Sea cucumber polysaccharide type sea cucumber extract and application of sea cucumber polysaccharide type sea cucumber extract in preparation of immunoregulation solid beverage

Abstract

The invention relates to the technical field of marine organism oligopeptide grading membrane filtration, in particular to a sea cucumber polysaccharide type sea cucumber extract and application thereof in preparation of an immunoregulation solid beverage. The small-molecular oligopeptide is obtained by taking sea cucumber as a raw material and carrying out directional enzyme digestion, degreasing and membrane filtration technologies. The immunoregulation solid beverage prepared from the sea cucumber polysaccharide type sea cucumber extract can significantly improve the immunity, and has an obvious dose-dependent effect; the product does not contain fat, and has good flavor, stable shelf life, and good fluidity and solubility.

Description

Technical Field

[0001] The invention relates to the technical field of marine biological oligopeptide graded membrane filtration, and in particular to a sea cucumber polysaccharide type sea cucumber extract and application thereof in the preparation of an immunomodulatory solid beverage. Background Art

[0002] The functions of the immune system are divided into immune defense, immune surveillance and immune self-stabilization, and play a key role in preventing and resisting diseases. The host's specific immune response to pathogens includes cellular immunity and humoral immunity. In order to cope with the decline of the body's immunity, a large number of researchers have now separated and extracted probiotics and active peptides with immunomodulatory activity. Bioactive peptides can enhance the body's immunity and exert immunomodulatory functions while affecting the proliferation of lymphocytes in the body and improving antiviral ability. Because bioactive polysaccharides have the advantages of wide sources, mild properties, easy digestion and absorption, diverse activities and extremely low side effects, their extraction and purification processes and biological functions have become a hot topic in the food field. Compared with other bioactive substances, low-fat, low-protein, low-salt and high-content active polysaccharides have a wide range of applications.

[0003] In recent decades, a variety of substances with important biological activities have been found in sea cucumbers, including sea cucumber extracts, sea cucumber polysaccharides, sea cucumber saponins, cerebrosides, sphingosine, phenolic substances, etc. Sea cucumber polysaccharides, as one of the important active substances in sea cucumbers, have multiple biological activities such as anticoagulation, anti-tumor, antioxidant, immunomodulation and improvement of insulin resistance. At present, there are many studies on the biological activity of sea cucumber polysaccharides, but they have not yet been industrialized.

[0004] Sea cucumber extract refers to a protein hydrolyzate obtained by separating and purifying sea cucumber after protease hydrolysis, which is mainly composed of small molecule peptides and has multiple functional characteristics. The sea cucumber polysaccharides contained therein have great application potential due to their multiple biological activities. However, in the industrial production of sea cucumber polysaccharides and their products, because the sea cucumber raw materials themselves contain a certain amount of fat and protein, if they are not removed in the process, it will affect the sea cucumber polysaccharide content in the sea cucumber extract and the shelf life flavor and stability of its products, thereby affecting the industrial market of sea cucumber extracts and their products. Traditional methods generally purify by chromatography, such as patent CN114316078A "A Preparation Method and Application of Sea Cucumber Polysaccharides", but this method is limited to raw materials. For sea cucumbers with high fat content, such as red sea cucumbers, in this method, due to the presence of fat in the sea cucumber enzymatic hydrolyzate, it will affect the ultrafiltration and sodium filtration of the enzymatic hydrolyzate, which is easy to block the membrane and is not conducive to industrial production. In addition, during the preparation of sea cucumber extracts, sea cucumber polysaccharides are severely lost, resulting in some sea cucumber extracts on the market being essentially collagen peptides. How to increase the yield of sea cucumber polysaccharides is also a problem that the present invention needs to solve. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the raw material of sea cucumber contains a certain amount of fat. If it is not removed in the process, it will have an adverse effect on the flavor and shelf life of the raw material of sea cucumber extract and its products, thereby affecting the industrial market of sea cucumber extract and its products. In addition, during the preparation process of sea cucumber extract, sea cucumber polysaccharide is seriously lost, resulting in some sea cucumber extracts on the market being essentially collagen peptides. How to improve the yield of sea cucumber polysaccharides is also a problem to be solved by the present invention.

[0006] In view of the problems of the prior art, the present invention discloses a sea cucumber polysaccharide type sea cucumber extract and its application in the preparation of an immunomodulatory solid beverage. Small molecule oligopeptides are obtained by using sea cucumber as raw material through directional enzyme cutting, defatting and membrane filtration technology; wherein, flour is used to absorb fat, and flour is used as a green "degreasing agent". According to the denaturation and gelatinization of protein and starch, the rheological properties and mesoscopic properties of the green, non-synthetic "degreasing agent" are changed, and enzyme inactivation and defatting are carried out simultaneously, and energy consumption is reduced while achieving defatting. The prepared immunomodulatory solid beverage can significantly improve immunity and has an obvious dose-dependent effect; it is fat-free, has a good flavor, a stable shelf life, and good fluidity and solubility.

[0007] To achieve the above object, the present invention is specifically implemented by the following technical scheme: A method for preparing a sea cucumber polysaccharide type sea cucumber extract comprises the following steps:

[0008] (0-1) Pretreatment: mince and grind the body wall of sea cucumbers; select the body wall of sea cucumbers without internal organs to remove the potential impact of heavy metal enrichment in the internal organs. The final product has a low heavy metal content, which expands the application range of the final product, such as special dietary sports nutrition foods, which have strict requirements on the content of heavy metal lead and total arsenic.

[0009] (0-2) Enzymatic hydrolysis: After the temperature is constant, add the neutral protease activated by warm water in advance, and the added amount is 0.1-0.3% of the weight of the sea cucumber grinding pulp. Neutral protease has a high efficiency of hydrolysis, which helps to extract the active ingredients in the sea cucumber and can also produce enzymatic products with various physiological activities. Stir the enzymatic hydrolysis for 4-6 hours. After 4 hours of enzymatic hydrolysis, measure the solid content every half an hour with a solids meter. When the solid content is basically stable, the enzymatic hydrolysis is completed;

[0010] (0-3) Degreasing and enzyme inactivation: Add 0.1-0.2% of the weight of the sea cucumber enzymatic hydrolysate to the flour weighed in advance. Dissolve it in warm water, put the dissolved flour solution into the enzymatic hydrolysate tank, stir and mix for 5 minutes, turn off the stirring, fully open the steam valve, heat the half-product after enzymatic hydrolysis to 98°C, keep it for 20 minutes, and perform denaturation, degreasing and enzyme inactivation; close the steam valve of the enzymatic hydrolysate tank, pour in ice water to cool it down to below 40°C; open the lower discharge valve, pass the cooled half-product through a 100-mesh sieve to remove sand, sea cucumber skin and other residues that cannot be enzymatically hydrolyzed, and pour the enzymatic hydrolysate after passing through the sieve into a temporary storage tank; the starch in the flour aqueous solution is dispersed with water, heated and dispersed into the sea cucumber enzymatic hydrolysate, and the whole is When heated to 50°C, the flour solution begins to absorb water and expand. When heated to 65°C, the flour solution protein denaturation can be observed, and white floccules appear in the hydrolyzate. When heated to 98°C-100°C, the hydrolyzate begins to boil, and the white floccules gather and float on the top of the hydrolyzate, turning yellow and smooth. During this process, the starch in the flour solution swells and gelatinizes, increases in volume, and dissolves in water. When added to the fat-containing hydrolyzate, the fat enters the starch solution micelles to form a fat-containing sol. At this time, it can be clearly observed that the color of the starch block after heating turns yellow. At the same time, the protein in the flour solution swells with water to form an area, and absorbs the fat in the hydrolyzate during the temperature rise and denaturation process.

[0011] (0-4) Separation: After cooling, the half-product is separated by a linked tubular centrifuge, 4000r / min, and the feed speed is 1.0m 3 / h, intercepting insoluble residues above 100 mesh, the obtained enzymatic solution filtrate is filtered through a plate and frame filter again, 0.8Mpa, 22 plates, intercepting the remaining insoluble residues (above 300 mesh), and the filtrate is transferred to a temporary storage tank;

[0012] (0-5) Membrane filtration: The semi-product in the temporary storage tank is filtered through a membrane filtration device and passed through a 10,000 Dalton membrane to obtain an enzymatic hydrolyzate with a molecular weight less than 10,000 Daltons. The filtrate is collected in a temporary storage tank;

[0013] (0-6) Concentration: The filtrate in the temporary storage tank is sent to a double-effect vacuum concentration device for concentration, with an evaporation pressure of 0.06 MPa and an evaporation temperature of 68-70°C for the first effect and 56-60°C for the second effect. After the filtrate is concentrated to a solid content of 15%-20%, it is injected into a blending tank through a pipeline for standby use;

[0014] (0-7) Drying: Drying to obtain sea cucumber polysaccharide type sea cucumber extract powder.

[0015] Furthermore, in step (0-1), the frozen sea cucumber plate is first thawed in a thawing tank, the sea cucumber viscera are removed for other uses, and the sea cucumber body wall is retained. The obtained body wall is first passed through a meat grinder and then connected to a colloid mill, minced and pumped to the colloid mill feed port through a vacuum pump and water is added in a ratio of 2:1, while the flow rate is controlled, and after passing through the colloid mill, it is pumped into an enzymolysis tank, and the steam valve in the interlayer of the enzymolysis tank is opened to increase the temperature, stirring is started, and the temperature is set to 56±2°C.

[0016] Furthermore, the method further comprises a deproteinization step: the enzymatic hydrolyzate after defatting and inactivating enzymes in step (0-3) is heated to 45-55° C., 3% sodium hydroxide is added, the pH value is adjusted to 7.8-8.2, and the temperature is kept and stirred for 2 hours.

[0017] Furthermore, the method further comprises a desalination step: in step (0-5), the temporary liquid after passing through the membrane is added with softened water at a ratio of 1:5 (w:w), the pH value is adjusted to 6.8-7.0, passed through a sodium membrane, and the retained liquid is collected.

[0018] Furthermore, step (0-7) adopts spray drying, and the specific steps are: the concentrated enzymatic hydrolysate passes through a homogenizer and then enters a spray tower for spray drying, the inlet temperature of the spray tower for spray drying is 170-190°C, and the outlet temperature is 95-105°C; a vibrating screen is set at the powder outlet, and the powder is screened once every 8-12 minutes to collect the sea cucumber polysaccharide type sea cucumber extract powder.

[0019] A sea cucumber polysaccharide type sea cucumber extract prepared by the above method is low in fat, protein and salt, and can systematically improve immunity from both cellular immunity and humoral immunity.

[0020] The above-mentioned sea cucumber polysaccharide type sea cucumber extract is used in the preparation of immune-regulating solid beverages, wherein the weight of the sea cucumber polysaccharide type sea cucumber extract powder accounts for 5-30%. The recommended daily intake of sea cucumber peptides is 1.0 g-2.0 g, and the amount added is customized according to the solid beverage packaging specifications of 5-30 g / bag

[0021] An immune-regulating solid beverage is composed of the following raw materials in percentage by weight: 5-30% of the above-mentioned sea cucumber polysaccharide type sea cucumber extract powder, 2-10% of chrysanthemum powder, a natural seasoning ingredient, 2-10% of yerba mate, 10-20% of xylitol, a natural seasoning ingredient, 3-7% of lemon powder, 0.1-0.5% of ginseng powder (artificially grown for less than five years), a new food raw material, 5-15% of resistant dextrin, a water-soluble dietary fiber, and 5-15% of inulin, a prebiotic.

[0022] The preparation method of the above-mentioned immunomodulatory solid beverage comprises the following steps:

[0023] (1) Weighing and mixing: Accurately weigh the raw materials according to the formula dosage, put the weighed raw materials into a three-dimensional mixer, seal it, and mix it for 20-40 minutes until it is uniform;

[0024] (2) Fluidized bed granulation: Turn on the fluidized bed granulation equipment, suck the mixed material into a closed container of the hopper, granulate, dry, and obtain granules with a moisture content of <5%;

[0025] (3) Granulation: The granulated particles are sieved through an 80-mesh sieve to obtain relatively uniform particles;

[0026] (4) Packaging: Put the semi-finished granules into the granule packing machine, code them, and pack them;

[0027] (5) Packaging: The packaged solid beverages are placed in coded packaging boxes and then sealed with cigarettes.

[0028] The beneficial effects of the present invention are:

[0029] (1) The obtained sea cucumber peptide has a molecular weight of <10000Da, high purity, a sea cucumber polysaccharide content of ≥100mg / g, does not contain fat and other raw materials, has good palatability, solubility and fluidity, a stable shelf life, and a wide range of applications.

[0030] (2) Compared with the prior art, the composition of the present invention is reasonably matched, ensuring the content of sea cucumber polysaccharides in sea cucumber extract while having good palatability. It has some characteristics such as zero added sucrose, zero added flavors, zero added preservatives, and no fat. Starting from both cellular immunity and humoral immunity, a systematic immune regulation animal experiment is carried out to verify that it can significantly improve immunity and has an auxiliary effect on improving the quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a flow chart of the method for preparing the immunomodulatory sea cucumber polysaccharide type sea cucumber extract solid beverage in a preferred embodiment of the present invention.

[0032] Figure 2 This is the molecular weight distribution of the immunomodulatory sea cucumber polysaccharide type sea cucumber extract in the preferred embodiment of the present invention.

[0033] Figure 3 This is the distribution of sea cucumber polysaccharide sequences in the immunomodulatory sea cucumber polysaccharide type sea cucumber extract in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0034] The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0035] The following examples can be understood as a part of the local structure or method of the present invention alone, and can also be understood as the combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are obtained from commercial sales.

[0036] Embodiment 1:

[0037] A method for preparing a sea cucumber polysaccharide type sea cucumber extract comprises the following steps:

[0038] (0-1) Pretreatment: The frozen sea cucumber plates are first thawed in a thawing tank, the sea cucumber viscera are removed for other uses, and the sea cucumber body wall is retained. The obtained body wall is first passed through a meat grinder and then connected to a colloid mill. After mincing, it is pumped to the colloid mill feed port through a vacuum pump and water is added in a ratio of 2:1. At the same time, the flow rate is controlled. After passing through the colloid mill, it is pumped into the enzymolysis tank. The steam valve in the interlayer of the enzymolysis tank is opened to increase the temperature, and stirring is started. The temperature is set to 56±2℃.

[0039] (0-2) Enzymolysis: After the temperature is constant, add the neutral protease activated by warm water in advance, the enzyme preparation added is 0.2% of the weight of the sea cucumber enzymolysis solution, stir and enzymolyze for 4-6 hours, and measure the solid content every half an hour with a solids analyzer after 4 hours of enzymolysis. When the solid content is basically stable, the enzymolysis ends;

[0040] (0-3) Degreasing and inactivating enzymes: The flour weighed in advance is added in an amount of 0.1% of the weight of the sea cucumber enzymatic hydrolysate, dissolved in warm water, and the dissolved flour solution is put into the enzymatic hydrolysis tank and stirred for 5 minutes. The stirring is turned off, and the steam valve is fully opened. The half-product after enzymatic hydrolysis is heated to 98°C and maintained for 20 minutes for denaturation, degreasing and enzyme inactivation. The steam valve of the enzymatic hydrolysis tank is closed, and ice water is poured in to cool it down to below 40°C. The lower discharge valve is opened, and the cooled half-product is sieved through a 100-mesh screen and poured into a temporary storage tank.

[0041] (0-4) Deproteinization: After defatting and inactivating the enzyme, heat the enzymatic hydrolyzate to 45-55°C, add 3% sodium hydroxide, adjust the pH to 7.8-8.2, keep warm and stir for 2 hours.

[0042] (0-5) Separation: After cooling, the half-product is separated by a linked tubular centrifuge, 4000r / min, and the feed speed is 1.0m 3 / h, intercepting insoluble residues above 100 mesh, the enzymatic hydrolyzate filtrate is filtered through a plate and frame filter again, 0.8Mpa, 22 plates, intercepting the remaining insoluble residues (above 300 mesh), and the filtrate is transferred to a temporary storage tank;

[0043] (0-6) Membrane: The half-product in the temporary storage tank is filtered through a membrane filtration device, passes through a 10,000 Dalton membrane, and the filtrate is collected in a temporary storage tank;

[0044] (0-7) Desalination: After passing through the membrane in step (0-5), softened water is added at a ratio of 1:5 (w:w), the pH is adjusted to 6.8-7.0, and the solution is passed through a sodium membrane to collect the retentate.

[0045] (0-6) Concentration: The filtrate in the temporary storage tank is sent to a double-effect vacuum concentration device for concentration, with an evaporation pressure of 0.06 MPa and an evaporation temperature of 68-70°C for the first effect and 56-60°C for the second effect. After the filtrate is concentrated to a solid content of 15%-20%, it is injected into a blending tank through a pipeline for standby use;

[0046] (0-7) Spray drying: The concentrated enzymatic hydrolysate is passed through a homogenizer and then fed into a spray tower for spray drying. The inlet temperature of the spray tower for spray drying is 170-190°C, and the outlet temperature is 95-105°C. A vibrating screen is set at the powder outlet, and the screen is performed once every 8-12 minutes to collect the sea cucumber polysaccharide type sea cucumber extract powder.

[0047]

[0048] According to GB5009.6-2016 "National Food Safety Standard Determination of Fat in Food - Soxhlet Extraction Method", the fat content of two batches of sea cucumber peptide powder without defatting process and sea cucumber peptide powder with flour defatting process was tested respectively. The fat content (wet basis) of one batch of sea cucumber peptide powder without defatting process was 3.02%, and the fat content (dry basis) was 3.30%. The fat content (wet basis) of sea cucumber peptide powder after flour defatting process was 0.11%, and the fat content (dry basis) was 0.15%; the fat content (wet basis) of the other batch of sea cucumber peptide powder without defatting process was 3.25%, and the fat content (dry basis) was 3.35%. The fat content (wet basis) of sea cucumber peptide powder after flour defatting process was 0.15%, and the fat content (dry basis) was 0.17%. Judging from the results of the fat content of sea cucumber peptide powder after defatting, the fat content of sea cucumber peptide powder after flour defatting process is 0.1-0.2%.

[0049] Embodiment 2:

[0050] The invention discloses an immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage, which is composed of the following raw materials in percentage by weight: 25% sea cucumber polysaccharide type sea cucumber extract powder, 10% chrysanthemum powder, 7.8% yerba mate tea, 20% xylitol, 7% lemon powder, 0.2% ginseng powder (artificially grown for less than five years), 15% resistant dextrin and 15% inulin.

[0051] Reference Figure 1 As shown, a method for preparing an immunomodulatory sea cucumber polysaccharide type sea cucumber extract solid beverage is as follows:

[0052] (1) Unpacking, weighing and mixing: Before entering the weighing room, remove the outer packaging bags of each raw material, replace them with sterile bags specially made for the workshop and mark them. Accurately weigh each raw material according to the formula dosage, put the weighed raw materials into the three-dimensional mixer, seal it, and mix it for 20-40 minutes until it is uniform;

[0053] (2) Fluidized bed granulation: Turn on the fluidized bed granulation equipment, suck the mixed material into a closed container of the hopper, granulate, dry, and obtain granules with a moisture content of <5%;

[0054] (3) Granulation: The granulated particles are sieved through an 80-mesh sieve to obtain relatively uniform particles;

[0055] (4) Packaging: Put the semi-finished granules into the granule packing machine, code them, and pack them;

[0056] (5) Packaging: The packaged solid beverages are placed in coded packaging boxes and then sealed with cigarettes.

[0057] The immunomodulatory sea cucumber polysaccharide type sea cucumber extract solid beverage prepared above was applied to experimental animals, and the experimental process and results are described as follows.

[0058] 1. Experimental Animals

[0059] 32 SPF male Kunming mice weighing 18-22 g were selected from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The experimental animal production license number is SYXK (Beijing) 2022-0052. The experimental environment temperature is 22±2°C and the relative humidity is 50±10%. The experimental animal use license number is SCXK (Lu) 20200022. The experimental animals were divided into a control group and low-, medium-, and high-dose groups, with 8 mice in each group. The test substance was dissolved in distilled water and administered to the mice by oral gavage once a day, with a gavage volume of 20 mL / kg·bw, for 30 consecutive days. The mice in the control group were gavaged with an equal volume of distilled water.

[0060] 2. Experimental Grouping

[0061] Table 1 Oral gavage dose and grouping

[0062]

[0063] 3. Experimental results

[0064] 3.1 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on body weight of mice

[0065] Table 1 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on body weight of mice

[0066]

[0067] As shown in Table 1, there was no significant difference in the initial body weight of mice among the groups (P>0.05). The weight gain values ​​of mice after oral administration of different doses of sea cucumber extract solid beverage for 30 days were not significantly different among the groups (P>0.05), that is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage had no effect on the weight of mice.

[0068] 3.2 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on organ-body weight ratios in mice

[0069] Table 2 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on spleen / body weight ratio of mice

[0070]

[0071] As can be seen from Table 2, mice were orally given different doses of sea cucumber extract solid beverage for 30 days. After statistical processing, there was no significant difference in the spleen / body weight ratio among the groups (P>0.05), that is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage had no effect on the spleen / body weight ratio of mice.

[0072] Table 3 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on thymus / body weight ratio in mice

[0073]

[0074] As can be seen from Table 3, when mice were orally given different doses of sea cucumber extract solid beverage for 30 days, there was no significant difference in the thymus / body weight ratio among the groups after statistical processing (P>0.05), that is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage had no effect on the thymus / body weight ratio of mice.

[0075] 3.3. Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on cellular immune function in mice

[0076] 3.3.1. Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on delayed-type hypersensitivity (DTH) in mice

[0077] Table 4 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on delayed-type hypersensitivity (DTH) in mice

[0078]

[0079] Compared with the control group, * P<0.05, ** P<0.01

[0080] As can be seen from Table 4, mice were orally given different doses of sea cucumber extract solid beverage for 30 days. After statistical processing, the differences in the swelling of the paws among the groups were significant (P<0.05). There was no significant difference between the control group and the low-dose group (P>0.05), and there was a significant difference between the control group and the medium- and high-dose groups (P<0.05, P<0.01). That is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage can enhance the delayed-type allergic reaction ability of mice.

[0081] 3.3.2 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on ConA-induced mouse lymphocyte transformation experiment

[0082] Table 5 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on ConA-induced mouse lymphocyte transformation experiment

[0083]

[0084] Compared with the control group, * P<0.05

[0085] As can be seen from Table 5, mice were orally given different doses of sea cucumber extract solid beverage for 30 days. After statistical processing, the lymphocyte proliferation ability was significantly different among the groups (P<0.05). There was no significant difference between the control group and the low-dose group (P>0.05), and there was a significant difference between the control group and the medium and high-dose groups (P<0.05, P<0.05), that is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage can enhance the lymphocyte transformation ability of mice.

[0086] 3.4. Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on humoral immunity in mice

[0087] 3.4.1. Effect of immunomodulatory sea cucumber polysaccharide extract solid beverage on the number of antibody-producing cells in mice

[0088] Table 6 Effect of immunomodulatory sea cucumber polysaccharide extract solid beverage on the number of antibody-producing cells in mice (x±SD)

[0089]

[0090] Compared with the control group, * P<0.05, ** P<0.01

[0091] As can be seen from Table 6, mice were orally given different doses of sea cucumber extract solid beverage for 30 days. After statistical processing, there was no significant difference in the number of antibody-producing cells among the groups (P>0.05), that is, the immune-regulating sea cucumber extract solid beverage had no effect on the number of antibody-producing cells in mice.

[0092] 3.4.2 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on serum hemolysin in mice

[0093] Table 7 Effects of immunomodulatory sea cucumber polysaccharide extract solid beverage on serum hemolysin levels in mice

[0094]

[0095] Compared with the control group, * P<0.05, ** P<0.01

[0096] As can be seen from Table 7, mice were orally given different doses of sea cucumber extract solid beverage for 30 days. After statistical processing, the antibody volume values ​​were significantly different among the groups (P<0.05). There was no significant difference between the control group and the low-dose group (P>0.05), but there was a significant difference between the control group and the medium- and high-dose groups (P<0.05, P<0.01). That is, the immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage can enhance the serum hemolysin level of mice.

[0097] This experiment shows that after oral administration of 0.833g / kg·bw, 1.67g / kg·bw and 5.01g / kg·bw sea cucumber extract solid beverage samples to mice for 30 consecutive days, there was no adverse effect on the weight gain of mice; there was no effect on the spleen weight ratio and thymus weight ratio of mice; the results of the cellular immune function and humoral immune function of mice were positive. According to the animal experiment judgment criteria for enhancing immunity in the "Draft for Comments on the Function Evaluation Method of Health Food (2020 Edition)", it can be considered that this immune-regulating sea cucumber polysaccharide type sea cucumber extract solid beverage has the function of enhancing immunity.

[0098] The various aspects, embodiments, and features of the present invention should be considered to be illustrative in all aspects and not limiting of the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0099] In the preparation method of the present invention, the order of each step is not limited to the order listed. For those skilled in the art, without creative work, the order of each step is also within the protection scope of the present invention. In addition, two or more steps or actions can be performed simultaneously.

[0100] Finally, it should be noted that the specific embodiments described herein are merely examples of the present invention, and are not intended to limit the implementation methods of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described, or replace them in a similar manner. It is not necessary and impossible to provide all examples of all implementation methods here. However, these obvious changes or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention, and interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A method for preparing a sea cucumber polysaccharide type sea cucumber extract, characterized in that The following steps are involved: (0-1) Pre-treatment: mincing and grinding of sea cucumber body wall; (0-2) Enzymatic hydrolysis: After the temperature is constant, add the neutral protease activated by warm water in advance, the addition amount is 0.1-0.3% of the weight of the sea cucumber grinding pulp, stir and enzymolyze for 4-6 hours, and measure the solid content every half an hour with a solids meter after 4 hours of enzymolysis. When the solid content is basically stable, the enzymolysis ends; (0-3) Degreasing and inactivating enzymes: The flour weighed in advance is added in an amount of 0.1-0.2% of the weight of the sea cucumber enzymatic hydrolysate, dissolved in warm water, and the dissolved flour solution is put into an enzymatic hydrolysis tank, stirred and mixed for 5 minutes, the stirring is turned off, the steam valve is fully opened, and the half-product after enzymatic hydrolysis is heated to 98° C. and maintained for 20 minutes for denaturation, degreasing and enzyme inactivation; the steam valve of the enzymatic hydrolysis tank is closed, and ice water is poured into it to cool it down to below 40° C.; Open the lower discharge valve, and the cooled semi-finished product is put into the temporary storage tank through a 100-mesh screen; (0-4) Separation: After cooling, the semi-product is separated by a tube centrifuge, 4000r / min, and the feed speed is 1.0m 3 / h, intercepting insoluble residues above 100 mesh, the enzymatic hydrolyzate filtrate is filtered through a plate and frame filter again, 0.8Mpa, 22 plates, intercepting the remaining insoluble residues, and the filtrate is transferred to a temporary storage tank; (0-5) Membrane: The half-product in the temporary storage tank is filtered through a membrane filtration device, passes through a 10,000 Dalton membrane, and the filtrate is collected in a temporary storage tank; (0-6) Concentration: The filtrate in the temporary storage tank is sent to a double-effect vacuum concentration device for concentration, with an evaporation pressure of 0.06 MPa and an evaporation temperature of 68-70°C for the first effect and 56-60°C for the second effect. After the filtrate is concentrated to a solid content of 15%-20%, it is injected into a blending tank through a pipeline for standby use; (0-7) Drying: Drying to obtain sea cucumber polysaccharide type sea cucumber extract powder.

2. The preparation method according to claim 1, characterized in that: Step (0-1) The frozen sea cucumber plate is first thawed in a thawing pool, the sea cucumber viscera are removed for other uses, and the sea cucumber body wall is retained; the obtained body wall is first passed through a meat grinder and then connected to a colloid mill, and after being minced, it is pumped to the feed port of the colloid mill through a vacuum pump and water is added in a ratio of 2:1, while the flow rate is controlled, and after passing through the colloid mill, it is pumped into an enzymolysis tank, and the steam valve in the interlayer of the enzymolysis tank is opened to increase the temperature, stirring is started, and the temperature is set to 56±2°C.

3. The preparation method according to claim 1, characterized in that: The method further comprises a deproteinization step: the enzymatic hydrolyzate after defatting and inactivating enzymes in step (0-3) is heated to 45-55° C., 3% sodium hydroxide is added, the pH value is adjusted to 7.8-8.2, and the temperature is kept and stirred for 2 hours.

4. The preparation method according to claim 1, characterized in that: The method also includes a desalination step: in step (0-5), the temporary liquid after passing through the membrane is added with softened water at a mass ratio of 1:5, the pH value is adjusted to 6.8-7.0, passed through a sodium membrane, and the intercepted liquid is collected.

5. The preparation method according to claim 1, characterized in that: Step (0-7) adopts spray drying, and the specific steps are: the concentrated enzymatic hydrolyzate passes through a homogenizer and then enters a spray tower for spray drying, the inlet temperature of the spray tower for spray drying is 170-190°C, and the outlet temperature is 95-105°C; a vibrating screen is set at the powder outlet, and the powder is screened once every 8-12 minutes to collect the sea cucumber polysaccharide type sea cucumber extract powder.

6. The sea cucumber polysaccharide type sea cucumber extract prepared by any method of claims 1-3.

7. The use of the sea cucumber polysaccharide type sea cucumber extract according to claim 4 in the preparation of an immunomodulatory solid beverage, characterized in that: The weight of the sea cucumber polysaccharide type sea cucumber extract powder in the solid beverage accounts for 5-30%.

8. An immunomodulatory solid beverage, characterized in that The invention is composed of the following raw materials in percentage by weight: 5-30% of the sea cucumber polysaccharide extract powder of claim 4, 2-10% of chrysanthemum powder, 2-10% of yerba mate tea, 10-20% of xylitol, 3-7% of lemon powder, 0.1-0.5% of ginseng powder (artificially grown for less than five years), 5-15% of resistant dextrin and 5-15% of inulin.

9. The method for preparing the solid beverage according to claim 6, characterized in that The following steps are involved: (1) Weighing and mixing: Accurately weigh the raw materials according to the formula dosage, put the weighed raw materials into a three-dimensional mixer, seal it, and mix it for 20-40 minutes until it is uniform; (2) Fluidized bed granulation: Turn on the fluidized bed granulation equipment, suck the mixed material into a closed container of the hopper, granulate, dry, and obtain granules with a moisture content of <5%; (3) Granulation: The granulated particles are sieved through an 80-mesh sieve to obtain relatively uniform particles; (4) Packaging: Put the semi-finished granules into the granule packing machine, code them, and pack them; (5) Packaging: The packaged solid beverages are placed in coded packaging boxes and then sealed with cigarettes.

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