Intelligence-promoting casein peptide and preparation method thereof

Abstract

The invention relates to an alpinia oxyphylla casein peptide and a preparation method thereof, and belongs to the field of small molecule active peptides, the alpinia oxyphylla casein peptide takes magnesium-chelated small molecule casein peptide as an active component, and the magnesium ion chelation rate is greater than 70%. The preparation method comprises the following steps: adding pure water into the casein powder, stirring and dissolving, adjusting the pH and temperature, adding protease for enzymolysis, and performing enzyme deactivation after enzymolysis is finished; carrying out ultrafiltration separation on supernate after enzymolysis by using an ultrafiltration membrane to obtain a casein peptide solution; adjusting the concentration of the casein peptide solution, and adding a magnesium-containing compound for chelation reaction; adding absolute ethyl alcohol into the solution to precipitate and separate out the chelated casein peptide; and centrifugally collecting casein peptide precipitate after alcohol precipitation, and freeze-drying to obtain the magnesium-chelated micromolecular casein peptide. The intelligence-promoting casein peptide is remarkable in physiological activity, has a good intelligence-promoting effect and is wide in application prospect; the preparation method is simple, reaction conditions are mild, and large-scale conversion into industrial production is easy.

Description

Technical Field

[0001] This invention belongs to the field of small molecule active peptide technology, and particularly relates to a brain-boosting casein peptide and its preparation method. Background Technology

[0002] Casein is the main protein component of mammalian milk, including that of cows, sheep, and humans. It is a large, hard, dense curd that is extremely difficult to digest and break down. Casein accounts for approximately 80% of milk, and its amino acid composition is relatively balanced, providing essential amino acids to support normal growth and development. Research has found that different enzymatic hydrolysis methods and processing conditions can yield casein peptides with different functional properties. In recent years, research on casein peptides has deepened, revealing various physiological functions, such as immune regulation, promoting mineral absorption, antioxidation, antibacterial activity, lowering blood pressure, and promoting sleep.

[0003] With societal development and the accelerating pace of life, there is a growing focus on brain health and intellectual enhancement. Whether it's students seeking to improve learning efficiency or working professionals aiming to boost performance, there is a significant demand for brain-boosting products. Various brain-enhancing health supplements and cognitive-enhancing foods are widely popular in the market, and the market size continues to expand.

[0004] Currently, there is relatively little research on the cognitive benefits of casein peptides. Only some studies have shown that casein peptides may have some impact on the development and function of the nervous system, but their specific mechanisms of action and effects still need to be further clarified.

[0005] Given the potential brain-boosting functions of casein peptides and the strong market demand for brain-boosting products, developing a brain-boosting casein peptide has broad market prospects. It can not only meet consumers' pursuit of safe and effective brain-boosting products and bring significant economic benefits to enterprises, but also help promote the development and innovation of the entire brain-boosting product industry and stimulate more research and application of functional peptides in the health field. Summary of the Invention

[0006] The purpose of this invention is to provide a brain-boosting casein peptide and its preparation method. The brain-boosting casein peptide prepared by this invention has brain-boosting effects, filling a gap in the field of casein peptides for brain enhancement.

[0007] The technical solution adopted by this invention to solve the technical problem is as follows: The present invention provides a brain-boosting casein peptide, which uses a small molecule casein peptide chelated with magnesium as the active ingredient.

[0008] As a preferred embodiment, the magnesium ion chelation rate of the nootropic casein peptide is >70%.

[0009] The present invention provides a method for preparing a brain-boosting casein peptide, which specifically includes the following steps: (1) Add 5-20 times the mass of pure water to casein powder, stir to dissolve, adjust pH to 6-10 and temperature to 40-60℃, add 0.3-1% protease for 3-6 hours of enzymatic hydrolysis, and then heat to inactivate the enzyme after the hydrolysis is completed. (2) After centrifugation, the supernatant of the enzymatic hydrolysate is obtained. The supernatant is then subjected to ultrafiltration using an ultrafiltration membrane to separate the casein peptide solution. (3) Adjust the concentration of casein peptide solution to 1-20%, add magnesium-containing compound, and carry out chelation reaction for 1-5 h at 35-45℃ and pH 6.5-7.5; (4) Add anhydrous ethanol to the chelated solution to make the final ethanol concentration 60-80%; let it stand at 4℃ for 5-12 hours to allow the chelated casein peptides to precipitate. (5) The casein peptide precipitate after alcohol precipitation was collected by centrifugation and then freeze-dried to obtain small casein peptides with chelated magnesium.

[0010] As a preferred embodiment, in step (1), after the enzymatic hydrolysis is completed, the liquid is heated to 80-90℃ and kept at that temperature for 10-15 minutes to inactivate the enzyme.

[0011] In a preferred embodiment, the protease is composed of Bacillus subtilis protease and chymotrypsin, wherein the mass ratio of Bacillus subtilis protease to chymotrypsin is (1-3):(1-3).

[0012] As a preferred embodiment, in step (2), the centrifugation conditions are: 5000-12000 rpm, 10-20 min.

[0013] In a preferred embodiment, in step (2), the molecular weight cutoff of the ultrafiltration membrane is 1000-2000 Da.

[0014] In a preferred embodiment, the magnesium-containing compound is selected from magnesium sulfate and magnesium chloride.

[0015] In a preferred embodiment, in step (3), the mass ratio of the casein peptide to the magnesium-containing compound is 1-10.

[0016] In a preferred embodiment, in step (3), the chelation reaction is carried out at a stirring speed of 200-600 rpm.

[0017] The beneficial effects of this invention are: 1. The casein peptide prepared by this invention has a magnesium-chelated small molecule casein peptide as its active ingredient. The magnesium ion chelation rate is >70%, and it has significant physiological activity, good brain-boosting effect, and broad application prospects.

[0018] 2. The method for preparing a brain-boosting casein peptide according to the present invention has a simple preparation process, mild reaction conditions, and is easy to scale up and transform into industrial production. Detailed Implementation

[0019] In a first aspect, the present invention provides a brain-boosting casein peptide.

[0020] This invention provides a brain-boosting casein peptide, which uses magnesium-chelated small-molecule casein peptides as its active ingredient. The magnesium ion chelation rate of this brain-boosting casein peptide is >70%, and experiments have proven that it has brain-boosting effects.

[0021] Secondly, the present invention provides a method for preparing brain-boosting casein peptides.

[0022] The present invention provides a method for preparing a brain-boosting casein peptide, which specifically includes the following steps: Step S1: Enzymatic hydrolysis; First, add 5 to 20 times the mass of pure water to the casein powder, stir to dissolve, and then adjust the pH of the solution to 6-10 and the temperature to 40℃-60℃. Then, add 0.3%-1% of protease for enzymatic hydrolysis for 3-6 hours. After the enzymatic hydrolysis is completed, raise the temperature of the solution to 80℃-90℃ (preferably 90℃) and keep it for 10-15 minutes (preferably 15 minutes) to inactivate the enzyme.

[0023] As a preferred embodiment, the method for preparing casein powder is as follows: Fresh milk is degreased by centrifugation (45℃-50℃, 15000r / min) to remove fat to a fat content of ≤0.08%, followed by pasteurization (72℃, 15 seconds) to inactivate microorganisms. Microfiltration is then performed three times using a 0.02μm stainless steel membrane at 3-5 bar pressure, with each concentration factor controlled at 4-7 times. Casein micelles are gradually separated using dilution filtration technology (water is added to half the original milk mass each time), resulting in a casein purity of over 80% in the final retentate. Vacuum concentration (50℃-70℃, vacuum degree 0.08MPa-0.09MPa) is then achieved to a solids content of 30%-35%, followed by drying in a spray dryer (inlet temperature 120℃, outlet temperature 75℃) to form casein powder.

[0024] As a preferred embodiment, the protease consists of Bacillus subtilis protease and chymotrypsin, with a mass ratio of Bacillus subtilis protease to chymotrypsin of (1-3):(1-3).

[0025] Among them, Bacillus subtilis protease and chymotrypsin are both commercial products. For example, Bacillus subtilis protease can be purchased from manufacturers such as Dongheng Huadao, Novozymes, and Longkote; chymotrypsin can be purchased from manufacturers such as Chongqing Jiangxia, Beijing Geyuan Tianrun, and Sichuan Deboer.

[0026] Step S2: Separation; First, centrifuge the enzymatic hydrolysate obtained in step S1 at 5000 rpm-12000 rpm for 10 min-20 min to obtain the supernatant; then, use an ultrafiltration membrane with a molecular weight cutoff of 1000 Da-2000 Da (preferably 1000 Da) to ultrafilter the obtained supernatant to separate casein peptide solutions with a molecular weight of less than 1000 Da.

[0027] Step S3: Chelate magnesium; First, adjust the concentration of the casein peptide solution obtained in step S2 to 1%-20%; then add a magnesium-containing compound, wherein the mass ratio of casein peptide to magnesium-containing compound is 1-10, and carry out the chelation reaction for 1 h-5 h at a stirring speed of 200 rpm-600 rpm under the conditions of temperature 35 ℃-45 ℃ and pH 6.5-7.5.

[0028] As a preferred embodiment, the magnesium-containing compound is selected from magnesium sulfate and magnesium chloride.

[0029] Step S4: Alcohol precipitation; First, slowly add anhydrous ethanol to the chelated solution to achieve a final ethanol concentration of 60% - 80%; then let it stand at 4℃ for 5h - 12h to allow the chelated casein peptides to precipitate.

[0030] Step S5: Drying; The casein peptide precipitate after alcohol precipitation was collected by centrifugation and then freeze-dried to obtain small molecule casein peptides with chelated magnesium.

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: A method for preparing a brain-boosting casein peptide

[0033] (1) Enzymatic hydrolysis: First, add 20 times the mass of pure water to the casein powder, stir to dissolve, adjust the pH of the solution to 9.0 and the temperature to 50℃; then add 1% of protease (the mass ratio of Bacillus subtilis protease to chymotrypsin is 2:1) for 3h of enzymatic hydrolysis; after the enzymatic hydrolysis is completed, heat the solution to 90℃ and keep it for 15min to inactivate the enzyme.

[0034] (2) Separation: First, centrifuge the enzymatic hydrolysate obtained in step (1) at 10,000 rpm for 10 min to obtain the supernatant; then use an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to perform ultrafiltration on the obtained supernatant to separate casein peptide solutions with a molecular weight of less than 1000 Da.

[0035] (3) Chelating magnesium: First, adjust the concentration of the casein peptide solution obtained in step (2) to 5%; then add magnesium sulfate, with a mass ratio of casein peptide to magnesium sulfate of 1. Under the conditions of temperature 40 ℃ and pH 7.0, carry out the chelation reaction at a stirring speed of 200 rpm for 4 h.

[0036] (4) Alcohol precipitation: First, slowly add anhydrous ethanol to the chelated solution to make the final ethanol concentration reach 70%; then let it stand for 5 hours at 4℃ to precipitate the chelated casein peptides.

[0037] (5) Drying: The casein peptide precipitate after alcohol precipitation is collected by centrifugation and then freeze-dried to obtain small molecule casein peptides with chelated magnesium.

[0038] Example 2: A method for preparing a brain-boosting casein peptide

[0039] (1) Enzymatic hydrolysis: First, add 10 times the mass of pure water to the casein powder, stir to dissolve, and then adjust the pH of the solution to 8.0 and the temperature to 45℃. Then add 0.8% of protease (the mass ratio of Bacillus subtilis protease to chymotrypsin is 1:1) and hydrolyze for 4 hours. After the enzymatic hydrolysis is completed, heat the solution to 90℃ and keep it for 15 minutes to inactivate the enzyme.

[0040] (2) Separation: First, centrifuge the enzymatic hydrolysate obtained in step (1) at 8000 rpm for 10 min to obtain the supernatant; then use an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to ultrafilter the obtained supernatant to separate casein peptide solutions with a molecular weight of less than 1000 Da.

[0041] (3) Chelating magnesium: First, adjust the concentration of the casein peptide solution obtained in step (2) to 7%; then add magnesium chloride, with a mass ratio of casein peptide to magnesium chloride of 3. Under the conditions of temperature 38 ℃ and pH 6.5, carry out the chelation reaction at a stirring speed of 300 rpm for 3 h.

[0042] (4) Alcohol precipitation: First, slowly add anhydrous ethanol to the chelated solution to make the final ethanol concentration reach 80%; then let it stand at 4℃ for 7h to precipitate the chelated casein peptides.

[0043] (5) Drying: The casein peptide precipitate after alcohol precipitation is collected by centrifugation and then freeze-dried to obtain small molecule casein peptides with chelated magnesium.

[0044] Experimental Example 1: Magnesium Ion Chelation Rate Test

[0045] The Mg content in the supernatant after alcohol precipitation in step (4) of Examples 1 and 2 was determined by atomic absorption spectrometry (AAS). 2+ Concentration, the chelation rate is calculated using the following formula: Chelation rate = (Initial total Mg) / (Mg) 2+ Concentration - Free Mg in supernatant 2+ Concentration) / Initial Total Mg 2+ (Concentration) × 100%. Magnesium ion chelation rate testing showed that the magnesium ion chelation rate of the casein peptides prepared in this invention was >70%. Details are as follows: The magnesium ion chelation rate of the casein peptide obtained in Example 1 was measured to be 73.6%.

[0046] The magnesium ion chelation rate of the casein peptide obtained in Example 2 was measured to be 75.8%.

[0047] Experimental Example 2 used casein peptides prepared in Examples 1 and 2 as test samples to determine the amino acid composition of the casein peptides.

[0048] The amino acid composition of the casein peptides prepared in Examples 1 and 2 was tested and is shown in Tables 1 and 2. The casein peptides prepared in Examples 1 and 2 contain all the essential amino acids required by the human body, with glutamate content exceeding 16%. Glutamate is closely related to learning, memory, and cognitive functions. During learning, glutamate can promote changes in synaptic plasticity, contributing to the formation of new neural connections and strengthening memory. It also plays an important role in maintaining and improving normal brain function.

[0049] Table 1. Amino acid composition test results of the casein peptides prepared in Example 1.

[0050] Table 2. Amino acid composition test results of the casein peptides prepared in Example 2.

[0051] Experimental Example 3 used the casein peptides prepared in Example 1 as the test sample for a cognitive function test.

[0052] Experimental animals: 50 adult male mice, weighing 18g-22g, were randomly assigned to 5 groups of 10 mice each.

[0053] Control group: normal mice; Model group and low, medium and high dose groups: memory impairment model mice (intraperitoneal injection of scopolamine 5mg / kg BW).

[0054] Administration time of low, medium, and high dose groups and test samples: The recommended oral dose of casein peptide for humans is 200 mg / day, which is equivalent to 3.34 mg / kg bw for an adult with a standard body weight of 60 kg. The experiment was divided into 5 groups (control group, model group, low-dose group, medium-dose group, and high-dose group). The control group and model group were given saline by gavage for 30 days, while the low, medium, and high-dose groups were given the test sample by gavage for 30 days. Mice in the low-dose group were fed 16.7 mg / kg bw, mice in the medium-dose group were fed 33.4 mg / kg bw, and mice in the high-dose group were fed 100.2 mg / kg bw, which are approximately 5 times, 10 times, and 30 times the human oral dose (3.34 mg / kg bw), respectively.

[0055] The test items and methods are as follows: 1. Diving platform experiment; Training began the day after the last gavage. Mice were placed in a reaction chamber (on and off the platform) for 3 minutes to acclimatize. Then, the mice were placed on a copper grid inside the chamber, and a 36V AC current was immediately applied. Upon receiving the electric shock, the mice's normal reaction was to jump back to the platform (the insulator) to avoid the noxious stimulus. Most mice may jump onto the copper grid again or multiple times, only to be shocked again and quickly jump back to the platform. Training lasted 3 minutes. A retest was conducted 24 hours later. The mice were placed on the platform, and the latency period for each mouse's first jump off the platform and the number of electric shocks within 3 minutes were recorded.

[0056] 2. Water maze experiment; The pool was divided into four quadrants. The platform was placed 2 cm below the horizontal plane of the second quadrant. The entry point was the middle position of each of the four quadrants. The reference objects outside the maze remained unchanged during the training period. The mice were allowed to swim freely for 2 minutes one day before the experiment to adapt to the environment.

[0057] The Morris water maze test consists of two parts: a navigation experiment and a spatial exploration experiment, lasting 5 days. For the first 4 days of the navigation experiment, mice were trained to locate the platform 4 times per day. Each mouse entered the water in the same order from each of the four quadrants. The time from entry to landing on the platform was the escape latency, and the result on day 4 was used as the final score. On day 5, the platform was removed, and the spatial exploration experiment was conducted. A random entry point was selected, and the number of times the mouse traversed the original platform location within 2 minutes and the effective time (the time spent in the quadrant where the original platform was located) were recorded to reflect the mouse's spatial memory of the platform location.

[0058] The test results are as follows: 1. Diving platform experiment; The results of the platform jumping experiment are shown in Table 3. Compared with the control group, the latency of mice in the model group was significantly reduced, and the number of errors was significantly increased. Compared with the model group, the latency of mice in the low, medium, and high dose groups was significantly prolonged, and the number of errors was reduced, indicating that casein peptides have a significant improving effect on mice with impaired memory, and the improvement effect is better with increasing dose.

[0059] Table 3. Effects of casein peptides on mouse learning in the platform jumping experiment ( ±S, n=10)

[0060] 2. Water maze experiment; The results of the water maze experiment are shown in Table 3. In the navigation experiment, the model group mice learned more slowly than the control group mice. Compared with the model group, the escape latency of mice in the low, medium, and high dose groups was significantly reduced, with the high dose group showing the most significant effect. In the spatial exploration experiment, the number of platform crossings and the effective time of the model group mice were significantly reduced compared with the control group. Compared with the model group, the number of platform crossings and the effective time of the low, medium, and high dose groups were significantly increased, with the high dose group showing the best effect. This indicates that casein peptides have a significant improving effect on mice with impaired memory, and the improvement effect is better with increasing dosage.

[0061] Table 4. Effects of casein peptides on mouse learning in the water maze experiment ( ±S, n=10)

[0062] Experiment 4 used the casein peptides prepared in Example 2 as the test sample for a cognitive function test.

[0063] The magnesium chelate casein peptide obtained in Example 2 was used to test its effect on mice with memory impairment using the same method as in Example 3. The results are shown in Tables 5 and 6, and it showed the same improvement effect as the magnesium chelate casein peptide in Example 1.

[0064] Table 5. Effects of casein peptides on mouse learning in the platform jumping experiment ( ±S, n=10)

[0065] Table 6. Effects of casein peptides on mouse learning in the water maze experiment ( ±S, n=10)

[0066] In summary, the casein peptides prepared by this invention have good brain-boosting effects.

[0067] This invention discloses a brain-boosting casein peptide and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The product of this invention has been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the product described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. A brain-boosting casein peptide, characterized in that, The active ingredient is a small molecule casein peptide that chelates magnesium.

2. The brain-boosting casein peptide according to claim 1, characterized in that, The magnesium ion chelation rate of the nootropic casein peptide is >70%.

3. The method for preparing a brain-boosting casein peptide as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Add 5-20 times the mass of pure water to casein powder, stir to dissolve, adjust pH to 6-10 and temperature to 40-60℃, add 0.3-1% protease for 3-6 hours of enzymatic hydrolysis, and then heat to inactivate the enzyme after the hydrolysis is completed. (2) After centrifugation, the supernatant of the enzymatic hydrolysate is obtained. The supernatant is then subjected to ultrafiltration using an ultrafiltration membrane to separate the casein peptide solution. (3) Adjust the concentration of casein peptide solution to 1-20%, add magnesium-containing compound, and carry out chelation reaction for 1-5 h at 35-45℃ and pH 6.5-7.5; (4) Add anhydrous ethanol to the chelated solution to make the final ethanol concentration 60-80%; let it stand at 4℃ for 5-12 hours to allow the chelated casein peptides to precipitate. (5) The casein peptide precipitate after alcohol precipitation was collected by centrifugation and then freeze-dried to obtain small casein peptides with chelated magnesium.

4. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, In step (1), after the enzymatic hydrolysis is completed, the liquid is heated to 80-90℃ and kept at 10-15 min to inactivate the enzyme.

5. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, The protease is composed of Bacillus subtilis protease and chymotrypsin, and the mass ratio of Bacillus subtilis protease to chymotrypsin is (1-3):(1-3).

6. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, In step (2), the centrifugation conditions are: 5000-12000 rpm, 10-20 min.

7. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, In step (2), the molecular weight cutoff of the ultrafiltration membrane is 1000-2000 Da.

8. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, The magnesium-containing compound is selected from magnesium sulfate and magnesium chloride.

9. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, In step (3), the mass ratio of the casein peptide to the magnesium-containing compound is 1-10.

10. The method for preparing a brain-boosting casein peptide according to claim 3, characterized in that, In step (3), the chelation reaction is carried out with a stirring speed of 200-600 rpm.

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