A silkworm protein composite powder for enhancing immunity

Abstract

The present invention belongs to the field of food technology, and specifically relates to a mulberry silkworm protein composite powder that can enhance immunity. The present invention uses silkworm pupae, pea protein powder and sea cucumber as raw materials, and uses alkaline protease and a composite protease composed of papain and neutral protease for enzymolysis to obtain a mulberry silkworm protein composite powder that can enhance immunity. It is found that the combination of silkworm pupa protein, pea protein and sea cucumber protein can enhance immunity and has a synergistic effect, and the composite powder prepared by the enzymolysis process of the present invention has a significantly better effect of synergistic immunity enhancement.

Description

Technical Field

[0001] The invention belongs to the technical field of food, and in particular relates to a silkworm protein composite powder capable of enhancing immunity. Background Art

[0002] The immune system of humans and other organisms is a complex system composed of many organs, tissues, cells, molecules, and lymphatic circulation that perform immune functions. Its main function is to identify and eliminate antigens and realize immune defense. Its basic role is to protect itself and resist the threats of foreign pathogens and its own cancerous cells.

[0003] Normal immune function and autoimmune response are very important to maintain the health of the body and the functions of various systems. When the immune function is disordered, various infectious diseases are prone to occur on the one hand, and on the other hand, infection can induce autoimmunity and even stimulate autoimmune diseases.

[0004] Competition in modern society is intensifying, the pace is accelerating, and modern people are in a tense living environment for a long time. They are under great work pressure, over-fatigue, disordered work and rest, too nervous, abuse of antibiotics, and abnormal living and eating habits, which cause the general weakening of immunity and endocrine, nervous system, and digestive system disorders, and the immunity is gradually reduced. A slight change in the weather will lead to various diseases such as colds, which seriously threaten the health of the body. With the development of science and technology, pollution in life has produced various harmful substances, such as peroxides released by drugs and food additives; radiation generated by mobile phones, televisions, computers, etc. These factors destroy the integrity and stability of human cells and eventually cause lesions in human organs.

[0005] Therefore, it is of great significance to develop a product that can enhance immunity, improve the body's low immunity, and thus prevent or treat the diseases that may be caused. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a silkworm protein composite powder for enhancing immunity, which comprises the following raw materials in parts by weight: 30-50 parts of silkworm pupae, 10-20 parts of pea protein powder, and 10-20 parts of sea cucumber.

[0007] Furthermore, the silkworm protein composite powder is prepared as follows:

[0008] (1) taking fresh silkworm pupae and sea cucumbers, washing each of them and homogenizing them in distilled water, combining the homogenates and adding pea protein powder, mixing them evenly to obtain a slurry;

[0009] (2) using alkaline protease and composite protease to enzymatically hydrolyze the slurry obtained in step (1);

[0010] (3) Centrifuging the enzymatic hydrolyzate obtained in step (2) to collect the supernatant, vacuum freeze-drying and pulverizing to obtain the silkworm protein composite powder.

[0011] Furthermore, in step (1), the volume mass ratio of distilled water to fresh silkworm pupae is 1-10:1, and the volume mass ratio of distilled water to sea cucumber is 1-10:1.

[0012] Furthermore, enzymolysis using alkaline protease comprises: adjusting the pH of the slurry obtained in step (1) to 8-10, adding 4000-5000 U / g of alkaline protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, enzymolyzing at 40-50° C. for 2-4 hours, and then inactivating the enzyme at 90-100° C.

[0013] Furthermore, the pH was adjusted using a 1M sodium hydroxide aqueous solution.

[0014] Furthermore, the composite protease is an equal mass mixture of papain and neutral protease.

[0015] Furthermore, the enzymatic hydrolysis using the composite protease includes: adjusting the pH of the slurry after enzymatic hydrolysis with alkaline protease to 6-7, adding 2000-3000U / g of the composite protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, enzymatic hydrolysis at 50-60°C for 2-4 hours, and then inactivating the enzyme at 90-100°C.

[0016] Furthermore, the pH was adjusted using a 0.5 M HCl solution.

[0017] Furthermore, the centrifugal speed is 8000-10000 rpm, and the time is 15-20 min.

[0018] Furthermore, the silkworm protein composite powder may also contain other nutrients that enhance immunity.

[0019] Furthermore, other nutrients that enhance immunity may include, for example, other protein substances, fat substances, carbohydrates, dietary fiber substances, vitamins and / or minerals, etc.

[0020] Beneficial effects of the present invention

[0021] The present invention compounds silkworm pupa protein, pea protein and sea cucumber protein for the first time, and finds that they can enhance immunity and have a synergistic effect. Furthermore, the present invention finds that instead of simply mixing them together, silkworm pupae, pea protein powder and sea cucumber are used as raw materials, and an enzymatic hydrolysis process is used to prepare a composite powder containing silkworm pupa protein peptides, pea protein peptides and sea cucumber protein peptides, and the synergistic immunity enhancement effect is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The cytotoxicity study results of the silkworm protein composite powder prepared in Example 1 of the present invention are shown.

[0023] Figure 2 The results of leukocyte counts in serum of mice taking the protein powders of Examples 1-3 of the present invention and Comparative Examples 1-6 are shown. DETAILED DESCRIPTION

[0024] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] Example 1

[0026] A silkworm protein composite powder for enhancing immunity comprises the following raw materials in parts by weight: 40 parts of silkworm pupae, 15 parts of pea protein powder and 15 parts of sea cucumber.

[0027] The silkworm protein composite powder is prepared as follows:

[0028] (1) taking a required amount by weight of fresh silkworm pupae and sea cucumber, washing each of them and homogenizing them in distilled water 3 times their own weight, combining the homogenates and adding a required amount by weight of pea protein powder, mixing them evenly to obtain a slurry;

[0029] (2) adjusting the pH of the slurry obtained in step (1) to 8.0 using a 1M sodium hydroxide aqueous solution, adding 4000U / g of alkaline protease (BOFIMECO, CAS No. 9014-01-1) based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 45°C for 4 hours, and then inactivating the enzyme at 90°C; cooling to room temperature, adjusting the pH to 6.5 using a 0.5M HCl solution, adding 3000U / g of a composite protease composed of equal masses of papain (BOFIMECO, CAS No. 9001-73-4) and neutral protease (BOFIMECO, CAS No. 9068-59-1) based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 50°C for 4 hours, and then inactivating the enzyme at 90°C;

[0030] (3) The enzymatic hydrolyzate obtained in step (2) is centrifuged at 10,000 rpm for 15 min to collect the supernatant, and the supernatant is vacuum freeze-dried and pulverized to obtain the silkworm protein composite powder.

[0031] Example 2

[0032] A silkworm protein composite powder for enhancing immunity comprises the following raw materials in parts by weight: 30 parts of silkworm pupae, 10 parts of pea protein powder and 20 parts of sea cucumbers.

[0033] The silkworm protein composite powder is prepared as follows:

[0034] (1) taking the required weight portions of fresh silkworm pupae and sea cucumbers, washing each of them and homogenizing them in distilled water 3 times their own weight, combining the homogenates and adding the required weight portions of pea protein powder, and mixing them evenly;

[0035] (2) adjusting the pH of the slurry obtained in step (1) to 9.0 using a 1M sodium hydroxide aqueous solution, adding 5000U / g of alkaline protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, enzymolysis at 50°C for 2 hours, and then inactivating the enzyme at 90°C; cooling to room temperature, adjusting the pH to 6.5 using a 0.5M HCl solution, adding 2500U / g of a composite protease composed of equal masses of papain and neutral protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, enzymolysis at 55°C for 3 hours, and then inactivating the enzyme at 90°C;

[0036] (3) The enzymatic hydrolyzate obtained in step (2) is centrifuged at 9000 rpm for 20 min to collect the supernatant, and the supernatant is vacuum freeze-dried and pulverized to obtain the silkworm protein composite powder.

[0037] Example 3

[0038] A silkworm protein composite powder for enhancing immunity comprises the following raw materials in parts by weight: 50 parts of silkworm pupae, 20 parts of pea protein powder and 10 parts of sea cucumbers.

[0039] The silkworm protein composite powder is prepared as follows:

[0040] (1) taking the required weight portions of fresh silkworm pupae and sea cucumbers, washing each of them and homogenizing them in distilled water 3 times their own weight, combining the homogenates and adding the required weight portions of pea protein powder, and mixing them evenly;

[0041] (2) adjusting the pH of the slurry obtained in step (1) to 9.0 using a 1M sodium hydroxide aqueous solution, adding 4500U / g of alkaline protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 40°C for 3 hours, and then inactivating the enzyme at 90°C; cooling to room temperature, adjusting the pH to 7.0 using a 0.5M HCl solution, adding 2000U / g of a composite protease composed of equal masses of papain and neutral protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 60°C for 2 hours, and then inactivating the enzyme at 90°C;

[0042] (3) The enzymatic hydrolyzate obtained in step (2) is centrifuged at 8000 rpm for 20 min to collect the supernatant, and the supernatant is vacuum freeze-dried and pulverized to obtain the silkworm protein composite powder.

[0043] Comparative Example 1

[0044] A protein powder for enhancing immunity, which differs from Example 1 in that in step (1), only the silkworm pupae are homogenized.

[0045] Comparative Example 2

[0046] A protein powder for enhancing immunity, which differs from Example 1 in that in step (1), only the sea cucumber is homogenized.

[0047] Comparative Example 3

[0048] A protein powder for enhancing immunity, which differs from Example 1 in that in step (1), only pea protein powder and distilled water are mixed.

[0049] Comparative Example 4

[0050] A protein powder for enhancing immunity, which differs from Example 1 in that pea protein powder is not added in step (1), but protein powder is added after pulverization and mixed evenly in step (3).

[0051] Comparative Example 5

[0052] A protein powder for enhancing immunity, which differs from Example 1 in that in step (2), only alkaline protease is used for enzymolysis, and subsequent composite protease enzymolysis is not performed.

[0053] Comparative Example 6

[0054] A protein powder for enhancing immunity, which differs from Example 1 in that in step (2), neutral protease is used instead of composite protease in the second enzymatic hydrolysis.

[0055] Experimental Example 1: Cytotoxicity Study

[0056] HepG2 cells in the logarithmic growth phase were taken and prepared into 10 5 100 μL of the suspension was inoculated into each well of a 96-well plate and incubated at 37°C and 5% CO 2 The cells were cultured under the conditions for 24 h until the cells adhered to the wall, and the original culture medium was discarded. A control group and an experimental group were set up respectively. 100 μL of serum-free DMEM culture medium was added to each well of the control group, and 100 μL of serum-free DMEM culture medium containing different concentrations of the silkworm protein composite powder prepared in Example 1 (final concentrations were 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL) was added to each well of the experimental group. Six duplicate wells were set up for each group, and the mixture was mixed and incubated at 37°C, 5% CO 2After culturing the cells for 24 h, 48 h, and 72 h under the same conditions, 10 μL of MTT solution was added to each well and incubation continued. After 3-4 h, the supernatant was discarded and 50 μL of DMSO was added to each well to dissolve the purple crystals. The cells were shaken for 15 min, and the absorbance of each group at 570 nm was measured with a microplate reader, and the cell morphology of each group was observed under an inverted microscope.

[0057] HepG2 cell viability calculation formula:

[0058] Cell viability (%) = [(A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 )]×100%;

[0059] Among them, A 实验组 is the absorbance value of the experimental group; A 对照组 is the absorbance value of the control group; A 空白组 is the absorbance value of the blank group (i.e., cells that were not treated in any way compared to the experimental and control groups).

[0060] The results are as follows Figure 1 As shown, it shows that the silkworm protein composite powder prepared by the present invention has no obvious toxic side effects.

[0061] Example 2: Study on Immunity Enhancement Function

[0062] The immunosuppressed mouse model was established by inducing cyclophosphamide (CTX). The mice taking levamisole hydrochloride were used as the positive control group, and the other groups were set up as the normal control group and the experimental groups taking the protein powders of Examples 1-3 and Comparative Examples 1-6 respectively. The mice were randomly divided into 12 groups in each group.

[0063] After the mice were adapted to the environment for 7 days, except for the normal control group, the other groups were gavaged with 80g / kg CTX for 3 days; from the fourth day, the experimental group was gavaged with 50mg / kg of protein powder of Example 1-3 or Comparative Example 1-6 once a day, the model group was gavaged with an equal amount of 0.9% saline, and the positive control group was gavaged with 20mg / kg levamisole hydrochloride for 15 days. 24h after the last administration, the treatment and related indexes were measured. The results are shown in Table 1 and Figure 2 shown.

[0064] Table 1

[0065]

[0066]

[0067] From Table 1 and Figure 2The results show that the silkworm protein composite powder of Examples 1-3 of the present invention reversed the weight loss trend of immunocompromised mice, and also promoted the increase of the thymus index and spleen index of mice, indicating that it has the effect of enhancing immunity, and its effect is significantly improved compared with the effect of the individual components (Comparative Examples 1-3), indicating that silkworm pupa protein, pea protein and sea cucumber protein can synergistically promote the enhancement of immunity. Comparative Examples 4-6 prove that the enzymatic hydrolysis process of the present invention can significantly improve the effect of the protein composite powder. When pea protein is not enzymatically hydrolyzed (Comparative Example 4), it can be seen that although the combination of the three also increases the immunity enhancement effect, it is still significantly insufficient compared to Example 1, and when only alkaline protease is used or papain is not used for enzymatic hydrolysis (Comparative Examples 5 and 6), the immunity enhancement effect of the protein composite powder is also significantly insufficient.

[0068] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A silkworm protein composite powder for enhancing immunity, which is made from the following raw materials in parts by weight: 40 parts of silkworm pupae, 15 parts of pea protein powder, and 15 parts of sea cucumber; The silkworm protein composite powder is prepared as follows: (1) taking a required amount by weight of fresh silkworm pupae and sea cucumber, washing each of them and homogenizing them in distilled water 3 times their own weight, combining the homogenates and adding a required amount by weight of pea protein powder, mixing them evenly to obtain a slurry; (2) adjusting the pH of the slurry obtained in step (1) to 8.0 using a 1M sodium hydroxide aqueous solution, adding 4000U / g of alkaline protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 45°C for 4 hours, and then inactivating the enzyme at 90°C; cooling to room temperature, adjusting the pH to 6.5 using a 0.5M HCl solution, adding 3000U / g of a composite protease composed of equal masses of papain and neutral protease based on the total mass of silkworm pupae, sea cucumber and pea protein powder, performing enzymolysis at 50°C for 4 hours, and then inactivating the enzyme at 90°C; (3) The enzymatic hydrolyzate obtained in step (2) is centrifuged at 10,000 rpm for 15 min to collect the supernatant, and the supernatant is vacuum freeze-dried and pulverized to obtain the silkworm protein composite powder.

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