Deuterium-depleted selenium-enriched kiwi fruit compound enzyme and preparation method thereof

Abstract

The invention discloses a preparation method of a deuterium-depleted selenium-enriched kiwi fruit compound enzyme. A production raw material formula comprises the following components in parts by mass: 1 part of a compound biological raw material and 1.2-1.8 parts of deuterium-depleted water, the composite biological raw material is prepared from a selenium-rich fermentation raw material and a composite selenium organic raw material according to the mass ratio of 1: (0.3-0.7); the selenium-rich fermentation raw materials comprise kiwi fruits; the composite selenium organic raw material is prepared from roxburgh rose, olive and okra according to the mass ratio of 1: (0.1-0.5): (0.2-0.4). The method has the advantages that the SOD enzyme activity and the selenium organification rate of the biological enzyme product can be remarkably improved, so that the human immunity can be improved on the whole.

Description

Technical Field

[0001] The present invention relates to a biological fermentation technology, in particular to a production technology of a composite enzyme. Background Art

[0002] Enzymes are healthy, green foods rich in trace elements, macroelements, enzymes, polyphenols, amino acids, and other nutrients. They are highly favored by researchers both domestically and internationally and hold great promise for development. Compared to consuming fruit directly, the active substances in enzymes are more easily absorbed and utilized by the body, and the organic small molecules are more concentrated and more readily accepted. Fermentation technology not only retains the nutritional value of the original substance but also produces new bioactive substances beneficial to human health. It also maximizes the release of organic matter, greatly improving bioavailability.

[0003] Recent studies have shown that enzymes are rich in substances that can prevent various diseases, supplement nutrition, and promote overall health. They have antioxidant properties, enhance immunity, lower blood lipids, promote weight loss, accelerate metabolism, and promote biocatalysis, effectively regulating human physiological functions. Enzymes are currently one of the most popular fermentation products both domestically and internationally, with enormous room for development and growth potential, and a broad market outlook. However, enzymes currently produced by traditional processes generally suffer from technical issues such as low SOD enzyme activity and low selenium organic conversion rates. Researchers have attempted to improve this process by adding selenium yeast or fermenting a mixture of various fruits. However, the organic selenium in the former is unstable and easily converted into ineffective inorganic selenium, while the latter often lacks effective synergy between the various raw materials and fails to achieve significant improvement.

[0004] Research has shown that the deuterium content in natural water is typically around 150 ppm. High deuterium levels can block mitochondrial metabolic pathways (blocking once every 4.2 seconds), hindering energy synthesis. Deuterium-depleted water (such as 122 ppm) reduces deuterium levels and optimizes mitochondrial function, thereby enhancing cellular energy metabolism and repair, and preventing diseases caused by metabolic disorders. Some studies have suggested that excessive deuterium concentrations may be a potential cause of cancer and chronic diseases (for example, research by Hungarian scholar Gábor Somlyai has sparked interest in the anti-cancer potential of deuterium-depleted water). High deuterium levels can disrupt mitochondrial function, leading to abnormal cell proliferation (such as cancer) or metabolic disorders (such as cardiovascular disease). Summary of the Invention

[0005] In order to improve the SOD enzyme activity and selenium organication rate of biological enzyme products, the present invention provides a low-deuterium and selenium-rich kiwifruit composite enzyme and a preparation method thereof.

[0006] The technical solution adopted by the present invention is: a preparation method of a low-deuterium, selenium-rich kiwifruit composite enzyme, wherein the production raw material formula includes the following components in the following mass ratios: 1 part of a composite biological raw material and 1.2 to 1.8 parts of deuterium-depleted water; the composite biological raw material is composed of a selenium-rich fermentation raw material and a composite selenium organic raw material in a mass ratio of 1:0.3 to 0.7; the selenium-rich fermentation raw material includes kiwifruit; and the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.1 to 0.5:0.2 to 0.4.

[0007] It is easy for those skilled in the art to understand that the deuterium-depleted water in the present invention is deuterium-depleted pure water or deuterium-depleted drinking water that can be used for enzyme production.

[0008] As a further improvement of the present invention, the selenium-enriched fermentation raw material consists of kiwi fruit and mulberry in a mass ratio of 1:0.2 to 0.4.

[0009] It is easy for those skilled in the art to understand that the specific production method of the present invention can be based on the existing enzyme production method. We provide a preferred solution here, which can be implemented in the following steps:

[0010] S1. pretreating the composite biological raw material to obtain a pretreated raw material;

[0011] S2, evenly placing the pretreated raw materials in a fermentation container, and adding a flavor regulator;

[0012] S3, dissolving the fermentation starter in the deuterium-depleted water and evenly pouring it into the fermentation container; ensuring that the total volume of the material accounts for 60% to 80% of the volume of the fermentation container;

[0013] S4, sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 5-7 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 12-18 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 7-14 days; obtaining a fermentation product;

[0014] S5. The obtained fermentation product is sequentially subjected to solid-liquid separation, aging, sterilization and filling to obtain a low-deuterium and selenium-rich kiwifruit composite enzyme product.

[0015] The pretreatment in the above method can be: peeling and slicing kiwifruit, and soaking it in a 0.4% to 0.6% citric acid solution for 8 to 12 minutes; removing the stems of mulberries and soaking them in a 0.08% to 0.12% food-grade sodium hypochlorite solution for 2 to 4 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -18 to -23°C for 1 to 3 hours, thawing, removing the thorns, halving, removing the seeds, and pulping the fruit; blanching the olives with steam for 2 to 4 minutes, quenching with cold water, removing the pits, and dicing; and cutting okra into 0.8 to 1.5 cm segments. Those skilled in the art can also choose other pretreatment methods according to actual conditions, as long as they are conducive to the rapid progress of the fermentation process.

[0016] It is easy to understand that flavor regulators can also be added according to the market positioning of the enzyme product, such as honey, brown sugar, rock sugar, white sugar and other sweeteners or other flavor regulators, which will not be elaborated here.

[0017] It is easy to understand that in order to ensure that the fermentation process can proceed quickly and efficiently, fermentation starters can also be added, such as common lactic acid bacteria powder, dry yeast, etc. Technicians can also choose other commonly used fermentation starters according to actual conditions, such as EM bacteria, etc.

[0018] As a further improvement of the present invention, the deuterium-depleted water used in the raw material should satisfy the deuterium content ≤ 50 ppm.

[0019] The kiwi fruit used in the present invention is preferably a selenium-rich kiwi fruit with a selenium content of ≥0.01 mg / kg, for example, Anhui yellow heart kiwi fruit, Chongqing Jiangjin Simianshan kiwi fruit, Guizhou green heart kiwi fruit and other varieties can be used, as long as the selenium content meets the above standards.

[0020] The present invention also discloses a low-deuterium-selenium-rich kiwifruit composite enzyme, which is prepared by the preparation method of the low-deuterium-selenium-rich kiwifruit composite enzyme of the present invention.

[0021] The beneficial effect of the present invention is that experiments show that the preparation method of the low-deuterium and selenium-rich kiwifruit composite enzyme of the present invention can significantly improve the SOD enzyme activity and selenium organication rate of the biological enzyme product, thereby being more conducive to improving the human immune ability as a whole. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to the embodiments.

[0023] Example 1:

[0024] Prepare enzyme products according to the following steps:

[0025] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material and 1.6 parts of low-deuterium purified water (the measured deuterium content is 44 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.4; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.027 mg / kg) and mulberry in a mass ratio of 1:0.3; the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.4:0.3.

[0026] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp; blanching the olives with steam for 3 minutes, quenching them with cold water, removing the cores, and dicing them; and cutting the okra into 1 cm segments to obtain the pre-treated raw material;

[0027] (3) The pretreated raw materials are evenly spread in a fermentation container, and brown sugar is added at 30% of the total weight of the production raw material formula;

[0028] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0029] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0030] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0031] Example 2:

[0032] Prepare enzyme products according to the following steps:

[0033] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material and 1.5 parts of low-deuterium purified water (deuterium content measured to be 47 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.5; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.023 mg / kg) and mulberry in a mass ratio of 1:0.25; the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.3:0.4.

[0034] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp; blanching the olives with steam for 3 minutes, quenching them with cold water, removing the cores, and dicing them; and cutting the okra into 1 cm segments to obtain the pre-treated raw material;

[0035] (3) The pretreated raw materials are evenly spread in a fermentation container, and honey is added at a weight ratio of 25% of the total weight of the production raw material formula;

[0036] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0037] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0038] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0039] Example 3:

[0040] Prepare enzyme products according to the following steps:

[0041] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material, 1.7 parts of low-deuterium purified water (deuterium content measured to be 44 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.6; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.024 mg / kg) and mulberry in a mass ratio of 1:0.2; the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.2:0.3.

[0042] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp; blanching the olives with steam for 3 minutes, quenching them with cold water, removing the cores, and dicing them; and cutting the okra into 1 cm segments to obtain the pre-treated raw material;

[0043] (3) The pretreated raw materials are evenly spread in a fermentation container, and yellow rock sugar is added at 35% of the total weight of the production raw material formula;

[0044] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0045] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0046] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0047] Comparative Example 1:

[0048] This comparative example is a control experiment of Example 1. It is carried out according to the same steps and conditions as Example 1. All raw materials are from the same batch as Example 1. The only difference is that ordinary pure water (measured deuterium content is 158 ppm) of the same mass ratio is used to replace the deuterium-depleted water in Example 1. The specific scheme is as follows:

[0049] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material, 1.6 parts of purified water (deuterium content measured to be 158 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.4; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.027 mg / kg) and mulberry in a mass ratio of 1:0.3; the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.4:0.3.

[0050] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp; blanching the olives with steam for 3 minutes, quenching them with cold water, removing the cores, and dicing them; and cutting the okra into 1 cm segments to obtain the pre-treated raw material;

[0051] (3) The pretreated raw materials are evenly spread in a fermentation container, and brown sugar is added at 30% of the total weight of the production raw material formula;

[0052] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0053] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0054] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0055] Comparative Example 2:

[0056] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1. All raw materials are from the same batch as Example 1. The only difference is that only roxburghii is used as the selenium-organizing raw material, and the total mass ratio of the selenium-organizing raw materials is kept consistent with that of Example 1. The specific scheme is as follows:

[0057] Prepare enzyme products according to the following steps:

[0058] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material and 1.6 parts of low-deuterium water (deuterium content measured to be 44 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and selenium-organization raw material in a mass ratio of 1:0.4; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.027 mg / kg) and mulberry in a mass ratio of 1:0.3; the selenium-organization raw material is sea buckthorn.

[0059] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp to obtain the pre-treated raw material;

[0060] (3) The pretreated raw materials are evenly spread in a fermentation container, and brown sugar is added at 30% of the total weight of the production raw material formula;

[0061] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0062] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0063] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0064] Comparative Example 3:

[0065] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1. All raw materials are from the same batch as in Example 1. The only difference is that only olives are used as the selenium-organization raw material, and the total mass ratio of the selenium-organization raw materials is kept consistent with that in Example 1. The specific scheme is as follows:

[0066] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material and 1.6 parts of low-deuterium water (deuterium content measured to be 44 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and selenium-organization raw material in a mass ratio of 1:0.4; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.027 mg / kg) and mulberry in a mass ratio of 1:0.3; the selenium-organization raw material is olive.

[0067] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; blanching the olives with steam for 3 minutes, quenching with cold water, removing the pits, and dicing; obtaining the pre-treated raw material;

[0068] (3) The pretreated raw materials are evenly spread in a fermentation container, and brown sugar is added at 30% of the total weight of the production raw material formula;

[0069] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0070] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0071] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0072] Comparative Example 4:

[0073] This comparative example is a control experiment of Example 1, and is carried out according to the same steps and conditions as Example 1. All raw materials are from the same batch as in Example 1. The only difference is that only okra is used as the selenium-organization raw material, and the total mass ratio of the selenium-organization raw materials is kept consistent with that in Example 1. The specific scheme is as follows:

[0074] (1) Prepare the raw materials according to the following production raw material formula: 1 part of composite biological raw material and 1.6 parts of low-deuterium water (deuterium content measured to be 44 ppm); the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.4; the selenium-rich fermentation raw material is composed of kiwi fruit (selenium content of the fruit is 0.027 mg / kg) and mulberry in a mass ratio of 1:0.3; the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.4:0.3.

[0075] (2) Pre-treating the composite biological raw material, specifically: peeling and slicing the kiwifruit, and soaking it in a 5% mass concentration citric acid solution for 10 minutes; removing the stems of the mulberries and soaking them in a 0.1% mass concentration food-grade sodium hypochlorite solution for 3 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -20°C for 2 hours, thawing it, removing the thorns, cutting it in half and removing the seeds, and beating the pulp; blanching the olives with steam for 3 minutes, quenching them with cold water, removing the cores, and dicing them; and cutting the okra into 1 cm segments to obtain the pre-treated raw material;

[0076] (3) The pretreated raw materials are evenly spread in a fermentation container, and brown sugar is added at 30% of the total weight of the production raw material formula;

[0077] (4) Dissolve 2% of the raw material formula of lactic acid bacteria powder in the deuterium-depleted water and evenly pour it into the fermentation container;

[0078] (5) sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 6 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 15 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 10 days; and obtaining a fermentation product;

[0079] (6) The fermentation product was squeezed through a 200-mesh filter cloth to obtain an enzyme stock solution. The enzyme stock solution was then transferred to an oak barrel, 0.01% potassium metabisulfite was added, and the solution was stored in the dark at a temperature of 12-15°C for 90 days. The solution was then filtered through a 0.22 μm membrane and filled into a brown glass bottle, which was then sealed with nitrogen to obtain the enzyme product.

[0080] Comparative experiment on SOD enzyme activity detection of enzyme products:

[0081] The SOD enzyme activity of the enzyme products of the above embodiments and comparative examples was detected by NBT photoreduction inhibition method, and the experimental parameters were as follows:

[0082] 1. Reaction mixture (prepare before use, protect from light):

[0083] 50 mM potassium phosphate buffer (pH = 7.8, containing 0.1 mM EDTA);

[0084] 13 mM L-methionine;

[0085] 75 μM NBT (nitroblue tetrazolium);

[0086] 2 μM riboflavin;

[0087] 2. Experimental equipment:

[0088] Spectrophotometer (560nm)

[0089] Constant temperature and light incubator (light intensity needs to be uniform, 5000 lux)

[0090] Constant temperature water bath

[0091] Pipettes and tips

[0092] Cuvette (good light transmittance)

[0093] Timer

[0094] Aluminum foil (for light protection)

[0095] 3. Detection method:

[0096] (1) Extract the enzyme product samples of each example and comparative example using pre-cooled 50 mM pH 7.8 potassium phosphate buffer (containing 0.1 mM EDTA) at a ratio of 1:10 (w / v). Centrifuge at 4°C (12,000 g, 20 minutes). Take the supernatant as the crude enzyme solution. Dilute the crude enzyme solution 10-fold using the same buffer. The diluted sample should be stored on ice until use.

[0097] (2) Mix the following ingredients in a brown bottle or aluminum foil-wrapped container in proportion: potassium phosphate buffer (pH 7.8, 50 mM, containing 0.1 mM EDTA); L-methionine (13 mM final concentration); NBT (75 μM final concentration); riboflavin (2 μM final concentration).

[0098] (3) Set up the reaction system (3 mL system, carried out in a test tube or cuvette): blank control: 2.9 mL reaction mixture (containing riboflavin) + 0.1 mL buffer; sample tube: 2.9 mL reaction mixture (containing riboflavin) + 0.1 mL diluted sample enzyme solution; sample control: 2.9 mL reaction mixture without riboflavin + 0.1 mL diluted sample enzyme solution (placed in the dark); dark control (DC): 2.9 mL reaction mixture (containing riboflavin) + 0.1 mL buffer (completely wrapped in aluminum foil to avoid light).

[0099] (4) Quickly place all test tubes / cuvettes except the sample control and dark control in a constant temperature light incubator preheated to 25°C. Simultaneously, start the timer and ensure that the light intensity is uniform and stable (5000 lux). Wrap the sample control tube in aluminum foil and place it in the dark. Wrap the dark control tube completely in aluminum foil and place it in the light box. Incubate the reaction for 20 minutes.

[0100] (5) Once the reaction time is up, immediately remove the cuvette from the light box and quickly place it in the dark. For samples reacting in a test tube, quickly transfer them to the cuvette. Using a spectrophotometer, at a wavelength of 560 nm, with air or water as a reference, measure the absorbance of the sample control, the absorbance of the dark control, the absorbance of the blank control, and the absorbance of each sample tube in sequence. Calculate the SOD enzyme activity of each sample based on the measured values. The results are shown in Table 1.

[0101] Table 1 SOD enzyme activity test results of enzyme products

[0102] SOD enzyme activity (U / mL) Example 1 12624 Example 2 11788 Example 3 12340 Comparative Example 1 7769 Comparative Example 2 10678 Comparative Example 3 11322 Comparative Example 4 12073

[0103] It can be seen from the test results of Examples 1 to 3 in Table 1 that the SOD enzyme activity of the low-deuterium and selenium-rich kiwifruit composite enzyme prepared by the method of the present invention can reach more than 12,000 U / mL, which has a very significant technical effect.

[0104] It can be seen from the difference in SOD enzyme activity between Example 1 and Comparative Example 1 in Table 1 that, on the basis of Comparative Example 1, Example 1 replaces ordinary pure water with deuterium-depleted water, and the SOD enzyme activity of the enzyme product is increased by 62.5%, indicating that the use of deuterium-depleted water in the present invention has a significant technical effect of improving the SOD enzyme activity of the selenium-rich kiwifruit composite enzyme product.

[0105] Comparative experiment on detection of selenium organication rate in enzyme products:

[0106] The selenium organication rate of the enzyme products of each embodiment and each comparative example was determined by HPLC-ICP-MS according to T / SATA 041-2023. The experimental parameters are as follows:

[0107] 1. Reagents and materials:

[0108] Standard product (purity ≥98%):

[0109] Selenocysteine ​​(SeCys2), methyl-selenocysteine ​​(L-SeMc), selenomethionine (SeMet).

[0110] Enzymatic hydrolysis reagent:

[0111] Proteinase K (50 mg / mL, prepared in 0.05 M MES-TRIS buffer, pH = 8.3).

[0112] Mobile phase:

[0113] Phase A: 10 mM citric acid solution; Phase B: ultrapure water (gradient elution).

[0114] other:

[0115] Ultrapure water (GB / T 6682 Level 1), 0.45μm organic filter membrane, 3mol / L acetic acid solution.

[0116] 2. Instruments and equipment

[0117] HPLC-ICP-MS coupled system (Shimadzu LC-20Ai + ICPMS-2030);

[0118] Chromatographic column: anion exchange column (PRP-X100, 250 × 4.1 mm);

[0119] Auxiliary equipment: high-speed centrifuge (8000rpm), ultrasonic instrument, vortex oscillator, pH meter.

[0120] 3. Sample processing:

[0121] 1.0 g of enzyme sample was weighed from each example and comparative example into a centrifuge tube, 10 mL of proteinase K solution was added, and the mixture was vortexed to mix. The enzyme was hydrolyzed in a 37°C water bath for 12 hours. 1 mL of 3 mol / L acetic acid solution was then added to terminate the reaction, and the mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane for testing.

[0122] 4. Chromatographic and mass spectrometry conditions:

[0123] Chromatographic conditions: flow rate 1.0 mL / min; column temperature 30°C; injection volume 20 μL;

[0124] Elution program: 0–5 min: 20% A → 5–15 min: 50% A;

[0125] ICP-MS conditions: RF power 1.5 kW; carrier gas flow rate 0.7 L / min;

[0126] Isotope monitoring: 78 Se, 80 Se.

[0127] The total selenium content was determined by ICP-MS, and the total organic selenium content was determined by HPLC-ICP-MS. The organic selenium rate was calculated based on the determination results, and the results are shown in Table 2.

[0128] Table 2 Test results of selenium organication rate of enzyme products

[0129] Total selenium (μg / 100mL) Total organic selenium (μg / 100mL) Selenium organicization rate Example 1 76.44 66.89 87.51% Example 2 83.50 70.47 84.40% Example 3 73.35 64.98 88.59% Comparative Example 1 77.01 57.10 74.15% Comparative Example 2 76.23 41.20 54.05% Comparative Example 3 81.37 37.81 46.47% Comparative Example 4 75.81 46.79 61.27%

[0130] It can be seen from the test results of Examples 1 to 3 in Table 2 that the selenium organication rate of the low-deuterium-selenium-rich kiwifruit composite enzyme prepared by the method of the present invention can reach more than 85%, which has a very significant technical effect.

[0131] From the comparison of Example 1 and Comparative Example 1 in Table 2, it can be seen that, under the same conditions, the selenium organication rate of the product of Comparative Example 1 using ordinary drinking water is 74.15%, while the selenium organication rate of the product of Example 1 using deuterium-depleted water is 87.51%. It can be seen that the deuterium-depleted water in the present invention has a significant technical effect of improving the selenium organication rate of the enzyme product.

[0132] From the comparison of Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 in Table 2, it can be seen that when only sea buckthorn, olive or okra is used as the selenium organic raw material, the selenium organication rates are 54.05%, 46.47% and 61.27% respectively; and under the premise that the total amount of selenium organic raw materials used remains unchanged, the selenium organication rate of the enzyme product prepared from the composite selenium organic raw material formed by compounding the three is 87.51%, which is significantly better than the effect when each of the three is used alone. It can be seen that the components of the composite selenium organic raw material of the present invention have a very obvious synergistic effect in improving the selenium organication rate of the enzyme.

Claims

1. A method for preparing a low-deuterium, selenium-rich kiwifruit composite enzyme, characterized by: The production raw material formula includes the following components in the following mass proportions: 1 part of composite biological raw material and 1.2-1.8 parts of deuterium-depleted water; the composite biological raw material is composed of selenium-rich fermentation raw material and composite selenium organic raw material in a mass ratio of 1:0.3-0.7; the selenium-rich fermentation raw material includes kiwi fruit; and the composite selenium organic raw material is composed of sea buckthorn, olive and okra in a mass ratio of 1:0.1-0.5:0.2-0.

4.

2. The method for preparing the deuterium-low selenium-rich kiwifruit composite enzyme according to claim 1, wherein: The selenium-enriched fermentation raw material consists of kiwi fruit and mulberry in a mass ratio of 1:0.2-0.

4.

3. The method for preparing the low-deuterium and selenium-rich kiwifruit composite enzyme according to claim 2, wherein: The steps include: S1. pretreating the composite biological raw material to obtain a pretreated raw material; S2, evenly placing the pretreated raw materials in a fermentation container, and adding a flavor regulator; S3, dissolving the fermentation starter in the deuterium-depleted water and evenly pouring it into the fermentation container; ensuring that the total volume of the material accounts for 60% to 80% of the volume of the fermentation container; S4, sealing the fermentation container, adjusting the fermentation temperature to 28-30° C. by a temperature control system, and fermenting for 5-7 days; then adjusting the fermentation temperature to 25-27° C. and fermenting for 12-18 days; then adjusting the fermentation temperature to 22-23° C. and fermenting for 7-14 days; obtaining a fermentation product; S5. The obtained fermentation product is sequentially subjected to solid-liquid separation, aging, sterilization and filling to obtain a low-deuterium and selenium-rich kiwifruit composite enzyme product.

4. The method for preparing the deuterium-low selenium-rich kiwifruit composite enzyme according to claim 3, wherein: The pretreatment in step S1 includes the following steps: peeling and slicing the kiwifruit, and soaking it in a citric acid solution with a mass concentration of 0.4% to 0.6% for 8 to 12 minutes; removing the stems of the mulberries and soaking them in a food-grade sodium hypochlorite solution with a mass concentration of 0.08% to 0.12% for 2 to 4 minutes, then rinsing with purified water and draining; freezing the sea buckthorn at -18 to -23°C for 1 to 3 hours, thawing, removing the thorns, halving and removing the seeds, and pulping the fruit; blanching the olives with steam for 2 to 4 minutes, quenching with cold water, removing the pits, and dicing; and cutting the okra into 0.8 to 1.5 cm segments.

5. The method for preparing the deuterium-low and selenium-rich kiwifruit composite enzyme according to claim 3, wherein: The flavor regulator is selected from one or more of honey, brown sugar, rock sugar, and white sugar.

6. The method for preparing the deuterium-low and selenium-rich kiwifruit composite enzyme according to claim 3, wherein: The fermentation starter is selected from one or both of lactic acid bacteria powder and dry yeast.

7. The method for preparing the deuterium-depleted and selenium-enriched kiwifruit composite enzyme according to any one of claims 1 to 6, characterized in that: The deuterium-depleted water satisfies: a deuterium content ≤ 50 ppm.

8. The method for preparing the deuterium-depleted and selenium-enriched kiwifruit composite enzyme according to any one of claims 1 to 6, characterized in that: The kiwi fruit meets the following requirements: the selenium content of the fruit is ≥ 0.01 mg / kg.

9. The low-deuterium-selenium-rich kiwifruit composite enzyme prepared by the preparation method of the low-deuterium-selenium-rich kiwifruit composite enzyme according to any one of claims 1 to 8.