Stropharia rugoso-annulata dietary fiber and deproteinization method and application thereof

Abstract

The invention belongs to the technical field of edible mushroom processing, and particularly relates to a stropharia rugoso-annulata dietary fiber and a deproteinization method and application thereof. The invention provides a Stropharia rugoso-annulata dietary fiber deproteinization method. Stropharia rugoso-annulata powder is sequentially subjected to amylase and saccharifying enzyme combined enzymolysis, bacillus velezensis WH-7 fermentation, further deproteinization through alkaline protease combined with neutral protease and helicase modification treatment; the insoluble dietary fibers and the soluble dietary fibers of the stropharia rugoso-annulata are obtained. According to the invention, bacillus velezensis WH-7 is adopted to carry out liquid state fermentation on the stropharia rugoso-annulata powder, so that the stropharia rugoso-annulata dietary fibers, especially protein impurities in insoluble dietary fibers, can be effectively removed; the dietary fibers are modified in combination with helicase, so that conversion from insoluble dietary fibers to soluble dietary fibers can be promoted, and meanwhile, the functional quality of the two dietary fibers is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus processing, and particularly relates to a stropharia dietary fiber and a deproteinization method and application thereof. Background Art

[0002] Stropharia rugosoannulata is not only tender, delicious and nutritious, but also can effectively decompose straw and promote the resource utilization of agricultural waste, so it has excellent commodity value and strong market demand. However, Stropharia rugosoannulata has a concentrated maturity period and strong metabolic function, and it is often picked in time and the mushrooms open, resulting in a large number of mushrooms being discarded and wasted. This situation not only reduces the actual income of Stropharia rugosoannulata, but also reflects the room for improvement in the processing and utilization of by-products of the Stropharia rugosoannulata industry.

[0003] After the mushrooms open, their tissues further become fibrotic, and the nutritional content decreases, but the dietary fiber content further accumulates, of which the soluble dietary fiber content accounts for 12% to 15% of the total dietary fiber, reaching the level of high-quality dietary fiber. However, the protein content in the mushrooms is rich, up to 30%, which hinders the purification of dietary fiber.

[0004] Currently, trifluoroacetic acid and Sevag reagents can be used to deproteinize the dietary fiber of Pleurotus ostreatus. However, both of the above reagents remove protein by denaturation and precipitation of protein. This method not only causes the loss of dietary fiber, but also fails to remove the protein in the dietary fiber.

[0005] Therefore, there is an urgent need for a method that can remove protein from Stropharia officinalis dietary fiber under mild conditions and improve the purity of dietary fiber. Summary of the invention

[0006] The purpose of the present invention is to provide a stropharia dietary fiber and a method and application of deproteinization thereof. The method of the present invention can effectively remove protein from the stropharia dietary fiber and improve the purity of the dietary fiber.

[0007] The present invention provides a method for deproteinizing stropharia dietary fiber, comprising the following steps:

[0008] The powder of Stropharia rugosa, amylase and saccharifying enzyme are mixed and subjected to a first enzymatic hydrolysis to obtain a pretreated mushroom powder solution;

[0009] Inoculating Bacillus Velez subsp. WH-7 into the pretreated mushroom powder solution to perform fermentation to obtain a mushroom powder fermentation liquid; performing solid-liquid separation on the mushroom powder fermentation liquid to obtain a first precipitate;

[0010] The first precipitate is subjected to a second enzymatic hydrolysis, a third enzymatic hydrolysis and solid-liquid separation in sequence to obtain a second supernatant and a second precipitate; the enzyme used in the second enzymatic hydrolysis is an alkaline protease; the enzyme used in the third enzymatic hydrolysis is a neutral protease;

[0011] The second precipitate is mixed with snail enzyme, and fourth enzymolysis, enzyme inactivation and solid-liquid separation are performed in sequence to obtain a third supernatant and a third precipitate;

[0012] The third precipitate is sequentially washed and solid-liquid separated to obtain a fourth supernatant and a fourth precipitate; the fourth precipitate is mixed with anhydrous ethanol and solid-liquid separated to obtain Stropharia capitis insoluble dietary fiber;

[0013] The second supernatant, the third supernatant and the fourth supernatant are combined to obtain a mixed supernatant; the mixed supernatant is successively subjected to reduced pressure concentration and solid-liquid separation to obtain a concentrated mixed supernatant; the concentrated mixed supernatant is mixed with anhydrous ethanol, and the mixture is allowed to stand for 12 to 18 hours to perform solid-liquid separation to obtain Stropharia capitis soluble dietary fiber.

[0014] As a preferred solution, the inoculation amount of the Bacillus Velez subtilis WH-7 is 3% to 10% of the mass of the pretreated mushroom powder solution; the fermentation temperature is 35 to 40° C.; and the fermentation time is 3 to 6 days.

[0015] As a preferred embodiment, the OD of the Bacillus Velezii WH-7 is 600 The value is 4.0~5.0.

[0016] As a preferred solution, the amount of snail enzyme added is 1% to 4% of the mass of the Stropharia officinalis powder.

[0017] As a preferred solution, the time of the fourth enzymolysis is 1 to 3 hours; the temperature of the fourth enzymolysis is 28 to 33°C.

[0018] As a preferred solution, the amount of alkaline protease added is 500-1000U alkaline protease per gram of Stropharia officinalis powder; the second enzymatic hydrolysis time is 2h; the second enzymatic hydrolysis temperature is 37-42°C;

[0019] The amount of neutral protease added is 500-1000U of neutral protease per gram of Stropharia officinalis powder; the time of the third enzymolysis is 1 hour; and the temperature of the third enzymolysis is 37-42°C.

[0020] As a preferred solution, the amount of amylase added is 100-150U of amylase per gram of Stropharia powder; the amount of saccharifying enzyme added is 150-200U of saccharifying enzyme per gram of Stropharia powder; the time of the first enzymolysis is 1-2h; the temperature of the first enzymolysis is 55-65°C.

[0021] As a preferred solution, the particle size of the Stropharia officinalis powder is 60-80 meshes.

[0022] The present invention also provides a stropharia dietary fiber obtained according to the method described in the above scheme, wherein the stropharia dietary fiber comprises stropharia insoluble dietary fiber and stropharia soluble dietary fiber; the protein content of the stropharia insoluble dietary fiber is ≤5.08%; and the protein content of the stropharia soluble dietary fiber is ≤4.45%.

[0023] The present invention also provides the use of the Stropharia officinalis dietary fiber described in the above scheme in the preparation of functional foods and / or health products.

[0024] Beneficial effects:

[0025] The present invention provides a method for deproteinizing stropharia dietary fiber, wherein stropharia powder is subjected to combined enzymolysis with amylase and saccharifying enzyme, fermentation with Bacillus Velez WH-7, further deproteinization with alkaline protease combined with neutral protease, and modification with snail enzyme to obtain stropharia insoluble dietary fiber and soluble dietary fiber. The present invention uses Bacillus Velez WH-7 to carry out liquid fermentation on mushroom powder, which can effectively remove protein impurities in stropharia dietary fiber, especially insoluble dietary fiber; and then the dietary fiber is modified with snail enzyme to promote the conversion of insoluble dietary fiber to soluble dietary fiber, and at the same time improve the functional quality of the two dietary fibers. The results of the embodiment show that the purity and yield of the stropharia dietary fiber obtained by the method of the present invention are improved, and the glucose adsorption capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity, sodium nitrite adsorption capacity, α-amylase inhibition, and α-glycosidase inhibition of the dietary fiber are all improved to varying degrees. The method of the present invention utilizes a bacterial enzyme synergistic treatment process with mild conditions and is green and environmentally friendly, and can fundamentally achieve the removal of protein impurities and functional modification, thereby promoting the high-value utilization of Pleurotus ostreatus by-products and promoting industrial upgrading. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 This is a plate culture diagram of Bacillus Velez WH-7 in Example 1;

[0028] Figure 2 This is a diagram of the dietary fiber of Pleurotus ostreatus in Example 1; wherein A is insoluble dietary fiber; B is soluble dietary fiber;

[0029] Figure 3 is the inhibition rate of the insoluble dietary fiber of Pleurotus ostreatus on α-amylase in Test Example 1;

[0030] Figure 4 is the inhibition rate of soluble dietary fiber of Stropharia rugosa on α-amylase in Test Example 1;

[0031] Figure 5 is the inhibition rate of the insoluble dietary fiber of Pleurotus ostreatus on α-glucosidase in Test Example 1;

[0032] Figure 6 is the inhibition rate of soluble dietary fiber of Stropharia rugosa on α-glucosidase in Test Example 1;

[0033] Figure 7 This is an electron microscope image of the Stropharia officinalis dietary fiber in Example 1; wherein A is insoluble dietary fiber; B is soluble dietary fiber, and the scale in the figure = 2 μm;

[0034] Figure 8 This is an electron microscope image of the Stropharia officinalis dietary fiber in Comparative Example 1; wherein A is insoluble dietary fiber; B is soluble dietary fiber, and the scale bar in the figure = 2 μm. DETAILED DESCRIPTION

[0035] The present invention provides a method for deproteinizing stropharia dietary fiber, comprising the following steps:

[0036] The powder of Stropharia rugosa, amylase and saccharifying enzyme are mixed and subjected to a first enzymatic hydrolysis to obtain a pretreated mushroom powder solution;

[0037] Inoculating Bacillus Velez subsp. WH-7 into the pretreated mushroom powder solution to perform fermentation to obtain a mushroom powder fermentation liquid; performing solid-liquid separation on the mushroom powder fermentation liquid to obtain a first precipitate;

[0038] The first precipitate is subjected to a second enzymatic hydrolysis, a third enzymatic hydrolysis and solid-liquid separation in sequence to obtain a second supernatant and a second precipitate; the enzyme used in the second enzymatic hydrolysis is an alkaline protease; the enzyme used in the third enzymatic hydrolysis is a neutral protease;

[0039] The second precipitate is mixed with snail enzyme, and fourth enzymolysis, enzyme inactivation and solid-liquid separation are performed in sequence to obtain a third supernatant and a third precipitate;

[0040] The third precipitate is sequentially washed and solid-liquid separated to obtain a fourth supernatant and a fourth precipitate; the fourth precipitate is mixed with anhydrous ethanol and solid-liquid separated to obtain Stropharia capitis insoluble dietary fiber;

[0041] The second supernatant, the third supernatant and the fourth supernatant are combined to obtain a mixed supernatant; the mixed supernatant is successively subjected to reduced pressure concentration and solid-liquid separation to obtain a concentrated mixed supernatant; the concentrated mixed supernatant is mixed with anhydrous ethanol, and the mixture is allowed to stand for 12 to 18 hours to perform solid-liquid separation to obtain Stropharia capitis soluble dietary fiber.

[0042] The present invention sequentially dries, crushes and sieves the stropharia to obtain stropharia powder. As an embodiment, the stropharia is an open stropharia. As an embodiment, the particle size of the stropharia powder is 60 to 80 meshes. In a specific embodiment of the present invention, the particle size of the stropharia powder can be 60, 70 or 80 meshes.

[0043] In the present invention, the stropharia powder, amylase and saccharifying enzyme are mixed, and the first enzymolysis is performed to obtain a pretreated mushroom powder solution. As an embodiment, water is added to the stropharia powder in a mass ratio of 1:10, and the pH is adjusted to 6.0 to obtain a stropharia solution; the stropharia solution is mixed with amylase and saccharifying enzyme, and the first enzymolysis is performed to obtain a pretreated mushroom powder solution. As an embodiment, the amount of the amylase added is 100-150U amylase per g of stropharia powder; as an embodiment, the amount of the saccharifying enzyme added is 150-200U saccharifying enzyme per g of stropharia powder; as an embodiment, the time of the first enzymolysis is 1-2h; in a specific embodiment of the present invention, the time of the first enzymolysis can be 1h or 2h; as an embodiment, the temperature of the first enzymolysis is 55-65°C; in a specific embodiment of the present invention, the temperature of the first enzymolysis can be any temperature value within the range of 55-65°C. The present invention does not limit the sources of the amylase and the saccharifying enzyme. In a specific embodiment of the present invention, the amylase is purchased from Shanghai Yuanye Biotechnology Co., Ltd. (50 U / mg, S10003); the saccharifying enzyme is purchased from Shanghai Yuanye Biotechnology Co., Ltd. (100 U / mg, S10017). The present invention uses amylase and saccharifying enzyme to perform a first enzymolysis on the Stropharia capensis powder, which can remove the starch components in the Stropharia capensis, which is beneficial to the purification of dietary fiber.

[0044] In the present invention, Bacillus Velez WH-7 is inoculated into the pretreated mushroom powder solution for fermentation to obtain a mushroom powder fermentation liquid. As an embodiment, the OD of the Bacillus Velez WH-7 in the present invention is 600 The value is 4.0 to 5.0; as an embodiment, the Velez subtilis WH-7 is a culture medium-free seed bacterial suspension. In a specific embodiment of the present invention, the preparation of the culture medium-free seed bacterial suspension includes: 600=4.0~5.0 of Bacillus Velez WH-7 bacterial liquid was centrifuged at 10000r / min for 10min at 4°C; the upper culture medium was poured off and the lower bacterial bodies were collected; the lower bacterial bodies were repeatedly washed with distilled water, centrifuged, and the culture medium was removed to obtain the lower bacterial bodies without the culture medium; distilled water equal to the original culture medium was added to the lower bacterial bodies without the culture medium, and the mixture was shaken to prepare the seed bacterial suspension without the culture medium, and the OD 600 It is 4.0~5.0.

[0045] As an embodiment, the inoculation amount of the Velez subtilis WH-7 of the present invention is 3% to 10% of the mass of the pretreated mushroom powder solution; in a specific embodiment of the present invention, the inoculation amount of the Velez subtilis WH-7 can be 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the mass of the pretreated mushroom powder solution. As an embodiment, the fermentation temperature is 35 to 40°C; in a specific embodiment of the present invention, the fermentation temperature can be 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. As an embodiment, the fermentation time is 3 to 6 days; in a specific embodiment of the present invention, the fermentation time can be 3, 4, 5 or 6 days. The natural dietary fiber has a tight structure and cannot fully expose the internal active groups. The complex enzyme system that can be produced by microbial fermentation can decompose the substrate macromolecular fiber into small molecules, improve the solubility of dietary fiber, expose more functional groups, and make the fiber structure more loose and porous. The invention utilizes liquid fermentation mushroom powder of Bacillus Velezii WH-7 to remove protein and modify fiber through a series of extracellular enzymes (protease, cellulase, hemicellulase, etc.) produced by the growth of the strain, so that the obtained dietary fiber has better structural and functional characteristics.

[0046] After obtaining the mushroom powder fermentation liquid, the present invention sterilizes and ultrasonically treats the mushroom powder fermentation liquid before solid-liquid separation to obtain a sterilized fermentation liquid. As an embodiment, the sterilization temperature is 121°C and the time is 20 minutes. As an embodiment, the power of the ultrasound is 250w and the time of the ultrasound is 0.5 to 1h. In the present invention, the mushroom powder fermentation liquid is subjected to solid-liquid separation, and the solid component is collected to obtain a first precipitate. As an embodiment, the solid-liquid separation is achieved by centrifugation. In the process of ultrasonically treating the mushroom powder fermentation liquid of the present invention, periodic changes of compression and expansion will occur, causing tiny vibrations and eddies in the fermentation liquid, making the mushroom powder tissue more fluffy and making it easier for the enzymatic hydrolysis products to be released, peeled off and removed from the mushroom powder tissue.

[0047] In the present invention, distilled water is added to the first precipitate at a mass ratio of 1:10, pH is adjusted to 9.0, and alkaline protease is added for the second enzymolysis to obtain the second enzymolysis product. As an embodiment, based on the mass of the giant puffball powder subjected to the first enzymolysis, the amount of alkaline protease added is 500-1000U alkaline protease per g of giant puffball powder; in a specific embodiment of the present invention, the amount of alkaline protease added can be 500, 600, 700, 800, 900 or 1000U alkaline protease per g of giant puffball powder. As an embodiment, the time of the second enzymolysis is 2h; the temperature of the second enzymolysis is 37-42°C; the speed of the second enzymolysis is 180r / min. In a specific embodiment of the present invention, the temperature of the second enzymolysis can be 37°C, 38°C, 39°C, 40°C, 41°C or 42°C. The present invention has no limitation on the source of the alkaline protease. In a specific embodiment of the present invention, the alkaline protease is purchased from Shanghai Yuanye Biotechnology Co., Ltd. (200 U / mg, S10154).

[0048] In the present invention, the pH of the second enzymolysis product is adjusted to 7.5, and a neutral protease is added to perform a third enzymolysis to obtain a third enzymolysis product. As an embodiment, based on the mass of the giant puffball powder subjected to the first enzymolysis, the amount of the neutral protease added is 500-1000U of neutral protease per g of giant puffball powder; in a specific embodiment of the present invention, the amount of the neutral protease added can be 500, 600, 700, 800, 900 or 1000U of neutral protease per g of giant puffball powder. As an embodiment, the time of the third enzymolysis is 1h; the temperature of the third enzymolysis is 37-42°C; the speed of the third enzymolysis is 180r / min. In a specific embodiment of the present invention, the temperature of the third enzymolysis can be 37°C, 38°C, 39°C, 40°C, 41°C or 42°C. The present invention does not limit the source of the neutral protease. In a specific embodiment of the present invention, the neutral protease is purchased from Shanghai Yuanye Biotechnology Co., Ltd. (100U / mg, S10013). The invention uses alkaline protease and neutral protease for step-by-step enzymolysis on the basis of fermenting and dredging mushroom powder tissue with Bacillus Velez WH-7, so as to further remove mushroom powder protein and purify dietary fiber.

[0049] In the present invention, the third enzymatic hydrolysate is subjected to a first enzyme inactivation treatment and solid-liquid separation in sequence to obtain a second supernatant and a second precipitate. As an embodiment, the temperature of the first enzyme inactivation treatment is 100° C. and the time is 10 min. As an embodiment, the solid-liquid separation is achieved by centrifugation, and the solid component and the liquid component are collected separately to obtain a second supernatant and a second precipitate.

[0050] In the present invention, distilled water is added to the second precipitate at a mass ratio of 1:10, and snail enzyme is added to perform the fourth enzymolysis to obtain the fourth enzymolysis product. As an embodiment, based on the mass of the giant puffball powder subjected to the first enzymolysis, the amount of the snail enzyme added is 1% to 4% of the mass of the giant puffball powder. In a specific embodiment of the present invention, the amount of the snail enzyme added can be 1%, 2%, 3% or 4% of the mass of the giant puffball powder. As an embodiment, the time of the fourth enzymolysis is 1 to 3 hours; the temperature of the fourth enzymolysis is 28 to 33°C. In a specific embodiment of the present invention, the time of the fourth enzymolysis can be 1, 2 or 3 hours, and the temperature of the fourth enzymolysis can be 28°C, 29°C, 30°C, 31°C, 32°C or 33°C. As an embodiment, the speed of the fourth enzymolysis is 180r / min. In the present invention, the fourth enzymolysis product is subjected to enzyme inactivation treatment and solid-liquid separation to obtain a third supernatant and a third precipitate. As an embodiment, the temperature of the enzyme inactivation is 100°C and the time is 10 minutes. As an embodiment, the solid-liquid separation is achieved by centrifugation, and the solid component and the liquid component are collected separately to obtain a third supernatant and a third precipitate. The present invention does not limit the source of the snail enzyme. In a specific embodiment of the present invention, the snail enzyme is purchased from Shanghai Yuanye Biological Co., Ltd. Snail enzyme contains more than 20 complex enzyme systems such as rich cellulase, β-glucanase, pectinase, etc., which can effectively enzymolyze a variety of insoluble polysaccharides, promote the conversion of insoluble dietary fiber into soluble dietary fiber, and make the structure of dietary fiber looser.

[0051] In the present invention, the third precipitate is sequentially washed and solid-liquid separated to obtain a fourth supernatant and a fourth precipitate. As an embodiment, the washing comprises: adding distilled water to the third precipitate at a mass ratio of 1:10, and washing twice. As an embodiment, the solid-liquid separation is achieved by centrifugation, and the solid component and the liquid component are collected separately to obtain a fourth supernatant and a fourth precipitate.

[0052] In the present invention, the fourth precipitate is mixed with anhydrous ethanol, and solid-liquid separation is performed to obtain the insoluble dietary fiber of Pleurotus ostreatus. As an embodiment, the mixing of the fourth precipitate with anhydrous ethanol includes: adding anhydrous ethanol to the fourth precipitate at a mass ratio of 1:10, and stirring at 120r / min for 30min at 40°C. As an embodiment, the solid-liquid separation is achieved by centrifugation, and the solid components are collected to obtain the insoluble dietary fiber of Pleurotus ostreatus. The present invention mixes the fourth precipitate with anhydrous ethanol, which can further dissolve and remove the soluble sugar in the residue and improve the purity of the dietary fiber.

[0053] In the present invention, the second supernatant, the third supernatant and the fourth supernatant are combined to obtain a mixed supernatant; the mixed supernatant is concentrated under reduced pressure to obtain a concentrated mixed supernatant. As an embodiment, the temperature of the reduced pressure concentration is 50°C; as an embodiment, the volume of the concentrated mixed supernatant is 10% to 20% of the mixed supernatant. In the present invention, the concentrated mixed supernatant is separated from the solid and liquid to obtain a concentrated mixed supernatant; as an embodiment, the solid-liquid separation is achieved by centrifugation, and the liquid component is collected to obtain a concentrated mixed supernatant. When the ethanol concentration reaches 80%, it is beneficial to remove soluble polysaccharides in the combined supernatant. The present invention concentrates the combined supernatant, which can reduce the amount of subsequent anhydrous ethanol added and save costs.

[0054] In the present invention, the concentrated mixed supernatant and anhydrous ethanol are mixed, left to stand for 12 to 18 hours, and solid-liquid separation is performed to obtain the soluble dietary fiber of Pleurotus ostreatus. As an embodiment, the concentrated mixed supernatant and anhydrous ethanol are mixed, including: adding anhydrous ethanol to the concentrated mixed supernatant at a mass ratio of 1:4. As an embodiment, the solid-liquid separation is achieved by centrifugation, and the solid components are collected to obtain the soluble dietary fiber of Pleurotus ostreatus. In a specific embodiment of the present invention, the standing time can be any value between 12 and 18 hours.

[0055] The present invention provides a stropharia dietary fiber obtained according to the method described in the above scheme, wherein the stropharia dietary fiber includes stropharia insoluble dietary fiber and stropharia soluble dietary fiber; the protein content of the stropharia insoluble dietary fiber is ≤5.08%; the protein content of the stropharia soluble dietary fiber is ≤4.45%. As an embodiment, the protein content of the stropharia insoluble dietary fiber is 4.35% to 5.08%; as an embodiment, the protein content of the stropharia soluble dietary fiber is 3.89% to 4.45%.

[0056] The present invention also provides the use of the Stropharia officinalis dietary fiber described in the above scheme in the preparation of functional foods and / or health products.

[0057] In order to further illustrate the present invention, a Stropharia officinalis dietary fiber and a method and application of protein removal provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] The Stropharia rugosa used in the embodiments and comparative examples of the present invention are from the same batch of Stropharia rugosa.

[0059] Example 1

[0060] (1) Processing of raw materials of Stropharia rugosa:

[0061] The opened Stropharia rugosa was dried, crushed, and passed through a 60-mesh sieve to obtain Stropharia rugosa powder (referred to as mushroom powder).

[0062] (2) Combined pretreatment with amylase and saccharifying enzyme:

[0063] Add 10 times the mass of water to the mushroom powder obtained in step (1), adjust the pH to 6.0, add 120 U / g of α-amylase (purchased from Shanghai Yuanye Biotechnology Co., Ltd., S10003) and 200 U / g of saccharifying enzyme (purchased from Shanghai Yuanye Biotechnology Co., Ltd., S10017), mix well, perform a first enzymolysis at 60° C. for 2 h, and sterilize at 121° C. for 20 min to obtain a pretreated mushroom powder solution for later use.

[0064] (3) Microbial fermentation:

[0065] ① Activation of strains: On a clean bench, take out the thawed glycerol tube preserved Bacillus velezensis WH-7 strain (published in the literature: Zhenying Han, Changwen Ye, Xinyu Dong, et al. Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis (2023), Frontiers in Bioengineering and Biotechnology, 12. doi: 10.3389 / fbioe.2024.1383083), streak it on a solid LB medium plate, culture it at 37°C for 12-15h, pick out a single colony that has grown, inoculate it into 5-10mL of liquid LB medium, and continue to culture it at 37°C and 180r / min for 12-15h. The components of the liquid LB medium are: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH 7.0-7.2; the components of the solid LB medium are: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L, pH 7.0-7.2; the LB medium is sterilized at 121°C for 30min. The picture of the Velez subtilis WH-7 on the LB medium plate is as follows Figure 1 shown.

[0066] ② Preparation of seed suspension: On a clean bench, pipette an appropriate amount of bacterial suspension from the activated strain into 50-100 mL of liquid LB medium and continue culturing at 37°C and 180 r / min until OD 600In the range of 4.0-5.0, a seed bacterial suspension is prepared and the bacterial suspension is stored at 4°C for subsequent inoculation and fermentation.

[0067] ③ Treatment of seed bacterial suspension to remove culture medium: In order to reduce the effect of peptone in the culture medium on the protein content in the fermentation substrate, centrifuge the seed bacterial suspension in step ② at 4°C and 10,000 r / min for 10 minutes. Pour out the upper culture medium, collect the lower bacterial cells, wash with distilled water, centrifuge, remove the culture medium, repeat the distilled water washing and centrifugation operation 3 times to obtain the lower bacterial cells. Add the same amount of distilled water as the original culture medium to the lower bacterial cells, shake well, and prepare the seed bacterial suspension to remove the culture medium. Maintain OD 600 In the range of 4.0 to 5.0.

[0068] ④ Liquid fermentation of mushroom powder: add the seed fungus suspension without culture medium prepared in step ③ to the pretreated mushroom powder solution of step (2) at an inoculum amount of 5% (w / w), culture at 37°C at 180r / min for 3 days, and sterilize at 121°C for 20min to obtain sterilized fermentation liquid.

[0069] (4) Protease combined with deproteinization:

[0070] ① The sterilized fermentation liquid in step (3) was subjected to ultrasound at 60°C, 250w (40kHz) for 1h, and centrifuged at 4000r / min for 10min to collect the solid component, which was recorded as the first precipitate (mushroom powder residue).

[0071] ② Add 10 times the mass of distilled water to the mushroom powder residue in step ① to adjust the pH to 9.0; based on the mass of the mushroom powder in step (1), add 600U / g of alkaline protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., S10154) for second enzymolysis, 180r / min at 40°C, and react for 2h to obtain a second enzymolysis product; after the reaction, adjust the pH of the second enzymolysis product to 7.5, based on the mass of the mushroom powder in step (1), add 500U / g of neutral protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., S10013) for third enzymolysis, 180r / min at 40°C for 1h to obtain a third enzymolysis product; after the reaction, inactivate the enzyme in the third enzymolysis product at 100°C for 10min, cool and centrifuge, collect the solid component and the liquid component, respectively, and record them as the second supernatant and the second precipitate (protease-treated mushroom powder residue).

[0072] (5) Snail enzyme modification treatment:

[0073] Add 10 times the mass of distilled water to the mushroom powder residue treated with protease in step (4), add snail enzyme (purchased from Shanghai Yuanye Biological Co., Ltd.) with a mushroom powder content of 1% (w / w) based on the mass of mushroom powder in step (1), perform enzymolysis at 180 r / min at 30° C. for 2 h, inactivate the enzyme at 100° C. for 10 min, cool and centrifuge, collect the solid component and the liquid component, respectively, and record them as the third supernatant and the third precipitate (solid residue).

[0074] (6) Preparation of insoluble dietary fiber:

[0075] The solid residue in step (5) was washed twice with 10 times the mass of distilled water, centrifuged, and the solid component and liquid component were collected respectively, which were recorded as the fourth supernatant and the fourth precipitate. 10 times the mass of anhydrous ethanol was added to the fourth precipitate, stirred at 120 r / min at 40°C for 30 minutes, centrifuged, and the solid component was collected and freeze-dried to obtain the insoluble dietary fiber of Stropharia rugosa, such as Figure 2 As shown in A.

[0076] (7) Preparation of soluble dietary fiber:

[0077] ① Combine the second supernatant, the third supernatant and the fourth supernatant in steps (4) to (6) to obtain a mixed supernatant; ② Concentrate the mixed supernatant in step ① at 50° C. under reduced pressure to 20% of the original volume, centrifuge, and discard the precipitate to obtain a concentrated mixed supernatant; ③ Add anhydrous ethanol to the concentrated mixed supernatant in step ② at a mass ratio of 1:4, let stand overnight at 4° C., and centrifuge to collect the solid component; ④ Freeze-dry the solid component in step ③ to obtain soluble dietary fiber, such as Figure 2 As shown in B.

[0078] Example 2

[0079] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0080] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0081] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0082] (3) Microbial fermentation: The fermentation was carried out in accordance with step (3) of Example 1, except that after inoculation with Bacillus Velez subtilis WH-7, the culture was continued for 4 days.

[0083] (4) Protease combined with deproteinization:

[0084] ①Proceed according to the operation of ① in step (3) of Example 1.

[0085] ② Add 10 times the mass of distilled water to the mushroom powder residue in step ①, adjust the pH to 9.0; add 700U / g mushroom powder alkaline protease, react at 40℃ and 180r / min for 2h; after the reaction, adjust the pH to 7.5, add 600U / g neutral protease, react at 40℃ and 180r / min for 1h; after the reaction, inactivate the enzyme at 100℃ for 10min, cool and centrifuge, collect the solid component and liquid component respectively, and record them as the second supernatant and the second precipitate (protease-treated mushroom powder residue).

[0086] (5) Snail enzyme modification treatment:

[0087] Add 10 times the mass of distilled water to the mushroom powder residue treated with protease in step (4), add snail enzyme with a mushroom powder content of 2% (w / w), perform enzymolysis at 180 r / min at 30° C. for 2 h, inactivate the enzyme at 100° C. for 10 min, cool and centrifuge, collect the solid component and the liquid component, respectively, and record them as the third supernatant and the third precipitate (solid residue).

[0088] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0089] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0090] Example 3

[0091] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0092] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0093] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0094] (3) Microbial fermentation: The process was carried out in accordance with step (3) of Example 1, except that after inoculation with Bacillus Velez subtilis WH-7, the culture was continued for 5 days.

[0095] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0096] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0097] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0098] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0099] Example 4

[0100] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0101] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0102] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0103] (3) Microbial fermentation: The fermentation was carried out in accordance with step (3) of Example 1, except that after inoculation with Bacillus Velez subtilis WH-7, the culture was continued for 4 days.

[0104] (4) Protease combined with deproteinization:

[0105] ② Add 10 times the mass of distilled water to the mushroom powder residue in step ①, adjust the pH to 9.0; add 800U / g mushroom powder alkaline protease, react at 40℃ and 180r / min for 2h; after the reaction, adjust the pH to 7.5, add 600U / g neutral protease, react at 40℃ and 180r / min for 1h; after the reaction, inactivate the enzyme at 100℃ for 10min, cool and centrifuge, collect the solid component and liquid component respectively, and record them as the second supernatant and the second precipitate (protease-treated mushroom powder residue).

[0106] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0107] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0108] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0109] Comparative Example 1

[0110] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0111] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0112] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0113] (3) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0114] (4) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0115] (5) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0116] (6) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0117] Comparative Example 2

[0118] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0119] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0120] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0121] (3) Microbial fermentation: The process was carried out according to step (3) of Example 1 to obtain a sterilized fermentation liquid.

[0122] (4) Protease combined with deproteinization:

[0123] ①Proceed according to the operation of ① in step (3) of Example 1.

[0124] ② Add 10 times the mass of distilled water to the mushroom powder residue in step ①, adjust the pH to 7.5, add 600U / g neutral protease for second enzymolysis, and react at 40°C and 180r / min for 1h to obtain a second enzymolysis product; after the reaction, inactivate the enzyme in the second enzymolysis product at 100°C for 10min, cool and centrifuge, and collect the solid component and liquid component respectively, which are recorded as the second supernatant and the second precipitate (protease-treated mushroom powder residue).

[0125] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0126] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0127] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0128] Comparative Example 3

[0129] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0130] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0131] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0132] (3) Microbial fermentation: The process was carried out according to step (3) of Example 1 to obtain a sterilized fermentation liquid.

[0133] (4) Protease combined with deproteinization:

[0134] ①Proceed according to the operation of ① in step (3) of Example 1.

[0135] ② Add 10 times the mass of distilled water to the mushroom powder residue in step ①, and adjust the pH to 9.0; add 600U / g mushroom powder alkaline protease for second enzymolysis, 180r / min at 40°C, and react for 2h to obtain a second enzymolysis product; after the reaction, inactivate the enzyme in the second enzymolysis product at 100°C for 10min, cool it, and centrifuge it to collect the solid component and the liquid component, respectively, which are recorded as the second supernatant and the second precipitate (protease-treated mushroom powder residue).

[0136] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0137] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0138] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0139] Comparative Example 4

[0140] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0141] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0142] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0143] (3) Microbial fermentation: The process was carried out according to step (3) of Example 1 to obtain a sterilized fermentation liquid.

[0144] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0145] (5) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0146] (6) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0147] Comparative Example 5

[0148] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0149] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0150] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0151] (3) Microbial fermentation: The operation was carried out in accordance with step (3) of Example 1, except that the inoculated strain was Bacillus licheniformis. After inoculation of Bacillus licheniformis, the culture was carried out at 37° C. and 180 r / min for 4 days.

[0152] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0153] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0154] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0155] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0156] Comparative Example 6

[0157] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0158] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0159] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0160] (3) Microbial fermentation: The operation was carried out in accordance with step (3) of Example 1, except that the inoculated strain was Bacillus amyloliquefaciens; after inoculation of Bacillus amyloliquefaciens, the culture was carried out at 37° C. and 180 r / min for 4 days.

[0161] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0162] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0163] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0164] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0165] Comparative Example 7

[0166] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0167] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0168] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0169] (3) Microbial fermentation: The process was carried out in accordance with step (3) of Example 1, except that the inoculation amount of Bacillus velezensis WH-7 was 1% (w / w).

[0170] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0171] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0172] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0173] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0174] Comparative Example 8

[0175] A method for preparing Stropharia officinalis dietary fiber similar to Example 1, comprising the following steps:

[0176] (1) Processing of Stropharia officinalis raw materials: The process is carried out according to step (1) of Example 1 to obtain Stropharia officinalis powder.

[0177] (2) Pretreatment with amylase and saccharifying enzyme: The process is carried out according to step (2) of Example 1 to obtain a pretreated mushroom powder solution.

[0178] (3) Microbial fermentation: The process was carried out in accordance with step (3) of Example 1, except that the inoculation amount of Bacillus velezensis WH-7 was 15% (w / w).

[0179] (4) Protease combined with deproteinization: The operation is carried out according to step (4) of Example 1 to obtain a second supernatant and a second precipitate.

[0180] (5) Snail enzyme modification treatment: The process is carried out according to step (5) of Example 1 to obtain a third supernatant and a third precipitate.

[0181] (6) Preparation of insoluble dietary fiber: The process was carried out according to step (6) of Example 1 to obtain Stropharia rugosa insoluble dietary fiber.

[0182] (7) Preparation of soluble dietary fiber: The process was carried out according to step (7) of Example 1 to obtain soluble dietary fiber from Pleurotus ostreatus.

[0183] Test Example 1

[0184] (1) Analysis of basic components of dietary fiber of Pleurotus ostreatus

[0185] The protein content of the Stropharia officinalis dietary fiber prepared in different embodiments and comparative examples was detected, and the protein content was detected by Kjeldahl nitrogen determination method GB5009.5-2016.

[0186] According to the test results of dietary fiber protein content of Pleurotus eryngii, it was found that in Comparative Examples 5 and 6, Bacillus licheniformis and Bacillus amyloliquefaciens were inoculated respectively, and the two bacteria had unsatisfactory ability to remove protein from Pleurotus eryngii, and the dietary fiber protein content after final treatment was 13% to 15%; in Comparative Example 7, the inoculation amount of Bacillus Velez was 1% to 2%, and the inoculation amount was small. After 5 days of cultivation, the deproteinization effect was still not obvious, and the protein content was 12% to 15%; in Comparative Example 8, the inoculation amount of Bacillus Velez was 12% to 15%, and due to the high inoculation amount, obvious odor was produced during the fermentation process. It can be seen that the treatment effect of Comparative Examples 5 to 8 on Pleurotus eryngii was not ideal, so no subsequent component and function analysis was performed.

[0187] The Stropharia officinalis dietary fibers of Examples 1 to 4 and Comparative Examples 1 to 4 were selected to determine the basic component indicators of the Stropharia officinalis dietary fibers: fat content, moisture content, ash content, extraction rate and purity; the above experiments were repeated 3 times, and the results were averaged. The test results are shown in Table 1.

[0188] Fat content detection: Soxhlet extraction method is used for detection GB5009.6-2016;

[0189] Moisture content detection: Direct drying method is used for detection GB5009.6-2016;

[0190] Ash content test: Test in accordance with GB5009.4-2016.

[0191] Dietary fiber purity was calculated according to the formula: 100-protein content-fat content-moisture content-ash content.

[0192] Table 1 Comparison of basic components of dietary fiber of Stropharia rugosa in Examples 1 to 4 and Comparative Examples 1 to 4

[0193]

[0194] According to Table 1, the method for efficient protein removal and functional modification of the stropharia dietary fiber provided by the present invention can effectively remove the protein in the dietary fiber, improve the purity of the dietary fiber, increase the extraction rate of soluble dietary fiber, and promote the conversion of insoluble dietary fiber to soluble dietary fiber. Among them, the protein content of the stropharia insoluble dietary fiber is 4.35% to 5.08%, the purity is 81.27% to 82.11%, and the extraction rate is 17.76% to 19.18%. The protein content of the stropharia soluble dietary fiber is 3.89% to 4.45%, the purity is 77.78% to 78.86%, and the extraction rate is 8.65% to 9.46%. The proportion of soluble dietary fiber to total dietary fiber is 31.08% to 33.39%. It is worth noting that the method of the present invention has little effect on the fat, ash and water content in the stropharia dietary fiber (Examples 1 to 4 and Comparative Examples 1 to 4). In addition, the extraction rate of insoluble dietary fiber in Comparative Examples 1 to 4 is higher than that in Examples 1 to 4, while the extraction rate of soluble dietary fiber is lower than that in Examples 1 to 4, which further verifies the modification effect of the extraction method on dietary fiber.

[0195] (2) Analysis of dietary fiber function indicators of Pleurotus ostreatus

[0196] The glucose adsorption capacity, cholesterol adsorption capacity, sodium cholate adsorption capacity, sodium nitrite adsorption capacity, α-amylase inhibition, and α-glycosidase inhibition of the dietary fibers in Examples 1 to 4 and Comparative Examples 1 to 4 were tested to evaluate the functional quality of the dietary fibers.

[0197] ① Determination of glucose adsorption capacity: Weigh 0.3g dietary fiber sample, add 30.0mL (25mmol / L) of glucose solution of different concentrations, and adsorb for 6h at 37℃. Centrifuge and take the supernatant, and determine the glucose content by DNS method. The calculation formula is as follows:

[0198] Where: M1 is the mass of glucose before adsorption (mg); M2 is the mass of glucose after adsorption (mg); M is the mass of the sample (g).

[0199] ② Determination of cholesterol adsorption capacity: A. Mix fresh egg yolk and pure water in a ratio of 1:9 to form a uniform emulsion; B. Weigh 0.5 g of dietary fiber sample and add 5 mL of the emulsion in step A to obtain a mixed solution; C. Adjust the pH value of the mixed solution to 2.0 or 7.0, incubate at 37°C for 100 min, centrifuge, take the supernatant and set aside; D. Determine the cholesterol content using the o-phthalaldehyde method, and the calculation formula is as follows:

[0200] Wherein: C1 is the cholesterol content in the control group (mg); C2 is the cholesterol content in the sample group (mg); M is the sample weight (g).

[0201] ③ Sodium cholate adsorption capacity: Weigh 0.1g dietary fiber sample, add 20mL of 0.2mg / mL sodium cholate solution, adjust the system pH to 7, shake at 37℃ for 90min, centrifuge, and determine the sodium cholate content by furfural colorimetry. Calculate the sodium cholate adsorption capacity of the sample according to the following formula:

[0202] Wherein: M1 is the sodium cholate content in the supernatant before adsorption (mg); M2 is the sodium cholate content in the supernatant after adsorption (mg); M0 is the sample mass (g).

[0203] ④ Determination of nitrite adsorption capacity: Weigh 0.2g dietary fiber sample, add 20mL NaNO2 solution (200μmol / L) and mix well, adjust pH to 7.0, react at 37℃ for 1h; centrifuge and take supernatant, determine nitrite content by naphthalene ethylenediamine hydrochloride method, and the control group is treated in the same way without adding sample. Calculate according to the following formula:

[0204] Where: M1 is the content of NaNO2 in the control group (mg); M2 is the content of NaNO2 in the sample (mg); M is the sample mass (g).

[0205] ⑤α-amylase inhibition ability: Take 0.4mL of sample solution of different concentrations (5, 10, 15, 20, 25mg / mL), add 0.4mL α-amylase solution (6U / mL) and 0.8mL PBS (pH7.0), mix; react at 37℃ for 15min, then add 0.8mL 1% starch solution, react at 37℃ for 15min, immediately add 2.0mL 3,5-dinitrosalicylic acid (DNS, purchased from Beijing Solebow Technology Co., Ltd., D7800) to terminate the reaction, boil for 5min, cool and centrifuge to obtain the supernatant; measure the absorbance at 400nm wavelength. At the same time, use acarbose solution for comparative test.

[0206] ⑥α-glucosidase inhibition: Take 0.4mL of sample solution of different concentrations (5, 10, 15, 20, 25mg / mL), add 0.4mL α-glucosidase solution (0.5U / mL) and 0.8mL PBS (pH7.0), mix; react at 37℃ for 15min, then add 0.8mL p-nitrophenyl-α-D-pyranoglucoside (PNPG, 10mmol / L) as substrate and react at 37℃ for 30min, then immediately add 2.0mL Na2CO3 (0.2mol / L) to terminate the reaction. Centrifuge and take the supernatant, and measure its absorbance at 400nm wavelength; at the same time, use acarbose solution for comparative test.

[0207] According to the enzyme inhibition rate formula

[0208]

[0209] Wherein: A1 is the absorbance of the sample with added α-amylase (or α-glucosidase), 1% starch solution (or PNPG) and dietary fiber; A2 is the absorbance of the sample with added 1% starch solution (or PNPG) and dietary fiber; A0 is the absorbance of the sample with added α-amylase (or α-glucosidase) and 1% starch solution (or PNPG).

[0210] The above experiments were repeated three times and the results were averaged. The dietary fiber function indicators are shown in Tables 2 to 4 and Figures 3 to 6 shown.

[0211] Table 2 Comparison of functional indicators of dietary fiber of Stropharia rugosa in Examples 1 to 4 and Comparative Examples 1 to 4

[0212]

[0213]

[0214] Table 3 Inhibition rate of α-amylase by Stropharia rugosa dietary fiber in Examples 1 to 4 and Comparative Examples 1 to 4

[0215]

[0216] Table 4 Inhibition rate of α-glucosidase by dietary fiber of Stropharia rugosa in Examples 1 to 4 and Comparative Examples 1 to 4

[0217]

[0218]

[0219] According to Table 2, the method for efficient deproteinization and functional modification of the stropharia dietary fiber provided by the present invention can effectively improve the adsorption effect of stropharia dietary fiber on glucose, cholesterol, sodium cholate and sodium nitrite. The glucose capacity of the insoluble dietary fiber of stropharia is 96.75-115.23 mg / g, the cholesterol adsorption capacity is 2.15-2.82 mg / g, the sodium cholate adsorption capacity is 11.87-14.89 mg / g, and the sodium nitrite adsorption capacity is 5.68-6.85 mg / g. The glucose capacity of the soluble dietary fiber of stropharia is 26.97-32.87 mg / g, the cholesterol adsorption capacity is 15.24-18.56 mg / g, the sodium cholate adsorption capacity is 8.97-10.25 mg / g, and the sodium nitrite adsorption capacity is 3.89-4.87 mg / g. The addition of snail enzyme plays an important role in the modification of the adsorption capacity of Pseudomonas australis dietary fiber. The various adsorption capacities in Comparative Example 4 are about 50% of those in the embodiment. At the same time, Bacillus Velezii WH-7 also shows a modification effect on the adsorption capacity of Pseudomonas australis dietary fiber. The various adsorption functions in Comparative Example 1 are slightly higher than those in Comparative Example 4, all lower than those in Comparative Examples 2-3, and significantly lower than those in the embodiment.

[0220] According to Tables 3-4 and Figures 3 to 6 It can be seen that the method for efficient deproteinization and functional modification of Pleurotus ostreatus dietary fiber provided by the present invention can effectively increase the inhibitory effect of Pleurotus ostreatus dietary fiber on α-amylase and α-glycosidase. In each treatment, the dietary fiber effect obtained in the embodiment is higher than that in the comparative example, among which the Pleurotus ostreatus dietary fiber in Example 2 has the best inhibitory effect on α-amylase and α-glycosidase. However, due to the lack of snail enzyme modification, the functional effect of comparative example 4 is the worst; due to the lack of fermentation of Bacillus Velez subtilis WH-7, the functional effect of comparative example 1 is also not ideal; although comparative examples 2 to 3 lack protease treatment and cannot further reduce the protein content and improve the purity of dietary fiber, due to the fermentation of Bacillus Velez subtilis WH-7 and snail enzyme modification, the corresponding functional effect is second only to the embodiment.

[0221] (3) Dietary fiber structure

[0222] Electron microscopic images of the Stropharia officinalis dietary fiber prepared in Example 1 and Comparative Example 1 were taken. Figures 7-8 shown.

[0223] Compared with Comparative Example 1, the insoluble dietary fiber ( Figure 7 In A), except for the insoluble dietary fiber ( Figure 8 In addition to having wrinkles and grooves on the surface, it also has a porous structure. Example 1 Soluble dietary fiber ( Figure 7 B) The surface of the honeycomb-like pores are formed, while the comparative example ( Figure 8The surface of B) is smooth and solid, which indicates that the dietary fiber prepared in Example 1 is more loose and porous, and such a morphology is more conducive to the retention of small molecular components.

[0224] In summary, the method of the present invention uses a bacterial-enzyme synergistic treatment process with mild conditions and green environmental protection, which can fundamentally achieve the removal of protein impurities and functional modification, thereby promoting the high-value utilization of Pleurotus ostreatus by-products and promoting industrial upgrading.

[0225] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for deproteinizing stropharia dietary fiber, characterized in that: The following steps are involved: The powder of Stropharia rugosa, amylase and saccharifying enzyme are mixed and subjected to a first enzymatic hydrolysis to obtain a pretreated mushroom powder solution; Inoculating Bacillus Velez subsp. WH-7 into the pretreated mushroom powder solution for fermentation to obtain a mushroom powder fermentation liquid; Performing solid-liquid separation on the mushroom powder fermentation liquid to obtain a first precipitate; The first precipitate is subjected to a second enzymatic hydrolysis, a third enzymatic hydrolysis and solid-liquid separation in sequence to obtain a second supernatant and a second precipitate; the enzyme used in the second enzymatic hydrolysis is an alkaline protease; the enzyme used in the third enzymatic hydrolysis is a neutral protease; The second precipitate is mixed with snail enzyme, and fourth enzymolysis, enzyme inactivation and solid-liquid separation are performed in sequence to obtain a third supernatant and a third precipitate; The third precipitate is sequentially washed and solid-liquid separated to obtain a fourth supernatant and a fourth precipitate; the fourth precipitate is mixed with anhydrous ethanol and solid-liquid separated to obtain Stropharia capitis insoluble dietary fiber; The second supernatant, the third supernatant and the fourth supernatant are combined to obtain a mixed supernatant; the mixed supernatant is successively subjected to reduced pressure concentration and solid-liquid separation to obtain a concentrated mixed supernatant; the concentrated mixed supernatant is mixed with anhydrous ethanol, and the mixture is allowed to stand for 12 to 18 hours to perform solid-liquid separation to obtain Stropharia capitis soluble dietary fiber.

2. The method according to claim 1, characterized in that: The inoculation amount of the Velez subtilis WH-7 is 3% to 10% of the mass of the pretreated mushroom powder solution; the fermentation temperature is 35 to 40° C.; and the fermentation time is 3 to 6 days.

3. The method according to claim 2, characterized in that The OD of the Bacillus velez WH-7 600 The value is 4.0~5.

0.

4. The method according to claim 1, characterized in that: The added amount of the snail enzyme is 1% to 4% of the mass of the Stropharia officinalis powder.

5. The method according to claim 1 or 4, characterized in that: The time of the fourth enzymolysis is 1 to 3 hours; the temperature of the fourth enzymolysis is 28 to 33°C.

6. The method according to claim 1, characterized in that The amount of alkaline protease added is 500-1000U alkaline protease per gram of Stropharia officinalis powder; the time of the second enzymolysis is 2h; the temperature of the second enzymolysis is 37-42°C; The amount of neutral protease added is 500-1000U of neutral protease per gram of Stropharia officinalis powder; the time of the third enzymolysis is 1 hour; and the temperature of the third enzymolysis is 37-42°C.

7. The method according to claim 1, characterized in that The amount of the amylase added is 100-150U of amylase per gram of Stropharia powder; the amount of the saccharifying enzyme added is 150-200U of saccharifying enzyme per gram of Stropharia powder; the time of the first enzymolysis is 1-2h; the temperature of the first enzymolysis is 55-65°C.

8. The method according to claim 1, characterized in that The particle size of the Stropharia officinalis powder is 60-80 meshes.

9. A Stropharia officinalis dietary fiber obtained according to the method according to any one of claims 1 to 8, characterized in that: The stropharia dietary fiber comprises stropharia insoluble dietary fiber and stropharia soluble dietary fiber; the protein content of the stropharia insoluble dietary fiber is ≤5.08%; and the protein content of the stropharia soluble dietary fiber is ≤4.45%.

10. Use of the Stropharia officinalis dietary fiber according to claim 9 in the preparation of functional foods and / or health products.

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