Use of PAYCS in regulation of intestinal flora, metabolites, and brain neurotransmitters

Abstract

The present disclosure belongs to the technical field of health food, and in particular, relates to the use of PAYCS in the regulation of intestinal flora, metabolites, and brain neurotransmitters. PAYCS remedies scopolamine-induced memory impairment in mice through target oxidation, inflammatory stress and regulation of the intestinal microorganism-metabolite-brain neurotransmitter axis through PAYCS. PAYCS improves the memory pathway through the intestinal microorganism-metabolite-neurotransmitter axis. PAYCS can significantly reduce serum MDA and LDH levels and significantly increase liver SOD content. PAYCS-H inhibits the increase of liver TNF-α and IL-1β, while PAYCS-L can only reduce the content of TNF-α in the liver. Thus, PAYCS-L changes the ratio of Bacteroides/Firmicutes and increases the relative abundance of plants such as Cactaceae and Prevotellaceae, improving the neurotransmitters associated with the metabolism of tryptophan in the brain.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit and priority of Chinese Patent Application No. 202210147703.1 filed with the China National Intellectual Property Administration on Feb. 17, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “2023-06-15 Sequence Listing-BGI018-001AUS.xml” created on Jun. 15, 2023, which is about 2,308 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure belongs to the technical field of health food, and in particular, relates to the use of PAYCS (the peptide Pro-Ala-Tyr-Cys-Ser, SEQ ID NO: 1) in the regulation of intestinal flora, metabolites, and brain neurotransmitters.

BACKGROUND

At present, the brain-gut axis is a bilateral/bi-directional information exchange system that integrates brain and intestinal functions, and the two-way interaction between the central nervous system, enteric nervous system, and gastrointestinal tract is also receiving increasing attention. Intestinal microorganisms participate in the functional response of the brain-gut axis and play a very important role in the exchange of information between the gut and the brain, thus medical scientists proposed a concept of the “microbiota-gut-brain axis”. Changes in the function of brain-gut axis would cause a variety of gastrointestinal tract diseases such as irritable bowel syndrome and related functional gastrointestinal tract diseases. Recent studies have also found that dysfunction of the brain-gut axis is also responsible for the development of many brain diseases, including autism, Parkinson's disease, mood and emotion disorders, and chronic pains.

Generally speaking, aging, menopause, Alzheimer's disease, brain ischemia, etc. will cause the body, especially the brain, to decline in function and memory. It is believed in modern medicine that this is associated with the decline in the function of hippocampus of the brain, and scopolamine is often used in medical trials to block its neural pathways. Studies on the regulation of the axis of intestinal microorganism-metabolism-neurotransmitter have not been reported.

Through the above analysis, the problems and defects of the prior arts lie in that studies on bioactive peptides that are capable of improving scopolamine-induced memory defects by improving the pathway of tryptophan neurotransmitter have not been reported in prior arts.

The difficulty of solving the above problem and defects is due to the complexity of Alzheimer's disease in which memory decline is caused by many factors. Most nutritional supplements are studied as antioxidants, but the relationship between memory decline and intestinal flora, metabolism, and neurotransmitter. In those studies, most plant extracts had desirable effects in memory improvement, but the processes for the preparation of the plant extracts were complicated, and the introduction of organic reagents was costly.

The significance of solving the above problems and defects is that the neuroprotective peptide PAYCS (SEQ ID NO: 1) is obtained through targeted isolation from anchovies. The preparation process is a green and simple one. By practicing the embodiments of the present disclosure, the improvement effect of the bioactive polypeptide on memory impairment through the regulation of intestinal flora, intestinal metabolism and brain neurotransmitters can be identified.

SUMMARY

In view of the problems existing in the prior arts, the present disclosure provides the use of PAYCS (SEQ ID NO: 1) in regulating intestinal flora, metabolites, and brain neurotransmitters.

The present disclosure is achieved by implementing the following technical proposal. Provided is the use of PAYCS (SEQ ID NO: 1) in regulation of intestinal flora, metabolites, and brain neurotransmitters, comprising: remedying scopolamine-induced memory impairment in mice through targeted oxidation, inflammatory stress, and regulation of the intestinal microorganism-metabolite-brain neurotransmitter axis by PAYCS (SEQ ID NO: 1).

PAYCS (SEQ ID NO: 1) may be prepared by using a process including the following conditions for enzymatic hydrolysis by protease:material-liquid ratio 1:3, enzyme dosage 1.3% by weight of the material, pH 7.2, reaction temperature 60° C., and reaction time 50 min. The conditions for enzymatic hydrolysis using an alkaline protease are: an enzyme dosage of 0.32%, a pH value of 7.3, a reaction temperature: 63° C., and a reaction time of 77 min.

In a further embodiment of the present disclosure, the alkaline protease is subjected to a second enzymatic hydrolysis. After the second enzymatic hydrolysis is completed, a heater temperature is adjusted, the pH of the resulting material is adjusted to the desired value using HCl, and glucoamylase is added to degrade the polysaccharide.

Upon completion of inactivation of the enzyme, the resulting materials are fully inactivated at 96° C., and the heater, the homogenization pump, and the valves are closed, and a cooling water circulating pump is started to promote cooling of the materials. When the temperature drops to 43° C., all the pumps and the valves are closed, the discharge valve is opened, and enzymatic hydrolysate is stored in a barrel.

In a further embodiment of the present disclosure, a decolorization step is conducted as follows: the enzymatic hydrolysate is centrifuged at 4,100×rpm for 22 min and the enzymatic hydrolysate is filtered using an 11-kDa ultrafiltration membrane. The supernatant is decolonized with an ultrafiltration membrane, with an inlet pressure of 0.09 MPa, an outlet pressure of 0.07 MPa, and a material temperature of 26° C. During the whole process, circulating cooling water is used to prevent temperature rise in the material, and a membrane throughput is determined by calculating the volume of outflow liquid within the measurement time.

In a further embodiment of the present disclosure, the method comprises cleaning the membrane using a forward cleaning process. Firstly, residues in the membrane system are removed with clean water, and a membrane cleaning agent is added to perform circulated cleaning for 61 min. The cleaning solution is removed, and then the membrane system is washed with clean water until the eluate becomes clear, contains no foams and the pH value becomes neutral. Then a reverse cleaning process is performed as follows: residues in the membrane system are removed with clean water, the ultrafiltration membrane is disassembled and placed and assembled in a direction opposite to the initial direction, and then the ultrafiltration membrane is cleaned using a forward cleaning procedure.

After a spray dryer is cleaned, a dryer and a heater are turned on to dry the water inside the dryer. An atomizer and a feed pump are turned on and the material is sprayed. An inlet temperature is 150° C., a frequency of a nebulizer is set at 370 Hz, and a speed of a peristaltic pump is set at 18 rpm.

In a further embodiment of the present disclosure, PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) exhibit different memory mitigation effects in Alzheimer's disease (AD) model mice.

In a further embodiment of the present disclosure, PAYCS (SEQ ID NO: 1) has a positive effect on both liver and serum deficits.

In a further embodiment of the present disclosure, PAYCS-H (higher dose of SEQ ID NO: 1) has good antioxidant and anti-inflammatory effects.

In a further embodiment of the present disclosure, PAYCS-L (lower dose of SEQ ID NO: 1) reconstitutes the abundance of intestinal microorganism population in amnesic mice.

In a further embodiment of the present disclosure, PAYCS (SEQ ID NO: 1) has a regulatory effect on brain neurotransmitters and fecal metabolites in amnesic mice.

Through combining all technical solutions as described above, the present disclosure provides the following advantages and positive effects: PAYCS (SEQ ID NO: 1) provided by the present disclosure is used to regulate intestinal flora, metabolites, and brain neurotransmitters. Through targeted oxidation, inflammatory stress, and regulation of the intestinal microorganism-metabolite-brain neurotransmitter axis, PAYCS (SEQ ID NO: 1) remedies scopolamine-induced memory impairment in mice.

The bioactive peptide PAYCS (Pro-Ala-Tyr-Cys-Ser) (SEQ ID NO: 1) derived from anchovy hydrolysate has shown an effect of memory improvement. The intestinal microbiota-brain axis plays an important role in brain functions which may be affected by nutritional supplements. In the scopolamine-induced mouse model, a better effect of memory enhancement was shown in the training part of the behavioral test following 3 weeks of treatment with PAYCS-L (0.2 g/kg bw, lower dose of SEQ ID NO: 1), while 3 weeks of treatment with PAYCS-H (0.4 g/kg bw, higher dose of SEQ ID NO: 1) showed better improvement in cognitive function in the test part. PAYCS (SEQ ID NO: 1) can significantly reduce the MDA and LDH levels in serum and significantly increase SOD content in the liver. PAYCS-H (higher dose of SEQ ID NO: 1) can inhibit the increase of both TNF-α and IL-1β in the liver, while PAYCS-L (lower dose of SEQ ID NO: 1) only reduces the content of TNF-α in the liver. The results of 16S rRNA analysis showed that PAYCS-L (lower dose of SEQ ID NO: 1) changed the ratio of Bacteroidetes/Firmicutes, and PAYCS (SEQ ID NO: 1) increased the relative abundance of plants such as Cacteroidaceae and Prevotellaceae. It is noted that memory-related metabolites (e.g., blumealactone C, thalictroidine, notoginsenoside M, 3a,7a,12b-trihydroxy-5b-cholanoic acid, sequiterpene lactone 326, helenalin, and cholic acid) and neurotransmitters (e.g., Ach, GABA, Glu, HisA, and Kyn) are significantly up-regulated. Therefore, different doses of PAYCS (SEQ ID NO: 1) exhibit memory-relieving effects through different pathways. PAYCS-L (lower dose of SEQ ID NO: 1) partially reverses memory deficits in amnesic mice by regulating the intestinal microorganism-metabolites-brain neurotransmitter axis. PAYCS-H (higher dose of SEQ ID NO: 1) reverses oxidative and inflammatory stress in the liver and serum, which may be a key pathway for improving memory. And PAYCS-H (higher dose of SEQ ID NO: 1) re-modulates intestinal microorganisms and fecal Metabolites. PAYCS (SEQ ID NO: 1) increases the relative abundance of microbita such as Cacteroidaceae and Prevotellaceae and improves the tryptophan metabolism-related neurotransmitters in the brain.

BRIEF DESCRIPTION OF THE DRAWINGS

To illustrate the technical solutions of embodiments of the present disclosure more clearly, a brief introduction will be made to the drawings required in the embodiments of the present disclosure. Apparently, the drawings described below are only provided for some embodiments of the present disclosure, those of ordinary skill in the art can further obtain other drawings according to these drawings without making creative efforts.

FIG. 1A-FIG. 1H show the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the behavior of amnesic mice with amnesia induced by scopolamine.

FIG. 1A shows the test results of the jumping test in an embodiment of the present disclosure.

FIG. 1B shows test results of the swimming speed test in an embodiment of the present disclosure.

FIG. 1C shows the test results of the swimming distance test in an embodiment of the present disclosure.

FIG. 1D shows the test results of the number of crossings on the target platform.

FIG. 1E shows test results of the number of crossings between the target quadrants.

FIG. 1F shows the test results of the escape latency and error times during the training.

FIG. 1G shows the test results of the measurement results of swimming distance, swimming speed, and the number of crossings between the target quadrants.

FIG. 1H shows the number of crossings and escape latency on the target platform.

FIG. 2A-FIG. 2D show the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the liver and serum oxidation indexes of amnesic mice induced by scopolamine.

FIG. 2A shows the measurement results of MDA in mouse liver and serum.

FIG. 2B shows the measurement results of LDH in mouse liver and serum.

FIG. 2C shows the measurement results of SOD in mouse liver and serum.

FIG. 2D shows the measurement results of GP(x) in mouse liver and serum.

FIG. 3A-FIG. 3B show the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the liver and serum inflammatory indexes of mice with amnesia induced by scopolamine.

FIG. 3A shows the measurement results of IL-1β content in mouse liver and serum.

FIG. 3B shows the measurement results of TNF-α content in mouse liver and serum.

FIG. 4A-FIG. 4F show the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the intestinal microflora in the mice with amnesia induced by scopolamine.

FIG. 4A shows a Venn diagram of a diversity at the OTU level.

FIG. 4B shows a diagram of the Student's test of Chao indexes.

FIG. 4C shows a histogram of community differences at the phylum level.

FIG. 4D shows the analysis of microflora difference between the control and the model at the species level.

FIG. 4E shows the analysis of microflora difference between PYACS-L and the model at the species level.

FIG. 4F shows the analysis of microflora difference between PYCS-H and the model at the species level.

FIG. 5A-FIG. 5E show the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the fecal metabolites of amnesic mice induced by scopolamine.

FIG. 5A shows a Venn diagram of different metabolites.

FIG. 5B shows a cluster heat map of the metabolites.

FIG. 5C shows the analysis of the metabolite difference between the control and the model.

FIG. 5D shows the analysis of the metabolite difference between the model and PAYCS-L (lower dose of SEQ ID NO: 1).

FIG. 5E shows the analysis of the metabolite difference between the model and PAYCS-H (higher dose of SEQ ID NO: 1).

FIG. 6 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on neurotransmitters of amnesic mice induced by scopolamine.

FIG. 7 shows an experiment designed to evaluate the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the learning and memory function of amnesic mice induced by scopolamine.

FIG. 8 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on neurotransmitters of amnesic mice induced by scopolamine.

FIG. 9 shows a histogram of community differences at a family level.

FIG. 10A-FIG. 10C show the correlation analysis between the intestinal flora and the fecal metabolites at a species level.

FIG. 11 shows the correlation analysis between the intestinal flora and the neurotransmitters at a genus level.

FIG. 12A-FIG. 12C show the correlation analysis between the fecal metabolites and the neurotransmitters.

FIG. 13 schematic diagram of the mechanism of action of PAYCS (SEQ ID NO: 1) in remedying scopolamine-induced memory defects in mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In order to make the objective, technical solutions, and advantages of the present disclosure clearer, the present disclosure is described below in more detail in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely used to explain the present disclosure and are not intended to limit the present disclosure.

Given the problems existing in the prior arts, the present disclosure provides the use of PAYCS (SEQ ID NO: 1) in the regulation of intestinal flora, metabolites, and brain neurotransmitters, and the present disclosure will be described in detail below in conjunction with specific embodiments. PAYCS (SEQ ID NO: 1) is a polypeptide derived from the enzymatic hydrolysate of the fish of anchovies and supplementation of PAYCS (SEQ ID NO: 1) can regulate the intestinal flora-metabolite-brain neurotransmitter axis by improving serum, liver oxidation stress, and inflammatory injuries to improve efficacy in scopolamine-induced memory impairment in mice. PAYCS (SEQ ID NO: 1) can be used as medicine or a supplement. Supplementing PAYCS (SEQ ID NO: 1) allows for remedying scopolamine-induced memory impairment in mice through targeted oxidation, inflammatory stress, and regulation of the intestinal microorganism-metabolite-brain neurotransmitter axis.

PAYCS (SEQ ID NO: 1) may be prepared by using a process including the following conditions for enzymatic hydrolysis by protease:material-liquid ratio 1:3, enzyme dosage 1.3%, pH 7.2, reaction temperature 60° C., and reaction time 50 min. The conditions for enzymatic hydrolysis using an alkaline protease includes an enzyme dosage of 0.32%, a pH value of 7.3, a reaction temperature of 63° C., and a reaction time of 77 min.

In a further embodiment of the present disclosure, the alkaline protease is subject to a second enzymatic hydrolysis. After the second enzymatic hydrolysis is completed, a heater temperature is adjusted, the pH of the resulting material is adjusted to the desired value using HCl, and glucoamylase is added to degrade the polysaccharide.

Upon completion of inactivation of the enzyme, the resulting materials are fully inactivated at 96° C., and the heater, the homogenization pump, and the valves are closed, and a cooling water circulating pump is started to promote cooling of the materials. When the temperature drops to 43° C., all the pumps and the valves are closed, the discharge valve is opened, and enzymatic hydrolysate is stored in a barrel.

A decolorization step is conducted as follows: the enzymatic hydrolysate is centrifuged at 4,100× rpm for 22 min and filtering enzymatic hydrolysate using an 11-kDa ultrafiltration membrane. The supernatant is decolonized with an ultrafiltration membrane, with an inlet pressure of 0.09 MPa, an outlet pressure of 0.07 MPa, and a material temperature of 26° C. During the whole process, circulating cooling water is used to prevent temperature rise in the material, and a membrane throughput is determined by calculating the volume of outflow liquid within the measurement time.

The use comprises cleaning of the membrane using a forward cleaning process. Firstly, residues in the membrane system are removed with clean water, and a membrane cleaning agent is added to perform circulated cleaning for 61 min. The cleaning solution is removed, and then the membrane system is washed with clean water until the eluent becomes clear, contains no foams and the pH value becomes neutral. Then a reverse cleaning process is performed as follows: residues in the membrane system are removed with clean water, the ultrafiltration membrane is disassembled and placed and assembled in a direction opposite to the initial direction, and then the ultrafiltration membrane is cleaned using a forward cleaning procedure.

After a spray dryer is cleaned, a dryer and a heater are turned on to dry the water inside the dryer. An atomizer and a feed pump are turned on and the material is sprayed. An inlet temperature is 150° C., a frequency of a nebulizer is set at 370 Hz, and a speed of a peristaltic pump is set at 18 rpm.

As shown in FIG. 13, PAYCS (SEQ ID NO: 1) remedies scopolamine-induced memory impairment in mice through targeted oxidation, inflammatory stress, and regulation of the intestinal microorganism-metabolite-brain neurotransmitter axis.

The bioactive peptide PAYCS (Pro-Ala-Tyr-Cys-Ser) (SEQ ID NO: 1) extracted from anchovy hydrolysate has shown an effect of memory improvement. The intestinal microbiota-brain axis plays an important role in brain functions which may be affected by nutritional supplements. In the scopolamine-induced mouse model, a better effect of memory enhancement was shown in the training part of the behavioral test following 3 weeks of treatment with PAYCS-L (0.2 g/kg bw, lower dose of SEQ ID NO: 1), while 3 weeks of treatment with PAYCS-H (0.4 g/kg bw, higher dose of SEQ ID NO: 1) showed better improvement in cognitive function in the test part. PAYCS (SEQ ID NO: 1) can significantly reduce the MDA and LDH levels in serum and significantly increase SOD content in the liver. PAYCS-H (higher dose of SEQ ID NO: 1) can inhibit the increase of both TNF-α and IL-1β in the liver, while PAYCS-L (lower dose of SEQ ID NO: 1) only reduces the content of TNF-α in the liver. The results of 16S rRNA analysis showed that PAYCS-L (lower dose of SEQ ID NO: 1) changed the ratio of Bacteroides/Firmicutes, and PAYCS (SEQ ID NO: 1) increased the relative abundance of plants such as Cacteroidaceae and Prevotellaceae. It is noted that memory-related metabolites (e.g., Panax notoginseng saponins and cholinergic acid) and neurotransmitters (e.g., Ach, GABA, Glu, HisA, and Kyn) are significantly up-regulated. Therefore, different doses of PAYCS (SEQ ID NO: 1) exhibit memory-relieving effects through different pathways. PAYCS-L (lower dose of SEQ ID NO: 1) partially reverses memory deficits in amnesic mice by regulating the intestinal microorganism-metabolite-brain neurotransmitter axis. PAYCS-H (higher dose of SEQ ID NO: 1) reverses oxidative and inflammatory stress in the liver and serum, which may be a key pathway for improving memory. And PAYCS-H (higher dose of SEQ ID NO: 1) re-modulates intestinal microorganisms and fecal Metabolites.

The present disclosure is described in detail below in conjunction with the accompanying drawings.

FIG. 1 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the behavior of mice with amnesia induced by scopolamine. The mice were tested for a jumping test (FIG. 1A), swimming speed (FIG. 1), swimming distance (FIG. 1C), the number of crossings on the target platform (FIG. 1D), the number of crossings on the target quadrants (FIG. 1E), the escape latency and the error times (FIG. 1F) during the training. In the test part, the swimming distance, the swimming speed, number of crossings on target quadrant (FIG. 1G), the number of crossings on the target platform, and escape latency (H) were determined. In these figures, #: compared with the control group, P<0.05; compared with the control group, P<0.01; *: compared model group, P<0.05; **: compared with the model group, P<0.01.

FIG. 2 shows the effects of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on liver and serum oxidation indexes of mice with amnesia induced by scopolamine and levels of MDA (FIG. 2A), LDH (FIG. 2B), SOD (FIG. 2C), and GP(x) (FIG. 2D) in the liver and the serum of mice were determined. #: compared with the control group, P<0.05; *: compared to the model group, P<0.05.

FIG. 3 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the inflammatory indexes in the liver and the serum of mice with amnesia induced by scopolamine, and the contents of IL-1β (FIG. 3A) and TNF-α (FIG. 3B) in mouse liver and serum were determined. #: compared with the control group, P<0.05; *: compared to the model group, P<0.05.

FIG. 4 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on the intestinal microflora in the mice with amnesia induced by scopolamine and provides a Venn diagram of a diversity (FIG. 4A) at OTU level, Student's test for Chao index (FIG. 4B), a histogram of community difference and controls and models (FIG. 4D) at the phylum level (FIG. 4C), and the microflora differences between the control and the model, between PYACS-L and the model (FIG. 4E), between PYCS-H and the model (FIG. 4F) at the species level. The symbols * and ** represent P<0.05 and P<0.01, respectively.

FIG. 5 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on fecal metabolites of mice with amnesia induced by scopolamine, showing a Venn diagram (FIG. 5A) of different metabolites, cluster heat map of metabolites (FIG. 5B), control versus model (FIG. 5C), model versus PAYCS-L (lower dose of SEQ ID NO: 1, FIG. 5D), and model versus PAYCS-H (higher dose of SEQ ID NO: 1, FIG. 5E). The scores of variable importance in projection (VIP) for symbol ** and symbol *** are P<0.05, P<0.01, and P<0.001 respectively.

FIG. 6 shows the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on brain neurotransmitters of mice with amnesia induced by scopolamine. #: compared with the control group, P<0.05; *: compared to the model group, P<0.05.

FIG. 7 is an experiment designed to evaluate the effect of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on learning and memory function in mice with amnesia induced by scopolamine.

FIG. 8 shows the effects of PAYCS-L (lower dose of SEQ ID NO: 1) and PAYCS-H (higher dose of SEQ ID NO: 1) on brain neurotransmitters in mice with amnesia induced by scopolamine.

FIG. 9 is a histogram of community indifference disparities at the household level.

FIG. 10A-FIG. 10C show the correlation analysis of intestinal microorganisms and fecal metabolites at the species level. FIG. 10A: control versus model; FIG. 10B: PAYCS-L (lower dose of SEQ ID NO: 1) versus model; FIG. 10C: PAYCS-H (higher dose of SEQ ID NO: 1) versus model. Symbols *, **, and *** represent P<0.05, P<0.01, and P<0.001s, respectively.

FIG. 11 shows the correlation analysis of the intestinal microorganism population and the brain neurotransmitters at the genus level. Symbols *, **, and *** represent P<0.05, P<0.01, and P<0.001, respectively.

FIG. 12A-FIG. 12C show the correlation analysis of the fecal metabolites and the brain neurotransmitters; FIG. 12A: control versus model; FIG. 12B: PAYCS-L (lower dose of SEQ ID NO: 1) versus model, FIG. 12C: PAYCS-H (higher dose of SEQ ID NO: 1) versus model. Symbols * and ** represent P<0.05, P<0.01, respectively P<0.001.

Described above are only some of the specific embodiments of the present disclosure and the protection scope of the present disclosure is not limited thereto, any person skilled in the art may make any modifications, equivalent substitutions, and improvements within the spirit and principles of the present disclosure, etc., and these modifications or improvements should be deemed to fall within the scope of protection of the present disclosure.

Claims

1. A method for regulating intestinal flora, metabolites, and brain neurotransmitters, comprising a step of administering a peptide to a subject to promote targeted oxidation, improve inflammatory stress, and regulate intestinal microorganism-metabolite-brain neurotransmitter axis; wherein the peptide is PAYCS (SEQ ID NO: 1) and is prepared by using a process including the following conditions for enzymatic hydrolysis by protease:material-liquid ratio 1:3, enzyme dosage 1.3% by weight of the material, pH 7.2, reaction temperature 60° C., and reaction time 50 min; and the conditions for enzymatic hydrolysis using an alkaline protease includes an enzyme dosage of 0.32%, a pH value of 7.3, a reaction temperature of 63° C., and a reaction time of 77 min.

2. The method according to claim 1, wherein the alkaline protease is subjected to a second enzymatic hydrolysis; after the second enzymatic hydrolysis is completed, a heater temperature is adjusted, the pH of the resulting material is adjusted to the desired value using HCl, and glucoamylase is added to degrade the polysaccharide; upon completion of inactivation of the enzyme, the resulting materials are fully inactivated at 96° C., and a heater, a homogenization pump, and valves are closed, and a cooling water circulating pump is started to promote cooling of the materials; when the temperature drops to 43° C., all the pumps and valves are closed, the discharge valve is opened, and enzymatic hydrolysate is stored in a barrel.

3. The method according to claim 2, wherein a decolorization step is conducted as follows: the enzymatic hydrolysate is centrifuged at 4,100×rpm for 22 min and filtering enzymatic hydrolysate using an 11-kDa ultrafiltration membrane; a supernatant is decolonized with an ultrafiltration membrane, with an inlet pressure of 0.09 MPa, an outlet pressure of 0.07 MPa, and a material temperature of 26° C.; during the whole process, circulating cooling water is used to prevent temperature rise in the material, and a membrane throughput is determined by calculating the volume of outflow liquid within the measurement time.

4. The method according to claim 3, further comprising cleaning of the membrane using a forward cleaning process; firstly, residues in the membrane system are removed with clean water, and a membrane cleaning agent is added to perform circulated cleaning for 61 min; the cleaning solution is removed, and then the membrane system is washed with clean water until eluent becomes clear, contains no foams and the pH value becomes neutral; then a reverse cleaning process is performed as follows: residues in the membrane system are removed with clean water, the ultrafiltration membrane is disassembled and placed and assembled in a direction opposite to the initial direction, and then the ultrafiltration membrane is cleaned using a forward cleaning procedure; after a spray dryer is cleaned, a dryer and a heater are turned on to dry the water inside the dryer; an atomizer and a feed pump are turned on and the material is sprayed; an inlet temperature is 150° C., a frequency of a nebulizer is set at 370 Hz, and a speed of a peristaltic pump is set at 18 rpm.

5. The method according to claim 1, wherein PAYCS (SEQ ID NO: 1) is administered at a lower dose PAYCS-L or at a higher dose PAYCS-H to an Alzheimer's disease (AD) model mice.

6. The method according to claim 1, wherein the method comprises administering PAYCS-H (higher dose of SEQ ID NO: 1) to improve antioxidant and anti-inflammatory effects.