A composition, preparation method, nutritional powder and application of branched-chain amino acid peptides from seabuckthorn seeds

Abstract

The present invention belongs to the technical field of the preparation of branched-chain amino acid peptides, and specifically relates to a composition, a preparation method, a nutritional powder and an application of branched-chain amino acid peptides from sea buckthorn seeds. The present invention provides a composition of branched-chain amino acid peptides from sea buckthorn seeds, and the branched-chain amino acid peptides include a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide. The composition of branched-chain amino acid peptides from sea buckthorn seeds provided by the present invention can increase muscle endurance, slow down muscle fatigue and accelerate the recovery of exercise fatigue, has the effects of anti-fatigue and increasing muscle endurance, and can be used for preparing anti-fatigue nutritional powder.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of branched-chain amino acid peptides, and particularly relates to a composition, a preparation method, a nutritional powder and an application of branched-chain amino acid peptides from sea buckthorn seeds. Background Art

[0002] Branched-chain amino acids (isoleucine, leucine and valine; BCAAs) are essential amino acids, accounting for about 21% of the total body protein content and 40% - 45% of the total essential amino acid content. Skeletal muscle is the largest storage depot of BCAAs in the human body, and BCAAs mainly stimulate protein synthesis in skeletal muscle (Sunil, L., et al. In silico designing of therapeutic protein enriched with branched-chain aminoacids for the dietary treatment of chronic liver dise ase [J]. Journal ofMolecular Graphics&Modelling, 2017). The growth of skeletal muscle depends on the positive balance (synthesis > degradation) of the dynamic turnover process of protein deposition, and the increase in protein synthesis plays a crucial role in skeletal muscle growth. Data shows that BCAAs are the only amino acids with relatively high metabolism in skeletal muscle, and the transamination reaction is dominant in the metabolism. BCAAs and the transamination products of BCAAs can significantly increase the activity of mTORC1, promote the initiation of protein translation and skeletal muscle protein synthesis (Bifari, et al. Branched-chain amino acids differently modulate catabolic and anabolicstates in mammals: a pharmacological point of view [J]. British Journal ofPharmaco logy, 2017.). During endurance exercise, BCAAs in muscles are consumed relatively quickly. Supplementing BCAAs can improve the body's exercise ability, delay the onset of fatigue, slow down muscle fatigue, and help the muscles recover to the normal BCAAs level (Liu Cheng. Anti-exercise fatigue effect of branched-chain amino acids from Agaricus blazei [J]. Edible Fungi of China, 2020.). Supplementing BCAAs can reduce the ratio of free tryptophan / BCAAs, inhibit the entry of tryptophan in the blood into the central nervous system, and has an anti-central fatigue effect (Xu Yunjie, et al. Anti-fatigue effect of branched-chain amino acids [J]. Amino Acids & Biotic Resources, 2008).

[0003] Currently, a large amount of evidence at home and abroad shows that supplementing BCAAs or a diet rich in BCAAs has a positive effect on anti-fatigue and regulating muscle synthesis. It is worth noting that compared with branched-chain amino acids, branched-chain amino acid oligopeptides have a better absorption mechanism and biological activity (Zhu, et al. Hypoglycemic effect of hydrophobic BCAAs peptides mediate via activating PI3K / Akt signaling in skeletal muscle of db / bd mice[J]. Journal of agriculture and food chemistry, 2021).

[0004] However, there is no report on the composition of seabuckthorn seed branched-chain amino acid peptides with anti-fatigue effects. Summary of the Invention

[0005] The object of the present invention is to provide a composition of seabuckthorn seed branched-chain amino acid peptides, which has an anti-fatigue effect.

[0006] To solve the above problems, the present invention provides the following technical solutions:

[0007] The present invention provides a composition of seabuckthorn seed branched-chain amino acid peptides, wherein the branched-chain amino acid peptides include a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide. The amino acid sequence of the first polypeptide is Ile-Pro or Leu-Pro, the amino acid sequence of the second polypeptide is Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val, the amino acid sequence of the third polypeptide is Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Ile or Leu-Pro-Leu, and the amino acid sequence of the fourth polypeptide is Asp-Leu-Val-Gly-Lys or Asp-Ile-Val-Gly-Lys.

[0008] Preferably, the composition of seabuckthorn seed branched-chain amino acid peptides is extracted from seabuckthorn seed meal.

[0009] Preferably, the molecular weight of the composition of seabuckthorn seed branched-chain amino acid peptides is less than 1000 Da.

[0010] The present invention provides a preparation method of the composition of seabuckthorn seed branched-chain amino acid peptides according to any one of the above technical solutions, including the following steps:

[0011] Treat seabuckthorn seed meal by the alkali solution-acid precipitation method to obtain seabuckthorn seed protein;

[0012] Hydrolyze seabuckthorn seed protein to obtain a hydrolysate;

[0013] Extract the hydrolysate to obtain a supernatant;

[0014] The dried supernatant yields a dried product of a composition containing seabuckthorn seed branched-chain amino acid peptides.

[0015] Preferably, the enzymes used in the hydrolysis include protease and / or pancreatin, and the protease includes one or more of acidic protease, trypsin, and flavor protease.

[0016] Preferably, the addition amount of acidic protease in the protease is 0.2 - 1.0% of the mass of the aqueous solution of seabuckthorn seed protein; the addition amount of trypsin in the protease is 0.5 - 1.5% of the mass of the aqueous solution of seabuckthorn seed protein; the addition amount of flavor protease in the protease is 0.2 - 1.5% of the mass of the aqueous solution of seabuckthorn seed protein; the addition amount of pancreatin is 0.2 - 1.5% of the mass of the aqueous solution of seabuckthorn seed protein.

[0017] Preferably, the temperature of the hydrolysis is 45 - 60°C and the time of the hydrolysis is 12 - 24 h.

[0018] Preferably, the extractant used in the extraction includes an ethanol aqueous solution; the volume ratio of the hydrolysate to the ethanol aqueous solution is 1:(6 - 9); the number of extractions is 1 - 3 times.

[0019] The present invention provides a nutritional powder, comprising the composition of seabuckthorn seed branched-chain amino acid peptides described in the above technical solution or the composition of seabuckthorn seed branched-chain amino acid peptides prepared by the preparation method described in the above technical solution.

[0020] The present invention provides the application of the composition of seabuckthorn seed branched-chain amino acid peptides described in the above technical solution or the composition of seabuckthorn seed branched-chain amino acid peptides prepared by the preparation method described in the above technical solution or the nutritional powder described in the above technical solution in anti-fatigue; the anti-fatigue includes one or several of 1) - 3);

[0021] 1) Slow down muscle fatigue;

[0022] 2) Increase muscle endurance;

[0023] 3) Accelerate the recovery of exercise fatigue.

[0024] Advantages of the present invention: The present invention provides a composition of branched-chain amino acid peptides from seabuckthorn seeds. The branched-chain amino acid peptides include a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. The amino acid sequence of the first polypeptide is Ile-Pro or Leu-Pro. The amino acid sequence of the second polypeptide is Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val. The amino acid sequence of the third polypeptide is Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Ile or Leu-Pro-Leu. The amino acid sequence of the fourth polypeptide is Asp-Leu-Val-Gly-Lys or Asp-Ile-Val-Gly-Lys.

[0025] The composition of branched-chain amino acid peptides provided by the present invention includes the above-mentioned polypeptides. The mode of action of the branched-chain amino acid peptides is to be completely hydrolyzed into free amino acids under the action of peptidases in the body and enter the human blood in the form of free amino acids. After the branched-chain amino acid peptides are decomposed, branched-chain amino acids are formed. Branched-chain amino acids are composed of valine, leucine, and isoleucine. Branched-chain amino acids can promote the synthesis of skeletal muscle proteins. Among them, valine mainly controls and regulates food intake to provide sufficient substrate concentration. Leucine mainly activates the translation initiation signal MTOR of proteins. Under the combined action of each amino acid, the polypeptide composition provided by the present invention can increase muscle endurance, slow down muscle fatigue, and accelerate the recovery of exercise fatigue, and has the effects of anti-fatigue and increasing muscle endurance. It can be used to prepare anti-fatigue medical foods or applied to the preparation of anti-fatigue nutritional powders. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0027] Figure 1 Secondary structure mass spectrometry diagram of the first polypeptide in the composition of branched-chain amino acid peptides from seabuckthorn seeds prepared in Example 1, I / L-P (Ile-Pro or Leu-Pro);

[0028] Figure 2 Secondary structure mass spectrometry diagram of the second polypeptide in the composition of branched-chain amino acid peptides from seabuckthorn seeds prepared in Example 1, I / L-P-E-D-V (Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val);

[0029] Figure 3 Secondary structure mass spectrometry diagram of the third polypeptide in the composition of branched-chain amino acid peptides from seabuckthorn seeds prepared in Example 1, I / L-P-I / L (Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Ile or Leu-Pro-Leu);

[0030] Figure 4 The secondary structure mass spectrometry diagram of the fourth polypeptide in the composition of the branched-chain amino acid peptide from seabuckthorn seeds prepared in Example 1: D-I / L-V-G-K (Asp-Leu-Val-Gly-Lys or Asp-Ile-Val-Gly-Lys);

[0031] Figure 5 The fatigue tolerance time of weight-bearing swimming of each group of mice in Test Example 1;

[0032] Figure 6 The weight-bearing swimming movement trajectory diagram of each group of mice in Test Example 1;

[0033] Figure 7 The fatigue tolerance time of the rotarod test of each group of mice in Test Example 2. Detailed implementation manners

[0034] The present invention provides a composition of a branched-chain amino acid peptide from seabuckthorn seeds. The branched-chain amino acid peptide includes a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. The amino acid sequence of the first polypeptide is Ile-Pro or Leu-Pro. The amino acid sequence of the second polypeptide is Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val. The amino acid sequence of the third polypeptide is Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Ile or Leu-Pro-Leu. The amino acid sequence of the fourth polypeptide is Asp-Leu-Val-Gly-Lys or Asp-Ile-Val-Gly-Lys.

[0035] In the present invention, the branched-chain amino acid peptide from seabuckthorn seeds is preferably extracted from seabuckthorn seed meal.

[0036] In the present invention, the branched-chain amino acid peptide of seabuckthorn seed protein is preferably artificially extracted from seabuckthorn seed meal. The molecular weight of the branched-chain amino acid peptide of seabuckthorn seed protein in the present invention is less than 1000 Da. The relative molecular weight of Ile-Pro or Leu-Pro in the present invention is 229.1546, and the relative molecular weight of Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val is 572.2921; the relative molecular weight of Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Leu or Leu-Pro-Ile is 342.2390; the relative molecular weight of Asp-Ile-Val-Gly-Lys or Asp-Leu-Val-Gly-Lys is 553.3346. The molecular weight of the composition of the branched-chain amino acid peptide of seabuckthorn seed in the present invention is less than 1000 Da. Most of the existing studies on protein branched-chain amino acid peptides are some macromolecular polypeptides, and there are still some blank areas in the research on oligopeptides. The composition of the branched-chain amino acid peptide of seabuckthorn seed in the present invention is an oligopeptide, which is more conducive to human digestion and absorption compared with proteins, more conducive to exerting its biological functions and faster absorption compared with amino acids, and oligopeptides have good processing and flavor characteristics. Moreover, the composition of the branched-chain amino acid peptide of seabuckthorn seed provided by the present invention has the effects of increasing muscle synthesis, increasing muscle endurance, and delaying muscle fatigue, and can be used for preparing anti-fatigue medical foods and anti-fatigue nutritional powders for athletes.

[0037] The present invention provides a preparation method of the composition of the branched-chain amino acid peptide of seabuckthorn seed described in the above technical solution, comprising the following steps:

[0038] Treat seabuckthorn seed meal by alkali solution extraction and acid precipitation to obtain seabuckthorn seed protein;

[0039] Hydrolyze the seabuckthorn seed protein to obtain a hydrolyzate;

[0040] Extract the hydrolyzate to obtain a supernatant;

[0041] The supernatant is vacuum freeze-dried to obtain a dry product containing the composition of the branched-chain amino acid peptide of seabuckthorn seed.

[0042] The present invention treats seabuckthorn seed meal by alkali solution extraction and acid precipitation to obtain seabuckthorn seed protein.

[0043] Before performing alkali solution and acid precipitation extraction on sea buckthorn seed meal, the present invention preferably further includes performing ultrafine pulverization treatment on the sea buckthorn seed meal. The material obtained by ultrafine pulverization is sieved, and the material passing through the sieve is subjected to alkali solution and acid precipitation. The present invention has no special limitation on the source of the sea buckthorn seed meal, and conventional methods can be used. The present invention has no special limitation on the parameters of the ultrafine pulverization, and conventional parameters can be used. The function of the ultrafine pulverization in the present invention is to increase the contact area in the alkali solution and acid precipitation reaction of the sea buckthorn seed meal, make the reaction more thorough and complete, and improve the yield of the subsequent produced sea buckthorn seed protein. The mesh number of the sieving in the present invention is preferably 70-100 meshes, more preferably 80-90 meshes, and even more preferably 85 meshes. The purpose of the sieving in the present invention is to remove impurities in the sea buckthorn seed meal and improve the purity of the sea buckthorn seed meal.

[0044] The present invention preferably performs alkali solution and acid precipitation on the material passing through the sieve obtained after sieving the material obtained by ultrafine pulverization. In the present invention, the preferred manner of the alkali solution includes: adding water to the sea buckthorn seed meal powder obtained after ultrafine pulverization and sieving, adjusting the pH value, followed by water bath and centrifugation to obtain a supernatant. The water in the present invention is preferably deionized water. The material-liquid ratio of the sea buckthorn seed meal powder and deionized water in the present invention is preferably 1 g:(8-10) mL, more preferably 1 g:9 mL. The adjusted pH value in the present invention is preferably 10.0-11.0, more preferably 10.5-11.0, and even more preferably 11.0. The temperature of the water bath in the present invention is preferably 40°C-60°C, more preferably 45°C-55°C, and even more preferably 50°C. The water bath time is preferably 1-2 h, more preferably 1.5 h. The function of the water bath in the present invention is to provide a suitable temperature and accelerate the speed of the alkali solution reaction.

[0045] After obtaining the supernatant, the present invention performs acid precipitation on the supernatant. In the present invention, the method of acid precipitation preferably includes: first adjusting the pH value of the supernatant and then centrifuging to obtain a precipitate; washing the precipitate successively, redissolving with deionized water and adjusting the pH value, and vacuum freeze-drying to obtain seabuckthorn seed protein. The pH value of the supernatant adjusted in the present invention is preferably 4.0 - 5.0, more preferably 5.0. The number of times of washing in the present invention is preferably 2 - 5 times, further preferably 4 - 5 times, and more preferably 5 times. In the present invention, it is preferred to adjust the pH value to 7 after adding ion water. The cold trap temperature of the vacuum freeze-drying in the present invention is preferably -65°C to -55°C, more preferably -60°C; the vacuum degree of the vacuum freeze-drying is preferably <10 Pa. The protein content in the seabuckthorn seed protein obtained in the present invention is 78% - 80%, and the protein content is high. Improper pH values in alkali solution-acid precipitation can cause elimination reactions and crosslinking reactions, and some toxic substances such as lysinoalanine will be produced. Residues such as serine, threonine, and cystine in the protein undergo elimination reactions to form dehydroalanine, and dehydroalanine can react with lysine residues to form the toxic substance lysinoalanine. The present invention uses the alkali solution-acid precipitation method to extract seabuckthorn seed protein, and sets specific pH, temperature, and solid-liquid ratio parameters to improve the purity of seabuckthorn seed meal protein and the protein content in the seed meal protein.

[0046] After obtaining the seabuckthorn seed protein, the present invention enzymatically hydrolyzes the seabuckthorn seed protein to obtain an enzymatic hydrolysate, and more preferably enzymatically hydrolyzes an aqueous solution of seabuckthorn seed protein to obtain an enzymatic hydrolysate. In the present invention, it is preferred to adjust the pH value of the aqueous solution of seabuckthorn seed protein after enzymatic hydrolysis, and the adjusted pH value is preferably 5.0 - 8.0, more preferably 6.0 - 7.0. The temperature of the enzymatic hydrolysis in the present invention is preferably 45°C to 60°C, further preferably 50°C to 58°C, and more preferably 55°C; the time of enzymatic hydrolysis is preferably 12 - 24 h, further preferably 15 - 22 h, and more preferably 20 h. The enzymatic hydrolysis in the present invention is preferably controlled enzymatic hydrolysis.

[0047] The enzymes used in the enzymatic hydrolysis of the present invention preferably include protease and / or pancreatin, and more preferably include protease and pancreatin. The protease in the present invention is preferably one or more of acidic protease, trypsin, and flavor protease, more preferably acidic protease and trypsin, or acidic protease and flavor protease, or acidic protease, or trypsin, or flavor protease. The enzymes used in the enzymatic hydrolysis in the present invention are more preferably two of acidic protease, trypsin, flavor protease, and pancreatin.

[0048] The addition amount of acidic protease in the protease in the present invention is preferably 0.2% - 1.0% of the mass percentage of the aqueous solution of seabuckthorn seed protein, further preferably 0.3% - 0.7%, and more preferably 0.6%. The enzyme activity of the acidic protease in the present invention is preferably 95,000 U / g - 105,000 U / g, and more preferably 100,000 U / g.

[0049] In the protease of the present invention, the addition amount of trypsin is preferably 0.5-1.0% by mass of the aqueous solution of seabuckthorn seed protein, more preferably 0.6-0.9%, and even more preferably 0.8%. The enzyme activity of the trypsin of the present invention is preferably 95,000 U / g to 105,000 U / g, and more preferably 100,000 U / g.

[0050] In the protease of the present invention, the addition amount of flavor protease is preferably 0.2-1.5% by mass of the aqueous solution of seabuckthorn seed protein, more preferably 0.3-1.3%, and even more preferably 1.0%. The enzyme activity of the flavor protease of the present invention is preferably 95,000 U / g to 105,000 U / g, and more preferably 100,000 U / g.

[0051] In the enzymatic hydrolysis of the present invention, the addition amount of pancreatin is preferably 0.2-1.5% by mass of the aqueous solution of seabuckthorn seed protein, more preferably 0.4-1.2%, and even more preferably 1.0%. The enzyme activity of the pancreatin of the present invention is preferably 95,000 U / g to 105,000 U / g, and more preferably 100,000 U / g.

[0052] The present invention does not limit the sources of protease and pancreatin, and conventional products can be used. The protease and pancreatin of the present invention are preferably food grade.

[0053] In the embodiments of the present invention, when the enzymes used in the enzymatic hydrolysis are preferably acidic protease and flavor protease, the addition amount of the acidic protease is preferably 1.0% by mass of the aqueous solution of seabuckthorn seed protein; the addition amount of the flavor protease is preferably 1.5% by mass of the aqueous solution of seabuckthorn seed protein. When the enzymes used in the enzymatic hydrolysis are preferably acidic protease and pancreatin, the addition amount of the acidic protease is preferably 1.5% by mass of the aqueous solution of seabuckthorn seed protein; the addition amount of the pancreatin is preferably 0.2% by mass of the aqueous solution of seabuckthorn seed protein. When the enzymes used in the enzymatic hydrolysis are preferably acidic protease and trypsin, the addition amount of the acidic protease is preferably 0.2% by mass of the aqueous solution of seabuckthorn seed protein, and the addition amount of the trypsin is preferably 1.5% by mass of the aqueous solution of seabuckthorn seed protein. The function of the enzymatic hydrolysis of the present invention is to enzymatically hydrolyze the seabuckthorn seed protein in the aqueous solution of seabuckthorn seed protein from the form of protein into the form of amino acids and polypeptides. The present invention obtains an oligopeptide with a molecular weight less than 1000 Da by controlling the dosage of the enzyme, the pH value during enzymatic hydrolysis, and the enzymatic hydrolysis temperature.

[0054] After the enzymatic hydrolysis, the present invention preferably further includes inactivating the enzyme solution. The present invention does not have special limitations on the temperature and time of the enzyme inactivation, and conventional parameters can be used.

[0055] The present invention extracts the enzymatic hydrolysate to obtain a supernatant. Before extraction, the present invention preferably performs cryogenic centrifugation on the enzymatic hydrolysate after inactivating the enzyme to obtain an enzymatic hydrolysate supernatant rich in branched-chain amino acids. The rotation speed of the cryogenic centrifugation is preferably 8000-12000 rpm, more preferably 10000 rpm. The time of cryogenic centrifugation is preferably 10-20 min, more preferably 15 min. The temperature of the cryogenic centrifugation is preferably 4°C.

[0056] To obtain an enzymatic hydrolysate supernatant rich in branched-chain amino acids, the present invention preferably extracts the enzymatic hydrolysate supernatant rich in branched-chain amino acids to obtain a supernatant. The extractant used in the extraction of the present invention preferably includes an ethanol aqueous solution. The volume ratio of the enzymatic hydrolysate supernatant rich in branched-chain amino acids to the ethanol aqueous solution in the present invention is preferably 1:(6-9), further preferably 1:(7-8.5), and more preferably 1:8. The number of extractions in the present invention is preferably 1-3 times, further preferably 2-3 times, and more preferably 3 times. The temperature of the extraction in the present invention is preferably room temperature. The time of each extraction is preferably the time for layering after adding the ethanol aqueous solution to the enzymatic hydrolysate supernatant rich in branched-chain amino acids and mixing well. After the first extraction in the extraction of the present invention, it is preferred to obtain an enzymatic hydrolysate supernatant rich in branched-chain amino acids and then add an ethanol aqueous solution for the second extraction. After the second extraction is completed, it is preferred to obtain an enzymatic hydrolysate supernatant rich in branched-chain amino acids and then add an ethanol aqueous solution for the third extraction. The present invention preferably uses the same volume of ethanol aqueous solution for the first extraction, the second extraction, and the third extraction. The volume concentration of the ethanol aqueous solution in the present invention is preferably 80%-95%, further preferably 90%-95%, and more preferably 95%. Since ethanol with different mass concentrations can precipitate peptides with different properties, the present invention selects the mass concentration of the ethanol aqueous solution to be preferably 80%-95% to precipitate peptides with strong polarity and large molecular weight.

[0057] The function of the extraction in the present invention is to extract branched-chain amino acid peptides in the enzymatic hydrolysate rich in branched-chain amino acids to a greater extent and extract more branched-chain amino acid peptides. Using high-concentration ethanol for extraction is beneficial to the extraction of the required branched-chain amino acid peptides and the improvement of the purity of the branched-chain amino acid peptides.

[0058] The present invention preferably performs centrifugation on the extraction product to obtain a supernatant. The centrifugation in the present invention is preferably cryogenic centrifugation. The temperature of the cryogenic centrifugation is preferably 4°C. The rotation speed of the cryogenic centrifugation is preferably 4500 rpm-8000 rpm, further preferably 5000 rpm-7000 rpm, and more preferably 6000 rpm. The cryogenic centrifugation time is preferably 10-20 min, further preferably 12-18 min, and more preferably 15 min. Using high-concentration ethanol for extraction increases the content of oligopeptides (i.e., branched-chain amino acid peptides) obtained.

[0059] A supernatant is obtained, and the present invention obtains a dry product of a composition containing seabuckthorn seed branched-chain amino acid peptides after vacuum freeze-drying the supernatant. Before vacuum freeze-drying, the present invention preferably performs vacuum distillation and concentration on the supernatant to 1 / 4 to 1 / 2 of the original supernatant volume, and more preferably 1 / 4. The present invention preferably performs vacuum freeze-drying on the supernatant obtained by vacuum distillation and concentration to obtain a dry product of a composition containing seabuckthorn seed branched-chain amino acid peptides. The dry product containing seabuckthorn seed branched-chain amino acid peptides obtained by the preparation method of the present invention can be directly used later without purification. The parameter range of the vacuum freeze-drying of the supernatant of the present invention is the same as the parameter range of the vacuum freeze-drying during the preparation of seabuckthorn seed protein. The present invention does not specifically limit the parameters of vacuum distillation and concentration, and conventional parameters can be used.

[0060] The preparation method of the seabuckthorn seed branched-chain amino acid peptide composition provided by the present invention first extracts seabuckthorn seed meal protein from the seabuckthorn seed meal and then enzymatically hydrolyzes the protein, so that the content and purity of the obtained branched-chain amino acid peptides are higher.

[0061] The method for preparing a seabuckthorn seed branched-chain amino acid peptide composition provided by the present invention adopts seabuckthorn seed meal byproducts of natural medicinal and edible plants and proteases permitted for use in the food industry to produce the seabuckthorn seed branched-chain amino acid peptide composition, has no fine chemical products, no secondary pollution, mild reaction conditions, low energy consumption and good safety.

[0062] The present invention provides a nutritional powder, comprising the seabuckthorn seed branched-chain amino acid peptide composition described in the above technical solution or the seabuckthorn seed branched-chain amino acid peptide composition prepared by the preparation method described in the above technical solution.

[0063] The present invention provides the application of the seabuckthorn seed branched-chain amino acid peptide composition described in the above technical solution or the seabuckthorn seed branched-chain amino acid peptide composition prepared by the preparation method described in the above technical solution or the nutritional powder described in the above technical solution in anti-fatigue, wherein the anti-fatigue includes one or more of 1) to 3);

[0064] 1) Reduce muscle fatigue;

[0065] 2) Increase muscle endurance;

[0066] 3) Accelerate recovery from sports fatigue.

[0067] The present invention uses a mouse weighted swimming test and a mouse rotarod test to verify that the seabuckthorn seed branched-chain amino acid peptide composition provided by the present invention can improve the anti-fatigue ability of mice, and the anti-fatigue effect is not significantly different from that of glutathione which has an anti-fatigue effect.

[0068] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0069] In the following examples, the enzyme activities of acid protease, trypsin, flavor protease, and pancreatin are all 100,000 U / g.

[0070] Example 1

[0071] (1) The by-product sea buckthorn seed meal is dried and then ultrafinely ground, and then sieved through a 70-mesh sieve, and then sea buckthorn seed protein is prepared by the method of alkali solution and acid precipitation.

[0072] Specific parameters of alkali solution: The sea buckthorn seed meal powder obtained by ultrafine grinding and sieving is added to deionized water according to the ratio of solid-liquid ratio of 1 g:8 mL to obtain a mixed solution. After mixing, a sodium hydroxide solution with a mass concentration of 6 mol / L is added to adjust the pH value of the mixed solution to 10. Then, it is water-bathed at a temperature of 40 °C for 1 h. The product obtained by water-bathing is centrifuged. The centrifugation temperature is 4 °C, the centrifugation speed is 8000 rpm, and the centrifugation time is 20 min. After centrifugation, the supernatant is taken.

[0073] Specific parameters of acid precipitation: The supernatant obtained by alkali solution is adjusted to a pH value of 4.0 with a hydrochloric acid solution with a mass concentration of 6 mol / L, and then centrifuged at a temperature of 4 °C. The centrifugation speed is 8000 rpm, and the centrifugation time is 20 min. After centrifugation, the supernatant is discarded to obtain a precipitate. The precipitate is washed twice with water, and then a little deionized water is added to adjust the pH value to 7.0, and stirred to dissolve the precipitate. The redissolved precipitate is subjected to vacuum freeze-drying to obtain sea buckthorn seed protein. The protein content in the prepared sea buckthorn seed protein is calculated by the Kjeldahl method. The protein content in the prepared sea buckthorn seed protein is 78%, and it is stored at 4 °C for later use.

[0074] (2) The sea buckthorn seed protein prepared in step (1) is added to deionized water to obtain an aqueous solution of sea buckthorn seed protein, and the pH is adjusted to 5.0. After adjustment, 0.2% acid protease based on the mass of the aqueous solution of sea buckthorn seed protein and 1.5% trypsin based on the mass of the aqueous solution of sea buckthorn seed protein are added, and it is enzymatically hydrolyzed at a temperature of 45 °C for 12 h and then inactivated.

[0075] (3) The enzymolysis product of sea buckthorn seed protein obtained in step (2) is subjected to freeze centrifugation. The freeze centrifugation temperature is 4 °C, the freeze centrifugation speed is 8000 rpm, and the freeze centrifugation time is 10 min. After freeze centrifugation, the precipitate is removed to obtain an enzymolysis supernatant rich in branched-chain amino acids.

[0076] (4) Mix the enzymatic hydrolysate supernatant rich in branched-chain amino acids obtained in step (3) with a 95% high-concentration ethanol aqueous solution at a volume ratio of 1:6, and then perform extraction once. The extraction time for each extraction is the time for layering after adding the ethanol aqueous solution to the enzymatic hydrolysate supernatant rich in branched-chain amino acids and mixing well. The extraction temperature is room temperature. The extract is subjected to freeze centrifugation at a temperature of 4°C, a rotational speed of 4500 rpm, and a time of 10 min. After freeze centrifugation, take the supernatant.

[0077] (5) Concentrate the supernatant obtained in step (4), and perform vacuum distillation under reduced pressure to concentrate it to one-fourth of the original supernatant volume. The product obtained by vacuum distillation under reduced pressure is subjected to vacuum freeze-drying. The specific parameters of vacuum freeze-drying are: freeze-drying area is 2.8 m 2 ; the cold trap temperature is -60°C; the vacuum degree is <10 Pa; the freeze-drying time is 24 h; the water capture capacity is 6 Kg / 24 h; the number of material tray layers is 4 - 6 layers; the material loading is 1800 ml; the material tray specification (ф*h mm): 240*20.

[0078] The secondary mass spectrum obtained by subjecting the dried product to high performance liquid chromatography-mass spectrometry is as Figures 1 to 4 shown. The parameters of high performance liquid chromatography-mass spectrometry are: a quaternary gradient pump system is used during detection, the temperature control range of the high-performance column oven is -5°C to 80°C, the maximum pressure is 600 bar, the maximum flow rate is 5 mL / min, the injection range of the autosampler is 0.1 - 100 μL, and the temperature range of the ultraviolet / differential detector is 5 - 55°C. According to Figures 1 to 4 it can be known that the composition of the branched-chain amino acid peptides of seabuckthorn seeds is composed of a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide. The amino acid sequence of the first polypeptide is Ile-Pro or Leu-Pro, the amino acid sequence of the second polypeptide is Ile-Pro-Glu-Asp-Val or Leu-Pro-Glu-Asp-Val, the amino acid sequence of the third polypeptide is Ile-Pro-Ile or Ile-Pro-Leu or Leu-Pro-Ile or Leu-Pro-Leu, and the amino acid sequence of the fourth polypeptide is Asp-Leu-Val-Gly-Lys or Asp-Ile-Val-Gly-Lys. According to the determination results of high performance liquid chromatography-mass spectrometry, it can be known that the dried product is a composition of branched-chain amino acid peptides of seabuckthorn seeds. The composition of the branched-chain amino acid peptides of seabuckthorn seeds is composed of four branched-chain amino acid peptides, and the molecular weight of each amino acid peptide is less than 1000 Da.

[0079] Example 2

[0080] (1) Dry the by-product seabuckthorn seed meal and then perform ultrafine grinding, then pass through an 80-mesh sieve, and then use the method of alkali dissolution and acid precipitation to prepare seabuckthorn seed protein;

[0081] Specific parameters for alkali dissolution: The sea buckthorn seed meal powder obtained by ultrafine pulverization and sieving was added to deionized water according to a solid-liquid ratio of 1 g:9 mL to obtain a mixed solution. After mixing, a sodium hydroxide solution with a mass concentration of 6 mol / L was added to adjust the pH value of the mixed solution to 10.5. Then, it was water-bathed at a temperature of 50 °C for 1.5 h. The product obtained from the water bath was centrifuged at a temperature of 4 °C, a centrifugal speed of 8000 rpm, and a centrifugation time of 20 min. After centrifugation, the supernatant was taken.

[0082] Specific parameters for acid precipitation: The pH value of the supernatant obtained from alkali dissolution was adjusted to 4.5 with a hydrochloric acid solution with a mass concentration of 6 mol / L. Then, it was centrifuged at a temperature of 4 °C, a centrifugal speed of 8000 rpm, and a centrifugation time of 20 min. After centrifugation, the supernatant was discarded to obtain a precipitate. After washing the precipitate 3 times with water, a little deionized water was added to adjust the pH value to 7.0, and it was stirred to redissolve the precipitate. The redissolved precipitate was subjected to vacuum freeze-drying to obtain sea buckthorn seed protein. The protein content in the prepared sea buckthorn seed protein was calculated by the Kjeldahl method. The protein content in the prepared sea buckthorn seed protein was 79%, and it was stored at 4 °C for later use.

[0083] The protein content in the prepared sea buckthorn seed protein was 79%, and it was stored at 4 °C for later use.

[0084] (2) The sea buckthorn seed protein prepared in step (1) was added to deionized water to obtain an aqueous solution of sea buckthorn seed protein, and the pH was adjusted to 6.0. After adjustment, 1.5% acidic protease based on the mass of the aqueous solution of sea buckthorn seed protein and 0.2% pancreatin based on the mass of the aqueous solution of sea buckthorn seed protein were added. After enzymatic hydrolysis at a temperature of 50 °C for 18 h, the enzyme was inactivated.

[0085] (3) The enzymatic hydrolysis product of sea buckthorn seed protein obtained in step (2) was subjected to freeze centrifugation at a temperature of 4 °C, a centrifugal speed of 10000 rpm, and a centrifugation time of 15 min. After freeze centrifugation, the precipitate was removed to obtain an enzymatic hydrolysis supernatant rich in branched-chain amino acids;

[0086] (4) The enzymatic hydrolysis supernatant rich in branched-chain amino acids obtained in step (3) was mixed with an ethanol aqueous solution with a volume concentration of 90% at a volume ratio of 1:8 and then extracted 2 times. The extraction time for each time was the time for layering after adding the ethanol aqueous solution to the enzymatic hydrolysis supernatant rich in branched-chain amino acids and mixing well. The extraction temperature was room temperature. The product obtained from extraction was subjected to freeze centrifugation at a temperature of 4 °C, a centrifugal speed of 6000 rpm, and a centrifugation time of 15 min. After freeze centrifugation, the supernatant was taken.

[0087] (5) Concentrate the supernatant obtained in step (4), and reduce the pressure to distill and concentrate it to one-fourth of the volume of the original supernatant. The product obtained by reduced pressure distillation and concentration is subjected to vacuum freeze-drying. The specific parameters of vacuum freeze-drying are the same as those in Example 1. Using high performance liquid chromatography - mass spectrometry (HPLC-MS) to detect the product obtained by drying, it is known that the composition of the branched-chain amino acid peptides of seabuckthorn seeds is an oligopeptide with a molecular weight less than 1000 Da. The HPLC-MS detection parameters are the same as those in Example 1.

[0088] Example 3

[0089] (1) Dry the by-product seabuckthorn seed meal and then perform ultrafine grinding. Then, sieve it through a 100-mesh sieve, and use the method of alkali dissolution and acid precipitation to prepare seabuckthorn seed protein.

[0090] Specific parameters of alkali dissolution: Add the seabuckthorn seed meal powder obtained by ultrafine grinding and sieving to deionized water according to the ratio of solid-liquid ratio of 1 g:10 mL to obtain a mixed solution. After mixing, add a sodium hydroxide solution with a mass concentration of 6 mol / L to adjust the pH value of the mixed solution to 11.0. Then, perform a water bath at a temperature of 60 °C for 2 h. The product obtained by the water bath is centrifuged. The temperature of centrifugation is 4 °C, the centrifugation speed is 8000 rpm, and the centrifugation time is 20 min. After centrifugation, take the supernatant.

[0091] Specific parameters of acid precipitation: Adjust the pH value of the supernatant obtained by alkali dissolution to 5.0 with a hydrochloric acid solution with a mass concentration of 6 mol / L. Then, perform centrifugation at a temperature of 4 °C. The centrifugation speed is 8000 rpm, and the centrifugation time is 20 min. After centrifugation, discard the supernatant to obtain a precipitate. The precipitate is washed 5 times with water, and then a little deionized water is added to adjust the pH value to 7.0, and stirred to dissolve the precipitate. The dissolved precipitate is subjected to vacuum freeze-drying to obtain seabuckthorn seed protein. Calculate the protein content in the prepared seabuckthorn seed protein by the Kjeldahl method. The protein content in the prepared seabuckthorn seed protein is 80%, and it is stored at 4 °C for later use.

[0092] (2) Add the seabuckthorn seed protein prepared in step (1) to deionized water to obtain an aqueous solution of seabuckthorn seed protein, and adjust the pH to 8.0. After adjustment, add an acidic protease accounting for 1.0% of the mass of the aqueous solution of seabuckthorn seed protein and a flavor protease accounting for 1.5% of the mass of the aqueous solution of seabuckthorn seed protein. Enzymatically hydrolyze at a temperature of 60 °C for 24 h and then inactivate the enzyme.

[0093] (3) Subject the seabuckthorn seed protein hydrolysate prepared in step (2) to freeze centrifugation. The freeze centrifugation temperature is 4 °C, the freeze centrifugation speed is 12000 rpm, and the freeze centrifugation time is 20 min. After freeze centrifugation, remove the precipitate to obtain an enzyme hydrolysis supernatant rich in branched-chain amino acids;

[0094] (4) Mix the enzymatic hydrolysate supernatant rich in branched-chain amino acids obtained in step (3) with an ethanol aqueous solution with a volume concentration of 95% at a volume ratio of 1:9, and then perform extraction; the number of extractions is 3 times. The volume of the ethanol aqueous solution added in the second extraction and the third extraction is the same as that added in the first extraction. The time for each extraction is the time for layering after adding the ethanol aqueous solution to the enzymatic hydrolysate supernatant rich in branched-chain amino acids and mixing well. The extraction temperature is room temperature. The extract is subjected to freeze centrifugation. The freeze centrifugation temperature is 4 °C, the freeze centrifugation speed is 8000 rpm, and the freeze centrifugation time is 20 min. After freeze centrifugation, take the supernatant.

[0095] (5) Concentrate the supernatant obtained in step (4), and perform vacuum distillation under reduced pressure to concentrate it to one-fourth of the original supernatant volume. The product obtained by vacuum distillation under reduced pressure is subjected to vacuum freeze-drying. The specific parameters of the vacuum freeze-drying are the same as those in Example 1. Use high-performance liquid chromatography-mass spectrometry to detect that the dried product is a composition of seabuckthorn seed branched-chain amino acid peptides. It can be known that this amino acid peptide is an oligopeptide with a molecular weight less than 1000 Da. The detection parameters of high-performance liquid chromatography-mass spectrometry are the same as those in Example 1.

[0096] Test Example 1 Effects of the composition of seabuckthorn seed branched-chain amino acid peptides on the weight-bearing swimming experiment of mice

[0097] SPF-grade ICR mice were raised under the conditions of a humidity of 50 ± 5% and a temperature of 24 ± 2 °C (6 mice per cage; the cage size was 320×215×170 mm, a total of 48 mice). Under standard laboratory conditions, the lighting during the breeding was 12 h / 12 h of day-night alternation. After adaptive breeding for 1 week, the mice were randomly assigned to 4 experimental groups, with 12 mice in each experimental group.

[0098] Normal control group, i.e., NC group: Intragastric administration of normal saline every day;

[0099] Positive control group, i.e., PC group: Intragastric administration of glutathione at 100 mg / kg·d every day;

[0100] High-dose group of the composition of seabuckthorn seed branched-chain amino acid peptides prepared in Example 1, i.e., SSPHH group: Intragastric administration of 500 mg / kg·d every day);

[0101] Low-dose group of the composition of seabuckthorn seed branched-chain amino acid peptides prepared in Example 1, i.e., SSPHL group: Intragastric administration of 250 mg / kg·d every day.

[0102] The intragastric administration treatment lasted for four weeks, with intragastric administration once a day. During the intragastric administration process, the mice were allowed to freely feed and drink water every day.

[0103] After 4 weeks of gavage treatment, during the day, the animal load-bearing swimming behavior experiment was conducted on all mice in the NC group, PC group, SSPHH group, and SSPHL group. During the entire test process, to obtain accurate results, it was necessary to ensure that the mouse behavioral experiment was carried out in a quiet and stable environment, and the temperature was maintained at 26 ± 1.0 °C throughout the experiment. This test was carried out within two days, specifically: First-stage test: Each mouse was gently placed alone into a plexiglass cylinder (diameter 15 cm × 30 cm high) filled with water (water level height: 15 cm) to allow each experimental mouse to adapt to swimming for 5 min; Second-stage test: 24 h after the adaptation swimming ended, the second-stage experiment began. After 30 min of gavage with normal saline (the mass concentration of normal saline was 75%, and the gavage volume was 1.5 mL per day) in the NC group, gavage with glutathione in the PC group, gavage with the composition of seabuckthorn seed branched-chain amino acid peptide prepared in Example 1 in the SSPHH group, and gavage with the composition of seabuckthorn seed branched-chain amino acid peptide prepared in Example 1 in the SSPHL group, a lead sheet with a load of 5% of the body weight was given at the tail root of the mouse for the load-bearing swimming experiment in the plexiglass cylinder. The load-bearing swimming experiment time was 5 min, and a digital imaging system was used to track and record the movement and behavior of the animals during the experiment. Pay attention to each mouse to keep its limbs moving continuously, and measure the fatigue tolerance (in seconds) of the mouse from the time of entering the water until its head is completely submerged and cannot surface for 8 seconds within 5 min. Standard for immobility during swimming: When the experimental mouse floats, it only gently paddles or slightly moves with one foot to keep its head above the water without struggling. After the test, the mouse was immediately taken out of the water and returned to the breeding cage.

[0104] The load-bearing swimming fatigue tolerance times of the mice in the NC group, PC group, SSPHH group, and SSPHL group are shown in Table 1 and Figure 5 、 Figure 6 。

[0105] Table 1 Load-bearing swimming fatigue tolerance times of mice in each group (unit: S)

[0106] NC group PC group SSPHH group SSPHL group Fatigue tolerance time 196.89 235.56 228.50 219.07

[0107] According to Table 1 and Figure 5 it can be seen that compared with the NC group, the load-bearing swimming fatigue tolerance times of the mice in the PC group, SSPHH group, and SSPHL group all increased to varying degrees, and among them, the load-bearing swimming fatigue tolerance times of the mice in the PC group, SSPHH group, and SSPHL group all increased significantly (p < 0.05). The trajectory diagrams of the load-bearing swimming of the mice in each group of the NC group, PC group, SSPHH group, and SSPHL group are shown in Figure 6, It can be seen from the trajectory diagram that compared with the mice in the NC group, the load-bearing swimming path and swimming distance of the mice in the PC group, SSPHH group, and SSPHL group increased, and the swimming trajectories were denser. It can be seen that after intragastric administration of the composition of seabuckthorn seed branched-chain amino acid peptides, the anti-fatigue ability of the mice was effectively improved.

[0108] Experimental Example 2 Effect of the composition of seabuckthorn seed branched-chain amino acid peptides on the rotarod test in mice

[0109] 24 hours after the end of the load-bearing swimming experiment in Experimental Example 1, all the mice in the NC group, PC group, SSPHH group, and SSPHL group of Experimental Example 1 were subjected to the rotarod test. The rotarod ability of the mice can be used to evaluate the degree of fatigue of the mice. The rotarod ability test was carried out during the day.

[0110] During the entire rotarod ability test, in order to obtain accurate results, it is necessary to ensure that the mouse behavioral experiment is carried out in a quiet and stable environment, and the temperature is maintained at 24 ± 2 °C throughout the experiment. The method of the mouse rotarod test is as follows: 4 weeks after the intragastric administration of the test articles to the mice in each group of Example 1, the mice were subjected to the rotarod experiment. The mice were placed on a rotarod with a diameter of 5 cm and a rotation speed of 15 r / min for crawling training. Each group of mice trained for 30 min every day for a total of 2 days. On the 3rd day, the rotation speed of the rotarod was increased to 35 r / min, and the time on the rod of each mouse was recorded. When the mouse crawls on the rod, the mouse's muscles are in a tense state. When the mouse reaches the fatigue state, it will fall off the rod. The falling time of the mouse was used as an evaluation index of fatigue, that is, the fatigue tolerance time.

[0111] The fatigue tolerance times of the rotarod experiments of the mice in the NC group, PC group, SSPHH group, and SSPHL group are shown in Table 2 and Figure 7 .

[0112] Table 2 Fatigue tolerance times of the rotarod experiments of mice in each group (unit: S)

[0113] NC group PC group SSPHH group SSPHL group Fatigue tolerance time 106.01 171.23 169.68 143.53

[0114] According to Table 2 and Figure 7 it can be seen that compared with the mice in the NC group, the fatigue tolerance times of the mice in the glutathione group (PC group, glutathione has an anti-fatigue effect), the high-dose treatment group of the composition of seabuckthorn seed branched-chain amino acid peptides (SSPHH group), and the low-dose treatment group of the composition of seabuckthorn seed branched-chain amino acid peptides (SSPHL group) all showed an increasing trend. The fatigue tolerance times of the PC group, SSPHH group, and SSPHL group were all significantly (p < 0.05) higher than those of the normal group; the fatigue tolerance times of the PC group and SSPHH group of mice were higher than those of the SSPHL group of mice.

[0115] In summary, after the composition of the branched-chain amino acid peptide from seabuckthorn seeds prepared by the present invention was intragastrically administered to mice, it could improve the fatigue state of the mice, enhance the anti-fatigue ability of the mice, prolong the fatigue tolerance time of the mice, and showed a trend of increasing in a dose-dependent manner. The composition of the branched-chain amino acid peptide from seabuckthorn seeds obtained in the present invention has the effects of anti-fatigue, increasing muscle endurance, and delaying muscle fatigue, and can be used for preparing anti-fatigue medical foods. Moreover, the composition of the branched-chain amino acid peptide from seabuckthorn seeds obtained has the effect of increasing muscle synthesis and can be used for preparing athlete nutritional powders.

[0116] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A preparation method of a composition of branched-chain amino acid peptides from sea buckthorn seeds, characterized in that, It includes the following steps: The seabuckthorn seed meal is treated by the alkali dissolution and acid precipitation method to obtain seabuckthorn seed protein; The seabuckthorn seed protein is enzymatically hydrolyzed to obtain an enzymatic hydrolysate; the enzymes used in the enzymatic hydrolysis are acid protease and trypsin; the addition amount of the acid protease is 0.2-1.0% of the mass of the aqueous solution of seabuckthorn seed protein; the addition amount of the trypsin is 0.5-1.5% of the mass of the aqueous solution of seabuckthorn seed protein; the temperature of the enzymatic hydrolysis is 45°C, and the time of the enzymatic hydrolysis is 12 h; The enzymatic hydrolysate is extracted to obtain a supernatant; the extractant used in the extraction is an ethanol aqueous solution; the volume ratio of the enzymatic hydrolysate to the ethanol aqueous solution is 1:(6-9); the volume concentration of the ethanol aqueous solution is 95%; The supernatant is dried to obtain a dried product of a composition containing seabuckthorn seed branched-chain amino acid peptides.

2. The preparation method according to claim 1, characterized in that, The number of times of the extraction is 1-3 times.

3. A composition of seabuckthorn seed branched-chain amino acid peptides prepared by the preparation method according to claim 1 or 2.

4. A nutritional powder, characterized in that, A composition containing seabuckthorn seed branched-chain amino acid peptides prepared by the preparation method according to claim 3.

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