CN113774101B - Animal and plant mixed protein peptide with memory improving function and preparation method and application thereof - Google Patents
Animal and plant mixed protein peptide with memory improving function and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention discloses animal and plant mixed protein peptide with a memory improving function, a preparation method and application, and belongs to the technical field of preparation of active peptide. The invention adopts degreased walnut protein powder and fresh fish heads as raw materials, degreases the fish heads by ethanol ultrasound, then fully hydrolyzes the walnut protein powder and the degreased fish head powder by an alkaline protease and papain two-step controlled enzymolysis technology to obtain walnut protein peptide and fish head protein peptide with high hydrolysis degree and high activity, and finally obtains mixed protein peptide by special proportion. The combined peptide has the advantages of simple formula, high safety and synergistic effect, can be used as a food-derived peptide, and provides a safe and reliable functional food with remarkable memory improving effect for consumers.
Description
Technical Field
The invention belongs to the technical field of preparation of active peptides, and particularly relates to animal and plant mixed protein peptide with a memory improving function, a preparation method and application thereof.
Background
The intense competition and the enormous pressure of the current society make the requirements of people on the learning, memory and cognitive functions of the people higher and higher. However, mechanical damage to the brain, intense learning, insufficient sleep or insomnia, and natural aging of the human body all cause a decline in cognitive ability and a decline in learning and memory functions. Meanwhile, the memory decline can be used as a clinical symptom to further cause various degenerative diseases, such as Alzheimer disease, parkinson disease, vascular dementia and the like. At present, no effective treatment means exists internationally for the diseases, and some synthetic drugs such as donepezil hydrochloride, piracetam, galantamine and the like in the market can improve learning and cognition capability to a certain extent, but can generate some serious adverse reactions such as hepatotoxicity, nervous disorder and the like. Therefore, it is very important to develop natural active substances having effects of preventing and improving memory deterioration and cognitive function deterioration.
Bioactive peptides have wide sources, and generally, hydrolysates with various functional activities, such as oxidation resistance, aging resistance, fatigue resistance, memory improvement and the like, can be obtained by utilizing certain naturally-occurring high-quality animal and plant protein resources through an enzyme engineering technology. Moreover, the efficacy of bioactive peptides from different sources in improving learning and memory has been verified in a plurality of in vivo cognition impairment animal models and in vitro related cell models, but the currently reported memory improvement related peptides are mainly some endogenous bioactive peptides or hydrolyzed peptides of a single species, and the research on the food-borne memory improvement peptide composition is relatively deficient.
The walnut is a traditional food used as both medicine and food in China, the brain-tonifying effect of the walnut is accepted and accepted by people, the walnut protein is a high-quality plant protein containing 18 amino acids and 8 essential amino acids for human bodies in complete varieties and balanced proportion, and the content of the nervous excitability amino acid glutamic acid is relatively high, so that the walnut protein is deeply developed by an enzyme hydrolysis technology, and the aim of efficiently releasing a potential memory peptide segment in a protein sequence is fulfilled, so that a way is provided for developing a food-borne polypeptide composition capable of preventing diseases such as low memory, cognitive impairment and the like.
The marine life contains some unique natural bioactive substances because the growth environment is completely different from that of land life, and the marine fish protein, polypeptide and amino acid are very important active substances, so that the research shows that part of the polypeptide from the marine fish has the activities of resisting cancer, resisting blood coagulation, lowering blood pressure and the like. In addition, the enzymolysis products of the marine fish processing by-products such as fish skin, fish head and the like contain rich bioactive peptides, and the marine fish proteolysis products with memory improving activity are prepared by utilizing a biological enzymolysis technology, so that the additional value of the fish processing by-products can be greatly improved. Because the fish head contains a large amount of amino acids essential to human body, especially the types and the content of the amino acids for regulating the nerve function are high, the biological active peptide with the function of improving the memory is screened after the biological active peptide is subjected to enzymolysis by a biological enzymolysis means, the requirements of consumer groups with low memory can be met, and the high-value utilization of byproducts generated in the fish processing process can be realized.
The food compounding technology can make up the defect that the nutrition and the function of a single raw material are not complete enough, and the reasonable food formula compounding can not only overcome the defect that the functionality of the single raw material is not strong, but also improve the bioavailability of the active ingredients and the like. Animal behavioral experiments show that the compounding of the walnut protein peptide and the fish head protein peptide can further improve the learning ability and the low memory, thereby achieving the purpose of exerting the synergistic effect. Therefore, it is very important to master a preparation method of natural animal and plant source mixed peptide with memory improvement.
Patent CN201910007547.7 discloses a process for preparing fish protein mixed peptide from crude fish protein, which comprises the steps of degreasing, removing fishy smell, enzymolysis, decoloring, ultrafiltration, drying and the like, so as to prepare the fish protein mixed peptide from crude fish protein powder, wherein the finished fish protein mixed peptide contains peptides with various physiological activities. However, the patent only relates to the preparation of fish protein mixed peptide, and does not examine the influence of the variety and the part of the fish on the final effect and solve the problems of improving the memory and the cognition.
Patent CN201811189789.4 discloses a brain-tonifying beef and walnut rice flour which comprises the following raw materials in parts by weight: 65-85 parts of rice, 8-14 parts of walnut, 5-10 parts of oat, 5-10 parts of red bean, 5-10 parts of hazelnut, 1-3 parts of black bean, 5-8 parts of black carp, 6-13 parts of beef, 4-8 parts of black fungus, 3-6 parts of polygonum multiflorum, 2-5 parts of red date, 1-3 parts of seasoning and 1-3 parts of nutritional additive, and has the health-care functions of strengthening brain, improving intelligence, improving immunity, improving metabolism, resisting aging and the like, and has good palatability. The patent has complex components, is difficult to define specific components, and has poor effects on improving memory and cognitive ability.
Disclosure of Invention
Therefore, the invention aims to provide a preparation method of natural animal and plant mixed peptide with the functions of obviously improving memory and cognitive ability.
The invention relates to a preparation method of natural animal and plant source mixed protein peptide with memory improving function, which comprises the following specific implementation steps:
a natural animal and plant mixed protein peptide with memory improving function comprises walnut protein peptide and fish head protein peptide.
Further, the walnut protein peptide and the fish head protein peptide are subjected to degreasing treatment; the fish head protein peptide is tilapia fish head protein peptide; the dosage ratio of the walnut protein peptide to the fish head protein peptide is 1.
Further, the dosage ratio of the walnut protein peptide to the fish head protein peptide is 1.
Furthermore, in the mixed protein peptide, the content of glutamic acid is more than 14.50g/100 g; the content of arginine is more than 6.90g/100 g; the content of glycine is more than 8.10g/100 g; the content of proline is more than 5.50g/100 g; the total content of hydrophobic amino acids is above 24g/100 g.
The invention relates to a preparation method of natural animal and plant source mixed protein peptide with memory improving function, which comprises the following specific implementation steps:
step 1, preparation of walnut protein peptide
Carrying out enzymolysis on the defatted walnut protein powder by adopting an alkaline protease and papain two-step enzymolysis method, and freeze-drying the enzymolysis liquid to obtain walnut protein peptide coarse powder.
Washing fresh fish heads with distilled water to remove impurities, beating into slurry, centrifuging to remove blood and partial grease, repeatedly performing ultrasonic extraction with anhydrous ethanol, performing suction filtration on the ethanol crude extract, repeating for 3 cycles, washing and drying fish head residues obtained by suction filtration, and finally crushing to obtain defatted fish head powder.
And (3) carrying out enzymolysis on the degreased fish head powder obtained in the step (2) by adopting an alkaline protease and papain two-step enzymolysis method, and freeze-drying the enzymolysis liquid to obtain fish head protein peptide coarse powder.
Step 4, compounding and combining walnut protein peptide and fish head protein peptide
And (3) mixing the walnut protein peptide coarse powder obtained in the step (1) and the fish head protein peptide coarse powder obtained in the step (3) according to a mass ratio to obtain a mixed peptide with a corresponding ratio, and verifying the activity of the mixed peptide for improving the memory function through subsequent experiments.
Further, the walnut protein peptide is prepared by the following method:
step 1: mixing defatted protein powder and distilled water, stirring, homogenizing with a homogenizer, and keeping the temperature with a constant temperature water bath;
step 2: adjusting pH, keeping pH constant, placing in water bath for 5min, adding alkaline protease, mixing, and performing enzymolysis at constant temperature;
and step 3: adjusting the pH value of the solution, keeping the pH value constant, putting the solution into a water bath for 5min, adding papain, uniformly mixing, oscillating at constant temperature for enzymolysis, and then inactivating enzyme;
and 4, step 4: cooling to room temperature, centrifuging, and freeze-drying the supernatant to obtain walnut protein peptide coarse powder.
Further, in the step 1, the mixing ratio of the defatted walnut protein powder to distilled water is 1;
in the step 2, the pH adjusting solution is a 1mol/L NaOH solution, the pH is adjusted to 9.5-10.5, the water bath temperature is 50-60 ℃, the adding amount of alkaline protease is 1-3%, the oscillation frequency is 150r/min, and the oscillation time is 4-6h;
in the step 3, the pH adjusting solution is 1mol/L NaOH or 1mol/L HCl solution, the pH is adjusted to 6.0-6.5, the water bath temperature is 50-60 ℃, the adding amount of papain is 1-3%, the oscillation frequency is 150r/min, the oscillation time is 4-6h, the enzyme deactivation temperature is 90-99 ℃, and the enzyme deactivation time is 10-15min;
in the step 4, the centrifugation speed is 8000rpm/min, and the centrifugation time is 20min.
Further, the pH value in the step 2,3 is the pH value at the temperature of the water bath.
Further, the fish head protein peptide is prepared by the following method:
step 1: preparing degreased fish head powder: taking fresh fish heads to remove fish gills and fish face meat, pulping the treated fish heads, centrifuging to remove an oil layer and a blood water layer, adding absolute ethyl alcohol into the centrifuged fish head slurry, performing an ultrasonic process, performing suction filtration to obtain fish head residues, adding absolute ethyl alcohol, repeating the ultrasonic process for 2 times, performing three-time circulation, freely washing the fish head residues obtained by suction filtration with distilled water, cleaning, drying in an oven at 55-60 ℃ for 12-15h, and crushing to obtain degreased fish head powder;
and 2, step: mixing the defatted fish head protein powder and distilled water, stirring, homogenizing with a homogenizer, sterilizing in a high-pressure steam sterilizer, and maintaining the temperature with constant-temperature water bath;
and step 3: adjusting pH, keeping pH constant, placing in water bath for 5min, adding alkaline protease, mixing, and performing enzymolysis at constant temperature under oscillation;
and 4, step 4: adjusting the pH value of the solution, keeping the pH value constant, putting the solution into a water bath for 5min, adding papain, uniformly mixing, oscillating at constant temperature for enzymolysis, and then inactivating enzyme;
and 5: cooling to room temperature, centrifuging, taking supernatant, and freeze-drying to obtain walnut protein peptide coarse powder.
Further, in the step 1, in the ultrasonic process, the solid-to-liquid ratio is 1:3-5, 25 ℃, 40kHz frequency, 80% power, 30min time, 3-4min stirring, and repeating the ultrasonic treatment 3 times in each process;
in the step 2, the mixing ratio of the defatted fish head protein powder to the distilled water is 1-10, the homogenizing time is 1-2min, the reaction temperature of the high-pressure steam sterilization pot is 115-125 ℃, the reaction time is 30min, the water bath temperature is 50-60 ℃, and the water bath time is 5-10min;
in the step 3, the pH adjusting solution is a 1mol/L NaOH solution, the pH is adjusted to 9.5-10.5, the water bath temperature is 50-60 ℃, the adding amount of alkaline protease is 1-3%, the oscillation frequency is 150r/min, and the oscillation time is 4-6h;
in the step 4, the pH adjusting solution is 1mol/L NaOH or 1mol/L HCl solution, the pH is adjusted to 6.0-6.5, the water bath temperature is 50-60 ℃, the adding amount of papain is 1-3%, the oscillation frequency is 150r/min, the oscillation time is 4-6h, the enzyme deactivation temperature is 90-99 ℃, and the enzyme deactivation time is 10-15min;
in the step 5, the centrifugation speed is 8000rpm/min, and the centrifugation time is 20min.
Further, the alkaline protease adopted in the enzymolysis process is 200U/mg; the papain used in the enzymolysis process is 30000USP U/mg.
Further, in the step 1, after the fish head is beaten, the centrifugal speed is 4000r and the time is 15min.
Further, the pH value in the step 3,4 is the pH value at the temperature of the water bath.
The invention has the beneficial effects that:
1) The invention mainly adopts an ethanol ultrasonic method to remove grease in the fish head, and the obtained mixed protein peptide has high content of effective components;
2) The invention adopts two steps of enzymolysis method of alkaline protease and papain to carry out enzymolysis on degreased fish head powder and degreased walnut protein powder to obtain fish head protein peptide and walnut protein peptide, and finally natural animal and plant mixed protein peptide is obtained by compounding and combining according to a certain mass ratio; the proportion is reasonable and scientific. The results of Morris water maze positioning navigation experiments show that the functions of the fish head protein peptide and the walnut protein peptide in the aspect of improving the memory can be mutually influenced and have synergistic effect, namely the mixed peptide has good functions of improving the cognitive ability and the memory.
Drawings
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a representative graph of the experimental path of localization for different groups of mice;
in the figure, a large black circle is a Morris water maze swimming pool, a cross-shaped line in the large circle indicates that the swimming pool is divided into four quadrants by a computer, the small black circle is a platform and is positioned 1-2cm below the water surface, the upper right area, where the platform is positioned, is a first quadrant, the upper left area is a second quadrant, the lower left area is a third quadrant, and the lower right area is a fourth quadrant. The cluttered curve trace in the figure is the swimming route trace of the mouse searching for the platform in the swimming pool, and the shorter the total distance of the curve indicates that the mouse finds the platform in shorter time, namely the escape latency of the mouse is shorter.
FIG. 3 is a representation of experimental paths for spatial exploration in different groups of mice;
in the figure, a large black circle is a Morris water maze swimming pool, a cross-shaped line in the large circle indicates that the swimming pool is divided into four quadrants by a computer, a small black circle is the position of an original platform, but the platform is moved at the moment, the position of the small black circle, namely an upper right area, an upper left area, a lower left area, a third quadrant and a lower right area are quadrants. The cluttered curved traces in the figure represent the swimming route of the mouse in the swimming pool to find the original platform, i.e. the mouse is put into the swimming pool facing the pool wall, the times of the mouse crossing the platform in 60s and the swimming time and distance in the quadrant of the original platform are observed.
Fig. 4 shows the number of platform crossings in the Morris water maze space exploration experiment of mice orally administered with the fish head protein peptide, the walnut protein peptide and the mixed peptide, which indicates the significance compared with the model group;
fig. 5 is a graph of the time of the target quadrant in the Morris water maze space exploration experiment for mice orally administered with the fish head protein peptide, the walnut protein peptide and the mixed peptide of the present invention, # # indicates a great significance compared to the normal control group, # # indicates a great significance compared to the model group, and # indicates a great significance compared to the model group;
fig. 6 is the target quadrant distance in the Morris water maze space exploration experiment for mice orally administered with the fish head protein peptide, the walnut protein peptide and the mixed peptide of the present invention, # # indicates a significant significance compared to the normal control group, # # indicates a significant significance compared to the model group.
Detailed Description
For clear and complete description of the technical solutions in the present invention, it is obvious that the inventor combines the embodiments to describe, but the following embodiments describe only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a preparation method of natural animal and plant source mixed protein peptide with memory improving function, which comprises the following specific implementation steps:
a natural animal and plant mixed protein peptide with memory improving function comprises walnut protein peptide and fish head protein peptide.
Further, the walnut protein peptide and the fish head protein peptide are subjected to degreasing treatment; the fish head protein peptide is tilapia fish head protein peptide; the dosage ratio of the walnut protein peptide to the fish head protein peptide is 1-3.
Further, the dosage ratio of the walnut protein peptide to the fish head protein peptide is 1.
Furthermore, in the mixed protein peptide, the content of glutamic acid is more than 14.50g/100 g; the content of arginine is more than 6.90g/100 g; the content of glycine is more than 8.10g/100 g; the content of proline is more than 5.50g/100 g; the total content of hydrophobic amino acids is above 24g/100 g.
The invention relates to a preparation method of natural animal and plant source mixed protein peptide with memory improving function, which comprises the following specific implementation steps:
step 1, preparation of walnut protein peptide
And carrying out enzymolysis on the degreased walnut protein powder by adopting a two-step enzymolysis method of alkaline protease and papain, and freeze-drying an enzymolysis solution to obtain walnut protein peptide coarse powder.
Washing fresh fish heads with distilled water to remove impurities, beating into slurry, centrifuging to remove blood and partial grease, repeatedly performing ultrasonic extraction with absolute ethanol, performing suction filtration on the ethanol crude extract, repeating for 3 cycles, washing and drying fish head residues obtained by suction filtration, and finally crushing to obtain degreased fish head powder.
And (3) carrying out enzymolysis on the degreased fish head powder obtained in the step (2) by adopting an alkaline protease and papain two-step enzymolysis method, and freeze-drying the enzymolysis liquid to obtain fish head protein peptide coarse powder.
Step 4, compounding and combining walnut protein peptide and fish head protein peptide
And (3) mixing the walnut protein peptide coarse powder obtained in the step (1) and the fish head protein peptide coarse powder obtained in the step (3) according to a mass ratio to obtain a mixed peptide with a corresponding ratio, and verifying the activity of the mixed peptide for improving the memory function through subsequent experiments.
Further, the walnut protein peptide is prepared by the following method:
step 1: mixing defatted protein powder and distilled water, stirring, homogenizing with a homogenizer, and keeping the temperature with a constant temperature water bath;
step 2: adjusting pH, keeping pH constant, placing in water bath for 5min, adding alkaline protease, mixing, and performing enzymolysis at constant temperature;
and step 3: adjusting the pH value of the solution, keeping the pH value constant, putting the solution into a water bath for 5min, adding papain, uniformly mixing, oscillating at constant temperature for enzymolysis, and then inactivating enzyme;
and 4, step 4: cooling to room temperature, centrifuging, taking supernatant, and freeze-drying to obtain walnut protein peptide coarse powder.
Further, in the step 1, the mixing ratio of the defatted walnut protein powder to the distilled water is 1;
in the step 2, the pH adjusting solution is a 1mol/L NaOH solution, the pH is adjusted to 9.5-10.5, the water bath temperature is 50-60 ℃, the adding amount of alkaline protease is 1-3%, the oscillation frequency is 150r/min, and the oscillation time is 4-6h;
in the step 3, the pH adjusting solution is 1mol/L NaOH or 1mol/L HCl solution, the pH is adjusted to 6.0-6.5, the water bath temperature is 50-60 ℃, the adding amount of papain is 1-3%, the oscillation frequency is 150r/min, the oscillation time is 4-6h, the enzyme deactivation temperature is 90-99 ℃, and the enzyme deactivation time is 10-15min;
in the step 4, the centrifugation speed is 8000rpm/min, and the centrifugation time is 20min.
Further, the fish head protein peptide is prepared by the following method:
step 1: preparing degreased fish head powder: taking fresh fish heads to remove fish gills and fish face meat, pulping the treated fish heads, centrifuging to remove an oil layer and a blood water layer, adding absolute ethyl alcohol into the centrifuged fish head slurry, performing an ultrasonic process, performing suction filtration to obtain fish head residues, adding absolute ethyl alcohol, repeating the ultrasonic process for 2 times, performing three-time circulation, freely washing the fish head residues obtained by suction filtration with distilled water, cleaning, drying in an oven at 55-60 ℃ for 12-15h, and crushing to obtain degreased fish head powder;
step 2: mixing the defatted fish head protein powder and distilled water, stirring, homogenizing with a homogenizer, sterilizing in a high-pressure steam sterilizer, and maintaining the temperature with constant-temperature water bath;
and 3, step 3: adjusting pH, keeping pH constant, placing in water bath for 5min, adding alkaline protease, mixing, and performing enzymolysis at constant temperature;
and 4, step 4: adjusting the pH value of the solution, keeping the pH value constant, putting the solution into a water bath for 5min, adding papain, uniformly mixing, oscillating at constant temperature for enzymolysis, and then inactivating enzyme;
and 5: cooling to room temperature, centrifuging, and freeze-drying the supernatant to obtain walnut protein peptide coarse powder.
Further, in the step 1, in the ultrasonic process, the solid-to-liquid ratio is 1:3-5, 25 ℃, 40kHz frequency, 80% power, 30min time, 3-4min stirring, and repeating the ultrasonic treatment 3 times in each process;
in the step 2, the mixing ratio of the defatted fish head protein powder to the distilled water is 1-10, the homogenizing time is 1-2min, the reaction temperature of the high-pressure steam sterilization pot is 115-125 ℃, the reaction time is 30min, the water bath temperature is 50-60 ℃, and the water bath time is 5-10min;
in the step 3, the pH adjusting solution is a 1mol/L NaOH solution, the pH is adjusted to 9.5-10.5, the water bath temperature is 50-60 ℃, the adding amount of alkaline protease is 1-3%, the oscillation frequency is 150r/min, and the oscillation time is 4-6h;
in the step 4, the pH adjusting solution is 1mol/L NaOH or 1mol/L HCl solution, the pH is adjusted to 6.0-6.5, the water bath temperature is 50-60 ℃, the adding amount of papain is 1-3%, the oscillation frequency is 150r/min, the oscillation time is 4-6h, the enzyme deactivation temperature is 90-99 ℃, and the enzyme deactivation time is 10-15min;
in the step 5, the centrifugation speed is 8000rpm/min, and the centrifugation time is 20min.
Further, the alkaline protease adopted in the enzymolysis process is 200U/mg; the papain used in the enzymolysis process is 30000USP U/mg.
Further, in the step 1, after the fish head is pulped, the centrifugal speed is 4000r, and the time is 15min.
And (3) mixing the obtained walnut protein peptide coarse powder with the tilapia mossambica head protein peptide coarse powder obtained in the step 3 according to the mass ratio of 1:1-3 to obtain mixed peptide with corresponding proportion, and verifying the activity of improving memory function through subsequent experiments.
Example 1
Step 1, preparation of walnut protein peptide
Taking 80g of defatted walnut protein powder, and mixing with distilled water according to a feed-liquid ratio of 1:8, uniformly stirring and homogenizing for 1.5min, then placing in a constant-temperature water bath at 55 ℃ for 5min, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping pH constant, placing in 55 deg.C water bath for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5 hr; taking out, adjusting pH of the solution to 6.0 with 1mol/L NaOH or 1mol/L HCl, placing in 55 deg.C constant temperature water bath for 5min, adding 2% papain (1.6 g), mixing, performing enzymolysis at 55 deg.C and 150r/min under constant temperature oscillation for 5h, and inactivating enzyme at 95 deg.C for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, collecting supernatant, and freeze drying to obtain walnut protein peptide coarse powder. The enzyme adding amount of the alkaline protease and the papain is calculated according to the amount of the walnut protein powder and the ratio, and the alkaline protease: 200U/mg; papain: min.30000USP U/mg.
Taking fresh tilapia mossambica head to remove fish gill and fish face meat, pulping the treated fish head, centrifuging for 15min at 4000r to remove an oil layer and a blood water layer, and centrifuging the fish head pulp in a ratio of 1:4, adding absolute ethyl alcohol into the mixture in a solid-to-liquid ratio, and carrying out ultrasonic treatment for 3 times at 25 ℃, 40kHz and 80% of ultrasonic power, wherein each time is 30min, and stirring is required for 3-4min; and after the ultrasonic treatment is finished, carrying out suction filtration to obtain fish head residues, and continuously mixing the fish head residues with the mixture of 1:4, adding absolute ethyl alcohol into the solid-liquid ratio, performing ultrasonic treatment again for 3 times at the same temperature and power, wherein each time is 30min, and stirring is required for 3-4min; this was repeated 3 times, i.e. a total of three cycles of sonication, 3 times per cycle, each for 30min, wherein each cycle required a new change of absolute ethanol. And after three cycles, freely washing the fish head residues obtained by suction filtration with distilled water, cleaning, placing in a 55 ℃ oven for drying for 12h to remove the residual absolute ethyl alcohol and part of the distilled water, drying, and crushing the fish head residues into powder by using a multifunctional crusher to obtain defatted tilapia head powder, and storing the defatted tilapia head powder in a drying dish for later use.
Taking 80g of degreased tilapia mossambica head powder obtained in the step 2, and mixing with distilled water according to a feed-liquid ratio of 1:8, uniformly stirring and homogenizing for 1.5min, placing in a high-pressure steam sterilization pot at 120 ℃ for reaction for 30min, placing in a constant-temperature water bath at 55 ℃ for 10min after the reaction is finished, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping pH constant, placing in 55 deg.C water bath for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5 hr; taking out, adjusting pH of the solution to 6.0 with 1mol/L NaOH or 1mol/L HCl, placing in 55 deg.C constant temperature water bath for 5min, adding 2% papain (1.6 g), mixing, performing enzymolysis at 55 deg.C and 150r/min under constant temperature oscillation for 5h, and inactivating enzyme at 95 deg.C for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, and freeze drying the supernatant to obtain coarse powder of tilapia head protein peptide. The enzyme adding amount of the alkaline protease and the papain is calculated according to the amount of the degreased tilapia mossambica head meal according to the proportion, and the alkaline protease: 200U/mg; papain: min.30000USP U/mg.
Step 4, compounding and combining walnut protein peptide and tilapia mossambica head protein peptide
And (2) mixing the walnut protein peptide coarse powder obtained in the step (1) with the tilapia mossambica head protein peptide coarse powder obtained in the step (3) according to the mass ratio of 1:1 to obtain the mixed peptide.
Example 2
Step 1, preparation of walnut protein peptide
Taking 80g of defatted walnut protein powder, and mixing with distilled water according to a feed-liquid ratio of 1:8, stirring uniformly, homogenizing for 1.5min, placing in a constant-temperature water bath at 55 ℃ for 5min, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping pH constant, placing in 55 deg.C water bath for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5 hr; taking out, adjusting pH of the solution to 6.0 with 1mol/L NaOH or 1mol/L HCl, placing in 55 deg.C constant temperature water bath for 5min, adding 2% papain (1.6 g), mixing, performing enzymolysis at 55 deg.C and 150r/min under constant temperature oscillation for 5h, and inactivating enzyme at 95 deg.C for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, and freeze drying the supernatant to obtain walnut protein peptide coarse powder. The enzyme adding amount of the alkaline protease and the papain is calculated according to the amount of the walnut protein powder and the ratio, and the alkaline protease: 200U/mg; and (3) papain: min.30000USP U/mg.
Taking fresh tilapia mossambica heads, removing fish gills and fish face meat, pulping the treated fish heads, centrifuging for 15min at 4000r to remove an oil layer and a blood water layer, and mixing the centrifuged fish head pulp with a ratio of 1:4, adding absolute ethyl alcohol, performing ultrasonic treatment for 3 times at 25 ℃, 40kHz and 80% of ultrasonic power for 30min each time, and stirring for 3-4min; and after the ultrasonic treatment is finished, carrying out suction filtration to obtain fish head residues, and continuously mixing the fish head residues with the mixture of 1:4, adding absolute ethyl alcohol into the solid-liquid ratio, performing ultrasonic treatment again for 3 times at the same temperature and power, wherein each time is 30min, and stirring is required for 3-4min; this was repeated 3 times, i.e. a total of three cycles of sonication, 3 times per cycle, each for 30min, wherein each cycle required a new change of absolute ethanol. And after three cycles, freely washing the fish head residues obtained by suction filtration with distilled water, cleaning, placing in a 55 ℃ oven for drying for 12h to remove the residual absolute ethyl alcohol and part of the distilled water, drying, and crushing the fish head residues into powder by using a multifunctional crusher to obtain defatted tilapia head powder, and storing the defatted tilapia head powder in a drying dish for later use.
Taking 80g of defatted tilapia mossambica head meal obtained in the step 2, and mixing with distilled water according to a feed-liquid ratio of 1:8, uniformly stirring and homogenizing for 1.5min, placing in a high-pressure steam sterilization pot at 120 ℃ for reaction for 30min, placing in a constant-temperature water bath at 55 ℃ for 10min after the reaction is finished, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping pH constant, placing in 55 deg.C water bath for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5 hr; taking out, adjusting the pH value of the solution to 6.0 by using 1mol/L NaOH or 1mol/L HCl, placing the solution in a constant-temperature water bath at 55 ℃ for 5min, adding 2% of papain (1.6 g), uniformly mixing, oscillating at the constant temperature of 55 ℃ and 150r/min for enzymolysis for 5h, and then placing the mixture at 95 ℃ for enzyme deactivation for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, and freeze drying the supernatant to obtain coarse powder of tilapia head protein peptide. The enzyme adding amount of the alkaline protease and the papain is calculated according to the amount of the degreased tilapia mossambica head meal according to the proportion, and the alkaline protease: 200U/mg; papain: min.30000USP U/mg.
Step 4, compounding and combining walnut protein peptide and tilapia mossambica head protein peptide
Mixing the walnut protein peptide coarse powder obtained in the step 1 and the tilapia mossambica head protein peptide coarse powder obtained in the step 3 according to the mass ratio of 1:2 to obtain the mixed peptide.
Example 3
Step 1, preparation of walnut protein peptide
Taking 80g of defatted walnut protein powder, and mixing with distilled water according to a feed-liquid ratio of 1:8, stirring uniformly, homogenizing for 1.5min, placing in a constant-temperature water bath at 55 ℃ for 5min, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping the pH value constant, placing in water bath at 55 deg.C for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5h; taking out, adjusting pH of the solution to 6.0 with 1mol/L NaOH or 1mol/L HCl, placing in 55 deg.C constant temperature water bath for 5min, adding 2% papain (1.6 g), mixing, performing enzymolysis at 55 deg.C and 150r/min under constant temperature oscillation for 5h, and inactivating enzyme at 95 deg.C for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, and freeze drying the supernatant to obtain walnut protein peptide coarse powder. Wherein the enzyme adding amount of the used alkaline protease and papain is calculated according to the amount of the walnut protein powder and the ratio, and the alkaline protease: 200U/mg; papain: min.30000USP U/mg.
Taking fresh tilapia mossambica head to remove fish gill and fish face meat, pulping the treated fish head, centrifuging for 15min at 4000r to remove an oil layer and a blood water layer, and centrifuging the fish head pulp in a ratio of 1:4, adding absolute ethyl alcohol, performing ultrasonic treatment for 3 times at 25 ℃, 40kHz and 80% of ultrasonic power for 30min each time, and stirring for 3-4min; and after the ultrasound is finished, carrying out suction filtration to obtain fish head residues, and continuously mixing the fish head residues with the mixture of 1:4, adding absolute ethyl alcohol into the solid-liquid ratio, performing ultrasonic treatment again for 3 times at the same temperature and power, wherein each time is 30min, and stirring is required for 3-4min; this was repeated 3 times, i.e. a total of three cycles of sonication, 3 times per cycle, each for 30min, wherein each cycle required a new change of absolute ethanol. And after three cycles, freely washing the fish head residue obtained by suction filtration with distilled water, washing, placing in a 55 ℃ oven for drying for 12 hours to remove the residual absolute ethyl alcohol and part of distilled water, drying, and crushing the fish head residue into powder by using a multifunctional crusher to obtain defatted tilapia head powder, and storing the defatted tilapia head powder in a drying dish for later use.
Taking 80g of degreased tilapia mossambica head powder obtained in the step 2, and mixing with distilled water according to a feed-liquid ratio of 1:8, uniformly stirring and homogenizing for 1.5min, placing in a high-pressure steam sterilization pot at 120 ℃ for reaction for 30min, placing in a constant-temperature water bath at 55 ℃ for 10min after the reaction is finished, and adjusting the pH of the solution to 10.0 by using 1mol/L NaOH; keeping pH constant, placing in 55 deg.C water bath for 5min, adding 2% alkaline protease (1.6 g), mixing, and performing constant temperature oscillation enzymolysis at 55 deg.C and 150r/min for 5 hr; taking out, adjusting pH of the solution to 6.0 with 1mol/L NaOH or 1mol/L HCl, placing in 55 deg.C constant temperature water bath for 5min, adding 2% papain (1.6 g), mixing, performing enzymolysis at 55 deg.C and 150r/min under constant temperature oscillation for 5h, and inactivating enzyme at 95 deg.C for 10min; cooling to room temperature, centrifuging at 8000rpm for 20min, and freeze-drying the supernatant to obtain coarse tilapia head protein peptide powder. The enzyme adding amount of the alkaline protease and the papain is calculated according to the amount of the defatted tilapia mossambica head meal according to a proportion, and the alkaline protease: 200U/mg; papain: min.30000USP U/mg.
Step 4, compounding and combining walnut protein peptide and tilapia head protein peptide
Mixing the walnut protein peptide coarse powder obtained in the step 1 and the tilapia mossambica head protein peptide coarse powder obtained in the step 3 according to the mass ratio of 1:3 to obtain the mixed peptide.
1. Amino acid composition analysis was performed on the walnut protein peptide and tilapia head protein peptide prepared in example 1, and the results are shown in table 1:
TABLE 1 analysis of amino acid composition of Tilapia mossambica head protein peptide and walnut protein peptide
As can be seen from Table 1, the tilapia mossambica head protein peptide and walnut protein peptide prepared by the method have the highest glutamic acid content, and the glutamic acid is a nerve excitation amino acid and is also an excitatory neurotransmitter with the highest central nervous system content and the widest distribution, and can be used as a supplement for cerebral cortex and the like, so that the tilapia mossambica head protein peptide and walnut protein peptide have certain curative effects on maintaining cerebral function, treating cerebral injury and improving memory. Proline may regulate synaptic transmission in the mammalian brain, arginine is one of the essential amino acids and may synergistically enhance the efficiency of improving memory with creatine. The tilapia head protein peptide has higher proline content and is obviously higher than the walnut protein peptide, and the walnut protein peptide has higher arginine content and is obviously higher than the tilapia head protein peptide, so that the tilapia head protein peptide and the tilapia head protein peptide have the following ratio of 1:1 has the functions of improving memory, maintaining brain function and the like, and can play a complementary and synergistic increasing role.
2. Animal behavioral experiments were performed on the mixed peptide prepared in example 1 to further verify its functional activity in improving memory decline and cognitive impairment
Animal experiment grouping: selecting BALB/c male mice, wherein the mice are 6-8 weeks old and 18-22g in weight, adaptively feeding for 1 week, randomly dividing the mice into 6 groups, wherein each group comprises 8-10 mice, weighing the mice once every two days during feeding, and performing intragastric administration according to the weights of the mice at 8 am every day, wherein the intragastric administration amount of a walnut protein peptide group is 400mg/kg.bw (preparing walnut protein peptide solution by using distilled water), the intragastric administration amount of a tilapia head protein peptide group is 400mg/kg.bw (preparing tilapia head protein peptide solution by using distilled water), the intragastric administration amount of a mixed peptide group is 200mg/kg.bw protein peptide and 200mg/kg.bw tilapia head protein peptide (preparing mixed peptide by using distilled water), the blank control group and a memory injury model group are perfused with distilled water with the same volume, the blank positive control group is perfused with 400mg/kg piracetame (preparing piracetame solution by using distilled water), after 35 days of continuous gastric administration, the mice are perfused with Morris water, namely 36 th day, the maze experiment is completed, the peritoneal injection of the blank positive control group is performed, and the intraabdominal cavity injection is divided into 3 mg/kg of 3 portions except for 3 portions of scopolamine: the method comprises a familiarity period of 1 day, a training period of 5 days (namely a positioning navigation experiment), and a testing period of 1 day (namely a space exploration experiment) (scopolamine is a muscarinic receptor antagonist and can block cholinergic function of a central nervous system, so that a mouse is impaired in memory in a short period, and piracetam has the capacity of combining with a cholinergic receptor, so that the acetylcholine level in the brain is improved, and the method has a positive treatment effect on memory-impaired animals).
An experimental instrument: the Morris water maze experiment system is composed of a circular water pool with the diameter of 1.2m and the height of 45cm, an automatic image acquisition and processing system and a SuperMaze animal behavior video analysis system, wherein the image acquisition and processing system and the video analysis system are mainly composed of a camera, a computer and an image acquisition card. During the experiment, water is changed every day, the water depth in the water pool is about 25-35cm, the diameter of the platform is 12cm and is located 1-2cm below the water surface, no mark is arranged in the water pool, 4 mark points are arranged on the upper edge of the water pool at equal intervals so that the direction of a mouse can be distinguished in the water pool, 3 points are left in the 4 points after the point of the quadrant where the platform is located is removed and are used as water inlet points during the mouse experiment, the water pool is equally divided into 4 quadrants by software during the observation on a computer, and the platform is placed in any quadrant according to the experiment requirements.
1. Morris water maze positioning navigation experiment
The method comprises the steps of carrying out a mouse positioning navigation experiment after the water maze familiarity period is finished, wherein the Morris water maze positioning navigation experiment is carried out for 5 days, each mouse carries out 3 experiments every day, namely, the mice are respectively put into water from the positions of other three quadrant markers except the quadrant where the platform is located, a computer automatically records the time required for the mouse to find the underwater platform within 60s during each training, namely the escape latency of each training of the mouse, if the mouse does not find the underwater platform within 60s, an experimenter guides the mouse to the platform by using a clean glass rod for placing for 20s, the escape latency of the mouse is recorded as 60s, the mouse is wiped by using a dry towel after each training, the body temperature of the mouse is baked to be normal, and the mouse is allowed to rest for 60s and then carries out the next training. Escape latency for training period of each group of mice is shown in table 2, and the mice underway at the 5 th day position represents a trajectory diagram as shown in fig. 2.
Table 2 escape latency of mouse localization experiment
According to the table 2, the escape latency of the model group mice is found to be almost unchanged after 5 days of training, which shows that the mice injected with scopolamine intraperitoneally are successfully modeled, the memory is damaged and the learning ability is obviously reduced, the mice of other groups have certain learning ability, the positive control group, the tilapia noggin group, the walnut noggin group and the mixed peptide group can obviously improve the damage of scopolamine to the memory of the mice and improve the learning ability of the mice, and the mice perfused with the mixed peptide have more obvious effect on improving the memory damage of the mice, and the improvement ability of the mice is stronger than that of the positive control group or the tilapia noggin group or the walnut noggin group separately perfused with the stomach. According to the graph shown in fig. 2, the swimming track of the mice in the model group is irregular, and a long time is needed to find the platform, and the mice in other groups can accurately find the platform in a short time like a normal control group after being trained for the previous days, so that the learning capacity of the four groups of mice except the normal group and the model group after being taken with the medicine is obviously improved, and the memory is also improved.
2. Morris water maze space exploration experiment
After the mouse water maze positioning navigation experiment is finished, the platform is moved away (but the area where the platform is located in the computer processing system still remains-namely a small circle in fig. 2 and 3), a Morris water maze space exploration experiment is carried out, each mouse carries out 3 times of space exploration during the experiment, namely the mouse is placed into water for exploration according to the positioning navigation experiment mode, the computer automatically records the times that the mouse passes through the area where the platform is located within 60s, the swimming distance of the quadrant where the mouse is located and the swimming time of the quadrant where the mouse is located, the results are respectively represented by fig. 4, 5 and 6, and fig. 3 represents a swimming track representative diagram of the mouse in the space exploration experiment. The experimental method is to detect the spatial memory capacity of the mouse to the original platform position by removing the underwater platform. As can be seen from FIG. 3, the swimming paths of the mice in the model group are disordered and the memory of the original platform position is completely lost; the positive control group, the fish head protein peptide group, the walnut protein peptide group and the mixed peptide group have certain repairing effect on memory damage of mice and can improve the learning ability of the mice, wherein the repairing effect and the learning ability improving effect of the mixed peptide are more remarkable. It can be seen from fig. 4, 5 and 6 that the frequency of crossing the platform by the mice injected with scopolamine intraperitoneally is almost 0, and the staying time and swimming distance in the target quadrant are both relatively short, which indicates that the mice have no memory of the position of the original platform and low cognitive ability; the times of observing the other groups of mice to pass through the platform are more, and the swimming time and distance around the target quadrant platform are longer, which shows that the mice in the groups have certain memory on the position of the original platform, the cognitive ability is stronger, the memory ability of the mice in the mixed peptide group on the original platform is stronger, each mouse can pass through the platform for 2-3 times on average, and the mixed peptide has better repairing effect on the memory damage of the mice.
In conclusion, the natural animal and plant mixed protein peptide can be prepared by the method, is food-borne peptide and high in safety, and animal behavior experiments show that the mixed peptide has the obvious effects of improving memory and cognitive impairment and improving learning ability, and the effect is stronger than that of any component forming the peptide, so that the experimental animal becomes more clever relative to a normal animal. The above-mentioned embodiments are preferred embodiments of the present invention, and the present invention is not limited in any way, and it will be apparent to those skilled in the art that the present invention can be modified or modified without departing from the spirit and principle of the present invention, and these should be included in the protection scope of the present invention.
Claims (5)
1. A natural animal and plant mixed protein peptide with a memory improving function is characterized by consisting of walnut protein peptide and fish head protein peptide with the dosage ratio of 1; the walnut protein peptide and the fish head protein peptide are subjected to degreasing treatment;
the walnut protein peptide is prepared by the following method:
step 1: mixing the defatted protein powder and distilled water according to a ratio of 1;
and 2, step: adjusting the pH value of the solution to be 1mol/L NaOH solution, adjusting the pH value to 9.5-10.5, keeping the pH value constant, putting the solution into a water bath with the temperature of 50-60 ℃ for 5min, adding 1-3% of alkaline protease, uniformly mixing, oscillating at constant temperature for enzymolysis, wherein the oscillation frequency is 150r/min, and the oscillation time is 4-6h;
and step 3: adjusting the pH value of the solution to 6.0-6.5 by using 1mol/L NaOH or 1mol/L HCl solution, keeping the pH value constant, putting the solution into a water bath with the temperature of 50-60 ℃ for 5min, adding 1-3% of papain, uniformly mixing, oscillating at constant temperature for enzymolysis, wherein the oscillation frequency is 150r/min and the oscillation time is 4-6h, and then inactivating the enzyme at the enzyme deactivation temperature of 90-99 ℃ for 10-15min;
and 4, step 4: cooling to room temperature, centrifuging at 8000r/min for 20min, and freeze drying the supernatant to obtain walnut protein peptide coarse powder;
the fish head protein peptide is prepared by the following method:
step 1: preparing degreased fish head powder: taking fresh fish heads to remove fish gills and fish face meat, pulping the treated fish heads, centrifuging to remove an oil layer and a blood water layer, adding absolute ethyl alcohol into the centrifuged fish head slurry, performing an ultrasonic process, performing suction filtration to obtain fish head residues, adding absolute ethyl alcohol, repeating the ultrasonic process for 2 times, performing three-time circulation, freely washing the fish head residues obtained by suction filtration with distilled water, cleaning, drying in an oven at 55-60 ℃ for 12-15h, and crushing to obtain degreased fish head powder; in the ultrasonic working procedure, the solid-liquid ratio is 1:3-5, 25 ℃, 40kHz frequency, 80% power, 30min time, 3-4min stirring, and repeating the ultrasonic treatment 3 times in each process;
step 2: mixing the defatted fish head protein powder and distilled water according to a ratio of 1 to 6-10, stirring, homogenizing for 1-2min by using a homogenizer, placing in a high-pressure steam sterilization pot for sterilization, wherein the reaction temperature of the high-pressure steam sterilization pot is 115-125 ℃, the reaction time is 30min, and the constant temperature is kept by using a constant-temperature water bath, the water bath temperature is 50-60 ℃, and the water bath time is 5-10min;
and step 3: adjusting pH to 9.5-10.5 with 1mol/L NaOH solution, keeping pH constant, placing in 50-60 deg.C water bath for 5min, adding 1-3% alkaline protease, mixing, oscillating at constant temperature for enzymolysis, with oscillation frequency of 150r/min and oscillation time of 4-6h;
and 4, step 4: adjusting the pH value of the solution to 6.0-6.5 by using 1mol/L NaOH or 1mol/L HCl solution, keeping the pH value constant, putting the solution into a water bath at 50-60 ℃ for 5min, adding 1-3% papain, uniformly mixing, oscillating at constant temperature for enzymolysis, wherein the oscillation frequency is 150r/min and the oscillation time is 4-6h, and then inactivating the enzyme at the enzyme temperature of 90-99 ℃ for 10-15min;
and 5: cooling to room temperature, centrifuging at 8000r/min for 20min, and freeze drying the supernatant to obtain fish head protein peptide coarse powder;
the alkaline protease adopted in the enzymolysis process is 200U/mg; the papain used in the enzymolysis process is 30000USP U/mg.
2. The natural animal and plant mixed protein peptide according to claim 1, wherein the fish head protein peptide is tilapia fish head protein peptide.
3. The natural animal and plant mixed protein peptide as claimed in claim 1, wherein the dosage ratio of the walnut protein peptide to the fish head protein peptide is 1.
4. The natural animal and plant mixed protein peptide according to claim 1, wherein the content of glutamic acid in the mixed protein peptide is 14.50g/100g or more; the content of arginine is more than 6.90g/100 g; the content of glycine is more than 8.10g/100 g; the content of proline is more than 5.50g/100 g; the total content of hydrophobic amino acids is above 24g/100 g.
5. Use of the natural animal and plant mixed protein peptide as defined in any one of claims 1 to 4 for the preparation of a food for preventing and/or improving memory impairment and cognitive impairment.
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