Tyr-Pro-casein peptide with sleep improving activity, and preparation method and application thereof
Technical Field
The invention relates to bioactive peptides for improving anxiety sleep disorder, in particular to a series of Tyr-Pro-type casein peptides with sleep improving activity, and a preparation method and application thereof.
Background
Insomnia refers to a series of phenomena such as difficulty in falling asleep, short sleep time, dreaminess, easy awakening, and the like, affects more than 35% of population worldwide, and is an important risk factor for causing mood disorder, fatigue, memory disorder, neuroinflammation, brain dysfunction and immunity decline.
Currently, sleep assessment can be divided into two main categories, objective sleep assessment and subjective sleep assessment methods. Because the mechanism or the target point of sleep disorder is complex, the sleeping disorder relates to a plurality of target points such as benzodiazepine receptors, 5-hydroxytryptamine, melatonin receptors and the like. Thus, there is currently no simple in vitro evaluation criteria that can represent a comprehensive sleep architecture. Sleep time is the most critical index for in vivo evaluation of insomnia and improvement of sleep activity by bioactive substances, and is usually measured by adopting a pentobarbital-induced mice sleep behavior test experiment. Therefore, the sleep improving effect of the active substance can be intuitively reflected by remarkably prolonging the sleep time of the pentobarbital sodium-induced mice.
Casein is an animal protein rich in Pro and Tyr, and has wide sources, rich resources and high nutritive value, and has great advantages for the development of bioactive products. Although different casein enzymatic products have been shown to have different sleep improving effects, the identification of sleep improving peptides is still limited. Representative alpha s 1-casein zymolyte prepared by trypsin shows sleep improving effect in a pentobarbital sodium-induced mouse sleep experiment, and a sleep improving peptide YLGYLEQLLR is obtained through separation and identification. In addition, casein obtained by enzymolysis with other specific commercial proteases has stronger effect on improving sleep enzymolysis products (patent CN114181292B and CN 112056453B). However, casein hydrolysates obtained with different proteases show different sleep improving effects, the underlying reasons of which are not clear. Further studies are needed to elucidate the detailed structural features of peptides from casein that are effective in improving sleep.
Therefore, by analyzing the structural characteristics of casein peptide, the method is helpful to develop more natural and safe sleep improving peptides. However, due to the limited data of the current sleep improving peptides, the prior art is still insufficient for the study of the structural features of the sleep improving peptides. Chinese patent (CN 112056453B) discloses an aromatic amino acid-rich sleep-improving zymolyte, and the casein zymolyte has a strong sleep-improving effect. However, how to improve the sleep improving effect of casein peptides rich in aromatic amino acids is not known, and the sequence characteristics of the existing sleep improving peptides are not reported sufficiently.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a series of Tyr-Pro-type casein peptides with strong sleep improving activity, and a preparation method and application thereof.
The Tyr-Pro-type casein peptide with the sleep improving activity is any one of Tyr-Pro,Tyr-Pro-Phe-Pro-Gly(SEQ.ID.NO.1),Tyr-Pro-Glu-Leu-Phe(SEQ.ID.NO.2),Tyr-Pro-Ser-Tyr-Gly-Leu-Asn(SEQ.ID.NO.3),,Tyr-Pro-Ser-Gly-Ala(SEQ.ID.NO.4),Tyr-Pro-Ser-Tyr-Gly(SEQ.ID.NO.5),Tyr-Pro-Ser-Gly-Ala-Trp-Tyr(SEQ.ID.NO.6),Tyr-Pro-Ser in sequence, and the Tyr-Pro-type casein peptide with the sleep improving activity is applied to preparation of functional foods or medicines for improving anxiety-type sleep disorder.
Further, the functional food is powder electuary, functional beverage, tabletting candy, gel soft candy or oral liquid, and the medicine is powder, tablet, liquid or capsule.
Furthermore, the functional food or the medicine also contains active ingredients and/or acceptable auxiliary materials which are used for improving sleep and are besides the Tyr-Pro-type casein peptide.
Further, the acceptable auxiliary materials in the functional food are milk powder, spina date seed, gamma-aminobutyric acid, whey protein peptide, collagen peptide, soybean peptide or melatonin, and the acceptable auxiliary materials in the medicine are sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, adsorption carriers, surfactants or lubricants.
Furthermore, the Tyr-Pro-type casein peptide with the sleep improving activity is a Tyr-Pro-based casein peptide with 5-7 amino acids extended from the C terminal, namely SEQ ID No. 4-6.
The invention provides a series of Tyr-Pro-type casein peptides with sleep improving activity, the amino acid sequence of the Tyr-Pro-type casein peptides with sleep improving activity is Tyr-Pro-Ser-Gly-Ala(SEQ.ID.NO.4),Tyr-Pro-Ser-Tyr-Gly(SEQ.ID.NO.5),Tyr-Pro-Ser-Gly-Ala-Trp-Tyr(SEQ.ID.NO.6),Tyr-Pro-Ser.
The preparation method for preparing the Tyr-Pro-type casein peptide with the sleep improving activity comprises the following steps:
(1) Adding casein and water, stirring and dissolving, and adjusting the pH to 6-8 to prepare casein dispersion;
(2) Adding serine proteinase and mixed proteinase M into the casein dispersion liquid in the step (1), carrying out synergistic enzymolysis, inactivating enzyme, and cooling;
(3) Centrifuging at 8000-10000 rpm and 4-10 ℃ for 10-20 min, and taking supernatant to obtain casein small molecule peptide solution containing SEQ ID No. 4-6 peptide fragments.
In the step (2), the serine protease is one of chymotrypsin and trypsin, the mixed protease M specifically comprises aminopeptidase and carboxypeptidase (preferably comprising 3:1 by mass ratio), the temperature of the synergistic enzymolysis is 35-55 ℃, and the time of the synergistic enzymolysis is 1-8h.
Further, the total protease addition amount of the serine protease and the mixed protease M in the step (2) is 0.2% -2.0% of the mass of casein, the mass ratio of the serine protease to the mixed protease M is (1-10): 10-19%, and the enzyme deactivation in the step (2) is to heat the enzymolysis liquid to 90-100 ℃ and boil for 15-30 min.
The casein enzymolysis product prepared by the preparation method, namely the casein small molecule peptide solution containing the peptide fragments of SEQ.ID.NO. 4-6, can prolong the sleep time by more than 2.37 times in the application of preparing the medicine and the health care product for improving the sleep.
The Tyr-Pro-type casein peptide and the enzymolysis product containing the Tyr-Pro-type casein peptide can obviously prolong the sleep time of a mouse induced by sodium pentobarbital, thereby showing stronger sleep improving activity. At the same time, sleep improvement is longer compared to other Tyr-type or different length Tyr-Pro-type peptides.
According to the Tyr-Pro-type casein peptide, the optimized Tyr-Pro-type 5-7 peptide intuitively reflects the sleep improving effect by prolonging the time effect of the pentobarbital sodium-induced mice. As reported in the prior art, the casein nonapeptide YPFGPPN (patent CN 117659154A) prolongs the time of inducing mice by pentobarbital sodium to 3186.83 +/-635.34 s, and compared with a positive pharmaceutical group, the sleeping activity is improved to 53.75%, and the casein Tyr-Pro-type 5-7 peptide can reach 57.57% -78.24%, so that the efficacy is remarkably improved.
In summary, proper addition of amino acids at the C-terminus of Tyr-Pro-monomers is an effective method for developing Tyr-type peptides with enhanced sleep activity, but is not necessarily more effective when extended, where a sequence length of 5-7 amino acids is more appropriate. Based on this, the Tyr-Pro-type casein peptide of the invention can be used for preparing functional foods or medicines with sleep improving effect.
Compared with the prior art, the invention has the following advantages and effects:
Compared with other Tyr-type peptides or Tyr-Pro-type peptides with other lengths, the Tyr-Pro-type 5-7 casein peptide provided by the invention has stronger sleep improving activity, remarkably prolongs the sleep time of mice induced by sodium pentobarbital, and can be applied to preparation of functional foods or medicines for improving sleep effectively.
Drawings
FIG. 1 is a graph showing the effect of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and other lengths of Tyr-Pro-type peptides on sodium pentobarbital-induced sleep time in mice.
FIG. 2 is a graph comparing the ratio of Tyr-Pro-type 5-7 peptides, other Tyr-type peptides, and other lengths of Tyr-Pro-type peptides to the time that the positive drug diazepam affects the sleep time of pentobarbital sodium-induced mice.
FIG. 3 is a graph showing the effect of different casein enzyme products in examples 9-10 and comparative example 3 on the sleep time of pentobarbital sodium-induced mice.
FIG. 4 is a mass spectrum identification BPC-MS of the different casein enzymatic hydrolysis products of examples 9-10 and comparative example 3.
FIGS. 5a to 5c are mass spectrometric identification diagrams of Tyr-Pro-type 5-7 peptides (SEQ. ID. NO.4 to 6) in examples 9 to 10 and in different casein enzymatic products of comparative example 3.
Detailed Description
Specific implementations of the invention are further described below with reference to the drawings and examples, but the implementation and protection of the invention are not limited thereto. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.
The experimental indexes and the method thereof related to the embodiment of the invention are as follows:
(1) Administration and treatment of mice
192 Kunming mice (6-8 weeks old) were placed in a controlled environment (25+ -2 ℃,12/12h light/dark cycle) and were free of diet. 144 of these were stressed for 2 weeks by randomized chronic stress (including tail clamping, restriction, water deprivation, fasted, inclined cage, diurnal inversion, wet cage, etc.), randomized group (n=12) normal group (normal mice, no stress stimulation), model group (stress stimulation but no drug administration), casein peptide group (each group was orally administered with 1.8mg/kg of gavage casein peptide while stress stimulation) and casein enzymatic products (each group was orally administered with 75mg/kg of gavage casein enzymatic product while stress stimulation). The casein peptide group mice were orally gavaged for 7 days while pressure stimulated, i.e., model group and casein peptide/hydrolysate group co-stimulated for 21 days. After the last lavage, sleep behaviours were determined.
(2) Sodium pentobarbital-induced mouse sleep behavioural assay
The detection is carried out by referring to a tenth chapter of a detection method for improving sleep function in the technical specification of health food detection and evaluation. Mice were started with sodium pentobarbital (42 mg/kg, intraperitoneal injection) 30 minutes after the last oral administration. The number of mice that fell asleep after the disappearance of the regular reflection of the inversion within 15 minutes was then recorded. Sleep latency and sleep duration were recorded in mice over 2 hours with a stopwatch.
(3) Mass spectrum identification method
The bioactive peptide is identified by using ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS/MS), gradient elution chromatography separation is adopted, and the bioactive peptide is identified and quantified by using secondary mass spectrometry, wherein the elution speed is 0.2mL/min, and the sample injection amount is 2 mu L. Mobile phase a was 0.1% formic acid water, B acetonitrile (chromatographic purity), elution procedure: 0-10 min, 22-47% B, 10-12 min, 47-90% B, 12-14 min,90% B, 14-15 min,90% B and (3) balancing the material in the range of percent to 5 percent of B for 15 to 18 minutes to an initial state. The mass spectrum parameters are that the energy of an electron bombardment ion source is 7eV, the capillary voltage is 4500V, the ion source temperature is 200 ℃, the drying gas flow is 8.0L/min, the atomizer pressure is 1.5bar, and the mass scanning range is m/z 50-1500.
Example 1
Pentapeptide Tyr-Pro-Ser-Tyr-Gly, when the gastric lavage amount is 1.8mg/kg, sodium pentobarbital induces the sleeping time of mice to be 2526.28 +/-220.32 s.
Example 2
Pentapeptide Tyr-Pro-Ser-Gly-Ala, when the gastric lavage amount is 1.8mg/kg, sodium pentobarbital induces the sleeping time of the mice to be 3433+/-613.69 s.
Example 3
Pentapeptide Tyr-Pro-Phe-Pro-Gly, and sodium pentobarbital induces mice to sleep for 3055.25 +/-428.78 s when the gastric lavage amount is 1.8 mg/kg.
Example 4
Pentapeptide Tyr-Pro-Glu-Leu-Phe, sodium pentobarbital induces mice to sleep for 3326.14 +/-323.47 s when the gastric lavage amount is 1.8 mg/kg.
Example 5
Heptapeptide Tyr-Pro-Ser-Gly-Ala-Trp-Tyr, sodium pentobarbital induces mice to sleep for 3219.38 +/-658.62 s when the gastric lavage amount is 1.8 mg/kg.
Example 6
Heptapeptide Tyr-Pro-Ser-Tyr-Gly-Leu-Asn, and sodium pentobarbital induces mice to sleep for 3251.38 +/-878.04 s when the gastric lavage amount is 1.8 mg/kg.
Comparative example 1
Hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu, sodium pentobarbital, when the gastric lavage amount is 1.8mg/kg, induces mice to sleep for 2024.0554 +/-672.99 s.
Comparative example 2
Pentapeptide Tyr-Tyr-Val-Pro-Leu, sodium pentobarbital induced mice sleep time is 1012.0277 + -72.13 s when the gastric lavage amount is 1.8 mg/kg.
Therefore, the Tyr-Pro-type prolonged pentobarbital sodium induces the mice to sleep for a longer time.
Example 7
Dipeptide Tyr-Pro, when the gastric lavage amount is 1.8mg/kg, sodium pentobarbital induces mice to sleep for 1792.69 +/-259.75 s.
Example 8
Tripeptide Tyr-Pro-Ser, when the gastric lavage amount is 1.8mg/kg, sodium pentobarbital induces mice to sleep for 1846.29 +/-398.16 s.
Therefore, the Tyr-Pro-type casein peptide can effectively improve the sleep time, and more preferable 5-7 peptides can obviously prolong the sleep time of the mice induced by sodium pentobarbital.
Example 9
(1) Respectively weighing and adding buffalo milk casein and distilled water according to the mass ratio of 1:10, moderately stirring and dissolving to prepare casein dispersion liquid, and regulating the pH value of a reaction system to 8.0 by using 4M NaOH solution;
(2) Adding trypsin with the mass percentage of 0.5% and mixed protease M (calculated by casein amount) with the mass percentage of 1.5% into the reaction system of the step (1), carrying out enzymolysis for 8 hours at 55 ℃ and inactivating enzyme for 15 minutes at 100 ℃, and cooling, wherein the mixed protease M comprises aminopeptidase and carboxypeptidase (the mass ratio is 3:1);
(3) Centrifuging at 10000rpm and 4 ℃ for 10min, and taking supernatant as casein small molecule peptide solution containing SEQ ID No. 5-7 peptide fragments.
Example 10
(1) Respectively weighing and adding buffalo milk casein and distilled water according to the mass ratio of 1:10, moderately stirring and dissolving to prepare casein dispersion liquid, and regulating the pH value of a reaction system to 6.0 by using 4M NaOH solution;
(2) Adding 0.1% of chymotrypsin and 0.1% of mixed proteinase M (calculated by casein) into the reaction system in the step (1), performing enzymolysis for 1h at 35 ℃, inactivating enzyme at 90 ℃ for 30min, and cooling;
(3) Centrifuging at 8000rpm and 4 ℃ for 20min, and taking supernatant as casein small molecule peptide solution containing SEQ ID No. 5-7 peptide fragments.
Comparative example 3
(1) Respectively weighing and adding buffalo milk casein and distilled water according to the mass ratio of 1:10, moderately stirring and dissolving to prepare casein dispersion liquid, and regulating the pH value of a reaction system to 7.0 by using 4M NaOH solution;
(2) Adding trypsin (calculated by casein amount) with the mass percentage of 0.2% into the reaction system of the step (1), carrying out enzymolysis for 8 hours at 55 ℃, inactivating enzyme for 15 minutes at 100 ℃, and cooling;
(3) Centrifuging at 10000rpm and 4 ℃ for 10min, and taking supernatant as casein small molecule peptide solution.
FIGS. 5a to 5c are mass spectrometric identification diagrams of Tyr-Pro-type 5 to 7 peptides (SEQ. ID. NO.5 to 7) in examples 9 to 10 and in different casein enzymatic products of comparative example 3.
As can be seen from fig. 1, the sleep time of the mice (model group) stimulated by chronic stress was drastically shortened to 735.80 ± 172.83s only by pentobarbital sodium, while the sleep time of the mice stimulated by chronic stress was remarkably prolonged to 2526.28 ±220.32s to 3539.65± 835.41s by pentobarbital sodium after oral administration of the peptide of the Tyr-Pro-type 5-7. And after being treated by dipeptide Tyr-Pro, tripeptide Tyr-Pro-Ser, hexapeptide Tyr-Phe-Tyr-Pro-Glu-Leu and pentapeptide Tyr-Tyr-Val-Pro-Leu, the sleeping time is only 1012.0277 +/-72.13 s-2024.0554+/-672.99 s. The casein Tyr-Pro-type 5-7 peptide can effectively prolong the sleep time of the pentobarbital sodium-induced mice, and has the potential of improving sleep.
As can be seen from fig. 2, comparing the sleep time of the oral gavage sample group with that of the positive drug group, the sleep improvement effect can be more intuitively compared. The result shows that the sleep improvement effect of the casein Tyr-Pro-type 5-7 peptide can reach 57.57% -78.24% of that of a positive control group, the sleep improvement effects of Tyr-Pro and Tyr-Pro-Ser are 40.86% and 42.08% of that of the positive control group, and the sleep improvement effects of the pentapeptides and hexapeptides of the comparative example are only 23.06% and 46.13%.
As can be seen from fig. 3, the sleep time of the mice (model group) induced by pentobarbital sodium was drastically shortened to 735.80 ± 172.83s, while the sleep time of the mice induced by pentobarbital sodium was significantly prolonged to 1745.52 ± 435.3s and 1965.93 ± 558.13s after oral administration of the casein enzymatic products of examples 9 and 10. And after the treatment of comparative example 3, the sleep time was 1312.06.+ -. 427.06s only. The casein enzymolysis product containing Tyr-Pro-type 5-7 peptide can effectively prolong the sleep time of the pentobarbital sodium-induced mice, and has the potential of improving sleep.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.