Organic solid fermented enzyme preparation, organic plant protein enzymatic hydrolysate and method for producing the same

Abstract

The present invention provides an organic solid-state fermentation enzyme preparation, an enzymatic hydrolyzate of organic plant protein, and methods for producing them. The organic solid-state fermentation enzyme preparation of the present invention is produced by solid-state fermentation of a protease-producing microorganism, and the organic solid-state fermentation enzyme preparation contains neutral protease, acid protease, alkaline protease, glucoamylase, and cellulase. The organic solid-state fermentation enzyme preparation is obtained by solid-state fermentation of an organic plant material using a protease-producing microorganism, and is rich in acid protease, neutral protease, alkaline protease, cellulase, and glucoamylase. A small amount of an external enzyme is then added to the preparation, resulting in a synergistic and complementary effect of the enzyme system, thereby enhancing the efficiency of enzymatic hydrolysis of plant protein.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and more particularly to an organic solid fermentation enzyme preparation, an organic plant protein enzymatic hydrolysate, and a method for producing the same. [Background technology]

[0002] Food safety is an issue that constantly attracts the attention of governments and society. As people's living standards improve, advances in agricultural products, fermented products, health foods, and other fields have rapidly progressed. Organic fruits and vegetables, organic enzymes, and organic products have become popular and beloved by many people. Certified organic products are being developed widely around the world, and with the concept of "returning to nature and protecting the environment," organic products are attracting the attention of many manufacturers as a future trend in the international market.

[0003] In recent years, the growing demand for organic fermentation products has led to the development of organically certified nitrogen sources in the fermentation industry. Plant protein is an important organically certified nitrogen source for fermentation. Currently, organically certified plant protein raw materials are expensive, and the hydrolysis efficiency of conventional protein enzymes is generally low, making the production cost of organic nitrogen sources a major issue. Furthermore, enzymatic hydrolysis requires the addition of large amounts of enzymes and the adjustment of pH with acids and bases, making it difficult for the hydrolysate to obtain organic certification. Therefore, it is necessary to solve the problems associated with the production of organic nitrogen sources by developing a process that can efficiently hydrolyze plant proteins while still meeting organic certification standards. Summary of the Invention [Problem to be solved by the invention]

[0004] The technical problem to be solved by the present invention is as follows: The present invention provides an organic solid fermentation enzyme preparation that can increase the enzymatic hydrolysis efficiency of proteins and reduce the production cost of enzymatic hydrolyzed organic plant proteins.

[0005] In response to the drawbacks of the prior art, a first object of the present invention is to provide an organic solid-state fermentation enzyme preparation, a second object of the present invention is to provide a method for producing the organic solid-state fermentation enzyme preparation, a third object of the present invention is to provide a method for producing an organic plant protein enzymatic hydrolysate, a fourth object of the present invention is to provide an organic plant protein enzymatic hydrolysate produced by the production method, and a fifth object of the present invention is to provide uses of the organic solid-state fermentation enzyme preparation or the organic plant protein enzymatic hydrolysate. [Means for solving the problem]

[0006] The following is the technical solution of the present invention. The present invention provides an organic solid-state fermentation enzyme preparation, which is produced by solid-state fermentation of a protease-producing microorganism, and which contains a neutral protease, an acid protease, an alkaline protease, a glucoamylase, and a cellulase.

[0007] Preferably, in the organic solid fermentation enzyme preparation, the enzymatic activity of the acidic protease is greater than 390 u / g, the enzymatic activity of the neutral protease is greater than 1500 u / g, the enzymatic activity of the alkaline protease is greater than 600 u / g, the enzymatic activity of the glucoamylase is greater than 400 u / g, and the enzymatic activity of the cellulase is greater than 130 u / g.

[0008] Preferably, in the organic solid-state fermentation enzyme preparation, the enzymatic activity of the acidic protease is greater than 800 u / g, the enzymatic activity of the neutral protease is greater than 4000 u / g, the enzymatic activity of the alkaline protease is greater than 1000 u / g, the enzymatic activity of the glucoamylase is greater than 1500 u / g, and the enzymatic activity of the cellulase is greater than 300 u / g.

[0009] Preferably, in the organic solid fermentation enzyme preparation, the enzymatic activity of the acid protease is greater than 1200 u / g, the enzymatic activity of the alkaline protease is greater than 2200 u / g, and the enzymatic activity of the cellulase is greater than 1200 u / g.

[0010] Preferably, in the organic solid fermentation enzyme preparation, the protease-producing microorganisms include one or more of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, preferably Aspergillus oryzae and Bacillus subtilis, and the weight ratio of Aspergillus oryzae to Bacillus subtilis is preferably 70-90:10-30.

[0011] The present invention also provides a method for producing a semiconductor device comprising: (1) culturing a protease-producing microorganism to obtain a solid seed; (2) performing solid fermentation of the solid seed prepared in step (1) to obtain an organic solid-state fermentation enzyme preparation.

[0012] Preferably, in the method for producing an organic solid-fermented enzyme preparation, the solid fermentation is solid-state variable temperature fermentation, which is performed by first fermenting at 36 to 40°C for 6 to 20 hours and then fermenting at 28 to 32°C for 30 to 50 hours, and preferably, the solid-state variable temperature fermentation is performed by first fermenting at 36 to 38°C for 8 to 15 hours and then fermenting at 30 to 32°C for 40 to 44 hours.

[0013] Preferably, in the method for producing an organic solid-fermented enzyme preparation, the seed medium in step (1) and the solid fermentation medium in step (2) both contain an organic starch agricultural by-product and / or an organic oil-pressed raw agricultural by-product. Preferably, the seed medium in step (1) and the solid fermentation medium in step (2) both contain an organic starch agricultural by-product and an organic oil-pressed raw agricultural by-product. More preferably, the content of the organic starch agricultural by-product is 9-40% by weight, and the content of the organic oil-pressed raw agricultural by-product is 60-91% by weight.

[0014] Preferably, the organic starch agricultural by-product comprises one or more of organic rice, organic wheat and organic corn, preferably organic corn.

[0015] Preferably, the organic oil-pressed raw agricultural by-product comprises organic bean cake and / or organic rapeseed cake, preferably organic bean cake.

[0016] Preferably, in the method for producing an organic solid-fermented enzyme preparation, both the seed medium in step (1) and the solid fermentation medium in step (2) further contain organic vegetable protein powder, and preferably, when the total weight of the medium is taken as 100%, the organic vegetable protein powder accounts for 6-10% and the organic starch agricultural by-product and / or organic oil-extracting raw material agricultural by-product accounts for 90-94%.

[0017] Preferably, in the method for producing an organic solid fermented enzyme preparation, the organic vegetable protein powder comprises organic rice protein powder and / or organic wheat protein powder, and more preferably, the organic vegetable protein powder comprises, by weight percentage, 60 to 80% organic rice protein powder and 20 to 40% organic wheat protein powder.

[0018] Preferably, in the above-mentioned method for producing an organic solid fermentation enzyme preparation, the inoculation amount of the seed culture described in step (1) is 0.3 to 0.5% based on the weight of the seed medium.

[0019] Preferably, in the above-mentioned method for producing an organic solid-fermentation enzyme preparation, the inoculation amount for solid fermentation described in step (2) is 0.3 to 0.5% by weight based on the weight of the solid fermentation medium.

[0020] Preferably, the method for producing an organic solid fermentation enzyme preparation further comprises a step of drying the organic solid fermentation enzyme preparation, and the drying temperature is preferably 37 to 40°C.

[0021] The present invention also provides a method for producing an enzymatic hydrolyzed organic vegetable protein, which comprises enzymatically hydrolyzing the above-mentioned organic solid-state fermentation enzyme preparation or an organic solid-state fermentation enzyme preparation produced by the above-mentioned method for producing an organic solid-state fermentation enzyme preparation, using an external enzyme.

[0022] Preferably, in the above-mentioned method for producing an enzymatic hydrolyzate of organic plant protein, the amount of the external enzyme used is 0.2 to 0.8% based on the dry weight of the organic solid-fermentation enzyme preparation, and preferably the external enzyme comprises one or more of papain, bromelain, neutral protease, acid protease, alkaline protease, glucanase, and cellulase, and more preferably the external enzyme comprises 0.1 to 0.3% papain, 0.04 to 0.08% neutral protease, 0.02 to 0.04% acid protease, 0.01 to 0.02% glucanase, and 0.01 to 0.02% cellulase based on the dry weight of the organic solid-fermentation enzyme preparation.

[0023] Preferably, in the method for producing an enzymatic hydrolysate of an organic vegetable protein, the enzymatic hydrolysis temperature is 45 to 55°C, the enzymatic hydrolysis time is 10 to 15 hours, and the enzymatic hydrolysis pH is preferably 4.5 to 7.5.

[0024] Preferably, the method for producing an enzymatic hydrolyzed organic vegetable protein further comprises a step of slurrying the organic solid fermentation enzyme preparation before the enzymatic hydrolysis, and preferably, the dry matter content of the slurry at the time of slurrying is 12 to 16% by weight.

[0025] Preferably, the method for producing an enzymatic hydrolyzate of an organic vegetable protein further comprises the steps of filtering, concentrating and drying the enzymatic hydrolyzate in this order.

[0026] The present invention also provides an organic plant protein enzymatic hydrolysate produced by the above-mentioned method for producing an organic plant protein enzymatic hydrolysate, and preferably, the total nitrogen content of the organic plant protein enzymatic hydrolysate is greater than 9% and the amino acid nitrogen content is greater than 4.5%, based on the dry weight of the organic plant protein enzymatic hydrolysate.

[0027] The present invention also provides use of an organic plant protein enzymatic hydrolysate produced by the above-mentioned method for producing an organic plant protein enzymatic hydrolysate or the above-mentioned organic plant protein enzymatic hydrolysate as an organic nitrogen source in the field of fermentation using yeast, lactic acid bacteria, or Bacillus subtilis.

[0028] The present invention also provides use of the organic vegetable protein enzymatic hydrolysate produced by the above-mentioned production method or the above-mentioned organic vegetable protein enzymatic hydrolysate as an organic umami seasoning or basic seasoning in the field of food seasoning.

[0029] The present invention also provides use of the above-mentioned organic solid fermentation enzyme preparation or an organic solid fermentation enzyme preparation produced by the above-mentioned method for producing an organic solid fermentation enzyme preparation as an organic protease in the enzymatic hydrolysis of vegetable proteins. [Effects of the Invention]

[0030] The beneficial effects of the present invention are as follows: The present invention uses protease-producing microorganisms to perform solid-state fermentation of organic plant materials to obtain an organic solid-state fermentation enzyme preparation rich in acid protease, neutral protease, alkaline protease, cellulase, and glucoamylase, to which a small amount of external enzymes is then added. The synergistic and complementary effects of the enzyme system result in a conversion rate of amino nitrogen in organic plant proteins of over 43%, significantly improving the efficiency of enzymatic hydrolysis of plant proteins. Furthermore, the resulting organic plant protein enzymatic hydrolysate can be used as an organic nitrogen source and flavor enhancer, and fully meets the European Union's organic certification standards, thereby increasing the added value of the product.

[0031] The following is the strain deposit information: The Bacillus subtilis strain (Bacillus subtilis ESP710) used in the present invention was deposited at the China Typical Culture Depository Center (CCTCC) on November 26, 2018, with deposit number CCTCC NO: M2018827 (i.e., CCTCC M2018827). The depository is located at Wuhan University, Wuhan, China, with postal code 430072 and telephone number (027)-68754052. (The Bacillus subtilis is described in a patent application with Chinese Application No. 201910686571.8 (Chinese Patent Application Publication No. 112300953).)

[0032] The Aspergillus niger ESP1023 strain used in the present invention has been deposited at the China Typical Culture Depository Center (CCTCC), located at Wuhan University, Wuhan, China, with a postal code of 430072 and a telephone number of 027-68754052. The date of deposit was November 26, 2018, and the deposit number is CCTCC NO: M2018829 (i.e., CCTCC M2018829). (The Aspergillus niger strain is described in Chinese Patent Application No. 201911268295.X (Chinese Patent Publication No. 112940943).) DETAILED DESCRIPTION OF THE INVENTION

[0033] In order to make the purpose, technical solution and technical effect of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be described clearly and completely. The embodiments described below are only some of the embodiments of the present invention, not all of them. Other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without inventive work are all within the protection scope of the present invention.

[0034] Currently, there are two main problems with producing enzymatically hydrolyzed plant proteins that meet organic certification standards. First, there is room for improvement in the technology for enzymatically hydrolyzing plant proteins. The enzymes lack effective synergistic effects, resulting in low enzymatic hydrolysis efficiency and a low amino nitrogen conversion rate of about 30%, which makes the product expensive to manufacture. Second, the plant enzymatic hydrolysis process requires the addition of large amounts of multiple enzymes and the use of strong acids and bases to adjust the pH, making it difficult for the process to meet organic certification standards.

[0035] In a specific embodiment of the present invention, an organic solid-state fermentation enzyme preparation is provided, which is produced using solid-state fermentation with a protease-producing microorganism, and the organic solid-state fermentation enzyme preparation contains a neutral protease, an acid protease, an alkaline protease, a glucoamylase, and a cellulase.

[0036] In a preferred embodiment of the present invention, in the above organic solid-state fermentation enzyme preparation, the enzymatic activity of the acidic protease is greater than 390 u / g, the enzymatic activity of the neutral protease is greater than 1500 u / g, the enzymatic activity of the alkaline protease is greater than 600 u / g, the enzymatic activity of the glucoamylase is greater than 400 u / g, and the enzymatic activity of the cellulase is greater than 130 u / g.

[0037] Furthermore, in the organic solid fermentation enzyme preparation, the enzymatic activity of the acidic protease is greater than 800 u / g, the enzymatic activity of the neutral protease is greater than 4000 u / g, the enzymatic activity of the alkaline protease is greater than 1000 u / g, the enzymatic activity of the glucoamylase is greater than 1500 u / g, and the enzymatic activity of the cellulase is greater than 300 u / g.

[0038] Furthermore, in the organic solid fermentation enzyme preparation, the enzyme activity of the acid protease is greater than 1200 u / g, the enzyme activity of the alkaline protease is greater than 2200 u / g, and the enzyme activity of the cellulase is greater than 1200 u / g.

[0039] However, the protease-producing microorganisms include one or more of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, preferably Aspergillus oryzae and Bacillus subtilis, and the weight ratio of Aspergillus oryzae to Bacillus subtilis is preferably 70-90:10-30.

[0040] The present invention also provides a method for producing a semiconductor device comprising: (1) culturing a protease-producing microorganism to obtain a solid seed; (2) performing solid fermentation of the solid seed prepared in step (2) to obtain an organic solid-state fermentation enzyme preparation.

[0041] However, both the seed medium in step (1) and the solid fermentation medium in step (2) contain organic starch agricultural by-products and / or organic oil-pressed raw agricultural by-products, for example, the organic starch agricultural by-products may be one or more of organic rice, organic wheat, and organic corn, and the organic oil-pressed raw agricultural by-products may be organic bean rice cakes and / or organic rapeseed rice cakes.

[0042] In a preferred embodiment of the present invention, the seed medium in step (1) and the solid fermentation medium in step (2) both contain organic starch agricultural by-products and organic oil-extracted raw agricultural by-products, and the content of the organic starch agricultural by-products is 9-40% by weight, and the content of the organic oil-extracted raw agricultural by-products is 60-91% by weight.

[0043] Both the seed medium in step (1) and the solid fermentation medium in step (2) further contain organic vegetable protein powder, which may be organic rice protein powder and / or organic wheat protein powder, and preferably, the organic vegetable protein powder contains, by weight percentage, 60-80% organic rice protein powder and 20-40% organic wheat protein powder.

[0044] In the seed medium and solid fermentation medium, the ratio of the organic vegetable protein powder is 6-10%, and the ratio of organic starch agricultural by-products and / or organic oil-extracted agricultural by-products is 90-94%, assuming that the total weight of the medium is 100%.

[0045] Organic rice protein powder and / or organic wheat protein powder have a high protein content, most of which is alkaline-soluble protein, which can stimulate the fungus to secrete large amounts of neutral protease and alkaline protease, accelerating the enzymatic decomposition of protein structures in plants and significantly increasing the conversion rate of amino acid nitrogen.

[0046] In another preferred embodiment of the present invention, the seed medium in step (1) and the solid fermentation medium in step (2) both contain 70-90% granular organic bean cake and 10-30% organic corn grain.

[0047] The solid-state fermentation described in step (2) is solid-state variable temperature fermentation, which includes first fermenting at 36 to 40°C for 6 to 20 hours, followed by fermenting at 28 to 32°C for 30 to 50 hours, and preferably includes first fermenting at 36 to 38°C for 8 to 15 hours, followed by fermenting at 28 to 30°C for 40 to 44 hours. The use of solid-state variable temperature fermentation can further enhance the enzymatic activity of the organic solid-state fermentation enzyme preparation and increase the conversion rate of amino acid nitrogen in plant proteins.

[0048] In another preferred embodiment of the present invention, the method for producing the organic solid fermentation enzyme preparation includes the steps of: (1) A spore suspension was prepared by adding a strain of protease-producing microorganism to 10 mL of sterile water, and the spore concentration was 10 7 ~10 9 cfu / mL, inoculating the spore suspension into a seed medium at an inoculum amount of 0.3 to 0.5% (based on the weight of the medium) and culturing at 32 to 38°C for 2 to 4 days to obtain a solid seed of the protease-producing microorganism; (2) The protease-producing microorganism solid seed prepared in step (1) is introduced into a mixing inoculator at an inoculation amount of 0.3 to 0.5% (based on the weight of the medium) to inoculate the solid fermentation medium, and finally sent to a disk koji-making machine for koji-making cultivation, with regular ventilation and turning over the raw materials for cultivation, and fermented for 6 to 20 hours at an ambient temperature of 36 to 40°C and humidity of 90 to 95%, and when the product temperature rises, the ambient temperature is increased to 28 to 32°C and humidity is increased to 90 to 95%, and fermented for 30 to 50 hours. (3) drying the solid fermented product at a low temperature of 37 to 40°C to obtain an organic solid fermentation enzyme preparation.

[0049] The method for preparing the seed medium and solid fermentation medium of the present invention both includes the steps of weighing out each ingredient according to the formulation, adding 85 to 100% (based on the total weight of the ingredients) of water, mixing uniformly, and sterilizing at 121°C for 50 minutes.

[0050] The present invention also provides a method for producing an enzymatic hydrolyzed organic vegetable protein, which comprises enzymatically hydrolyzing the above-mentioned organic solid-state fermentation enzyme preparation or the organic solid-state fermentation enzyme preparation produced by the above-mentioned production method using an exogenous enzyme.

[0051] In the present invention, the term "external enzyme" refers to an enzyme of another origin that is added to the organic solid-state fermentation enzyme preparation, and may be, for example, one or more of papain, bromelain, neutral protease, acid protease, alkaline protease, glucanase, and cellulase. Preferably, the external enzyme comprises one or more of papain, acid protease, neutral protease, glucanase, and cellulase, and more preferably, papain, acid protease, neutral protease, glucanase, and cellulase. Preferably, the amount of the external enzyme used is 0.2 to 0.8%, based on the dry weight of the organic solid-state fermentation enzyme preparation, and more preferably, the external enzyme comprises 0.1 to 0.3% papain, 0.04 to 0.08% neutral protease, 0.02 to 0.04% acid protease, 0.01 to 0.02% glucanase, and 0.01 to 0.02% cellulase. The key to the enzymatic hydrolysis of vegetable proteins is the synergistic effect of multiple enzymes. The main purpose of adding complex enzymes is to further enrich the enzyme system of protease and cellulase and strengthen the synergistic effect. For example, papain, an endopeptidase, can effectively compensate for the shortcomings of the solid koji digestive enzyme system.

[0052] In the above method for producing an enzymatic hydrolysate of an organic vegetable protein, the enzymatic hydrolysis temperature is 45 to 55° C., the enzymatic hydrolysis time is 10 to 15 hours, and the enzymatic hydrolysis pH is preferably 4.5 to 7.5.

[0053] However, the pH adjuster for enzymatic decomposition is citric acid and / or sodium carbonate.

[0054] The method for producing the enzymatic hydrolyzed organic plant protein further comprises a step of slurrying the organic solid fermentation enzyme preparation before the enzymatic hydrolysis, and preferably, the dry matter content of the slurry at the time of slurrying is 12 to 16% by weight.

[0055] The method for producing the enzymatic hydrolyzate of organic vegetable protein further includes the steps of filtering, concentrating, and drying the enzymatic hydrolyzate in this order.

[0056] In a preferred embodiment of the present invention, the method for producing the organic plant protein enzymatic hydrolysate further comprises the following steps: (1) Water is added to the above organic solid fermentation enzyme preparation to prepare a reaction solution with a dry weight concentration of 12 to 16%, which is then subjected to a slurry treatment using a colloid mill. 0.2 to 0.8% of an external enzyme is added based on the dry weight of the reaction solution to carry out enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45 to 55°C, the enzymatic hydrolysis pH is 4.5 to 7.5, and the enzymatic hydrolysis time is 10 to 15 hours. (2) Perlite is added to each of the above enzyme hydrolyzed solutions and mixed uniformly. The mixture is filtered through a plate and frame filter press, and the filtrate is concentrated by distillation under reduced pressure to obtain a concentrate with a dry matter content of 25 to 35%. Finally, organic maltodextrin is added to the concentrate with a mass fraction of 5 to 10%, and after stirring and dissolving, the concentrate is spray-dried to obtain an organic vegetable protein enzyme hydrolyzed product.

[0057] The present invention also provides an organic vegetable protein enzymatic hydrolysate produced by the above-mentioned production method, which has a total nitrogen content of more than 9% and an amino acid nitrogen content of more than 4.5%, based on the dry weight of the organic vegetable protein enzymatic hydrolysate.

[0058] This invention uses protease-producing microorganisms to carry out solid-state variable-temperature fermentation to obtain a fermentation product rich in acid protease, neutral protease, alkaline protease, cellulase, and glucoamylase. By adding a small amount of external enzymes, the synergistic and complementary effects of the enzyme system can be achieved, greatly improving the enzymatic degradation efficiency of plant proteins. The entire process essentially does not require the addition of chemicals such as hydrochloric acid, sulfuric acid, or sodium hydroxide, and can meet the requirement of less than 5% of external non-organic certified substances, thereby meeting the European Union's organic certification standards.

[0059] The present invention also provides use of the above-mentioned enzymatic hydrolysate of organic plant protein as an organic nitrogen source in the field of fermentation using yeast, lactic acid bacteria or Bacillus subtilis.

[0060] The present invention also provides use of the above-mentioned enzymatic hydrolysate of organic vegetable protein as an organic umami seasoning or basic seasoning in the field of food seasoning.

[0061] The present invention also provides use of the above organic solid fermentation enzyme preparation as an organic protease in the enzymatic hydrolysis of vegetable proteins.

[0062] The beneficial effects of the organic solid fermentation enzyme preparation, the organic plant protein enzymatic hydrolysate and the method for producing the same described in the present invention will be further explained below with specific examples.

[0063] See Table 1 for the suppliers of raw materials and equipment used in the examples and comparative examples of the present invention. [Table 1]

[0064] Example 1 1. Preparation of Solid Seeds 80g of granular organic bean cake and 20g of organic corn kernels were weighed, and 8g of composite organic vegetable protein powder (consisting of 80% by weight of organic rice protein powder and 20% by weight of organic wheat protein powder) was added. Then, 90g of water was added and mixed uniformly, and the mixture was sterilized at 120°C for 50 minutes to obtain a solid seed medium.

[0065] Add the Aspergillus oryzae strain stored in a slant medium to 10 mL of sterilized water to obtain a spore concentration of 10 7 ~10 8 A spore suspension containing 0.3% cfu / mL of the spores was prepared, and the spore suspension was inoculated onto a solid seed medium at an inoculum amount of 0.3% (based on the weight of the solid seed medium). The solid seed medium was then cultured at 32°C for 3 days to prepare a solid seed of Aspergillus oryzae.

[0066] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, and 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added. Then, 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0067] The solid fermentation medium was inoculated with Aspergillus oryzae solid seeds at an inoculum amount of 0.4% (based on the weight of the medium), and then sent to a disk koji-making machine for koji cultivation. The medium was ventilated regularly, and the raw materials were turned upside down during cultivation. The ambient temperature was set at 36°C and the humidity at 94% for 15 hours of high-temperature fermentation. Once the product temperature had risen, the ambient temperature was set at 30°C and the humidity at 90% for 44 hours of low-temperature fermentation. Finally, the product was dried at low temperature at 38°C to obtain an organic solid-state fermentation enzyme preparation rich in multiple enzymes.

[0068] The enzyme activities of protease, glucoamylase, and cellulase in the organic solid fermentation enzyme preparation were measured using the following method, and the results are shown in Table 2.

[0069] (1) Protease activity measurement Measurements were performed according to the method in Appendix B (Protease activity measurement: Folin-Ciocalteu method) of GB / T 23527-2009 (Protease preparations). Phosphate buffer was used to measure neutral protease activity, sodium lactate buffer to measure acidic protease activity, and borate buffer to measure alkaline protease activity.

[0070] (2) Measurement of glucoamylase activity Measurements were made in reference to the method for measuring saccharification power in 5.6 of QB / T 4527-2011 (General analysis method for koji (large koji) for sake brewing).

[0071] (3) Cellulase activity measurement Measurements were performed with reference to the method in Appendix A (method for measuring enzyme activity in filter paper) of QB / T 2583-2003 (cellulase preparations).

[0072] 3. Preparation of organic plant protein enzymatic hydrolysate The organic solid-state fermentation enzyme preparation prepared in step 2 above was mixed with water to a dry matter concentration of 16% by weight, and then emulsified using a colloid mill. The mixture was then subjected to enzymatic hydrolysis with 0.2% Angel Complex protease and 0.3% papain (by dry weight) at 50°C, maintaining the pH at 5.0-6.5 with sodium carbonate solution for 12 hours. The resulting organic plant protein enzymatic hydrolyzate was obtained. The Angel Complex protease contained, by weight, 40% neutral protease, 20% acid protease, 10% glucanase, and 10% cellulase.

[0073] Perlite was added to the above organic vegetable protein enzymatic hydrolyzate and mixed uniformly, then the mixture was filtered under pressure using a plate and frame filter press to obtain a filtrate, which was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 10% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0074] The total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the organic plant protein enzymatic hydrolyzate were measured using the following method, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0075] (1) Measurement of total nitrogen content Total nitrogen was measured in accordance with the method described in GB 5009.5-2016 (Determination of Protein in Foods), Section 3, Combustion Method.

[0076] (2) Measurement of amino acid nitrogen content Measurements were made with reference to GB 5009.235-2016 (Determination of Amino Acid Nitrogen in Foods), Method 1, Acidity Meter Method, 5.2, Sauce and Chinese Soybean Miso samples.

[0077] (3) Conversion rate of amino acid nitrogen Conversion rate of amino acid nitrogen = Amino acid nitrogen content in enzyme hydrolyzed solution / Total nitrogen content in enzyme hydrolyzed solution × 100%

[0078] Example 2 1. Preparation of Solid Seeds 70g of granular organic bean cake and 30g of organic corn kernels were weighed, and 8g of composite organic vegetable protein powder (consisting of 80% by weight of organic rice protein powder and 20% by weight of organic wheat protein powder) was added. Then, 100g of water was added and mixed uniformly, and the mixture was sterilized at 120°C for 50 minutes to obtain a solid seed medium.

[0079] Add 10 mL of sterile water to the Bacillus subtilis (ESP710) cells stored in a slant culture medium until the Bacillus concentration in the culture solution is 10 8 ~10 9 A bacterial solution containing cfu / mL of Bacillus was prepared, and Bacillus subtilis was inoculated onto a solid seed medium at an inoculum amount of 0.1% (based on the weight of the medium) and cultured at 38°C for 2 days to prepare a solid seed of Bacillus subtilis.

[0080] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added, 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0081] The solid fermentation medium was inoculated with a 0.4% inoculum amount (based on the weight of the medium) of Bacillus subtilis solid seed, which was then sent to a disk koji-making machine for koji cultivation. The mixture was periodically ventilated, and the raw materials were turned over during cultivation. The ambient temperature was set to 40°C and the humidity was limited to 95% for high-temperature fermentation for 20 hours. Once the product temperature had risen, the ambient temperature was set to 28°C and the humidity was limited to 90% for low-temperature fermentation for 50 hours. Finally, the mixture was dried at low temperature at 38°C to obtain an organic solid-state fermentation enzyme preparation rich in multiple enzymes.

[0082] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid fermentation enzyme preparation were measured, and the results are shown in Table 2.

[0083] 3. Preparation of organic plant protein enzymatic hydrolysate Water was added to the organic solid fermentation enzyme preparation prepared in step 2 above to adjust the dry matter concentration to 16%, and a colloid mill was used to make a slurry to form an emulsion. 0.2% papain was added based on the dry weight for enzymatic hydrolysis. The enzymatic hydrolysis temperature was 55°C, and the pH was maintained at 4.5-5.0 with a citric acid solution. The enzymatic hydrolysis was continued for 15 hours to obtain an organic vegetable protein enzymatic hydrolysis liquid.

[0084] Perlite was added to the above organic vegetable protein enzymatic hydrolyzate and mixed uniformly, then the mixture was filtered under pressure using a plate and frame filter press to obtain a filtrate, which was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 10% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0085] Using the method described in Example 1, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0086] Example 3 1. Preparation of Solid Seeds 90g of organic bean cake and 10g of organic corn were weighed, and 8g of composite organic vegetable protein powder (consisting of 80% by weight of organic rice protein powder and 20% by weight of organic wheat protein powder) was added. Then, 95g of water was added and mixed uniformly, and the mixture was sterilized at 120°C for 50 minutes to obtain a solid seed medium.

[0087] Aspergillus niger ESP1023 strain stored in a slant medium was added to 10 mL of sterilized water to obtain a spore concentration of 10 7 ~10 8 A spore suspension containing cfu / mL was prepared, and the spore suspension was inoculated onto a solid seed medium at an inoculum amount of 0.3% (based on the weight of the medium) and cultured at 32°C for 3 days to prepare a solid seed of Aspergillus niger.

[0088] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added, 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0089] The solid fermentation medium was inoculated with Aspergillus niger solid seeds at an inoculation rate of 0.4% (based on the weight of the medium), and then sent to a disk koji-making machine for koji cultivation. The medium was ventilated regularly, and the raw materials were turned over during cultivation. The ambient temperature was set at 38°C and the humidity at 94% for 20 hours of high-temperature fermentation. Once the product temperature had risen, the ambient temperature was set at 30°C and the humidity at 90% for 30 hours of low-temperature fermentation. Finally, the product was dried at low temperature at 38°C to obtain an organic solid-state fermentation enzyme preparation rich in multiple enzymes.

[0090] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid fermentation enzyme preparation were measured, and the results are shown in Table 2.

[0091] 3. Preparation of organic plant protein enzymatic hydrolysate Water was added to the organic solid fermentation enzyme preparation prepared in step 2 above to adjust the dry matter concentration to 16%, and a colloid mill was used to make a slurry to form an emulsion. 0.8% bromelain was added based on the dry weight and enzymatically hydrolyzed. The enzymatic hydrolysis temperature was 55°C, and the pH was maintained at 7.0-7.5 with sodium carbonate solution for 10 hours, yielding an organic vegetable protein enzymatic hydrolysis liquid.

[0092] Perlite was added to the above organic vegetable protein enzymatic hydrolyzed solution and mixed uniformly, then the solution was filtered under pressure using a plate and frame filter press to obtain a filtrate, which was then concentrated under reduced pressure until the dry matter concentration reached 35% to obtain a concentrate.Finally, 5% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzed product.

[0093] Using the method described in Example 1, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0094] Example 4 1. Preparation of Solid Seeds The solid Aspergillus oryzae species prepared in Example 1 and the solid Bacillus subtilis species prepared in Example 2 were mixed in a weight ratio of 90:10 to obtain a composite solid bacterial species.

[0095] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic rapeseed rice cake with a particle size of 6-14 mesh was weighed, 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added, 900g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0096] The above solid fermentation medium was inoculated with a 0.5% inoculum amount (based on the weight of the medium) of the complex bacterial solid strain, and then sent to a disk koji-making machine for koji cultivation. The mixture was periodically ventilated, and the raw materials were turned over during cultivation. The ambient temperature was set to 36°C and the humidity was limited to 94%, and high-temperature fermentation was carried out for 15 hours. Once the product temperature rose, the ambient temperature was set to 30°C and the humidity was limited to 90%, and low-temperature fermentation was carried out for 44 hours. Finally, it was dried at 40°C, and an organic solid-state fermentation enzyme preparation rich in multiple enzymes was obtained.

[0097] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid fermentation enzyme preparation were measured, and the results are shown in Table 2.

[0098] 3. Preparation of organic plant protein enzymatic hydrolysate Water was added to the organic solid-state fermentation enzyme preparation prepared in step 2 above to adjust the dry matter concentration to 12%, and the mixture was slurried using a colloid mill to form an emulsion. 0.1% Angel Complex protease and 0.1% papain were added, based on the dry weight, for enzymatic hydrolysis at 50°C. The pH was maintained at 5.0-5.5 with sodium carbonate solution for 10 hours, yielding an organic vegetable protein enzymatic hydrolyzate.

[0099] Perlite was added to the organic vegetable protein enzymatic hydrolyzate and mixed uniformly, then the mixture was filtered under pressure using a plate and frame filter press to obtain a filtrate. The filtrate was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 10% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0100] Using the method described in Example 1, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0101] Example 5 1. Preparation of Solid Seeds The solid strain of Aspergillus oryzae prepared in Example 1 and the solid strain of Bacillus subtilis prepared in Example 2 were mixed in a weight ratio of 70:30 to obtain a composite solid strain of bacteria.

[0102] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 800g of granular organic bean cake with a particle size of 6-14 mesh and 200g of organic corn kernels with a particle size of 10-30 mesh were weighed, and 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added. 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0103] The above solid fermentation medium was inoculated with a 0.3% inoculum amount (based on the weight of the medium) of the complex bacterial solid strain, and then sent to a disk koji-making machine for koji cultivation. The medium was periodically ventilated, and the raw materials were turned over during cultivation. The ambient temperature was set to 38°C and the humidity was limited to 95% for high-temperature fermentation for 15 hours. Once the product temperature rose, the ambient temperature was set to 32°C and the humidity was limited to 95% for low-temperature fermentation for 44 hours. Finally, it was dried at 37°C to obtain an organic solid-state fermentation enzyme preparation rich in multiple enzymes.

[0104] The enzyme activities of protease, glucoamylase, and cellulase in the organic solid fermentation enzyme preparation were measured using the following method, and the results are shown in Table 2.

[0105] 3. Preparation of organic plant protein enzymatic hydrolysate Water was added to the organic solid fermentation enzyme preparation prepared in step 2 above to adjust the dry matter concentration to 12%, and a colloid mill was used to make a slurry to form an emulsion. 0.15% complex protease and 0.2% papain were added, based on the dry weight, to carry out enzymatic hydrolysis. The enzymatic hydrolysis temperature was 50°C, and the pH was maintained at 5.5 to 6.5 with sodium carbonate solution. The enzymatic hydrolysis was carried out for 10 hours to obtain an organic plant protein enzymatic hydrolysis liquid.

[0106] Perlite was added to the organic vegetable protein enzymatic hydrolyzate and mixed uniformly, followed by pressure filtration using a plate and frame filter press to obtain a filtrate. The filtrate was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 5% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0107] Using the method described in Example 1, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0108] Example 6 This example differs from Example 1 in that neither the solid seed medium in Step 1 nor the solid fermentation medium in Step 2 contains vegetable protein powder, and the specific preparation method is as follows. 1. Preparation of Solid Seeds 80g of granular organic bean cake and 20g of organic corn kernels were weighed, then 90g of water was added and mixed evenly, and sterilized at 120°C for 50 minutes to obtain a solid seed medium.

[0109] Add the Aspergillus oryzae strain stored in a slant medium to 10 mL of sterilized water to obtain a spore concentration of 10 7 ~10 8 A spore suspension containing 0.3% cfu / mL of the spores was prepared, and the spore suspension was inoculated onto a solid seed medium at an inoculum amount of 0.3% (based on the weight of the medium) and cultured at 32°C for 3 days to prepare a solid seed of Aspergillus oryzae.

[0110] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, and then 950g of water was added and mixed uniformly, followed by sterilization at 121℃ for 50 minutes to obtain a solid fermentation medium.

[0111] The solid fermentation medium was inoculated with Aspergillus oryzae solid seeds at an inoculum amount of 0.4% (based on the weight of the medium), and then sent to a disk koji-making machine for koji cultivation. The medium was ventilated regularly, and the raw materials were turned upside down during cultivation. The ambient temperature was set at 36°C and the humidity at 94% for 15 hours of high-temperature fermentation. Once the product temperature had risen, the ambient temperature was set at 30°C and the humidity at 90% for 44 hours of low-temperature fermentation. Finally, the product was dried at low temperature at 38°C to obtain an organic solid-state fermentation enzyme preparation rich in multiple enzymes.

[0112] 3. Preparation of organic plant protein enzymatic hydrolysate The organic solid-state fermentation enzyme preparation prepared in step 2 above was mixed with water to a dry matter concentration of 16% by weight, and then emulsified using a colloid mill. The mixture was then subjected to enzymatic hydrolysis with 0.2% Angel Complex protease and 0.3% papain (by dry weight) at 50°C, maintaining the pH at 5.0-6.5 with sodium carbonate solution for 12 hours. The resulting organic plant protein enzymatic hydrolyzate was obtained. The Angel Complex protease contained, by weight, 40% neutral protease, 20% acid protease, 10% glucanase, and 10% cellulase.

[0113] Perlite was added to the above organic vegetable protein enzymatic hydrolyzate and mixed uniformly, then the mixture was filtered under pressure using a plate and frame filter press to obtain a filtrate, which was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 10% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0114] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid-state fermentation enzyme preparation, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Tables 2 and 3.

[0115] Example 7 This example differs from Example 1 in that the preparation process of the organic solid-fermented enzyme preparation in Step 2 involves solid-state fermentation at an ambient temperature of 36°C and humidity of 90% for 50 hours. The specific production method is as follows. 1. Preparation of Solid Seeds The manufacturing method was the same as in Example 1.

[0116] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, and 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added. Then, 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0117] The solid fermentation medium was inoculated with Aspergillus oryzae solid seeds at an inoculation rate of 0.4% (based on the weight of the medium), and then sent to a disk koji-making machine for koji cultivation. The medium was periodically ventilated, and the raw materials were turned upside down during cultivation. The ambient temperature was set at 36°C and the humidity was limited to 94%, and the mixture was fermented for 50 hours. Finally, the mixture was dried at low temperature at 38°C to obtain an organic solid fermentation enzyme preparation rich in multiple enzymes.

[0118] 3. Preparation of organic plant protein enzymatic hydrolysate Using the above organic solid fermentation enzyme preparation, an organic vegetable protein enzymatic hydrolyzate and an organic vegetable protein enzymatic hydrolyzate were prepared according to the method described in step 3 of Example 1.

[0119] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid-state fermentation enzyme preparation, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Tables 2 and 3.

[0120] Example 8 This example differs from Example 1 in that the preparation process of the organic solid-fermented enzyme preparation in Step 2 involves solid-state fermentation at an ambient temperature of 30°C and humidity of 90% for 50 hours. The specific production method is as follows. 1. Preparation of Solid Seeds The manufacturing method was the same as in Example 1.

[0121] 2. Preparation of Organic Solid Fermentation Enzyme Preparation 1000g of granular organic bean cake with a particle size of 6-14 mesh was weighed, and 80g of composite organic vegetable protein powder (consisting of 60% by weight of organic rice protein powder and 40% by weight of organic wheat protein powder) was added. Then, 950g of water was added and mixed uniformly, and the mixture was sterilized at 121°C for 50 minutes to obtain a solid fermentation medium.

[0122] The solid seeds of Aspergillus oryzae were inoculated into the above solid fermentation medium at an inoculation rate of 0.4% (based on the weight of the medium), and then sent to a disk koji-making machine for koji cultivation. The medium was periodically ventilated, and the raw materials were turned upside down during cultivation. The ambient temperature was limited to 30°C and humidity to 90%, and the mixture was fermented for 50 hours. Finally, it was dried at low temperature at 38°C to obtain an organic solid fermentation enzyme preparation rich in multiple enzymes.

[0123] 3. Preparation of organic plant protein enzymatic hydrolysate Using the above organic solid fermentation enzyme preparation, an organic vegetable protein enzymatic hydrolyzate and an organic vegetable protein enzymatic hydrolyzate were prepared according to the method described in step 3 of Example 1.

[0124] Using the method described in Example 1, the enzyme activities of protease, glucoamylase, and cellulase in the organic solid-state fermentation enzyme preparation, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Tables 2 and 3.

[0125] (Comparative Example 1) This example differs from Example 1 in that instead of using an organic solid-state fermented enzyme preparation, powdered organic bean cake and composite organic vegetable protein powder are used for enzymatic hydrolysis to obtain an organic vegetable protein enzymatic hydrolyzate, of which the composite organic vegetable protein powder is composed of 80% by weight of organic rice protein powder and 20% by weight of organic wheat protein powder. The specific preparation method is as follows: 1000g of powdered organic bean cake and 80g of complex organic vegetable protein powder were weighed and added to water to a dry matter concentration of 16% by weight. The mixture was then slurried in a colloid mill to form an emulsion. The resulting mixture was then enzymatically hydrolyzed with 0.2% Angel Complex protease and 0.3% papain, based on dry weight, at 50°C. The pH was maintained at 5.0-6.5 with sodium carbonate solution for 12 hours to obtain an organic vegetable protein enzymatic hydrolyzate. The Angel Complex protease, by weight, contained 40% neutral protease, 20% acid protease, 10% glucanase, and 10% cellulase.

[0126] Perlite was added to the above organic vegetable protein enzymatic hydrolyzate and mixed uniformly, then the mixture was filtered under pressure using a plate and frame filter press to obtain a filtrate, which was then concentrated under reduced pressure until the dry matter concentration reached 25% to obtain a concentrate. Finally, 10% by weight of organic maltodextrin (based on the mass of the concentrate) was added to the concentrate, which was stirred to dissolve, and then spray-dried to obtain an organic vegetable protein enzymatic hydrolyzate.

[0127] Using the method described in Example 1, the total nitrogen content and amino acid nitrogen content in the organic plant protein enzymatic hydrolyzate and the plant protein enzymatic hydrolyzate were measured, and the conversion rate of amino acid nitrogen in the organic plant protein enzymatic hydrolyzate was used as an index for determining the enzymatic hydrolysis efficiency. The results are shown in Table 3.

[0128] [Table 2]

[0129] As can be seen from Table 2, the organic solid-state fermentation enzyme preparations prepared in Examples 1 to 8 of the present invention contained neutral protease, acid protease, alkaline protease, glucoamylase, and cellulase, of which the enzymatic activity of the acid protease was 391-2792 u / g, the enzymatic activity of the neutral protease was 1752-4906 u / g, the enzymatic activity of the alkaline protease was 629-3298 u / g, the enzymatic activity of the glucoamylase was 421-1872 u / g, and the enzymatic activity of the cellulase was 131-2639 u / g. Compared to Example 6, Example 1 used a vegetable protein powder as the medium, and the enzymatic activities of the various enzymes were all much higher than those of Example 6. Compared to Examples 7 and 8, Example 1 used solid-state variable-temperature fermentation, and the enzymatic activities of the various enzymes were all higher than those of Examples 7 and 8.

[0130] [Table 3]

[0131] As can be seen from Table 3, the organic vegetable protein enzymatic hydrolyzed solutions prepared in Examples 1 to 8 of the present invention had amino acid nitrogen conversion rates of 43.44 to 73.38%, while Comparative Example 1, in which the enzymatic hydrolyzed solution was prepared by directly enzymatically hydrolyzing the plant material using an external enzyme, had an amino acid nitrogen conversion rate of only 23.72%. Compared to Example 6, Example 1, in which the medium contained vegetable protein powder, had an amino acid nitrogen conversion rate of 68.92%, and the enzymatic hydrolysis efficiency was approximately 25% higher than Example 6. Compared to Examples 7 and 8, Example 1, which utilized solid-state variable temperature fermentation, had enzymatic hydrolysis efficiencies approximately 60% and 14% higher than those of Examples 7 and 8, respectively.

[0132] Finally, it should be noted that the above preferred embodiments are intended to illustrate the technical solutions of the present invention, and are not intended to limit the scope thereof. Although the present invention has been described in detail by the above preferred embodiments, those skilled in the art will understand that various changes in form and details may be made thereto without departing from the scope of the present invention as defined by the claims.

[0133] (Addendum) (Appendix 1) The present invention provides an organic solid-state fermentation enzyme preparation produced by solid-state fermentation of a protease-producing microorganism, characterized in that the preparation contains neutral protease, acid protease, alkaline protease, glucoamylase, and cellulase.

[0134] (Appendix 2) 2. The organic solid-state fermentation enzyme preparation of claim 1, wherein the enzymatic activity of the acidic protease is greater than 390 u / g, the enzymatic activity of the neutral protease is greater than 1500 u / g, the enzymatic activity of the alkaline protease is greater than 600 u / g, the enzymatic activity of the glucoamylase is greater than 400 u / g, and the enzymatic activity of the cellulase is greater than 130 u / g.

[0135] (Appendix 3) 3. The organic solid-state fermentation enzyme preparation according to claim 2, wherein the enzymatic activity of the acidic protease is greater than 800 u / g, the enzymatic activity of the neutral protease is greater than 4000 u / g, the enzymatic activity of the alkaline protease is greater than 1000 u / g, the enzymatic activity of the glucoamylase is greater than 1500 u / g, and the enzymatic activity of the cellulase is greater than 300 u / g.

[0136] (Appendix 4) The organic solid fermentation enzyme preparation according to claim 3, wherein the enzymatic activity of the acidic protease is greater than 1200 u / g, the enzymatic activity of the alkaline protease is greater than 2200 u / g, and the enzymatic activity of the cellulase is greater than 1200 u / g.

[0137] (Appendix 5) 5. The organic solid fermentation enzyme preparation according to any one of appendices 1 to 4, wherein the protease-producing microorganisms comprise one or more of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, preferably Aspergillus oryzae and Bacillus subtilis, and preferably the weight ratio of Aspergillus oryzae to Bacillus subtilis is 70-90:10-30.

[0138] (Appendix 6) (1) culturing a protease-producing microorganism to obtain a solid seed; (2) a step of performing solid fermentation of the solid seed prepared in step (1) to obtain an organic solid-state fermentation enzyme preparation.

[0139] (Appendix 7) The solid-state fermentation is a solid-state variable temperature fermentation, and the solid-state variable temperature fermentation is performed by first fermenting at 36 to 40°C for 6 to 20 hours, followed by fermenting at 28 to 32°C for 30 to 50 hours, preferably by first fermenting at 36 to 38°C for 8 to 15 hours, followed by fermenting at 30 to 32°C for 40 to 44 hours.

[0140] (Appendix 8) 8. The method of claim 6 or 7, wherein the seed medium in step (1) and the solid fermentation medium in step (2) both contain organic starch agricultural by-products and / or organic oil-pressed raw agricultural by-products, preferably the seed medium in step (1) and the solid fermentation medium in step (2) both contain organic starch agricultural by-products and organic oil-pressed raw agricultural by-products, more preferably the content of the organic starch agricultural by-products is 9-40% by weight and the content of the organic oil-pressed raw agricultural by-products is 60-91% by weight.

[0141] (Appendix 9) 9. The method of claim 8, wherein the organic starch agricultural by-product comprises one or more of organic rice, organic wheat, and organic corn, preferably organic corn.

[0142] (Appendix 10) The method of claim 8 or 9, wherein the organic oil-pressed raw agricultural by-product comprises organic bean rice cake and / or organic rapeseed rice cake, preferably organic bean rice cake.

[0143] (Appendix 11) The method according to any one of Appendixes 6 to 10, wherein the seed medium in step (1) and the solid fermentation medium in step (2) both further comprise organic vegetable protein powder, and preferably, when the total weight of the medium is taken as 100%, the organic vegetable protein powder accounts for 6-10% and the organic starch agricultural by-products and / or organic oil-extracting raw agricultural by-products accounts for 90-94%.

[0144] (Appendix 12) 12. The method of claim 11, wherein the organic vegetable protein powder comprises organic rice protein powder and / or organic wheat protein powder, and preferably, the organic vegetable protein powder comprises, by weight percentage, 60-80% organic rice protein powder and 20-40% organic wheat protein powder.

[0145] (Appendix 13) 13. The method according to any one of Appendices 6 to 12, wherein the inoculation amount of the seed culture described in step (1) is 0.3 to 0.5% based on the weight of the seed medium.

[0146] (Appendix 14) 14. The method according to any one of claims 6 to 13, wherein the inoculum amount for the solid fermentation in step (2) is 0.3 to 0.5% by weight of the solid fermentation medium.

[0147] (Appendix 15) 15. The production method according to any one of appendices 6 to 14, further comprising a step of drying the organic solid fermentation enzyme preparation, preferably at a drying temperature of 37 to 40°C.

[0148] (Appendix 16) A method for producing an enzymatic hydrolysate of an organic vegetable protein, comprising enzymatically hydrolyzing an organic solid fermentation enzyme preparation according to any one of Appendices 1 to 5 or an organic solid fermentation enzyme preparation produced by the production method according to any one of Appendices 6 to 13, using an external enzyme.

[0149] (Appendix 17) The production method according to Appendix 16, wherein the amount of the external enzyme used is 0.2 to 0.8% based on the dry weight of the organic solid-state fermentation enzyme preparation, and preferably the external enzyme comprises one or more of papain, bromelain, neutral protease, acid protease, alkaline protease, glucanase, and cellulase, and more preferably the external enzyme comprises 0.1 to 0.3% papain, 0.04 to 0.08% neutral protease, 0.02 to 0.04% acid protease, 0.01 to 0.02% glucanase, and 0.01 to 0.02% cellulase based on the dry weight of the organic solid-state fermentation enzyme preparation.

[0150] (Appendix 18) The method for producing according to appendix 16 or 17, characterized in that the enzymatic decomposition temperature is 45 to 55°C, the enzymatic decomposition time is 10 to 15 hours, and preferably the enzymatic decomposition pH is 4.5 to 7.5.

[0151] (Appendix 19) 19. The method according to any one of claims 16 to 18, further comprising a step of slurrying the organic solid fermentation enzyme preparation before the enzymatic degradation, wherein the dry matter content of the slurry is preferably 12 to 16% by weight.

[0152] (Appendix 20) 20. The production method according to any one of Appendices 16 to 19, further comprising the steps of filtering, concentrating, and drying the enzymatic degradation product in this order.

[0153] (Appendix 21) An enzymatic hydrolysate of organic plant protein produced by the manufacturing method described in any one of Appendices 16 to 20, preferably characterized in that the total nitrogen content is greater than 9% and the amino acid nitrogen content is greater than 4.5%, based on the dry weight of the enzymatic hydrolysate of organic plant protein.

[0154] (Appendix 22) Use of an organic plant protein enzymatic hydrolysate produced by the production method described in any one of Appendixes 16 to 21 or an organic plant protein enzymatic hydrolysate described in Appendix 22 as an organic nitrogen source in the field of fermentation using yeast, lactic acid bacteria, or Bacillus subtilis.

[0155] (Appendix 23) Use of an organic vegetable protein enzymatic hydrolysate produced by the production method according to any one of Appendices 16 to 21 or an organic vegetable protein enzymatic hydrolysate according to Appendices 22 as an organic umami seasoning or basic seasoning in the food seasoning field.

[0156] (Appendix 24) Use of the organic solid-fermentation enzyme preparation according to any one of Appendices 1 to 5 or the organic solid-fermentation enzyme preparation produced by the production method according to any one of Appendices 6 to 15 as an organic protease in the enzymatic hydrolysis of vegetable proteins.

Claims

1. The present invention provides an organic solid-state fermentation enzyme preparation produced by solid-state fermentation of a protease-producing microorganism, characterized in that the preparation contains neutral protease, acid protease, alkaline protease, glucoamylase, and cellulase.

2. 2. The organic solid fermentation enzyme preparation according to claim 1, wherein the enzymatic activity of the acidic protease is greater than 390 u / g, the enzymatic activity of the neutral protease is greater than 1500 u / g, the enzymatic activity of the alkaline protease is greater than 600 u / g, the enzymatic activity of the glucoamylase is greater than 400 u / g, and the enzymatic activity of the cellulase is greater than 130 u / g.

3. 3. The organic solid fermentation enzyme preparation according to claim 2, wherein the enzymatic activity of the acidic protease is greater than 800 u / g, the enzymatic activity of the neutral protease is greater than 4000 u / g, the enzymatic activity of the alkaline protease is greater than 1000 u / g, the enzymatic activity of the glucoamylase is greater than 1500 u / g, and the enzymatic activity of the cellulase is greater than 300 u / g.

4. 4. The organic solid fermentation enzyme preparation according to claim 3, wherein the enzymatic activity of the acidic protease is greater than 1200 u / g, the enzymatic activity of the alkaline protease is greater than 2200 u / g, and the enzymatic activity of the cellulase is greater than 1200 u / g.

5. The organic solid fermentation enzyme preparation according to any one of claims 1 to 4, wherein the protease-producing microorganisms comprise one or more of Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis, preferably Aspergillus oryzae and Bacillus subtilis, and preferably the weight ratio of Aspergillus oryzae to Bacillus subtilis is 70-90:10-30.

6. (1) culturing a protease-producing microorganism to obtain a solid seed; (2) performing solid fermentation of the solid seed prepared in step (1) to obtain an organic solid fermentation enzyme preparation. A method for producing an organic solid fermentation enzyme preparation according to any one of claims 1 to 5.

7. The solid-state fermentation is a solid-state variable temperature fermentation, which is performed by first fermenting at 36-40°C for 6-20 hours, and then fermenting at 28-32°C for 30-50 hours, preferably by first fermenting at 36-38°C for 8-15 hours, and then fermenting at 30-32°C for 40-44 hours. The method of claim 6,

8. 8. The method of claim 6 or 7, wherein the seed medium in step (1) and the solid fermentation medium in step (2) both comprise organic starch agricultural by-products and / or organic oil-pressed raw agricultural by-products, preferably the seed medium in step (1) and the solid fermentation medium in step (2) both comprise organic starch agricultural by-products and organic oil-pressed raw agricultural by-products, more preferably the content of the organic starch agricultural by-products is 9-40% by weight and the content of the organic oil-pressed raw agricultural by-products is 60-91% by weight.

9. 9. The method of claim 8, wherein the organic starch agricultural by-product comprises one or more of organic rice, organic wheat and organic corn, preferably organic corn.

10. The method according to claim 8 or 9, wherein the organic oil-pressed raw agricultural by-product comprises organic bean cake and / or organic rapeseed cake, preferably organic bean cake.

11. The method according to any one of claims 6 to 10, characterized in that the seed medium in step (1) and the solid fermentation medium in step (2) both further contain organic vegetable protein powder, preferably, when the total weight of the medium is taken as 100%, the organic vegetable protein powder accounts for 6-10% and the organic starch agricultural by-products and / or organic oil-extracting raw agricultural by-products accounts for 90-94%.

12. 12. The method of claim 11, wherein the organic vegetable protein powder comprises organic rice protein powder and / or organic wheat protein powder, and preferably, the organic vegetable protein powder comprises, by weight percentage, 60-80% organic rice protein powder and 20-40% organic wheat protein powder.

13. The method according to any one of claims 6 to 12, wherein the inoculation amount of the seed culture in step (1) is 0.3 to 0.5% by weight of the seed medium.

14. The method according to any one of claims 6 to 13, wherein the inoculation amount of the solid fermentation in step (2) is 0.3 to 0.5% by weight of the solid fermentation medium.

15. The method according to any one of claims 6 to 14, further comprising a step of drying the organic solid fermentation enzyme preparation, wherein the drying temperature is preferably 37 to 40°C.

16. The organic solid fermentation enzyme preparation according to any one of claims 1 to 5, which is produced by using an exogenous enzyme. Or, a method for producing an enzymatic hydrolyzed organic plant protein, comprising enzymatically hydrolyzing an organic solid fermentation enzyme preparation produced by the production method according to any one of claims 6 to 13.

17. The amount of the external enzyme used is 0.2 to 0.8% based on the dry weight of the organic solid fermentation enzyme preparation, and preferably the external enzyme comprises one or more of papain, bromelain, neutral protease, acid protease, alkaline protease, glucanase, and cellulase, and more preferably the external enzyme comprises 0.1 to 0.3% papain, 0.04 to 0.08% neutral protease, 0.02 to 0.04% acid protease, 0.01 to 0.02% glucanase, and 0.01 to 0.02% cellulase based on the dry weight of the organic solid fermentation enzyme preparation. The production method according to claim 16, characterized in that

18. The method according to claim 16 or 17, wherein the enzymatic decomposition temperature is 45 to 55°C, the enzymatic decomposition time is 10 to 15 hours, and the enzymatic decomposition pH is preferably 4.5 to 7.

5.

19. The method according to any one of claims 16 to 18, further comprising a step of slurrying the organic solid fermentation enzyme preparation before the enzymatic degradation, preferably characterized in that the dry matter content of the slurry is 12 to 16% by weight percent at the time of the slurrying.

20. The method according to any one of claims 16 to 19, further comprising the steps of filtering, concentrating and drying the enzymatic degradation product in this order.

21. An enzymatic hydrolysate of organic plant protein produced by the method of any one of claims 16 to 20, preferably characterized in that the total nitrogen content is greater than 9% and the amino acid nitrogen content is greater than 4.5%, based on the dry weight of the enzymatic hydrolysate of organic plant protein.

22. Use of an organic plant protein enzymatic hydrolysate produced by the production method according to any one of claims 16 to 21 or an organic plant protein enzymatic hydrolysate according to claim 22 as an organic nitrogen source in the field of fermentation using yeast, lactic acid bacteria, or Bacillus subtilis.

23. Use of an organic vegetable protein enzymatic hydrolysate produced by the production method according to any one of claims 16 to 21 or an organic vegetable protein enzymatic hydrolysate according to claim 22 as an organic umami seasoning or basic seasoning in the field of food seasoning.

24. Use of the organic solid fermentation enzyme preparation according to any one of claims 1 to 5 or the organic solid fermentation enzyme preparation produced by the production method according to any one of claims 6 to 15 as an organic protease in the enzymatic degradation of vegetable proteins.

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