Environment-friendly high-water-solubility low-permeability soybean peptide protein prepared through solid state fermentation and application of high-water-solubility low-permeability soybean peptide protein

Abstract

The invention discloses environment-friendly high-water-solubility low-permeability soybean peptide protein prepared through solid state fermentation and application, and relates to the technical field of biological fermention.The preparation method specifically comprises the following steps that firstly, soybean meal is pretreated, then zymophyte is inoculated for solid state fermentation, and a fermentation product is obtained; then carrying out flash evaporation drying on the fermentation product to obtain a dried product; dissolving the dried product in water, and carrying out centrifugal separation to respectively obtain a supernatant and a precipitate; and finally, carrying out low-temperature vacuum drying on the supernate and the precipitate to respectively obtain high-water-solubility low-permeability soybean peptide protein and functional byproducts. According to the environment-friendly high-water-solubility low-permeability soybean peptide protein prepared through solid state fermentation, a chemical-reagent-free enzymolysis technology is adopted, the pH value is naturally maintained in the fermentation process, a low-energy-consumption separation technology is combined, the problems that in a traditional chemical method, pollution is high, the COD emission amount is only 0.09 ton / ton, chloride residues are smaller than 0.1 ppm are solved, and the environment-friendly advantage is remarkable.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation technology, and in particular to a highly water-soluble and low-osmotic soybean peptide protein prepared by environmentally friendly solid-state fermentation and its application. Background Art

[0002] Soybean meal is a by-product of soybean oil extraction. It is rich in protein, fat, carbohydrates and a variety of essential amino acids, and is a high-quality plant-based protein feed source. However, due to its large molecular weight and complex structure, its protein has a low digestion and absorption rate when fed directly to animals, resulting in a waste of resources and increased costs. Existing soybean peptide preparation mainly relies on chemical acids and alkalis (such as hydrochloric acid and sodium hydroxide) to regulate the pH of enzymatic hydrolysis. This process not only produces a large amount of chlorine-containing wastewater, causing serious environmental pollution, but also limits the quality of the resulting protein product. The proportion of peptides <1000Da is low, the osmotic pressure is high, and it cannot meet the needs of specific applications.

[0003] Compared to traditional chemical enzymatic hydrolysis, microbial fermentation can enhance the utilization value of soybean meal. Respiratory, fermentative, and hydrolytic enzymes secreted by microorganisms can degrade soybean meal protein into small peptides, significantly improving its nutritional properties and expanding its application range. Although microbial solid-state fermentation technology has demonstrated advantages in reducing chemical pollution, existing technologies still suffer from strong strain dependence, poor universality, insufficient protease activity of a single fermenting bacterium, difficulty in naturally maintaining pH during the fermentation process, imprecise control of product water solubility, high osmotic pressure, and low by-product utilization efficiency. This leads to significant resource waste and limits its application in large-scale industrial feed production. Therefore, it is urgent to develop an environmentally friendly solid-state fermentation-prepared, highly water-soluble, low-osmotic soybean peptide protein and its application to ensure the environmentally friendly and efficient development of soybean peptide protein production and application. Summary of the Invention

[0004] The purpose of the present invention is to provide an environmentally friendly solid-state fermentation-prepared highly water-soluble and low-osmotic soybean peptide protein and its application, so as to solve the shortcomings of the existing soybean peptide solid-state fermentation technology, such as strong strain dependence, poor universality, imprecise control of product water solubility, high osmotic pressure, low by-product utilization efficiency, and waste of resources.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing highly water-soluble and low-osmotic soybean peptide protein prepared by environmentally friendly solid-state fermentation, which specifically comprises the following steps:

[0006] S1. First, pretreating the soybean meal, and then inoculating fermentation bacteria to perform solid-state fermentation on the pretreated soybean meal to obtain a fermentation product;

[0007] S2. flash drying the fermentation product to obtain a dried product;

[0008] S3, dissolving the dried product in water to obtain a fermentation mixture, then centrifuging the fermentation mixture to obtain a supernatant and a precipitate respectively;

[0009] S4. The supernatant and the precipitate are respectively subjected to low-temperature vacuum drying to obtain highly water-soluble low-osmotic soybean peptide protein and functional by-products.

[0010] Furthermore, the method for pre-treating the soybean meal in S1 is to grind the soybean meal and then pass it through an 80-mesh sieve.

[0011] Furthermore, the fermentation bacteria in S1 is at least one of Bacillus velezensis, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus lentus, Bacillus laterosporus, and Saccharomyces cerevisiae.

[0012] Furthermore, the fermentation time of the solid-state fermentation in S1 is set to 24-28 hours, the fermentation temperature is set to 30-37° C., and the system pH is set to 6.0-7.5.

[0013] Furthermore, the air inlet temperature of the flash drying in S2 is set to 120-150°C, and the air outlet temperature is set to 60-80°C.

[0014] Furthermore, the weight-to-volume ratio of the dried product to water in S3 is 1-10:10; and the centrifugal speed in S3 is set to 3000-5000 rpm.

[0015] Furthermore, the low-temperature vacuum drying temperature in S4 is set to 40-50° C., and the pressure is set to -0.1-0.01 MPa.

[0016] The present invention also discloses a highly water-soluble low-osmotic soybean peptide protein, which is prepared by the aforementioned preparation method. The crude protein content is 65% to 83%, the peptide segments less than 1000 Da account for 70% to 76%, and the osmotic pressure is 220 to 300 mOsm / kg.

[0017] Furthermore, the crude protein content of the functional by-product is 30% to 34%, the dietary fiber content is 31% to 33%, and the water-soluble fiber content is 50% to 52%.

[0018] The invention also discloses application of highly water-soluble low-osmotic soybean peptide protein in animal feed.

[0019] Compared with the prior art, the highly water-soluble and low-osmotic soybean peptide protein prepared by environmentally friendly solid-state fermentation and its application provided by the present invention have the following beneficial effects:

[0020] 1. This invention utilizes a composite bacterial system of Bacillus Velez-Zeitung and Bacillus subtilis to achieve synergistic effects, breaking through the performance bottleneck of a single bacterial strain and achieving complementary protease activity. The hydrolysis rate is significantly increased to 30%, a 15% increase over traditional technologies, and the peptide protein yield is increased by 8%. Furthermore, small molecule peptides (<1000Da) account for 70% to 76% of the product, and the osmotic pressure is controlled at 220 to 300 mOsm / kg, a 20% to 30% improvement over existing technologies.

[0021] 2. This invention uses chemical-free enzymatic hydrolysis technology, utilizes the fermentation process to naturally maintain pH, and combines it with a low-energy separation process to avoid the high pollution levels of traditional chemical methods. COD emissions are only 0.09 tons per ton of raw material (compared to 2.5 tons for traditional methods), and chloride residues are less than 0.1 ppm (compared to 85 ppm for traditional methods). This offers significant environmental advantages and considerable ecological benefits.

[0022] 3. The highly water-soluble and low-osmotic soybean peptide protein prepared by the method of the present invention is more easily absorbed by organisms, and the functional by-products are rich in dietary fiber. Adding them can improve the quality of pet food, and is suitable for high-end markets such as young animal feed and improved pet food functions. The investment in solid-state fermentation equipment is reduced by 40% compared with the liquid process, the energy consumption of flash drying is reduced by 55%, and the profit per ton of product is increased by 500 yuan. It is suitable for low-cost transformation of small and medium-sized enterprises, has strong market competitiveness, and high economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0024] Figure 1 A process flow chart of the preparation method of highly water-soluble and low-osmotic soybean peptide protein provided by an embodiment of the present invention;

[0025] Figure 2 A diagram showing changes in pH during solid-state fermentation of a highly water-soluble, low-osmotic soybean peptide protein according to an embodiment of the present invention;

[0026] Figure 3 This is an HPLC test chart of the molecular weight distribution of highly water-soluble and low-osmotic soybean peptide protein provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Comparative Example 1: A method for preparing soybean peptide protein by fermentation with a single brewer's yeast, specifically comprising the following steps:

[0029] S1. First, 100 kg of soybean meal was crushed, and then passed through an 80-mesh sieve. Then, Saccharomyces cerevisiae CGMCC2.1189 was inoculated. The soybean meal after passing through the 80-mesh sieve was subjected to solid-state fermentation. The fermentation time of the solid-state fermentation was set to 24 h, the fermentation temperature was set to 37° C., and the pH of the system was naturally maintained at 6.0-7.5 to obtain a fermentation product;

[0030] The above-mentioned Saccharomyces cerevisiae CGMCC2.1189 contains 520 U / g of neutral protease and 310 U / g of acid protease.

[0031] S2. Flash drying the fermentation product with the air inlet temperature set to 140° C. and the air outlet temperature set to 70° C. to obtain a dried product.

[0032] S3. The dried product was dissolved in water at a weight-to-volume ratio of the dried product to water of 1:1.8 to obtain a fermentation mixture, and then the fermentation mixture was centrifuged at 4000 rpm for 20 min, separated after centrifugation, and finally a supernatant was obtained.

[0033] S4. The supernatant is subjected to low-temperature vacuum drying, the low-temperature vacuum drying temperature is set to 45° C., and the pressure is set to -0.08 MPa, to obtain soybean peptide protein.

[0034] The soybean peptide protein has a crude protein content of 58.2%, peptide segments less than 1000 Da account for 48.3%, an osmotic pressure of 352 mOsm / kg, a peptide protein yield of 25.6%, and a hydrolysis degree of 18%.

[0035] Example 1:

[0036] See also Figure 1 A method for preparing highly water-soluble and low-osmotic soybean peptide protein prepared by environmentally friendly solid-state fermentation, specifically comprising the following steps:

[0037] S1. First, soybean meal is pretreated, and then fermentation bacteria is inoculated to perform solid-state fermentation on the pretreated soybean meal to obtain a fermentation product; the method for pretreating the soybean meal is to crush the soybean meal and then pass it through an 80-mesh sieve; the fermentation bacteria is at least one of Bacillus velez, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus lentus, Bacillus laterosporus, and Saccharomyces cerevisiae; the fermentation time of the solid-state fermentation is set to 24 to 28 hours, the fermentation temperature is set to 30 to 37° C., and the system pH is set to 6.0 to 7.5.

[0038] The specific implementation method is as follows: first, 100 kg of soybean meal is crushed, then passed through an 80-mesh sieve, and then inoculated with Bacillus Velez CCTCC M 20251162. The soybean meal after passing through the 80-mesh sieve is subjected to solid-state fermentation. The fermentation time of the solid-state fermentation is set to 26 hours, the fermentation temperature is set to 35°C, no chemical acid or alkali reagent is added throughout the process, and the pH of the system is naturally maintained at 6.0-7.5 to obtain a fermentation product.

[0039] The above-mentioned Bacillus Velez CCTCC M 20251162 contains 800 U / g of neutral protease and 500 U / g of acid protease.

[0040] S2. Flash-dry the fermentation product to obtain a dried product; the flash drying air inlet temperature is set to 120-150° C., and the air outlet temperature is set to 60-80° C.

[0041] The specific implementation method is to flash dry the fermentation product, set the air inlet temperature to 130° C., and set the air outlet temperature to 60° C. to obtain a dried product.

[0042] The air outlet temperature can also be set to 65℃, 70℃, 75℃, and 80℃.

[0043] S3. Dissolving the dried product in water to obtain a fermentation mixture, then centrifuging the fermentation mixture to obtain a supernatant and a precipitate, respectively; the weight-to-volume ratio of the dried product to water is 1-10:10; and the centrifugal speed in S3 is set to 3000-5000 rpm.

[0044] The specific implementation method is to dissolve the dry product in water with a weight volume ratio of the dry product to water of 1:1.8 to obtain a fermentation mixture, and then centrifuge the fermentation mixture at 4000 rpm for 20 minutes, separate after centrifugation, and finally obtain a supernatant and a precipitate respectively.

[0045] S4. The supernatant and the precipitate are respectively subjected to low-temperature vacuum drying to obtain a highly water-soluble low-osmotic soybean peptide protein and a functional by-product, respectively. The low-temperature vacuum drying temperature is set at 40 to 50° C. and the pressure is set at -0.1 to -0.01 MPa.

[0046] The specific implementation method is to perform low-temperature vacuum drying on the supernatant and the precipitate respectively, wherein the low-temperature vacuum drying temperature is set to 45° C. and the pressure is set to -0.08 MPa, to obtain highly water-soluble low-osmotic soybean peptide protein and functional by-products respectively.

[0047] The highly water-soluble low-osmotic soybean peptide protein has a crude protein content of 68.5%, peptide segments less than 1000 Da account for 76.1%, an osmotic pressure of 285 mOsm / kg, water-soluble protein accounts for 45%, and a peptide protein yield of 31.2%.

[0048] The crude protein content of the functional by-product is 30.7%, the dietary fiber content is 31.3%, the water-soluble fiber content is 51%, and the water holding capacity is 3.6 g / g.

[0049] Example 2:

[0050] See also Figures 1 to 3 This embodiment provides a technical solution based on the first embodiment, which specifically includes the following steps:

[0051] S1. First, 100 kg of soybean meal is crushed, then passed through an 80-mesh sieve, and then inoculated with a composite bacterial liquid of Bacillus subtilis and Bacillus velez. The soybean meal after passing the 80-mesh sieve is subjected to solid-state fermentation. The fermentation time of the solid-state fermentation is set to 24 hours, the fermentation temperature is set to 37°C, and no chemical acid or alkali reagents are added throughout the process. The pH of the system is naturally maintained at 6.0-7.5 to obtain a fermentation product.

[0052] The composite bacterial solution is a mixture of Bacillus subtilis and Bacillus velez in a volume ratio of 1:1, and the total protease activity meets the hydrolysis degree of 30%.

[0053] S2. Flash drying the fermentation product with the air inlet temperature set to 140° C. and the air outlet temperature set to 80° C. to obtain a dried product.

[0054] S3. The dried product was dissolved in water at a weight-to-volume ratio of the dried product to water of 1:1.7 to obtain a fermentation mixture, and then the fermentation mixture was centrifuged at 4000 rpm for 20 min. After centrifugation, the supernatant and the precipitate were respectively obtained.

[0055] S4. The supernatant and the precipitate are respectively subjected to low-temperature vacuum drying, the low-temperature vacuum drying temperature is set to 45° C., and the pressure is set to -0.08 MPa, to obtain highly water-soluble low-osmotic soybean peptide protein and functional by-products, respectively.

[0056] The highly water-soluble low-osmotic soybean peptide protein has a crude protein content of 75.8%, peptide segments less than 1000 Da account for 76.5%, an osmotic pressure of 278 mOsm / kg, water-soluble protein accounts for 52%, and a peptide protein yield of 32.8%.

[0057] The crude protein content of the functional by-product is 31.5%, the dietary fiber content is 31.8%, the water-soluble fiber content is 50.8%, and the water holding capacity is 2.9 g / g.

[0058] Table 1 shows the results of chromatography-molecular weight analysis, which displays the chromatographic information such as retention time, molecular weight distribution, peak area, peak area percentage, and peak height of the eight components in the sample. Figure 3 The HPLC test chart of peptide protein molecular weight distribution is shown in Figure 2. Figure 3As shown in the results of Table 1, peptide segments less than 1000 Da account for more than 76%, proving that they are the main components of highly water-soluble, low-osmotic soybean peptide protein, suitable for rapid absorption, and beneficial for application in sports nutrition and young animal feed; while the high molecular weight region (>2000 Da) accounts for only 21.53%, indicating that the preparation method provided by the present invention has an efficient hydrolysis process and extremely low residual macromolecular protein.

[0059] Table 1 Peptide protein chromatography-molecular weight analysis results

[0060]

[0061]

[0062] In order to study the effect of microorganisms on the pH of the reaction system during solid-state fermentation, 2g of fermented soybean meal sample was taken into a beaker, 100mL of distilled water was added, and the solution was fully dissolved. The pH was then accurately measured using a calibrated pH meter to observe the pH changes during the fermentation process. The pH time curve during the fermentation process is shown in Figure 2. Figure 2 As shown in the figure, the pH of the system remained around 7 throughout the solid-state fermentation process, indicating that the synergistic effect of Bacillus Velez and Bacillus subtilis can stably maintain the required pH for enzymatic hydrolysis without the addition of any chemical reagents. This pH change curve fully demonstrates that microbial metabolism and substrate conversion can achieve a dynamic balance during the fermentation process, providing a key parameter basis for the natural maintenance of the system pH.

[0063] In addition, as shown in Table 2, compared with the traditional chemical enzymatic hydrolysis method, the indices of the highly water-soluble and low-osmotic soybean peptide protein product prepared in this example are significantly improved.

[0064] Table 2 Comparative analysis of process performance of Example 2 and traditional chemical enzymatic hydrolysis

[0065] index Example 2 Traditional chemical enzymatic hydrolysis Peptide protein yield (%) 32.8 - Peptide protein crude protein (%) 75.8 68.5 <1000Da peptides (%) 76.5 48.2 Osmolality (mOsm / kg) 278 350 COD emissions (tons / tons of raw materials) 0.09 2.5 Chloride residue (ppm) ＜0.1 85

[0066] As can be seen from Table 2, the crude protein content of Example 2 is 75.8%, which is higher than 68.5% of the traditional method, indicating that the process of Example 2 has a stronger ability to enrich protein and a higher product purity; the small molecule peptide segment of Example 2 accounts for 76.5%, far exceeding the 48.2% of the traditional method. Small molecule peptide segments are more easily absorbed by organisms and can improve the bioavailability or functional activity of the product; the osmotic pressure of Example 2 is 278mOsm / kg, which is significantly lower than 350mOsm / kg of the traditional method. Low osmotic pressure can reduce osmotic damage to cells or tissues and is suitable for pet feed; the COD emission of Example 2 is only 0.09 tons / ton of raw material, which is only 3.6% of the traditional method (2.5 tons / ton of raw material), indicating that the process of Example 2 has extremely low water pollution and meets green environmental protection requirements; the chloride residue of Example 2 is <0.1ppm, which is much lower than 85ppm of the traditional method. Low residue can reduce the risk of equipment corrosion, reduce environmental pollution, and improve product safety.

[0067] Table 3 Wastewater discharge and pollutant emissions of the project

[0068]

[0069] Table 3 shows the wastewater discharge and pollutant discharge of the project. It can be seen that the preparation method of highly water-soluble low-osmotic soybean peptide protein provided by the present invention generates a production wastewater discharge of 1,568.6m 3 / a. After treatment at the terminal sewage treatment plant, the COD emission concentration is 50 mg / L, meeting the Level A standard (COD ≤ 50 mg / L) of the Pollutant Discharge Standard for Municipal Wastewater Treatment Plants (GB18918-2002); the NH3-N emission concentration is 5 mg / L, meeting the Level A standard (NH3-N ≤ 5 mg / L (water temperature > 12°C)). The compliance of pollutant concentrations in the production wastewater indicates that the production process can effectively control organic matter and ammonia nitrogen emissions.

[0070] In addition, COD emissions are: 1568.6m 3 / a×50mg / L×10 -6 =0.078t / a, far below the implicit limit of 0.1t / a; annual NH3-N emissions are: 1568.6m 3 / a×5mg / L×10 -6 =0.0078t / a, which is significantly lower than the implicit limit of 0.01t / a. The emission volume is extremely low, and the impact on the receiving water environment is controllable, which meets the environmental protection requirements.

[0071] In summary, the highly water-soluble and low-osmotic soybean peptide protein preparation process provided by the embodiments of the present invention is significantly superior to the traditional chemical enzymatic hydrolysis method in terms of peptide protein yield, product purity, small molecule peptide segment ratio, environmental protection (low COD and chloride) and osmotic pressure control. It has the technical advantages of high efficiency, environmental protection and safety, and is suitable for industrial promotion and application.

[0072] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A method for preparing highly water-soluble low-osmotic soybean peptide protein prepared by environmentally friendly solid-state fermentation, characterized in that: The specific steps include: S1. First, pretreating the soybean meal, and then inoculating fermentation bacteria to perform solid-state fermentation on the pretreated soybean meal to obtain a fermentation product; S2. flash drying the fermentation product to obtain a dried product; S3, dissolving the dried product in water to obtain a fermentation mixture, then centrifuging the fermentation mixture to obtain a supernatant and a precipitate respectively; S4. The supernatant and the precipitate are respectively subjected to low-temperature vacuum drying to obtain highly water-soluble low-osmotic soybean peptide protein and functional by-products.

2. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method according to claim 1, characterized in that: The method for pre-treating the soybean meal in S1 is to grind the soybean meal and then pass it through an 80-mesh sieve.

3. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method according to claim 1, characterized in that: The fermentation bacteria in S1 is at least one of Bacillus velezensis, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus lentus, Bacillus laterosporus, and Saccharomyces cerevisiae.

4. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method according to claim 1, characterized in that: The fermentation time of the solid-state fermentation in S1 is set to 24-28 hours, the fermentation temperature is set to 30-37° C., and the system pH is set to 6.0-7.

5.

5. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method according to claim 1, characterized in that: In the flash drying step S2, the air inlet temperature is set to 120-150°C, and the air outlet temperature is set to 60-80°C.

6. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method according to claim 1, characterized in that: The weight-to-volume ratio of the dried product to water in S3 is 1-10:10; the centrifugal speed in S3 is set to 3000-5000 rpm.

7. The method for preparing a highly water-soluble and low-osmotic soybean peptide protein prepared by solid-state fermentation according to claim 1, characterized in that: The low-temperature vacuum drying temperature in S4 is set to 40-50° C., and the pressure is set to -0.1-0.01 MPa.

8. A highly water-soluble low-osmotic soybean peptide protein, characterized in that: The invention is applicable to the preparation method of the highly water-soluble and low-osmotic soybean peptide protein prepared by an environmentally friendly solid-state fermentation method as described in any one of claims 1 to 7, wherein the crude protein content is 65% to 83%, the peptide segments less than 1000 Da account for 70% to 76%, and the osmotic pressure is 220 to 300 mOsm / kg.

9. A highly water-soluble low-osmotic soybean peptide protein according to claim 8, characterized in that: The crude protein content of the functional by-product is 30% to 34%, the dietary fiber content is 31% to 33%, and the water-soluble fiber content is 50% to 52%.

10. Use of the highly water-soluble and low-osmotic soybean peptide protein according to claim 8 in animal feed.

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