is to provide a Sargassum enzymatic hydrolysate and a preparation method thereof.

[0006] The present invention also aims at using the above-mentioned Sargassum enzymolysate in improving the antioxidant capacity of grass carp and using the above-mentioned Sargassum enzymolysate in improving the survival rate of grass carp under ammonia nitrogen stress.

[0007] The first object of the present invention can be achieved by the following technical scheme: A method for preparing a Sargassum hydrolysate comprises the following steps:

[0008] (1) Select fresh Sargassum, wash to remove impurities and salt, dry, grind into powder, and sieve;

[0009] (2) cooking the sieved Sargassum powder at high temperature;

[0010] (3) adding the matured Sargassum powder into a phosphate buffer solution for soaking, and adjusting the pH value with an acid or a base to obtain a Sargassum solution;

[0011] (4) adding a composite enzyme to the Sargassum solution for enzymolysis, wherein the composite enzyme comprises cellulase, pectinase, papain and alginate lyase;

[0012] (5) After enzymatic hydrolysis, the enzyme is inactivated and freeze-dried to obtain the Sargassum enzymatic hydrolysate.

[0013] In the method for preparing the above-mentioned Sargassum hydrolysate:

[0014] Optionally, in step (1), the product is sieved through a 60-80 mesh sieve.

[0015] Optionally, in step (2), the sieved Sargassum powder is cooked at 90-100° C. for 1-3 hours.

[0016] Optionally, in step (3), the cooked Sargassum powder is added to a phosphate buffer and soaked for 3 to 6 hours, wherein the amount of the phosphate buffer is 5 to 10 times the total mass of the cooked Sargassum powder.

[0017] Optionally, in step (3), an acid or a base is used to adjust the pH value to 4.5 to 6.5.

[0018] Optionally, the acid is a hydrochloric acid aqueous solution or an acetic acid aqueous solution, and its mass percentage is 8-12%, and the alkali is a sodium hydroxide aqueous solution, and its mass percentage is 1-3%.

[0019] Optionally, in step (4), the amount of cellulase added is 1 to 3% of the total mass of the Sargassum solution, the amount of pectinase added is 1 to 3% of the total mass of the Sargassum solution, the amount of papain added is 1 to 3% of the total mass of the Sargassum solution, and the amount of alginate lyase added is 1 to 3% of the total mass of the Sargassum solution.

[0020] Optionally, the cellulase includes one or more of exoglucanase, endoglucanase, β-glucosidase and cellobiase, and the pectinase includes one or more of protopectinase, lyase and polygalacturonase.

[0021] Optionally, in step (4), the enzymolysis temperature is 40-60° C. and the enzymolysis time is 3-7 h.

[0022] The present invention also provides the application of the method in improving the availability of Sargassum protein, optimizing the composition of small molecule compounds and improving the degradation efficiency of Sargassum.

[0023] Optionally, the Sargassum protein includes crude protein, total amino acids, essential amino acids and non-essential amino acids, the small molecule compounds include phenols, terpenes, organic acids and derivatives thereof, alkaloids, flavonoids, phenylpropanoids, ketones, aldehydes, acids, lipids and lipid molecules, steroids and steroid derivatives, sugars and analogs thereof, nucleosides, nucleotides and analogs, vitamins and derivatives thereof, quinones, short peptides and polyketides; and the method of improving the degradation efficiency of Sargassum includes reducing the content of cellulose and hemicellulose, and increasing the content of polysaccharides, alginate and seaweed oligosaccharides calculated as glucose.

[0024] The present invention further provides a Sargassum hydrolysate prepared by the method.

[0025] The above second object of the present invention can be achieved by the following technical scheme: application of the Sargassum enzymatic hydrolysate in improving the antioxidant capacity of grass carp.

[0026] Optionally, the improvement of the antioxidant capacity of grass carp includes: the Sargassum enzymolyte aqueous solution enhances the ability to scavenge free radicals, the Sargassum enzymolyte aqueous solution enhances the antioxidant capacity of grass carp liver cell line, and the Sargassum enzymolyte aqueous solution enhances the antioxidant capacity of grass carp liver; wherein the free radical scavenging ability includes enhancing the ability to scavenge hydroxyl radicals and superoxide anion radicals, the antioxidant capacity of the grass carp liver cell line includes enhancing the activity of superoxide dismutase and glutathione peroxidase in liver cells, reducing the level of malondialdehyde and improving the total antioxidant capacity, and the antioxidant capacity of the fish liver includes enhancing the activity of superoxide dismutase and glutathione peroxidase in grass carp liver, reducing the level of malondialdehyde and improving the total antioxidant capacity.

[0027] The present invention also provides the use of the Sargassum enzymatic hydrolysate in improving the survival rate of grass carp under ammonia nitrogen stress.

[0028] The method comprises dissolving the obtained Sargassum hydrolysate in double distilled water, fully soaking and stirring, and obtaining an aqueous solution of the Sargassum hydrolysate. The aqueous solution is filtered and sterilized, and its free radical scavenging level is determined. Meanwhile, the sterilized aqueous solution is added to a culture solution of grass carp liver cells L8824, and the levels of oxidation products and antioxidant enzymes of the grass carp liver cells L8824 are determined; the Sargassum aqueous solution is intraperitoneally injected into the grass carp, and the levels of oxidation products and antioxidant enzymes of the grass carp liver are determined; and an ammonia nitrogen stress experiment is performed to observe the survival of the grass carp intraperitoneally injected with the Sargassum aqueous solution under a high ammonia nitrogen concentration in the water body.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The present invention provides a method for efficiently enzymatically hydrolyzing Sargassum, which significantly reduces the content of macromolecular polysaccharides, improves the bioavailability of protein, and optimizes the composition of small molecule compounds in Sargassum, that is, the method of the present invention improves the availability of Sargassum protein, optimizes the composition of small molecule compounds, and improves the degradation efficiency of Sargassum;

[0031] (2) The Sargassum hydrolysate obtained by the present invention can enhance the antioxidant properties of grass carp and improve the survival rate of grass carp under ammonia nitrogen stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the crude protein level of Sargassum in Experimental Example 2;

[0033] Figure 2 The distribution of small molecule compounds of Sargassum in Experimental Example 3;

[0034] Figure 3 The changes of the beneficial functional molecules of Sargassum in Experimental Example 3;

[0035] Figure 4 is the free radical scavenging ability of the Sargassum hydrolysate in Experimental Example 4;

[0036] Figure 5 The effect of the Sargassum hydrolysate on the antioxidant capacity of the grass carp L8824 liver cell line in Experimental Example 5;

[0037] Figure 6 The effect of Sargassum hydrolysate on the antioxidant capacity of grass carp liver in Experimental Example 6;

[0038] Figure 7 This is the effect of Sargassum enzymatic hydrolysate on the survival rate of grass carp under ammonia nitrogen stress in Experimental Example 7. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments and drawings of the present invention to clearly and completely describe the technical solutions of the present invention. 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.

[0040] In the following examples, unless otherwise specified, all conventional methods are used, and the raw materials used in the following examples are commercially available products unless otherwise specified.

[0041] Example 1

[0042] The method for preparing the Sargassum hydrolysate provided in this embodiment comprises the following steps:

[0043] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0044] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0045] (3) adding phosphate buffered saline (PBS) in an amount of 8 times the mass of the high-temperature-ripened Sargassum powder and fully soaking the powder for 4 h to allow the Sargassum powder to be soaked and softened;

[0046] 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.

[0047] (4) adding 10 wt % acetic acid to adjust the pH to 5.0 to obtain a Sargassum solution;

[0048] (5) adding a composite enzyme, based on the total mass of the Sargassum solution, including 1 wt% of cellulase, 1 wt% of pectinase, 1 wt% of papain and 1 wt% of alginate lyase, and performing enzymolysis at 45° C. for 4 h;

[0049] (6) heating to 105° C. to sterilize the enzyme, and then freeze-drying to obtain the Sargassum enzymatic hydrolysate.

[0050] Example 2

[0051] The method for preparing the Sargassum hydrolysate provided in this embodiment comprises the following steps:

[0052] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0053] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0054] (3) adding phosphate buffer solution (PBS) in an amount of 8 times the total mass of the high-temperature-ripened Sargassum powder, and soaking the powder for 4 h to soften the Sargassum powder;

[0055] (4) adding 2 wt % NaOH or 10 wt % acetic acid to adjust the pH to 5.0 to obtain a Sargassum solution;

[0056] (5) adding a composite enzyme, based on the total mass of the Sargassum solution, 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;

[0057] (6) heating to 105° C. to sterilize the enzyme, and then freeze-drying to obtain the Sargassum enzymatic hydrolysate.

[0058] Example 3

[0059] The method for preparing the Sargassum hydrolysate provided in this embodiment comprises the following steps:

[0060] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0061] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0062] (3) adding phosphate buffer solution (PBS) in an amount of 8 times the total mass of the high-temperature-ripened Sargassum powder, and soaking the powder for 4 h to soften the Sargassum powder;

[0063] (4) adding 2 wt % NaOH or 10 wt % acetic acid to adjust the pH to 5.0 to obtain a Sargassum solution;

[0064] (5) adding complex enzymes, based on the total mass of the Sargassum solution, including 3 wt% of cellulase, 3 wt% of pectinase, 3 wt% of papain and 3 wt% of alginate lyase, and performing enzymolysis at 55° C. for 4 h;

[0065] (6) heating to 105° C. to sterilize the enzyme, and then freeze-drying to obtain the Sargassum enzymatic hydrolysate.

[0066] Comparative Example 1

[0067] (1) washing fresh Sargassum to remove impurities and surface salt, drying and grinding into powder, and passing through a 60-mesh sieve;

[0068] (2) curing the Sargassum powder in a high temperature environment of 95°C for 2 hours;

[0069] (3) adding phosphate buffer solution (PBS) in an amount of 8 times the total mass of the high-temperature-ripened Sargassum powder, and soaking the powder for 4 h to soften the Sargassum powder;

[0070] (4) adding 2 wt % NaOH or 10 wt % acetic acid to adjust the pH to 5.0 to obtain a Sargassum solution;

[0071] (5) Add the complex enzyme that is fully inactivated at 105°C and perform enzymatic hydrolysis at 50°C for 4 h;

[0072] (6) heating to 105° C. to sterilize the enzyme, and then freeze-drying to obtain the Sargassum enzymatic hydrolysate.

[0073] Blank example 1

[0074] Sterile phosphate buffered saline (PBS) was used as blank example 1.

[0075] Test Example 1

[0076] In this test example 1, the content of lignin, hemicellulose and cellulose was determined by implementing a series of specific detection methods. These methods follow the detailed steps in FUT-SOP (L) -37.1. In addition, in order to determine the polysaccharide content (in glucose), a method that meets the SN / T 4260-2015 standard was adopted. The content of alginic acid was detected by following the method of HG / T 5050-2016 standard, and the content of seaweed oligosaccharides was detected according to the method of Q / 371083WX002-2016 standard. The test results obtained are summarized in Table 1 for further analysis and discussion.

[0077] Table 1 Distribution of sugars in Sargassum

[0078]

[0079] 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. The composite enzyme in the present invention can degrade them into seaweed oligosaccharides with better water solubility.

[0080] As can be seen from Table 1, in this Experimental Example 1, by treating Sargassum with the composite enzyme in the concentration range of 1wt% to 3wt%, it was observed that the contents of cellulose and hemicellulose were significantly reduced. At the same time, the contents of polysaccharides (in terms of glucose), alginic acid, and algal oligosaccharides were significantly increased. These results show that the composite enzyme effectively improves the degradation efficiency of Sargassum within the above concentration range. In particular, when the composite enzyme concentration is 2%, the degradation efficiency of Sargassum reaches the optimal level.

[0081] Test Example 2

[0082] The crude protein level of the enzymatic hydrolysate of Sargassum was detected according to the method in GBT6432-2018; the amino acid level of the enzymatic hydrolysate of Sargassum was detected according to the method in GBT18246-2019. The results are shown in Table 2 and Figure 1 .

[0083] Table 2 Amino acid distribution of Sargassum

[0084] (g / 100g) Comparative Example 1 Example 1 Example 2 Example 3 Threonine <![CDATA[0.3000±0.0013 b ]]> <![CDATA[0.2709±0.0007 a ]]> <![CDATA[0.3600±0.0015 c ]]> <![CDATA[0.3006±0.0012 b ]]> Methionine <![CDATA[0.0799±0.0006 b ]]> <![CDATA[0.0804±0.0007 b ]]> <![CDATA[0.1001±0.0006 c ]]> <![CDATA[0.0704±0.0006 a ]]> Lysine <![CDATA[0.2301±0.0004 d ]]> <![CDATA[0.2001±0.0005 b ]]> <![CDATA[0.2202±0.0004 c ]]> <![CDATA[0.1801±0.0006 a ]]> Isoleucine <![CDATA[0.2501±0.0006 c ]]> <![CDATA[0.2206±0.0009 a ]]> <![CDATA[0.2704±0.0007 d ]]> <![CDATA[0.2406±0.0007 b ]]> Histidine <![CDATA[0.2803±0.0008 c ]]> <![CDATA[0.1705±0.0007 a ]]> <![CDATA[0.2902±0.0004 d ]]> <![CDATA[0.2705±0.0009 b ]]> Valine <![CDATA[0.3004±0.0009 c ]]> <![CDATA[0.2703±0.0006 a ]]> <![CDATA[0.3402±0.0005 d ]]> <![CDATA[0.2904±0.0008 b ]]> Leucine <![CDATA[0.3901±0.0004 c ]]> <![CDATA[0.3603±0.0006 a ]]> <![CDATA[0.4501±0.0003 d ]]> <![CDATA[0.3804±0.0008 b ]]> Phenylalanine <![CDATA[0.2300±0.0005 c ]]> <![CDATA[0.2205±0.0006 b ]]> <![CDATA[0.0501±0.0003 a ]]> <![CDATA[0.2306±0.0007 c ]]> Tryptophan <![CDATA[0.2402±0.0004 a ]]> <![CDATA[0.4803±0.0004 c ]]> <![CDATA[0.6500±0.0002 d ]]> <![CDATA[0.4005±0.0009 b ]]> Arginine <![CDATA[0.2400±0.0003 b ]]> <![CDATA[0.2402±0.0004 b ]]> <![CDATA[0.3004±0.0008 c ]]> <![CDATA[0.2306±0.0007 a ]]> Aspartic acid <![CDATA[0.5707±0.0378 a ]]> <![CDATA[0.5887±0.0337 a ]]> <![CDATA[0.6897±0.0427 b ]]> <![CDATA[0.5494±0.0364 a ]]> Serine <![CDATA[0.2323±0.0156 a ]]> <![CDATA[0.2535±0.0190 a ]]> <![CDATA[0.3133±0.0176 b ]]> <![CDATA[0.2300±0.0129 a ]]> Glutamate <![CDATA[1.2841±0.0826 a ]]> <![CDATA[1.5100±0.0937 b ]]> <![CDATA[1.6800±0.0853 c ]]> <![CDATA[1.2901±0.0718 a ]]> Glycine <![CDATA[0.2646±0.0168 a ]]> <![CDATA[0.2740±0.0177 a ]]> <![CDATA[0.3326±0.0222 b ]]> <![CDATA[0.2593±0.0200 a ]]> Alanine <![CDATA[0.3496±0.0226 a ]]> <![CDATA[0.3727±0.0248 a ]]> <![CDATA[0.4252±0.0293 b ]]> <![CDATA[0.3456±0.0254 a ]]> Cysteine <![CDATA[0.0495±0.0038 b ]]> <![CDATA[0.0400±0.0023 a ]]> <![CDATA[0.0509±0.0037 b ]]> <![CDATA[0.0705±0.0048 c ]]> Tyrosine <![CDATA[0.1619±0.0096 a ]]> <![CDATA[0.1613±0.0121 a ]]> <![CDATA[0.2227±0.0143 b ]]> <![CDATA[0.1622±0.0132 a ]]> Proline <![CDATA[0.0710±0.0048 a ]]> <![CDATA[0.1411±0.0092 b ]]> <![CDATA[0.2127±0.0142 c ]]> <![CDATA[0.1426±0.0094 b ]]> Essential amino acids <![CDATA[2.5411±0.0054 b ]]> <![CDATA[2.5141±0.0054 a ]]> <![CDATA[3.0316±0.0029 d ]]> <![CDATA[2.5947±0.0066 c ]]> Not required <![CDATA[2.9836±0.1908 a ]]> <![CDATA[3.3414±0.2108 a ]]> <![CDATA[3.9271±0.2288 b ]]> <![CDATA[3.0497±0.1916 a ]]> Total amino acids <![CDATA[5.5247±0.1876 a ]]> <![CDATA[5.8555±0.2080 a ]]> <![CDATA[6.9587±0.2261 b ]]> <![CDATA[5.6444±0.1875 a ]]>

[0085] From Table 2 and Figure 1It can be seen that: in Experimental Example 2, after treating Sargassum with a composite enzyme at a concentration of 1wt% to 3wt%, it was observed that the levels of crude protein, total amino acids, essential amino acids and non-essential amino acids increased significantly. Specifically, the concentrations of tryptophan, glutamic acid and proline increased significantly, while the level of amino acids decreased significantly. These results show that composite enzyme treatment can effectively improve the protein availability of Sargassum. When the composite enzyme concentration was 2wt%, the protein availability of Sargassum reached the highest level. This discovery provides important technical support for improving the nutritional value of Sargassum.

[0086] Test Example 3

[0087] Thermo Vanquish ultra-high performance liquid chromatography (HPLC) system was used to analyze the HSS T3 column, and Thermo Orbitrap Exploris 120 mass spectrometry analysis were performed to detect the small molecule compound composition of Sargassum. The results are shown in Figure 2 , Figure 3 and Table 3.

[0088] Table 3 Changes of small molecule compounds in Sargassum

[0089] ％ Comparative Example 1 Example 1 Example 2 Example 3 Phenols <![CDATA[1.71±0.06 a ]]> <![CDATA[2.88±0.10 c ]]> <![CDATA[3.64±0.10 d ]]> <![CDATA[2.71±0.08 b ]]> Terpenes <![CDATA[26.09±0.70 c ]]> <![CDATA[24.55±0.59 b ]]> <![CDATA[16.07±0.36 a ]]> <![CDATA[16.92±0.43 a ]]> Organic acids and their derivatives <![CDATA[3.68±0.10 ab ]]> <![CDATA[3.57±0.08 a ]]> <![CDATA[4.61±0.13 c ]]> <![CDATA[3.87±0.10 b ]]> Alkaloids <![CDATA[1.63±0.04 b ]]> <![CDATA[1.67±0.04 bc ]]> <![CDATA[1.73±0.05 c ]]> <![CDATA[1.36±0.04 a ]]> Flavonoids <![CDATA[4.26±0.11 a ]]> <![CDATA[4.41±0.11 ab ]]> <![CDATA[4.76±0.12 c ]]> <![CDATA[4.55±0.12 bc ]]> Phenylpropanoids <![CDATA[5.80±0.16 c ]]> <![CDATA[4.54±0.13 a ]]> <![CDATA[4.89±0.12 b ]]> <![CDATA[4.80±0.12 b ]]> Ketones, aldehydes, acids <![CDATA[3.17±0.10 c ]]> <![CDATA[1.85±0.05 a ]]> <![CDATA[2.17±0.07 b ]]> <![CDATA[1.98±0.06 a ]]> Lipids and lipid molecules <![CDATA[4.74±0.13 c ]]> <![CDATA[2.07±0.06 a ]]> <![CDATA[3.03±0.09 b ]]> <![CDATA[6.53±0.14 d ]]> Steroids and steroid derivatives <![CDATA[1.48±0.05 c ]]> <![CDATA[0.21±0.01 a ]]> <![CDATA[0.25±0.01 a ]]> <![CDATA[0.34±0.01 b ]]> Sugars and their analogs <![CDATA[6.31±0.16 a ]]> <![CDATA[6.65±0.15 a ]]> <![CDATA[8.92±0.23 c ]]> <![CDATA[8.14±0.22 b ]]> Nucleosides, nucleotides and analogs <![CDATA[0.72±0.02 a ]]> <![CDATA[0.84±0.03 b ]]> <![CDATA[1.23±0.03 d ]]> <![CDATA[0.96±0.03 c ]]> Vitamins and their derivatives <![CDATA[0.64±0.02 c ]]> <![CDATA[0.49±0.01 a ]]> <![CDATA[0.71±0.02 d ]]> <![CDATA[0.57±0.02 b ]]> Quinones <![CDATA[0.16±0.00 a ]]> <![CDATA[0.20±0.00 b ]]> <![CDATA[0.20±0.01 b ]]> <![CDATA[0.24±0.01 c ]]> Short peptide <![CDATA[0.04±0.00 b ]]> <![CDATA[0.03±0.00 a ]]> <![CDATA[0.03±0.00 a ]]> <![CDATA[0.03±0.00 a ]]> Polyketides <![CDATA[0.02±0.00 b ]]> <![CDATA[0.01±0.00 a ]]> <![CDATA[0.04±0.00 d ]]> <![CDATA[0.03±0.00 c ]]>

[0090] from Figure 2 , Figure 3 As can be seen from Table 3, after the 1% to 3% concentration of the composite enzyme was used to treat the Sargassum, the levels of phenolic substances in Examples 1, 2 and 3 increased by an average of about 110%, organic acids and their derivatives increased by 25%, sugars and their analogs increased by 40%, and nucleosides, nucleotides and analogs increased by 70%. At the same time, terpenoids decreased by 40%, and steroids and steroid derivatives decreased by 80%.

[0091] Further analysis found that in Example 2, the level of myristicin increased by about 240 times, 10-deacetylbaccatin III increased by about 2900 times, baccatin III increased by about 1050 times, pheophytin A increased by about 390 times, nicotinic acid increased by about 50 times, taurine increased by about 20 times, and N-acetylneuraminic acid increased by about 200 times. In addition, the complex enzyme treatment significantly reduced the level of quercetin + 3-galactoside + 7-rhamnoside, which decreased by about 45 times. It is worth noting that 10-deacetylbaccatin III and baccatin III are important precursors for the synthesis of paclitaxel and have significant anti-cancer activity. Taurine is widely recognized as a compound with extremely strong biological activity.

[0092] These results show that the composition of small molecule compounds in Sargassum has changed significantly after 1% to 3% compound enzyme treatment, especially the content of beneficial functional molecules has been improved. At a concentration of 2% compound enzyme, the composition of small molecule compounds in Sargassum showed the best state, which provides new possibilities for improving the application value of Sargassum.

[0093] Test Example 4

[0094] The Sargassum hydrolysates in Examples 1-3, Comparative Example 1 and Blank Example 1 were dissolved in double distilled water, the mass of which was 5 times the total mass of the Sargassum hydrolysate, and the products were fully soaked and stirred. The magnetic stirring speed was 500 rpm and the soaking time was 4 h to obtain an aqueous solution of Sargassum hydrolysate. The aqueous solution was filtered and sterilized with a filter membrane of 0.22 μm, and its free radical level was measured.

[0095] The scavenging ability of Sargassum on hydroxyl radicals and superoxide anion free radicals was tested according to the instructions of the hydroxyl radical and superoxide anion free radical determination kit. The results are shown in Figure 4 .

[0096] from Figure 4 It can be seen that after treatment with 1wt% to 3wt% of the compound enzyme, the free radical scavenging ability of Sargassum was significantly enhanced. Under 2wt% compound enzyme treatment, the free radical scavenging ability reached the highest level. This enhanced free radical scavenging ability may be related to the increase in the content of phenolic substances, nucleotides and other active ingredients, providing a wider application prospect for the functional utilization of Sargassum.

[0097] Test Example 5

[0098] The sterilized Sargassum aqueous solution in Examples 1-3, Comparative Example 1 and Blank Example 1 was added to the culture medium of the grass carp L8824 hepatocyte cell line (purchased from China Center for Type Culture Collection, Wuhan), cultured for 48 hours, and the levels of oxidation products and antioxidant enzymes in the grass carp hepatocytes were determined. The evaluation of antioxidant capacity included the levels of superoxide dismutase, glutathione peroxidase and malondialdehyde, as well as the total antioxidant capacity.

[0099] The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the liver cells of grass carp L8824, as well as the total antioxidant capacity (T-AOC), were determined using commercial kits. Figure 5 .

[0100] The activities of SOD, GSH-Px and T-AOC in Examples 1, 2 and 3 were significantly increased. This indicates that the function of the antioxidant enzyme system in the cell is enhanced, and it can more effectively resist oxidative damage. At the same time, the level of MDA is significantly reduced, indicating that the degree of lipid peroxidation is reduced and the oxidative stress state of the cell is improved. In general, after 1% to 3% compound enzyme treatment, the enzymatic hydrolysate of Sargassum significantly enhanced the antioxidant capacity of grass carp liver cells. Especially in the 2% compound enzyme treatment, the antioxidant capacity of grass carp liver cells reached the highest level.

[0101] Test Example 6

[0102] 100 μL of the sterilized Sargassum aqueous solution in Examples 1-3, Comparative Example 1 and Blank Example 1 was intraperitoneally injected into a grass carp weighing about 100 g. The action time was 48 hours. After 48 hours, the levels of oxidation products and antioxidant enzymes in the liver of the grass carp were measured. The evaluation of antioxidant capacity included the levels of superoxide dismutase, glutathione peroxidase and malondialdehyde, as well as the total antioxidant capacity.

[0103] The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in grass carp liver, as well as the total antioxidant capacity (T-AOC) were determined using commercial kits. Figure 6 .

[0104] Compared with Comparative Example 1, the antioxidant enzyme activity of the grass carp liver in Examples 1, 2 and 3 was significantly enhanced, while the content of oxidation products was significantly reduced. In general, the antioxidant capacity of the grass carp liver was enhanced after the Sargassum was treated with 1wt% to 3wt% compound enzymes. In particular, in the 2wt% compound enzyme treatment, the antioxidant capacity of the liver reached the highest level.

[0105] Test Example 7

[0106] A 48-hour ammonia nitrogen stress test was conducted on grass carp injected intraperitoneally with the Sargassum aqueous solution in Examples 1-3, Comparative Example 1 and Blank Example 1. The ammonia nitrogen concentration in the water was adjusted to 0.5 mg / L, and the mortality of the grass carp was observed every 6 or 12 hours.

[0107] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that, whether at each observation time point or after the stress experiment, the treatment with Sargassum enzymatic hydrolysate greatly improved the survival rate of grass carp, especially in the 2wt% compound enzyme treatment group, the grass carp's ability to resist stress reached the highest.

[0108] It should be pointed out that the above embodiments are only further explanations of the present invention rather than limitations, and any adjustments or changes by those skilled in the art within the meaning and scope equivalent to the technical solution of the present invention should be deemed to be included in the protection scope of the present invention.

Claims

1. A method for preparing a Sargassum hydrolysate, characterized in that: The following steps are involved: (1) Select fresh Sargassum, wash to remove impurities and salt, dry, grind into powder, and sieve; (2) cooking the sieved Sargassum powder at high temperature; (3) adding the matured Sargassum powder into a phosphate buffer solution for soaking, and adjusting the pH value with an acid or a base to obtain a Sargassum solution; (4) adding a composite enzyme to the Sargassum solution for enzymolysis, wherein the composite enzyme comprises cellulase, pectinase, papain and alginate lyase; (5) After enzymatic hydrolysis, the enzyme is inactivated and freeze-dried to obtain the Sargassum enzymatic hydrolysate.

2. The method for preparing the Sargassum hydrolysate according to claim 1, characterized in that: In step (2), the sieved Sargassum powder is matured at 90-100° C. for 1-3 hours.

3. The method for preparing the Sargassum hydrolysate according to claim 1, characterized in that: In step (3), the cooked Sargassum powder is added to a phosphate buffer and soaked for 3 to 6 hours, wherein the amount of the phosphate buffer is 5 to 10 times the total mass of the cooked Sargassum powder; in step (3), an acid or a base is used to adjust the pH value to 4.5 to 6.

5.

4. The method for preparing the Sargassum hydrolysate according to claim 1, characterized in that: In step (4), the amount of cellulase added is 1 to 3% of the total mass of the Sargassum solution, the amount of pectinase added is 1 to 3% of the total mass of the Sargassum solution, the amount of papain added is 1 to 3% of the total mass of the Sargassum solution, and the amount of alginate lyase added is 1 to 3% of the total mass of the Sargassum solution. In step (4), the enzymolysis temperature is 40 to 60° C. and the enzymolysis time is 3 to 7 hours.

5. Use of the method according to any one of claims 1 to 4 in improving the availability of Sargassum protein, optimizing the composition of small molecule compounds, and improving the degradation efficiency of Sargassum.

6. The use according to claim 5, characterized in that: The Sargassum protein includes crude protein, total amino acids, essential amino acids and non-essential amino acids; the small molecule compounds include phenols, terpenes, organic acids and derivatives thereof, alkaloids, flavonoids, phenylpropanoids, ketones, aldehydes, acids, lipids and lipid molecules, steroids and steroid derivatives, sugars and analogs thereof, nucleosides, nucleotides and analogs, vitamins and derivatives thereof, quinones, short peptides and polyketides; and the method for improving the degradation efficiency of Sargassum includes reducing the content of cellulose and hemicellulose, and increasing the content of polysaccharides, alginic acid and seaweed oligosaccharides calculated as glucose.

7. A Sargassum hydrolysate, characterized in that: The method is prepared by any one of claims 1 to 4.

8. Use of the Sargassum enzymatic hydrolysate according to claim 7 in improving the antioxidant capacity of grass carp.

9. The use according to claim 8, characterized in that: The method for improving the antioxidant capacity of grass carp includes: the aqueous solution of Sargassum enzymolyte enhances the ability to scavenge free radicals, the aqueous solution of Sargassum enzymolyte enhances the antioxidant capacity of grass carp liver cell line, and the aqueous solution of Sargassum enzymolyte enhances the antioxidant capacity of grass carp liver; wherein the ability to scavenge free radicals includes enhancing the ability to scavenge hydroxyl radicals and superoxide anion radicals, the antioxidant capacity of the grass carp liver cell line includes enhancing the activities of superoxide dismutase and glutathione peroxidase in liver cells, reducing the level of malondialdehyde and improving the total antioxidant capacity, and the antioxidant capacity of fish liver includes enhancing the activities of superoxide dismutase and glutathione peroxidase in grass carp liver, reducing the level of malondialdehyde and improving the total antioxidant capacity.

10. Use of the Sargassum hydrolysate according to claim 7 in improving the survival rate of grass carp under ammonia nitrogen stress.

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