Preparation method of xanthoceras sorbifolia bunge protein peptide
Through a preparation method including pretreatment, subcritical extraction and enzymatic lysis, the problem of low yield of Wenguan Fructose protein peptide is solved, the yield and biological activity of protein peptides is improved, and its application in related industries has been promoted.
Patent Information
- Application Number
- CN202510119856.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-25
- Publication Date
- 2025-04-25
AI Technical Summary
The yield of the existing technology of Chinese cinnabar protein peptide is relatively low, which has affected its application prospects in industries such as food, medicine and health products.
A preparation method including pretreatment, subcritical extraction and enzymatic lysis is used. The specific steps include: mixing venison fruit with triethanolamine oleate and isoamyl alcohol for pretreatment, obtaining the oil meal through subcritical extraction, and adding protease at an appropriate pH value and temperature for enzymatic decomposition, and finally obtaining venison fruit protein peptide.
This method significantly improves the yield of Wenguan Fructose peptide, reduces protein loss, and improves the biological activity and application efficiency of the product.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of Xanthoceras sorbifolia protein peptides, and in particular to a method for preparing Xanthoceras sorbifolia protein peptides. Background Art
[0002] Xanthoceras sorbifolia is a deciduous shrub or small tree of the genus Xanthoceras in the Sapindaceae family, widely distributed in northern China. It has strong adaptability and can effectively prevent wind and sand, and conserve water and soil. The seeds of Xanthoceras sorbifolia can be called "treasures". Not only can high-quality edible oil be extracted, but the oil it contains can also be used in the preparation of biodiesel, playing an important role in the energy field. Xanthoceras sorbifolia is rich in protein. The Xanthoceras sorbifolia protein peptides obtained by further hydrolysis of these proteins have multiple biological activities, which can lower blood pressure, enhance immunity, and help the human body resist disease invasion. It has broad application prospects in industries such as food, medicine, and health products.
[0003] At present, the common method for preparing Xanthoceras sorbifolia protein peptides is the alkaline extraction and acid precipitation method. Although this method has a relatively simple process and can achieve protein dissolution and precipitation by adjusting the pH of the solution, the alkaline environment easily causes some proteins to lose their biological activity, reducing the final protein peptide yield and affecting the efficacy of subsequent protein products. The other is the enzymatic method, which has gradually evolved into a water enzyme method. The water enzyme method has the advantages of mild conditions, less protein denaturation, and safe operation, but the price of the enzyme currently used is relatively high, and the production cost is relatively high. In addition, for Xanthoceras sorbifolia, directly treating Xanthoceras sorbifolia with the water enzyme method will obtain oil and protein at the same time, which will increase the difficulty of subsequent separation and purification of Xanthoceras sorbifolia protein, thereby affecting the yield of Xanthoceras sorbifolia protein peptides.
[0004] Therefore, a method for preparing Xanthoceras sorbifolia protein peptide is needed to improve the yield of Xanthoceras sorbifolia protein peptide, which is of great significance for promoting the development of Xanthoceras sorbifolia protein peptide industry. Summary of the invention
[0005] The invention provides a method for preparing Xanthoceras sorbifolia protein peptide, which solves the problem of low yield of Xanthoceras sorbifolia protein peptide in the related art.
[0006] The technical solution of the present invention is as follows: The present invention provides a method for preparing Xanthoceras sorbifolia protein peptide, comprising the following steps: S1. Mixing Xanthoceras sorbifolia and a reagent, soaking, and separating to obtain a pretreated product; S2, subjecting the pretreated product to subcritical extraction and unloading to obtain oil meal; S3, adding water to the oil meal, adjusting the pH to 6.8-7.5, adding protease, enzymolysis, separation, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagents include triethanolamine oleate and isoamyl alcohol.
[0007] As a further technical solution, the volume ratio of triethanolamine oleate to isoamyl alcohol is 1:3-4.
[0008] As a further technical solution, the reagent also includes an aqueous solution of sodium cocoyl isethionate.
[0009] As a further technical solution, the mass fraction of the sodium cocoyl isethionate aqueous solution is 50% to 65%.
[0010] In the present invention, an aqueous solution of sodium cocoyl isethionate is introduced into the reagent in step S1, which increases the action of triethanolamine oleate and isoamyl alcohol, and can strengthen the penetration of triethanolamine oleate and isoamyl alcohol into Xanthoceras sorbifolia, making the structure of Xanthoceras sorbifolia more loose, facilitating subsequent enzymatic hydrolysis, thereby further improving the yield of Xanthoceras sorbifolia protein peptides.
[0011] As a further technical solution, the volume ratio of the sum of the volumes of the triethanolamine oleate and isoamyl alcohol to the volume of the sodium cocoyl isethionate aqueous solution is 4-9:1.
[0012] When the volume ratio of triethanolamine oleate and isopentanol to the volume ratio of sodium cocoyl isethionate aqueous solution is 4-9:1, this ratio can optimize the synergistic effect of the three reagents and further improve the yield of Xanthoceras sorbifolia protein peptide. However, when the volume ratio of triethanolamine oleate and isopentanol to the volume ratio of sodium cocoyl isethionate aqueous solution is outside the range of 4-9:1, the synergistic effect of the three reagents decreases.
[0013] As a further technical solution, the protease includes Bacillus subtilis neutral protease.
[0014] As a further technical solution, the protease also includes bromelain.
[0015] In the present invention, bromelain is introduced into the protease. When the Bacillus subtilis neutral protease and bromelain act together, the action sites of the two enzymes can complement each other and regulate the process and degree of enzymolysis, so that the protein can be decomposed more finely, thereby increasing the probability of generating small molecule protein peptides.
[0016] As a further technical solution, the mass ratio of the Bacillus subtilis neutral protease to the bromelain is 1-3:1.
[0017] When the mass ratio of Bacillus subtilis neutral protease and bromelain is 1-3:1, the probability of generating small molecule protein peptides can be further improved.
[0018] As a further technical solution, in step S1, the soaking temperature is 40-50°C and the soaking time is 1-2 hours.
[0019] As a further technical solution, in step S1, after the separation, it also includes drying and crushing.
[0020] As a further technical solution, in step S1, the particle size of the pretreated material is 325-400 mesh.
[0021] As a further technical solution, in step S2, liquid butane is used during the subcritical extraction, and the mass ratio of the liquid butane to the pretreated material is 2.5-3.5:1.
[0022] The mass ratio of liquid butane to pretreated material is limited to 2.5~3.5:1. At this ratio, the liquid butane is guaranteed to be in full contact with the pretreated material, making the oil extraction more thorough, increasing the relative content of Xanthoceras sorbifolia protein in the oil meal, and laying the foundation for the subsequent increase in the yield of Xanthoceras sorbifolia protein peptides.
[0023] As a further technical solution, in step S2, during the subcritical extraction, the extraction temperature is 25-32°C, the extraction pressure is 0.9-1.4 MPa, and the extraction time is 2-4 h.
[0024] In the present invention, the pretreated Xanthoceras sorbifolia is subjected to subcritical extraction to obtain the oil meal of Xanthoceras sorbifolia, and then the Xanthoceras sorbifolia protein is extracted from the oil meal, which has many advantages compared to directly extracting protein from Xanthoceras sorbifolia. On the one hand, subcritical extraction can efficiently separate the oil in Xanthoceras sorbifolia to obtain relatively pure oil meal, reduce the interference of impurities on protein extraction and enzymatic hydrolysis, and help to improve the yield of Xanthoceras sorbifolia protein peptides. On the other hand, the subcritical extraction process has mild conditions, causes less damage to the protein structure in Xanthoceras sorbifolia, and can retain the activity of the protein to the greatest extent.
[0025] As a further technical solution, in step S3, the adding of the protease is divided into a first adding and a second adding; When adding for the first time, 2 / 3 of the mass of protease is added, the enzymolysis time is 1-2 hours, and the enzymolysis temperature is 35-40°C; During the second addition, the remaining protease is added, the enzymolysis time is 0.5-1h, and the enzymolysis temperature is 35-40°C.
[0026] During the enzymatic hydrolysis process, a step-by-step addition method is adopted. The amount of enzyme added for the first time is relatively sufficient, which can quickly perform a preliminary decomposition of the Xanthoceras sorbifolia protein in the oil meal. The remaining enzyme is added for the second enzymatic hydrolysis, which can further process the incompletely enzymatically hydrolyzed protein and make the enzymatic hydrolysis more thorough. This step-by-step addition method can better control the enzymatic hydrolysis process, improve the enzymatic hydrolysis efficiency, and avoid the waste and excessive enzymatic hydrolysis caused by adding too much enzyme at one time.
[0027] As a further technical solution, in step S1, the mass volume ratio of the Xanthoceras sorbifolia to the reagent is 10 g: 80-100 mL; In step S3, the amount of water added is 8 to 10 times the mass of the oil meal, and the amount of protease added is 0.03% to 0.12% of the mass of the Xanthoceras sorbifolia.
[0028] The working principle and beneficial effects of the present invention are: In the present invention, in the process of obtaining Xanthoceras sorbifolia protein peptide, the Xanthoceras sorbifolia is pretreated, then subjected to subcritical extraction, and then coordinated with an effective enzymatic hydrolysis process, and the yield of the Xanthoceras sorbifolia protein peptide is improved under the effective coordination of each process. Among them, the Xanthoceras sorbifolia is pretreated in triethanolamine oleate and isoamyl alcohol, and the synergistic use of triethanolamine oleate and isoamyl alcohol can provide a relatively stable environment for the protein, reduce the protein loss in the Xanthoceras sorbifolia, and also make the structure of the Xanthoceras sorbifolia itself loose, increase the penetration of the solvent, and lay a foundation for the subsequent extraction and enzymatic hydrolysis process, thereby improving the yield of the Xanthoceras sorbifolia protein peptide. DETAILED DESCRIPTION
[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] In the following examples and comparative examples, the enzyme activity of Bacillus subtilis neutral protease is 20,000 u / g; the enzyme activity of bromelain is 50,000 u / g.
[0031] Example 1 A method for preparing Xanthoceras sorbifolia protein peptide comprises the following steps: S1. Mix 10 g of Xanthoceras sorbifolia and 80 mL of the reagent, soak at 40° C. for 2 h, separate, dry, and crush to obtain a pretreated product with a particle size of 325 mesh; S2, inputting liquid butane into the pretreated material (the mass ratio of liquid butane to pretreated material is 2.5:1), performing subcritical extraction at 25°C and 0.9MPa for 4h, unloading, and obtaining oil meal; S3, adding water to the oil meal (the amount of water added is 8 times the mass of the oil meal), adjusting the pH to 6.8, adding 0.002g of protease, and performing enzymolysis at 35°C for 2h, then adding 0.001g of protease, and performing enzymolysis at 35°C for 1h, separating, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagent includes triethanolamine oleate and isoamyl alcohol in a volume ratio of 1:3; In step S3, the protease includes Bacillus subtilis neutral protease.
[0032] Example 2 A method for preparing Xanthoceras sorbifolia protein peptide comprises the following steps: S1. Mix 10 g of Xanthoceras sorbifolia and 90 mL of the reagent, soak at 45 ° C for 1.5 h, separate, dry, and crush to obtain a pretreated product with a particle size of 350 mesh; S2, inputting liquid butane into the pretreated material (the mass ratio of liquid butane to pretreated material is 3:1), performing subcritical extraction at 28°C and 1.2MPa for 3h, unloading, and obtaining oil meal; S3, adding water to the oil meal (the amount of water added is 9 times the mass of the oil meal), adjusting the pH to 7.0, adding 0.006g of protease, and enzymolyzing at 40°C for 1h, then adding 0.003g of protease, and enzymolyzing at 40°C for 0.5h, separating, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagent includes triethanolamine oleate and isoamyl alcohol in a volume ratio of 2:7; In step S3, the protease includes Bacillus subtilis neutral protease.
[0033] Example 3 A method for preparing Xanthoceras sorbifolia protein peptide comprises the following steps: S1. Mix 10 g of Xanthoceras sorbifolia and 100 mL of the reagent, soak at 50° C. for 1 h, separate, dry, and crush to obtain a pretreated product with a particle size of 400 mesh; S2, inputting liquid butane into the pretreated material (the mass ratio of liquid butane to pretreated material is 3.5:1), performing subcritical extraction at 32°C and 1.4MPa for 2h, unloading, and obtaining oil meal; S3, adding water to the oil meal (the amount of water added is 10 times the mass of the oil meal), adjusting the pH to 7.5, adding 0.008g of protease, and enzymolyzing at 40°C for 1h, then adding 0.004g of protease, and enzymolyzing at 40°C for 0.5h, separating, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagent includes triethanolamine oleate and isoamyl alcohol in a volume ratio of 1:4; In step S3, the protease includes Bacillus subtilis neutral protease.
[0034] Example 4 The only difference between this embodiment and embodiment 3 is that in step S1 of this embodiment, the reagents include 14 mL of triethanolamine oleate, 56 mL of isoamyl alcohol and 30 mL of a 60% by mass aqueous solution of sodium cocoyl isethionate, that is, the volume ratio of the sum of the volumes of triethanolamine oleate and isoamyl alcohol to the 60% by mass aqueous solution of sodium cocoyl isethionate is 7:3.
[0035] Example 5 The only difference between this embodiment and embodiment 3 is that in step S1 of this embodiment, the reagents include 19 mL of triethanolamine oleate, 76 mL of isoamyl alcohol and 5 mL of a 60% by mass aqueous solution of sodium cocoyl isethionate, that is, the volume ratio of the sum of the volumes of triethanolamine oleate and isoamyl alcohol to the 60% by mass aqueous solution of sodium cocoyl isethionate is 19:1.
[0036] Example 6 The only difference between this embodiment and embodiment 3 is that in step S1 of this embodiment, the reagents include 16 mL of triethanolamine oleate, 64 mL of isoamyl alcohol and 20 mL of a 60% by mass aqueous solution of sodium cocoyl isethionate, that is, the volume ratio of the sum of the volumes of triethanolamine oleate and isoamyl alcohol to the 60% by mass aqueous solution of sodium cocoyl isethionate is 4:1.
[0037] Example 7 The only difference between this embodiment and embodiment 3 is that in step S1 of this embodiment, the reagents include 18 mL of triethanolamine oleate, 72 mL of isoamyl alcohol and 10 mL of a 60% by mass aqueous solution of sodium cocoyl isethionate, that is, the volume ratio of the sum of the volumes of triethanolamine oleate and isoamyl alcohol to the 60% by mass aqueous solution of sodium cocoyl isethionate is 9:1.
[0038] Example 8 The only difference between this embodiment and embodiment 7 is that, in this embodiment, the protease further comprises bromelain, and the mass ratio of Bacillus subtilis neutral protease to bromelain is 0.5:1.
[0039] Example 9 The only difference between this embodiment and embodiment 7 is that, in this embodiment, the protease further comprises bromelain, and the mass ratio of Bacillus subtilis neutral protease to bromelain is 4:1.
[0040] Example 10 The only difference between this embodiment and embodiment 7 is that, in this embodiment, the protease further comprises bromelain, and the mass ratio of Bacillus subtilis neutral protease to bromelain is 1:1.
[0041] Embodiment 11 The only difference between this embodiment and embodiment 7 is that, in this embodiment, the protease further comprises bromelain, and the mass ratio of Bacillus subtilis neutral protease to bromelain is 3:1.
[0042] Comparative Example 1 The only difference between this comparative example and Example 1 is that in step S1 of this comparative example, the reagent only includes isoamyl alcohol.
[0043] Comparative Example 2 The only difference between this comparative example and Example 1 is that in step S1 of this comparative example, the reagent only includes triethanolamine oleate.
[0044] Comparative Example 3 The difference between this comparative example and Example 1 is that in step S1 of this comparative example, the reagent only includes a 60% by mass aqueous solution of sodium cocoyl isethionate.
[0045] Comparative Example 4 A method for preparing Xanthoceras sorbifolia protein peptide comprises the following steps: S1. Grind 10 g of Xanthoceras sorbifolia to 325 mesh, input liquid butane (the mass ratio of liquid butane to Xanthoceras sorbifolia is 2.5:1), perform subcritical extraction at 25°C and 0.9 MPa for 4 h, and unload to obtain oil meal; S2, adding water to the oil meal (the amount of water added is 8 times the mass of the oil meal), adjusting the pH to 6.8, adding 0.002g of protease, and hydrolyzing at 35°C for 2h, then adding 0.001g of protease, and hydrolyzing at 35°C for 1h, separating, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S2, the protease includes Bacillus subtilis neutral protease.
[0046] Comparative Example 5 A method for preparing Xanthoceras sorbifolia protein peptide comprises the following steps: S1. Mix 10 g Xanthoceras sorbifolia and 80 mL of the reagent, soak at 40° C. for 2 h, separate, dry, and crush to obtain a 325-mesh pretreated product; S2, adding water to the pretreated material (the amount of water added is 8 times the mass of the pretreated material), adjusting the pH to 6.8, adding 0.002 g of protease, and performing enzymolysis at 35°C for 2 h, then adding 0.001 g of protease, and performing enzymolysis at 35°C for 1 h, separating, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagent includes triethanolamine oleate and isoamyl alcohol in a volume ratio of 1:3; In step S2, the protease includes Bacillus subtilis neutral protease.
[0047] Experimental Example 1 The yields of Xanthoceras sorbifolia protein peptides prepared in Examples 1 to 7 and Comparative Examples 1 to 5 are shown in Table 1 below, wherein Xanthoceras sorbifolia protein peptide yield (%) = Xanthoceras sorbifolia protein peptide mass / oil meal × 100%.
[0048] Table 1 Yields of Xanthoceras sorbifolia protein peptides from Examples 1 to 7 and Comparative Examples 1 to 5
[0049] As can be seen from Table 1, compared with Comparative Examples 1 to 5, the yield of the Xanthoceras sorbifolia protein peptide obtained in Examples 1 to 7 is greater than 49%, indicating that the Xanthoceras sorbifolia protein peptide obtained by this scheme has a higher yield. Compared with Example 3, the yields of Examples 4 to 7 are improved, indicating that by introducing the sodium cocoyl isethionate aqueous solution into the reagent of step S1, under the joint action of the sodium cocoyl isethionate aqueous solution, triethanolamine oleate and isopentanol, the yield of the Xanthoceras sorbifolia protein peptide can be further improved. Compared with Examples 4 to 5, the yields of Examples 6 to 7 are improved, indicating that when the volume ratio of triethanolamine oleate and isopentanol to the sodium cocoyl isethionate aqueous solution is 4 to 9:1, the yield of the Xanthoceras sorbifolia protein peptide can be further improved.
[0050] Experimental Example 2 The proportion of protein peptides with a molecular weight of 1200Da to 2000Da in the Xanthoceras sorbifolia protein peptides of Examples 7 to 11 was tested. During the test, a gel chromatography system (GPC), a TSK gel GMPWXL aqueous gel chromatography column, and a RID-20 differential refractive index detector were used to analyze the molecular weight distribution in the Xanthoceras sorbifolia protein peptides. The test results are shown in Table 2 below: Table 2 Test results of the proportion of protein peptides with molecular weight of 1200Da~2000Da in Xanthoceras sorbifolia protein peptides of Examples 7~11
[0051] As can be seen from Table 2, the proportion of protein peptides with a molecular weight of 1200Da~2000Da in Examples 7~11 can reach more than 94%, indicating that the molecular weight distribution of the Xanthoceras sorbifolia protein peptide obtained in this scheme is relatively concentrated, mainly distributed in the molecular weight stage of 1200Da~2000Da, and belongs to small molecule peptides. Among them, compared with Example 7, the proportion of protein peptides with a molecular weight of 1200Da~2000Da in Examples 8~11 is increased, indicating that when Bacillus subtilis neutral protease and bromelain act together, the probability of generating small molecule protein peptides can be increased. Compared with Examples 8~9, the proportion of protein peptides with a molecular weight of 1200Da~2000Da in Examples 10~11 is increased, indicating that when the mass ratio of Bacillus subtilis neutral protease and bromelain is 1~3:1, the probability of generating small molecule protein peptides can be further increased.
[0052] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing Xanthoceras sorbifolia protein peptide, characterized in that: The following steps are involved: S1. Mixing Xanthoceras sorbifolia and a reagent, soaking, and separating to obtain a pretreated product; S2, subjecting the pretreated product to subcritical extraction and unloading to obtain oil meal; S3, adding water to the oil meal, adjusting the pH to 6.8-7.5, adding protease, enzymolysis, separation, and drying to obtain Xanthoceras sorbifolia protein peptide; In step S1, the reagents include triethanolamine oleate and isoamyl alcohol.
2. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: The volume ratio of the triethanolamine oleate to isoamyl alcohol is 1:3-4.
3. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: The reagents also include an aqueous solution of sodium cocoyl isethionate; The volume ratio of the triethanolamine oleate and isoamyl alcohol to the sodium cocoyl isethionate aqueous solution is 4-9:
1.
4. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: The protease includes Bacillus subtilis neutral protease.
5. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 4, characterized in that: The proteases also include bromelain.
6. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 5, characterized in that: The mass ratio of the Bacillus subtilis neutral protease to the bromelain is 1-3:
1.
7. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: In step S2, liquid butane is used during the subcritical extraction, and the mass ratio of the liquid butane to the pretreated material is 2.5-3.5:
1.
8. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: In step S2, during the subcritical extraction, the extraction temperature is 25-32° C., the extraction pressure is 0.9-1.4 MPa, and the extraction time is 2-4 h.
9. The method for preparing a Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: In step S3, the adding of protease is divided into a first adding and a second adding; When adding for the first time, 2 / 3 of the mass of protease is added, the enzymolysis time is 1-2 hours, and the enzymolysis temperature is 35-40°C; During the second addition, the remaining protease is added, the enzymolysis time is 0.5-1h, and the enzymolysis temperature is 35-40°C.
10. The method for preparing the Xanthoceras sorbifolia protein peptide according to claim 1, characterized in that: In step S1, the mass volume ratio of the Xanthoceras sorbifolia to the reagent is 10 g: 80-100 mL; In step S3, the amount of water added is 8 to 10 times the mass of the oil meal, and the amount of protease added is 0.03% to 0.12% of the mass of the Xanthoceras sorbifolia.