Preparation method of sea cucumber with immunity enhancing function

Abstract

The invention discloses a preparation method of sea cucumber with an immunity enhancing function, which comprises the following steps: taking out viscera of fresh sea cucumber, removing sand mouths, cleaning body walls and the viscera respectively, and performing enzymolysis on the viscera; adding comprehensive fruit and vegetable powder into the enzymatic hydrolysate, steaming the body walls of the sea cucumbers, soaking the sea cucumbers in the comprehensive fruit and vegetable powder enzymatic hydrolysate, directly packaging and sterilizing, or soaking the sea cucumbers in the comprehensive fruit and vegetable powder enzymatic hydrolysate, drying the sea cucumbers and packaging. The sea cucumber prepared by the preparation method disclosed by the invention effectively protects the structure of functional protein, reduces the loss of nutritional ingredients, and has a prominent immunity enhancing function. In addition, sea cucumber viscera waste is comprehensively utilized, the defect that the vitamin content in sea cucumbers is low is overcome, the nutritive value of the sea cucumbers is improved, and the product is easier to store.

Description

Technical Field

[0001] The invention relates to a method for preparing sea cucumber with the function of enhancing immunity, and belongs to the technical field of food processing. Background Art

[0002] Sea cucumbers are rich in active ingredients such as protein, polysaccharides, phenols, saponins and unsaturated fatty acids, with low fat content and high nutritional value, and are often used as a tonic for strengthening the body. However, freshly caught sea cucumbers will undergo autolysis under the action of endogenous enzymes and are not easy to preserve, so more than 80% of sea cucumbers are processed into dried products and circulated on the market. However, in the process of processing from fresh sea cucumbers to dried products, a large amount of sea cucumber viscera are discarded, and these discarded sea cucumber viscera contain rich nutrients such as protein, polysaccharides, fat, saponins, etc., which causes the loss of nutrients and a great waste of marine biological resources.

[0003] In recent years, ready-to-eat sea cucumbers have been very popular among consumers for their strong elasticity, good flexibility, good taste and convenient eating. However, they have a limited storage period and are easy to soften, become sticky and spoil. Sea cucumbers are rich in protein and minerals, which help improve the body's immune ability and promote the generation of immune cells. Therefore, a sea cucumber with the function of enhancing immunity has been developed to meet the urgent needs of the market. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing sea cucumbers with the function of enhancing immunity. The sea cucumbers in the present invention effectively protect the structure of functional proteins, reduce the loss of nutrients, comprehensively utilize the sea cucumber viscera waste, add comprehensive fruit and vegetable powder, improve the immune regulation function, and improve the product's room temperature storage stability.

[0005] Compared with the prior art, the present invention has the following beneficial effects:

[0006] 1. Recycle and reuse sea cucumber viscera by enzymatic hydrolysis to reduce the loss of nutrients and promote comprehensive utilization of resources.

[0007] 2. Adding vitamin-rich mixed fruit and vegetable powder increases the product's storage stability at room temperature.

[0008] 3. Enhanced the immunoregulatory function of the product.

[0009] 4. The application of ultra-high pressure sterilization not only has a sterilization effect, but also reduces the loss of nutrients in sea cucumbers during processing. DETAILED DESCRIPTION

[0010] A method for preparing sea cucumber with immunity enhancing function comprises the following steps:

[0011] 1) After the fresh sea cucumber is washed with water, the sea cucumber abdomen is opened, the sea cucumber internal organs are taken out, the sand mouth is removed, and the body wall and internal organs are cleaned respectively to obtain the clean fresh sea cucumber body wall and internal organs;

[0012] 2) clean the viscera, homogenize, add protease, enzymolyze, heat inactivate, centrifuge, take the supernatant, obtain the sea cucumber viscera enzymolyzate, and adjust to neutral;

[0013] 3) Add the comprehensive fruit and vegetable powder to the enzymatic solution and stir to dissolve to obtain an immersion solution;

[0014] 4) taking clean fresh sea cucumber bodies and steaming them to obtain cooked sea cucumbers;

[0015] 4) placing the cooked sea cucumber in the soaking liquid and soaking it in a shaking manner;

[0016] 5) Packaging:

[0017] a) packaging the sea cucumber body and the soaking liquid together to form one piece / bag of ready-to-eat sea cucumber;

[0018] b) drying the sea cucumber bodies to obtain dried sea cucumbers, 1 piece / bag;

[0019] 6) Sterilize 5a).

[0020] Example 1

[0021] 200 parts by weight of fresh sea cucumber (about 25g each), after washing with water, open the abdomen of the sea cucumber, take out the sea cucumber viscera, remove the sand mouth, clean the body wall and viscera separately, obtain the clean fresh sea cucumber body wall and viscera, homogenize the viscera, add 80 parts by weight of water, add 0.1mol / L sodium hydroxide to adjust the pH to 8.0, add 10000U / g of trypsin, 37℃, enzymolysis for 5h, heat and boil for 5min, 6000r / min, centrifuge for 10min, take the supernatant, add citric acid solution to neutral, cool to room temperature, add 20 parts by weight of comprehensive fruit and vegetable powder, stir evenly, and obtain the impregnation liquid. Steam the fresh sea cucumber body wall for 10min to obtain the cooked sea cucumber body, soak it in the impregnation liquid, and continue to shake during the soaking process, the frequency is 150r / min, and the time is 3h. The cooked sea cucumber is combined with the remaining impregnation liquid and packaged. Ultra-high pressure sterilization was performed at a pressure of 400 MPa, a time of 10 min, and a temperature of 25° C. to obtain Example 1(a); the sea cucumber body was taken out, placed in an oven at 50° C., and dried to obtain Example (b).

[0022] Comparative Example 1

[0023] 200 parts by weight of fresh sea cucumber, after washing with water, open the abdomen of the sea cucumber, take out the sea cucumber viscera, remove the sand mouth, clean the body wall and viscera separately, get the clean fresh sea cucumber body wall, add 20 parts by weight of comprehensive fruit and vegetable powder, dissolve in 100 parts by weight of water, stir evenly, and get the immersion liquid. Steam the fresh sea cucumber body wall for 10 minutes to get the cooked sea cucumber body, immerse it in the immersion liquid, and continue to shake during the immersion process, the frequency is 150r / min, and the time is 3h. The cooked sea cucumber is combined with the remaining immersion liquid and packaged. Ultra-high pressure sterilization, pressure 400MPa, time 10min, temperature 25℃.

[0024] Comparative Example 2

[0025] 200 parts by weight of fresh sea cucumber, after washing with water, open the abdomen of the sea cucumber, take out the viscera of the sea cucumber, remove the sand mouth, clean the body wall and viscera separately, obtain the clean body wall and viscera of fresh sea cucumber, homogenize the viscera, add 100 parts by weight of water, add 0.1 mol / L sodium hydroxide to adjust the pH to 8.0, add 10000U / g of trypsin, 37°C, enzymolysis for 5h, heat and boil for 5min, 6000r / min, centrifuge for 10min, take the supernatant, add citric acid solution to neutral, cool to room temperature, and obtain the immersion liquid. Steam the body wall of the fresh sea cucumber for 10min to obtain the cooked sea cucumber body, immerse it in the immersion liquid, and continue to shake during the immersion process, the frequency is 150r / min, and the time is 3h. The cooked sea cucumber is combined with the remaining immersion liquid and packaged. Ultra-high pressure sterilization, pressure 400MPa, time 10min, temperature 25°C.

[0026] Comparative Example 3

[0027] 200 parts by weight of fresh sea cucumbers are washed with water, and then the sea cucumber abdomen is opened to remove the sea cucumber viscera, the sand mouth is removed, the body wall and the viscera are cleaned separately to obtain a clean fresh sea cucumber body wall, the fresh sea cucumber body wall is steamed for 10 minutes to obtain a cooked sea cucumber body, 120 parts by weight of water is added, and the body is shaken at a frequency of 150 r / min for 3 hours, and then packaged. Ultra-high pressure sterilization is performed at a pressure of 400 MPa for 10 minutes at a temperature of 25°C.

[0028] Index determination:

[0029] 1. Determination of nutritional ingredients

[0030] 1.1 Determination of protein content

[0031] Based on the Kjeldahl nitrogen determination method in GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food".

[0032] 1.2 Determination of sea cucumber saponins

[0033] Based on GB / T 33108-2016 "Determination of sea cucumber saponins in sea cucumbers and their products - High performance liquid chromatography".

[0034] 1.3 Determination of soluble polypeptide mass fraction: Folin-phenol reagent method

[0035] Take the enzymatic solution, make it up to 100mL volumetric flask, take 5.0mL solution, add 5.0mL trichloroacetic acid solution with a mass fraction of 10%, mix and shake, centrifuge (6000r / min, 20min); take 1.0mL solution of supernatant, add 5.0mL of Folin phenol reagent A solution, mix, and place at 30℃ for 10min; then, add 0.5mL Folin phenol reagent B solution, shake and mix immediately, and keep warm at 30℃ for 30min. Take the solution in the test tube without adding standard as blank, measure the absorbance value (D650) at 650nm, substitute it into the standard curve drawn with bovine serum albumin as standard, and the soluble mass fraction can be obtained. The results of protein, saponin and soluble peptide determination are shown in Table 1.

[0036] Table 1 Results of protein, saponin and soluble peptide (per bag)

[0037] project Example 1 (a) Example 1 (b) Comparative Example 1 Comparative Example 2 Comparative Example 3 Protein (g) 1.70±0.21 1.53±0.36 1.41±0.27 1.61±0.25 1.35±0.20 Saponin(g) 0.019±0.004 0.016±0.006 0.011±0.003 0.017±0.005 0.010±0.004 Soluble peptide (g) 0.070±0.008 0.060±0.008 0.031±0.004 0.067±0.010 0.040±0.006

[0038] 1.4 Nutritional analysis

[0039] Protein: The protein content in Example 1(a) and Comparative Example 2, in which visceral enzymatic hydrolysate was added, was relatively high, because the protein in the viscera was recycled. Saponin: The saponin content in Example 1(a) and Comparative Example 2, in which visceral enzymatic hydrolysate was added, was relatively high, because the saponin in the viscera was recycled.

[0040] The soluble peptide content is higher in Example 1 (a) and Comparative Example 2. Since sea cucumber viscera hydrolysate was added in Example 1 and Comparative Example 2, the hydrolysate is mostly soluble peptides, so the content is relatively high.

[0041] In general, the protein, saponin and soluble peptide contents of Example 1(a) in which visceral enzymatic hydrolysate and mixed fruit and vegetable powder were added were higher than those of the comparative example, indicating that the nutritional components thereof were richer.

[0042] 2. Experiment on enhancing immunity function

[0043] 2.1 Experimental animals: SPF grade ICR mice, female, weighing 18-22 g.

[0044] 2.2 Experimental Materials and Reagents

[0045] RPMI 1640 complete culture medium, Hank's solution (pH 7.2-7.4), Giemsa stain, Con A (Con A), MTT: India ink, oxidized coenzyme I, sheep red blood cells (SRBC), chicken red blood cells, penicillin, streptomycin, SA buffer, complement (guinea pig serum).

[0046] 2.3 Experimental instruments and equipment

[0047] Carbon dioxide incubator (Series8000), electronic balance, microplate reader (PowerWave340), centrifuge (L2540A), spectrophotometer (Ultra-6100), microscope (CX23), etc.

[0048] 2.4 Experimental methods

[0049] (1) Animals and groups: The mice were randomly divided into five groups, each with 60 mice. The first group was immunized for carbon clearance test and determination of animal organ / body ratio; the second group was immunized for half hemolysis value (HC 50 ) determination and antibody-producing cell detection; delayed-type hypersensitivity reaction experiment was performed in immunized group three; phagocytosis of chicken red blood cells by mouse peritoneal macrophages was performed in immunized group four; ConA-induced mouse spleen lymphocyte transformation experiment and NK cell activity determination were performed in immunized group five.

[0050] (2) Dosage design: Take the samples of Example 1(a), Example 1(b), Comparative Examples 1, 2, and 3, and give them to mice at 4.5 g / kg·BW (1 times the recommended dose for adults). At the same time, set up a negative control group (gavage with equal volume of distilled water), and animals were gavaged with 2% of body weight, and the solvent was distilled water. After continuous gavage for 30 days, various immune indicators were measured.

[0051] (3) Inspection items and methods: Inspections were conducted in accordance with the inspection items and methods for enhancing immunity in the Technical Specifications for Inspection and Evaluation of Health Foods issued by the Ministry of Health. For the requirements of the test items for enhancing immunity, delayed-type hypersensitivity (plantar thickening method) (DTH), carbon clearance test in mice, determination of organ / body weight ratio, phagocytosis of chicken red blood cells by mouse peritoneal macrophages (semi-in vivo method), ConA-induced transformation test of mouse spleen lymphocytes (MTT method), antibody-producing cell detection (Jerne modified slide method), and half hemolysis value (HC 50 ) determination, NK cell activity determination (lactate dehydrogenase LDH determination method).

[0052] (4) Data processing and result judgment: Use SPSS analysis software for data statistics and analysis.

[0053] 2.5 Results and Analysis

[0054] 2.5.1 Effects of each group on the ratio of mouse organs to body weight

[0055] The effects on the organ / body weight ratio of mice after administration of each group of samples for 30 days are shown in Table 2.

[0056] Table 2 Effects of each experimental group on the ratio of mouse organs to body weight

[0057] Group Number of animals Spleen / body weight ratio / (mg / g) Thymus / body weight ratio / (mg / g) Negative control group 10 0.46±0.01 0.33±0.03 Example 1 (a) 10 0.46±0.05 0.32±0.02 Example 1(b) 10 0.45±0.08 0.35±0.02 Comparative Example 1 10 0.47±0.04 0.34±0.01 Comparative Example 2 10 0.45±0.02 0.35±0.02 Comparative Example 3 10 0.44±0.05 0.33±0.04

[0058] As shown in Table 2, after oral administration to different experimental groups of mice for 30 days, there was no significant difference in the spleen / body weight ratio and thymus / body weight ratio among the groups (P>0.05), that is, each group had no effect on the organ / body weight ratio of mice.

[0059] 2.5.2 Effects of each group on delayed-type hypersensitivity (DTH) in mice

[0060] The effects on delayed-type hypersensitivity (DTH) in mice after administration of each group for 30 days are shown in Table 3.

[0061] Table 3 Effects of each group on delayed-type hypersensitivity (DTH) in mice

[0062]

[0063] Note: * Compared with the negative control group, there is a significant difference P<0.05; ** Compared with the negative control group, there is a significant difference P<0.01.

[0064] As can be seen from Table 3, after oral administration of samples to different groups of mice for 30 days, the differences in the degree of swelling of the paw (plantar thickening) before and after the injection of SRBC were significantly different between the comparison groups 1, 2, and 3 and the negative control group (P<0.05), and there were extremely significant differences between the Example 1(a) and Example 1(b) groups and the negative control group, indicating that Examples 1(a) and (b) most significantly enhanced the delayed-type hypersensitivity of mice.

[0065] 2.5.3 Effects of each group on ConA-induced mouse lymphocyte transformation

[0066] The effects of the samples in each group on the transformation of mouse spleen lymphocytes 30 days after administration are shown in Table 4.

[0067] Table 4 Effects of each group on ConA-induced mouse lymphocyte transformation

[0068] Group Number of animals OD difference Negative control group 10 0.240±0.025 Example 1 (a) 10 0.239±0.020 Example 1(b) 10 0.232±0.051 Comparative Example 1 10 0.231±0.026 Comparative Example 2 10 0.242±0.031 Comparative Example 3 10 0.235±0.036

[0069] As shown in Table 4, there was no significant difference in the transformation function of mouse spleen lymphocytes between each dose group and the negative control group (P>0.05), indicating that each group of samples could not affect the transformation function of mouse spleen lymphocytes.

[0070] 2.5.4 The hemolysis value (HC) of each group on mice 50 )

[0071] After 30 days of administration of each group of samples to mice, the hemolysis value (HC 50 ) is shown in Table 5.

[0072] Table 5 The half hemolysis value (HC) of each group on mice 50 )

[0073] Group Number of animals <![CDATA[Half hemolysis value / (HC 50 )]]> Negative control group 10 170.43±16.34 Example 1 (a) 10 213.44±14.59* Example 1(b) 10 208.98±8.15* Comparative Example 1 10 182.19±10.80 Comparative Example 2 10 189.10±8.36 Comparative Example 3 10 175.35±10.56

[0074] Note: * Compared with the negative control group, there is a significant difference P < 0.05

[0075] As shown in Table 5, the half hemolysis values ​​of Example 1 (a) and (b) groups were significantly different from those of the negative control group (P<0.05), while there were no significant differences between the comparative examples 1, 2, and 3 groups and the negative control group.

[0076] 2.5.5 Effects of each group of powder on mouse antibody-producing cells

[0077] The effects of each group of samples on mouse antibody-producing cells 30 days after administration are shown in Table 6.

[0078] Table 6 Effects of each group on mouse antibody-producing cells

[0079] Group Number of animals <![CDATA[Plaque number / (10 6 spleen cells)]]> Negative control group 10 19.55±1.24 Example 1 (a) 10 19.62±1.50 Example 1(b) 10 18.02±2.36 Comparative Example 1 10 19.58±1.23 Comparative Example 2 10 18.54±2.31 Comparative Example 3 10 18.64±0.98

[0080] As shown in Table 6, there was no significant difference in the number of hemolytic plaques in each group compared with the negative control group (P>0.05), indicating that each group could not enhance the function of mouse antibody-producing cells.

[0081] 2.5.6 Effects of each group on carbon clearance function of mice

[0082] The effects of each group of samples on the carbon clearance ability of mice after 30 days of administration are shown in Table 7 。

[0083] Table 7 Effects of each group on carbon clearance function of mice

[0084] Group Number of animals Phagocytic index / (α) Negative control group 10 6.55±0.30 Example 1 (a) 10 6.60±0.42 Example 1(b) 10 6.49±0.21 Comparative Example 1 10 6.63±0.38 Comparative Example 2 10 6.64±0.53 Comparative Example 3 10 6.54±0.10

[0085] As shown in Table 7, there was no significant difference in the phagocytic index between each dose group and the negative control group (P>0.05), indicating that each group could not increase the carbon clearance function of mice.

[0086] 2.5.7 Effects of each group on phagocytosis of chicken red blood cells by mouse peritoneal macrophages

[0087] The effects of the samples in each group on the phagocytosis of chicken red blood cells by mouse peritoneal macrophages 30 days after administration are shown in Table 8.

[0088] Table 8 Effects of each group on the phagocytosis of chicken red blood cells by mouse macrophages

[0089] Group Number of animals Phagocytosis rate / (%) Phagocytic index Negative control group 10 26.11±2.08 0.352±0.019 Example 1 (a) 10 27.04±2.00 0.398±0.029** Example 1(b) 10 27.11±2.65 0.390±0.020** Comparative Example 1 10 26.49±1.52 0.372±0.016* Comparative Example 2 10 27.05±1.81 0.388±0.020** Comparative Example 3 10 26.89±1.26 0.366±0.016*

[0090] Note: * Compared with the negative control group, there is a significant difference P<0.05; ** Compared with the negative control group, there is a significant difference P<0.01.

[0091] As shown in Table 8, the phagocytic index of Example 1 (a), Example 1 (b), and Comparative Example 2 groups were significantly different from that of the negative control group (P<0.01). Comparative Example 1 and Comparative Example 3 groups were significantly different from the negative control group (P<0.05), indicating that each group was able to enhance the phagocytic function of mouse peritoneal macrophages.

[0092] 2.5.8 Effects of each group on mouse NK cell activity

[0093] The effects of the samples in each group on the NK cell activity of mice 30 days after administration are shown in Table 9.

[0094] Table 9 Effects of each group on mouse NK cell activity

[0095] Group Number of animals NK cell activity / (%) Negative control group 10 30.04±1.33 Example 1 (a) 10 39.69±1.02* Example 1(b) 10 37.11±1.16* Comparative Example 1 10 31.24±1.76 Comparative Example 2 10 33.13±1.57 Comparative Example 3 10 32.24±1.62

[0096] Note: * Compared with the negative control group, there is a significant difference P<0.05; ** Compared with the negative control group, there is a significant difference P<0.01.

[0097] As shown in Table 9, the NK cell activity of each dosage group was increased compared with the negative control group, and there was a significant difference between Example 1 (a) and (b) groups and the negative control group (P<0.05).

[0098] The experiment found that the Example 1 (a) and (b) groups can significantly improve the delayed-type hypersensitivity reaction (plantar thickening method) (DTH), the half hemolysis value (HC 50 ) determination, mouse peritoneal macrophage phagocytosis of chicken red blood cells experiment, and NK cell activity, the effect was better than that of the control groups.

[0099] 3. Texture analysis

[0100] The TA.XT Plus physical property tester was used to analyze and measure the texture profile analysis (TPA) characteristics of the ready-to-eat sea cucumber samples. The main indicators include hardness, adhesiveness, elasticity, cohesion, resilience and chewiness. The experiment used a P / 0.5 cylindrical probe, the test deformation was 50%, and the induction force was 5×g. The pre-measurement speed was 1.0 mm / s, the measurement speed was 0.5 mm / s, and the post-measurement speed was 0.5 mm / s. Each group was measured in parallel 6 times. Samples with different storage times were taken for measurement, and the results are shown in Table 10.

[0101] Table 10 Texture analysis results

[0102]

[0103]

[0104] Four indicators, namely hardness, elasticity, chewiness and resilience, were selected to further analyze the changing rules of the texture characteristics of sea cucumbers during storage. Within 0 to 30 days, the hardness, elasticity, chewiness and resilience of each group showed a gradual downward trend, and Example 1 had the smallest decline. The decline in Example 2 was lower than that in Example 1, indicating that the storage stability of adding visceral enzymatic hydrolysate was higher than that of adding comprehensive fruit and vegetable powder. The stability effect of adding visceral enzymatic hydrolysate and comprehensive fruit and vegetable powder at the same time was significantly higher than that of single application.

Claims

1. A method for preparing sea cucumber having the function of enhancing immunity, characterized in that: The following steps are involved: 1) After the fresh sea cucumber is washed with water, the sea cucumber abdomen is opened, the sea cucumber internal organs are taken out, the sand mouth is removed, and the body wall and internal organs are cleaned respectively to obtain the clean fresh sea cucumber body wall and internal organs; 2) clean the viscera, homogenize, add protease, enzymolyze, centrifuge, take the supernatant, obtain the sea cucumber viscera enzymolyzate, heat inactivate, and adjust to neutral; 3) taking clean fresh sea cucumber body wall and steaming it to obtain cooked sea cucumber; 4) The cooked sea cucumber is placed in an enzymatic soaking tank and soaked in a shaking manner; 5) Packaging: a) packaging the sea cucumber body and the soaking liquid together to form one piece / bag of ready-to-eat sea cucumber; b) drying the sea cucumber bodies to obtain dried sea cucumbers, 1 piece / bag; 6) Sterilize 5a).

2. A method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that The protease is one or more of trypsin, neutral protease and alkaline protease.

3. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: Enzymatic hydrolysis conditions: enzyme dosage 5000-1000U / g, time 4-6h, temperature 35-50℃, pH 7-11.

4. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: The steaming time of fresh sea cucumber is 10 to 20 minutes.

5. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: Comprehensive fruit and vegetable powder includes pineapple, sweet potato, apple, papaya, banana, green plum, bitter gourd, water spinach, asparagus, grapes, orange, carrot, watermelon, chrysanthemum, guava, spinach, passion fruit, celery, pumpkin, etc.

6. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: The cooked sea cucumber is oscillated in the immersion liquid at a frequency of 100 to 200 r / min for 2 to 4 hours.

7. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: The drying temperature of sea cucumber body is 40~60℃.

8. The method for preparing a sea cucumber having the function of enhancing immunity according to claim 1, characterized in that: Sterilization is carried out by ultra-high pressure sterilization, with a pressure of 400-600MPa, a time of 10-30min, and a temperature of 20-45°C.

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