Sheep milk powder having improved memory activity and a method of preparing the same

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a sheep milk powder with improved memory activity and a preparation method thereof. The present application provides a sheep milk powder with improved memory activity, wherein the sheep milk powder contains a memory improving peptide; the memory improving peptide is an acetylcholinesterase inhibitory peptide; the present application adopts compound protease to enzymatically hydrolyze sheep milk casein, and then adopts ultrafiltration, gel chromatography and preparative chromatography to separate and purify the acetylcholinesterase inhibitory peptide, so as to obtain the acetylcholinesterase inhibitory peptide, the acetylcholinesterase (AChE) inhibitory rate of which is 86.06+ / -0.04%, and the acetylcholinesterase inhibitory peptide is added to sheep milk, and a sheep milk powder is obtained through spray drying. The present application provides theoretical and technical support for deep processing of sheep milk casein, and also provides the acetylcholinesterase inhibitory peptide for research and development of food with improved memory activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a sheep milk powder with memory-enhancing activity and its preparation method. Background Technology

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease associated with memory impairment and cognitive deficits. It impairs memory, learning, cognition, and behavioral functions, leading to a decline in self-care abilities. It is also often accompanied by mental and emotional control disorders, as well as the decline in health that occurs during the normal aging process, which has a serious negative impact on the quality of life of patients and their caregivers.

[0003] Acetylcholinesterase inhibitors (AChEIs) work by inhibiting the activity of acetylcholinesterase (AChE), reducing the breakdown of acetylcholine, increasing cholinergic activity, and increasing the concentration of acetylcholine in the synaptic cleft, thereby enhancing cholinergic neurotransmission and improving cognitive function. Memantine, on the other hand, exerts a neuroprotective effect by blocking NMDA receptors, reducing glutamate-mediated excitatory neurotoxicity. However, the effectiveness of these drugs varies from person to person and is limited in duration; they only relieve symptoms but cannot alter the disease course, and they have serious side effects. For example, tacorlina has severe hepatotoxicity and gastrointestinal side effects and is now banned by the FDA. Other drugs have side effects such as bradycardia, orthostatic hypotension, diarrhea, vomiting, nausea, fatigue, insomnia, loss of appetite, and weight loss. Therefore, it is essential to find a safe and effective food-derived acetylcholinesterase inhibitor.

[0004] Memory-enhancing peptides refer to peptides that are related to improving memory. Acetylcholinesterase inhibitory peptides are one type of memory-enhancing peptides. The main methods for their preparation are enzymatic hydrolysis and fermentation. Reported raw materials for enzymatic hydrolysis include anchovies, salmon, walnuts, pea protein, whey protein, anchovy protein, and oyster protein. There are no reports of using sheep casein as a raw material. Summary of the Invention

[0005] The purpose of this invention is to provide a sheep milk powder with memory-enhancing activity and its preparation method, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a sheep milk powder with memory-enhancing activity, wherein the milk powder contains a memory-enhancing peptide; the memory-enhancing peptide is an acetylcholinesterase inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

[0007] Optionally, the acetylcholinesterase inhibitory peptide has an acetylcholinesterase inhibition rate of 86.06 ± 0.04%.

[0008] The present invention provides a method for preparing the above-mentioned sheep milk powder, comprising the steps of enzymatic hydrolysis of sheep milk casein using a complex protease, ultrafiltration, gel chromatography separation and purification, preparation of chromatographic separation and purification and freeze drying to obtain acetylcholinesterase inhibitory peptide powder, adding the acetylcholinesterase inhibitory peptide powder, nutritional fortifier, prebiotic and probiotic to sheep milk powder, and aseptically mixing evenly to obtain the memory-improving peptide sheep milk powder; The complex protease includes bromelain and flavor protease; the enzyme activity ratio of bromelain and flavor protease in the complex protease is 4:1.

[0009] Optionally, the pH of the enzymatic hydrolysis is 6.5-7.0, the enzyme dosage is 8000-10000 U / g; the sheep milk casein concentration is 4wt%-6wt%, the enzymatic hydrolysis temperature is 50-55℃, and the enzymatic hydrolysis time is 1-3h; the acetylcholinesterase inhibition rate of the sheep milk casein hydrolysate prepared by the enzymatic hydrolysis is 22.15%-33.98%. The ultrafiltration includes the step of collecting components with a molecular weight <3kDa and freeze-drying them to obtain crude acetylcholinesterase inhibitory peptide powder M1.

[0010] Optionally, the gel chromatography is Sephadex G-15 dextran gel chromatography, the concentration of the crude peptide powder M1 during purification is 80 mg / mL, the sample loading volume is 1 mL, the eluent is pure water, the elution rate is 1 mL / min, and the component M12 of the second peak is collected.

[0011] Optionally, the preparative chromatography is a preparative reversed-phase high-performance liquid chromatography, with mobile phase A being an acetonitrile aqueous solution containing 0.1% TFA and mobile phase B being ultrapure water containing 0.1% TFA; a linear gradient elution program is used: 0-45 min, with phase A increasing from 5% to 85%. The injection volume is 0.5 mL, and component M09 is collected, then freeze-dried to obtain the acetylcholinesterase inhibitor lyophilized peptide powder.

[0012] Optionally, the amount of the acetylcholinesterase inhibitory peptide lyophilized peptide powder added is 0.50g-2.0g / kg sheep milk powder, and the nutritional fortifier includes phytosterol esters, phosphatidylserine, and N-acetylneuraminic acid; the amount of the phytosterol esters added is 1g-5g / kg sheep milk powder, the amount of the phosphatidylserine added is 0.5g-2.0g / kg sheep milk powder, the amount of the N-acetylneuraminic acid added is 0.5g-1.5g / kg sheep milk powder; and the amount of the prebiotic added is 8g-40g / kg sheep milk powder. The prebiotics include one or more of inulin, isomaltooligosaccharide, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, stachyose, mannose, and chitosan oligosaccharide. The amount of probiotics added is 0.3g-1.2g / kg of sheep milk powder. The probiotics are one or more of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Lactobacillus fermentum, and Lactobacillus plantarum.

[0013] The aseptic mixing process employs a three-dimensional mixed culture, with a mixing speed of 10-20 r / min and a mixing time of 15-25 min.

[0014] This invention provides the application of acetylcholinesterase inhibitory peptide in the preparation of food or pharmaceuticals with memory-enhancing activity, wherein the amino acid sequence of the acetylcholinesterase inhibitory peptide is shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

[0015] The present invention provides a product having memory-enhancing activity, the product comprising an acetylcholinesterase inhibitory peptide; the amino acid sequence of the acetylcholinesterase inhibitory peptide is shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

[0016] The present invention discloses the following technical effects: This invention provides a sheep milk powder with memory-enhancing activity, wherein the milk product contains bioactive peptides; the amino acid sequences of the bioactive peptides are shown in SEQ ID NO.1~SEQ ID NO.45, PPQ, and PLQ; this invention employs a complex protease to enzymatically hydrolyze sheep milk casein, followed by ultrafiltration, dextran gel electrophoresis, and preparative chromatography to separate and purify the cholinesterase-inhibiting peptides, ultimately obtaining the sheep milk powder. The AChE inhibition rate of the bioactive peptides is 86.06±0.04%. This invention provides a new approach for the high-value utilization of sheep milk casein and also provides theoretical and technical support for the development of foods that prevent and improve memory. Attached Figure Description

[0017] Figure 1The effects of single and compound proteases on the activity and degree of hydrolysis of acetylcholinesterase inhibitory peptides in sheep casein hydrolysate; Figure 2 The effect of the ratio of protease BL to FW on the activity and degree of hydrolysis of acetylcholinesterase inhibitory peptides in sheep milk casein hydrolysate; Figure 3 The effect of temperature on the activity and degree of hydrolysis of acetylcholinesterase inhibitory peptides in sheep casein hydrolysate; Figure 4 The effect of pH on the inhibitory activity of acetylcholinesterase and the degree of hydrolysis of sheep casein hydrolysate; Figure 5 The effect of enzyme dosage on the activity of acetylcholinesterase inhibitory peptides and degree of hydrolysis in sheep casein hydrolysate; Figure 6 Effects of sheep milk casein concentration on the activity of acetylcholinesterase inhibitory peptides and degree of hydrolysis in its enzymatic hydrolysate Figure 7 The effect of enzymatic hydrolysis time on the activity of acetylcholinesterase inhibitory peptides and the degree of hydrolysis in sheep casein hydrolysate; Figure 8 The effect of ultrafiltration membranes with different molecular weight cutoffs on the separation of acetylcholinesterase-inhibiting peptides from sheep casein hydrolysate; Figure 9 The effects of eluents Tris-HCl (a), phosphoric acid (b), and water (c) on the separation of acetylcholinesterase-inhibiting peptides in ultrafiltration fraction M1; Figure 10 The effect of loading volume on the separation of acetylcholinesterase inhibitory peptides in ultrafiltration fraction M1 was investigated; where (a)-(c) were 0.8 mL, 1.0 mL and 1.2 mL respectively. Figure 11 The effect of loading concentration on the separation of acetylcholinesterase inhibitory peptides in ultrafiltration fraction M1 was investigated; where (a)-(c) were 60 mg / mL, 80 mg / mL and 100 mg / mL, respectively. Figure 12 The effect of elution rate on the separation of acetylcholinesterase inhibitory peptides in ultrafiltration fraction M1 was investigated; where (a)-(c) were 0.6 mL / min, 0.8 mL / min and 1 mL / min, respectively. Figure 13 The effect of each component of Sephadex G-15 on the inhibitory activity of acetylcholinesterase; Figure 14 The preparative chromatogram of component M12 obtained by Sephadex G-15 separation; Figure 15 To prepare the acetylcholinesterase inhibitory activity of each component obtained by chromatography; Figure 16The effect of pH on the inhibitory activity of component M09 acetylcholinesterase; Figure 17 Effect of temperature on the inhibitory activity of component M09 acetylcholinesterase Figure 18 The effects of simulated gastrointestinal fluid (a) and simulated intestinal fluid (b) on the inhibitory activity of component M09 acetylcholinesterase. Detailed Implementation Example 1: Process optimization for preparing cholinesterase inhibitory peptides by enzymatic hydrolysis of sheep milk casein 1. Raw materials, enzyme preparations and reagents The raw materials and enzyme preparations are shown in Table 1.

[0018] Table 1 Raw materials, enzyme preparations and reagents 2. Experimental Methods 2.1 Preparation of sheep milk casein Accurately weigh whole sheep milk powder, mix it with distilled water at a ratio of 1g:8mL, and centrifuge at 6500r / min for 15min to obtain skimmed sheep milk. Skimmed sheep milk was heated to 45°C in a constant temperature water bath, and 1 mol / L hydrochloric acid was added dropwise to adjust the pH to 4.6, causing sheep milk casein to precipitate. The precipitate was centrifuged at 6500 rpm for 15 min, the supernatant was discarded, and the resulting precipitate was freeze-dried for later use, denoted as sheep milk casein.

[0019] 2.2 Determination of Acetylcholinesterase Inhibition Rate The microplate method was used, with the following conditions: 40 μL of PBS (50 mM), 30 μL of AChE (0.2 U / mL), and 50 μL of sample solution were added to a 96-well plate. After pre-incubation at 37°C for 5 min, 50 μL of 3 mM DTNB solution and 30 μL of 7.5 mM ATCI substrate solution were added. The reaction was carried out at 37°C for 20 min, and the OD was measured at 405 nm. This was recorded as the sample group. Considering the color of the sample, interference needed to be eliminated, so an equal volume of PBS was used instead of AChE to prepare the sample background group. An equal volume of sample solvent was used instead of the sample solution for the blank group, and a blank background group was also prepared. The inhibition rate was calculated, and all experiments were repeated three times. The formula for calculating the acetylcholinesterase inhibition rate is as follows: Acetylcholinesterase inhibition rate (%) = [1-(OD S -OD S0 ) / (OD B -OD B0 )]×100; in, OD S For sample groups; OD S0This is the background group of the sample; OD B This is a blank group; OD B0 This is the blank background group.

[0020] 2.3 Determination of degree of hydrolysis A certain amount of sheep milk casein was pre-wetted with a small amount of water, and then 0.1 mol / L NaOH solution was added to promote its dissolution. After complete dissolution, the solution was diluted with distilled water to the required concentration. The resulting solution was placed in a constant temperature water bath, and the system temperature and pH were adjusted to the optimal hydrolysis conditions for each protease. The protease was then added to allow it to react fully with the substrate. During hydrolysis, the pH of the system was maintained by continuously adding 0.1 mol / L NaOH solution, and the volume of NaOH consumed was recorded at set time intervals. Finally, the degree of protein hydrolysis (DH) was calculated using the pH-Stat method, as shown in the following formula: Degree of hydrolysis DH (%) = (B × M) b ) / (α×M p ×8.2); Wherein, B: volume of NaOH (mL); M b NaOH concentration (mol / L); α: degree of amino dissociation; M p 8.2: Protein mass (g); 8.2: Millimoles of peptide bonds in casein (mmol).

[0021] 2.4. Protease Screening and Compounding 2.4.1 Screening of single and complex proteases Sheep milk casein was prepared into a 5 wt% substrate solution. The dosage of single and complex proteases was 8000 U / g, the dosage of proteases with two enzymes was 4000 U / g each, and the dosage of proteases with three enzymes was 2666.7 U / g each. Enzymatic hydrolysis was carried out at pH 7.0 and 50℃ for 3 h. During the reaction, the pH was maintained constant using 0.1 mol / L NaOH solution, and samples were taken every 30 min. After hydrolysis, the samples were heated in a 98℃ water bath for 15 min to terminate the reaction, then cooled to room temperature and the pH was adjusted to 7.0. The samples were then centrifuged at 8000 r / min for 10 min at 4℃, and the supernatant was collected for determining the degree of hydrolysis (DH) and acetylcholinesterase inhibitory activity to screen suitable single or combined proteases. The proteases and their optimal pH and temperature are shown in Table 2.

[0022] Table 2. Proteases and their optimal pH and temperature 2.4.2 Optimization of protease ratio The proteases BL and FW were combined, with a total enzyme addition of 8000 U / g. They were then mixed evenly at enzyme activity ratios of 4:1, 3:2, 1:1, 2:3, and 1:4, and then enzymatically hydrolyzed at pH 7.0 and 50℃ for 3 hours. The degree of hydrolysis (DH) and acetylcholinesterase inhibitory activity were measured to obtain a suitable protease ratio.

[0023] 2.5 Optimization of the enzymatic hydrolysis process for preparing acetylcholinesterase inhibitory peptides from sheep milk casein Based on a BL to FW enzyme activity ratio of 4:1, the effects of five factors on the preparation of acetylcholinesterase inhibitory peptide and degree of hydrolysis of sheep milk casein were investigated, namely, temperature (40, 45, 50, 55, 60℃), pH (6, 6.5, 7, 7.5, 8), enzyme dosage (6000, 7000, 8000, 9000, 10000 U / g), casein concentration (3, 4, 5, 6, 7wt%), and time (1, 2, 3, 4, 5 h), to obtain suitable enzymatic hydrolysis conditions.

[0024] 2.6 Data Analysis and Statistics All experiments were repeated three times, and the results are expressed as mean ± standard deviation. Origin 2021 and Design Expert 2010 software were used for data processing and graphing, and SPSS 26.0 software was used for statistical analysis.

[0025] 3. Results and Analysis 3.1 Effects of single and complex proteases on the inhibition rate and degree of hydrolysis of acetylcholinesterase in sheep milk casein The effects of single and complex proteases on the inhibition rate and degree of hydrolysis of acetylcholinesterase in sheep casein were shown in the following results. Figure 1 As shown. By Figure 1 It was found that the acetylcholinesterase inhibitory peptide activity of the sheep milk casein hydrolysate containing the compound protease BL+FW was superior to that of single and other compound protease hydrolysates, with an AChE inhibition rate of 21.48±0.4%, compared to 20.49±0.95% for the sheep milk casein BL hydrolysate. Therefore, the compound protease BL+FW was selected for hydrolyzing sheep milk casein, and further experiments are needed to determine the ratio of sheep milk compound protease BL+FW.

[0026] 3.2 Effect of the ratio of protease BL to FW on the inhibition rate and degree of hydrolysis of acetylcholinesterase in sheep milk casein The effect of the ratio of protease BL to FW on the inhibition rate and degree of hydrolysis of sheep milk casein acetylcholinesterase is shown in the figure. Figure 2 .Depend on Figure 2It can be seen that when the ratio of protease BL:FW is 4:1, the AChE inhibition rate and DH of sheep casein hydrolysate reach the maximum, which are 22.18±0.36% and 11.84±0.37%, respectively. Therefore, the ratio of BL:FW is selected to optimize the subsequent sheep casein hydrolysis conditions.

[0027] 3.3 Effects of enzymatic hydrolysis conditions on the inhibition rate of acetylcholinesterase and the degree of hydrolysis of sheep milk casein hydrolysate 3.4.1 Enzymatic hydrolysis temperature like Figure 3 As shown, the DH of sheep milk casein increases with increasing temperature. The acetylcholinesterase inhibition rate of sheep milk casein hydrolysate shows a trend of first increasing and then decreasing with increasing temperature, reaching its maximum at 50℃ and 55℃. Therefore, the suitable enzymatic hydrolysis temperature is 50-55℃.

[0028] 3.4.2, pH Depend on Figure 4 It can be seen that the DH of sheep milk casein hydrolysate increases with increasing pH, while the acetylcholinesterase inhibition rate shows a trend of first increasing and then decreasing. When the pH is 7.0, the AChE inhibition rate of sheep milk casein is 23.04±0.30%, and the DH is also the highest at pH 7.0. With further increase in pH, the acetylcholinesterase inhibition rate gradually decreases, and the suitable pH is 6.5-7.0.

[0029] 3.4.3. Increase the dosage of complex protease Depend on Figure 5 It was found that the acetylcholinesterase inhibition rate and degree of hydrolysis of sheep casein protein hydrolysate increased with the increase of the amount of complex protease added, indicating that within the experimental range, increasing the enzyme dosage can effectively promote casein degradation and the release of highly active acetylcholinesterase inhibitory peptides. When the enzyme dosage was 9000 U / g, the AChE inhibition rate and DH of sheep casein protein hydrolysate were 28.64±0.58% and 12.36±0.29%, respectively. With further increases in enzyme dosage, the increase gradually decreased, and the suitable enzyme dosage was 8000-10000 U / g.

[0030] 3.4.4 Sheep milk casein concentration Depend on Figure 6 It can be seen that the acetylcholinesterase inhibition rate and DH showed opposite trends with increasing casein concentration. When the casein concentration was 6 wt%, the AChE inhibition rate of sheep milk casein hydrolysate reached its maximum value, which was 29.61±0.95% and 30.141±1.00%, respectively. With further increase in casein concentration, the acetylcholinesterase inhibition rate tended to remain unchanged. The suitable casein concentration for sheep milk was 4 wt%-6 wt%.

[0031] 3.4.5 Enzymatic hydrolysis time Depend on Figure 7 It can be seen that the AChE inhibition rate and DH (hydrolysate) initially increased rapidly and then tended to stabilize with increasing enzymatic hydrolysis time. At 3 hours of enzymatic hydrolysis, the sheep milk casein reaction was nearly stable, with the AChE inhibition rate and degree of hydrolysis being 29.90±0.92% and 11.64±0.16%, respectively. Further extending the time resulted in a decrease in the acetylcholinesterase inhibition rate; therefore, the optimal enzymatic hydrolysis time is 1-3 hours.

[0032] With an AChE inhibition rate higher than 22%, the optimal pH for enzymatic hydrolysis was 6.5-7.0, the enzyme dosage was 8000-10000 U / g, the sheep milk casein concentration was 4wt%-7wt%, the hydrolysis temperature was 50-55℃, and the hydrolysis time was 1-3h. The prepared sheep milk casein hydrolysate showed an acetylcholinesterase inhibition rate of 22.15%-33.98%.

[0033] Example 2: Preparation, stability and identification of acetylcholinesterase inhibitory peptide 1. Experimental materials Example 1: Sheep milk casein hydrolysate prepared under optimal conditions (pH 7.0 for hydrolysis, enzyme dosage 10000 U / g; sheep milk casein concentration 6 wt%, hydrolysis temperature 55℃, hydrolysis time 3 h).

[0034] 2. Experimental Methods 2.1 Ultrafiltration separation of cholinesterase inhibitory peptides Before ultrafiltration, the polyethersulfone membrane was washed with 0.1 mol / L NaOH solution for 30 min, and then washed with purified water until neutral. The supernatant of sheep casein hydrolysate (M0) was passed sequentially through polyethersulfone membranes with pore sizes of 10 kDa and 3 kDa at 25 °C and 0.15 MPa. The retentate and permeate were collected to obtain three components: M1 (3 kDa ultrafiltration membrane permeate), M2 (10 kDa ultrafiltration membrane permeate and 3 kDa ultrafiltration membrane retention solution), and M3 (10 kDa ultrafiltration membrane retention solution). Both M1 and M0 were freeze-dried to obtain lyophilized powder, which was then prepared into 50 mg / mL solutions to determine the acetylcholinesterase inhibition rate.

[0035] 2.2 Purification of acetylcholinesterase inhibitory peptides using Sephadex G-15 dextran gel. Weigh an appropriate amount of Sephadex G-15 dextran gel and soak it in purified water for 24-36 hours with occasional stirring to ensure full expansion. Repeatedly remove suspended particles from the surface. Use a glass rod to guide the pretreated gel into a 1.0 × 40 cm glass chromatography column and allow it to settle for 1 hour to form a stable column bed. Then, continuously flush the column with eluent to remove air bubbles and equilibrate the column bed. Adjust the baseline at an absorbance of 220 nm until it stabilizes before loading the sample. Optimize the gel column chromatography conditions by comparing chromatograms under different eluent types, loading amounts, loading concentrations, and flow rates. After determining the optimal conditions, perform Sephadex G-15 gel column separation and collect the eluted fractions for subsequent analysis.

[0036] 2.3 Preparative Chromatographic Separation Fraction M12, obtained by Sephadex G-15 gel electrophoresis, was prepared into a 100 mg / mL solution with ultrapure water and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Mobile phase A was acetonitrile (containing 0.1% TFA), and mobile phase B was ultrapure water (containing 0.1% TFA). A linear gradient elution program was used: 0-45 min, with phase A increasing from 5% to 85%. The injection volume was 0.5 mL. The target fraction was collected based on the chromatographic peaks, and the eluents were combined and lyophilized for later use.

[0037] 2.4 Stability Study of Acetylcholinesterase Inhibitory Peptide Food processing methods, storage environment, and gastrointestinal digestion can affect its inhibitory activity. Therefore, to explore the optimal processing method and storage conditions, this example analyzes the stability changes of the prepared chromatographic component M09 under different pH, temperature, and simulated gastrointestinal fluid conditions.

[0038] 2.4.1 pH stability M09 was prepared into a 10 mg / mL solution, and its pH was adjusted to 2.5, 4.5, 6.5, 8.5, 10.5 and 12.5 with 1 mol / mL HCl or NaOH, respectively. The solutions were then treated in a 37℃ water bath for 0, 2 and 4 h, and the cholinesterase inhibition rate was then measured.

[0039] 2.4.2 Thermal stability M09 was prepared into a 10 mg / mL solution and placed in water baths at 55℃, 65℃, 75℃, 85℃, and 95℃. The cholinesterase inhibition rate was measured at 20 min, 40 min, and 60 min.

[0040] 2.4.3 Simulating gastrointestinal stability The stability of samples under digestive conditions was determined by simulating gastric and intestinal digestion processes in vitro. Samples were prepared into 10 mg / mL solutions, and the pH was adjusted to 2.0 with 1 mol / L HCl. 2% pepsin (E / S ratio) was added, and the solutions were reacted in a 37℃ water bath for 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min, respectively. After the reaction, the pH was adjusted to 7.5 with 1 mol / L NaOH, and the effect of gastric simulation on cholinesterase inhibition rate was measured. Then, 2% trypsin (E / S ratio) was added, and the reaction was continued at 37℃ for 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. After the reaction, the solutions were heated in a 98℃ water bath for 15 min, and the cholinesterase inhibition rate was measured.

[0041] 2.5 Identification of acetylcholinesterase inhibitory peptides 1 mg of M09 obtained from preparative chromatography was dissolved in 100 μL of 0.1% TFA solution by sonication. The supernatant was collected by centrifugation at 14000g for 10 min and then centrifuged at 13500g for 10 min using a 3 kDa ultrafiltration centrifuge tube. The filtrate was quantified using a UV-Vis spectrophotometer. Subsequently, desalting was performed using a C18 solid-phase extraction column, and peptides were eluted with 70% acetonitrile. Each fractionated sample was separated using a nano-flow rate HPLC system (EasynLC). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution (acetonitrile content 84%). The chromatographic column was equilibrated with 95% solution A. Samples were loaded via an autosampler onto the loading column (Thermoscientific EASY column, 100 μm × 2 cm, 5 μm, C18), and then separated onto the analytical column (Thermo Scientific EASY column, 75 μm × 10 cm, 3 μm, C18) at a flow rate of 250 nL / min. The relevant HPLC gradients were as follows: 0 min–50 min, linear gradient of solution B from 0%–35%; 50 min–58 min, linear gradient of solution B from 35%–100%; 58 min–60 min, solution B maintained at 100%. Mass spectrometry analysis was then performed using a Q Exactive HF-X mass spectrometer (Thermo Fisher) for 60 min, with positive ion detection. Data were acquired and retrieved using MaxQuant software for database searching.

[0042] 2.6 Data Analysis and Statistics Same as Example 1.

[0043] 3. Results and Discussion 3.1 Effects of ultrafiltration on the components of cholinesterase inhibitory peptides A lyophilized powder of the supernatant, ultrafiltration permeate, and retentate of sheep casein protein hydrolysate was prepared into a solution with a concentration of 50 mg / mL. The cholinesterase inhibition rate was then determined, and the results are as follows: Figure 8 As shown. By Figure 8 It was found that after ultrafiltration fractionation of sheep casein hydrolysate, the smaller the molecular weight, the higher the cholinesterase inhibitory activity. In the sheep casein hydrolysate, the AChE inhibition rates of each molecular weight fraction ranged from 26.47% to 29.91%, with the <3 kDa fraction exhibiting the highest inhibition rate at 29.91 ± 0.61%, significantly higher than the original hydrolysate's 26.47 ± 0.07% (p < 0.05). As the molecular weight increased, the cholinesterase inhibitory activity gradually decreased, indicating that the active peptides were mainly concentrated in the low molecular weight region, which is consistent with the conclusion that <3 kDa had the highest proportion in the GPC molecular weight distribution. In summary, the peptide cholinesterase inhibitory activity is closely related to its molecular weight, and ultrafiltration can significantly improve the specific cholinesterase activity of the unfractionated hydrolysate. Therefore, in this example, the <3 kDa fraction was selected for subsequent Sephadex G-15 gel separation and purification.

[0044] 3.2 Purification of acetylcholinesterase inhibitory peptides using Sephadex G-15 dextran gel. 3.2.1 Effect of eluent on the separation of acetylcholinesterase inhibitory peptides The sheep milk caseinate was ultrafiltered to obtain fraction M1 with a molecular weight <3 kDa, which was prepared at a concentration of 80 mg / mL. A loading volume of 0.8 mL was used, and the ultrafiltered fraction was eluted with three eluents: Tris-HCl, phosphoric acid, and water at a rate of 0.8 mL / min. The results are as follows: Figure 9 As shown. By Figure 9 It was found that when Tris-HCl and phosphoric acid were used as eluents, there was significant overlap between adjacent peaks, resulting in broad peak shapes and pronounced tailing. When water was used as the eluent, the sheep casein protein hydrolysate consistently exhibited clear peak shapes and separation, with well-defined boundaries between the main and secondary peaks and relatively weak tailing, which was more conducive to subsequent fraction collection. Therefore, deionized water was chosen as the eluent for gel column chromatography.

[0045] 3.2.2 Effect of sample loading amount on the separation of acetylcholinesterase inhibitory peptides Two fractions with molecular weights <3 kDa were obtained by ultrafiltration of sheep casein hydrolysate and prepared at a concentration of 80 mg / mL. Pure water was used as the eluent, and the ultrafiltration fractions were eluted with loading volumes of 0.8 mL, 1 mL, and 1.2 mL, respectively, at an elution rate of 0.8 mL / min. The results are as follows: Figure 10 As shown. By Figure 10It can be seen that when the sample loading volume is 1.0 mL, the main peak signal is strong, the peak shape is clear, and the interpeak separation is good; when the sample loading volume is 0.8 mL, the peak height is low and the signal is weak; when the sample loading volume is 1.2 mL, slight peak overlap occurs. In summary, 1.0 mL is selected as the optimal sample loading volume of sheep milk casein hydrolysate for subsequent experiments.

[0046] 3.2.3 Effect of sample loading concentration on the separation of acetylcholinesterase inhibitory peptides Two fractions with molecular weights <3 kDa were obtained by ultrafiltration of sheep casein hydrolysate and prepared into concentrations of 60 mg / mL, 80 mg / mL, and 100 mg / mL, respectively. Pure water was used as the eluent, with a loading volume of 1 mL and an elution rate of 0.8 mL / min. The results of eluting the ultrafiltered fractions are as follows: Figure 11 As shown. By Figure 11 It can be seen that when the sample concentration is 80 mg / mL, the peak height is moderate, the peak shape is clear, and the interpeak separation is good, which is the best condition for sheep milk casein hydrolysate; when the sample concentration is 60 mg / mL, the signal is weak, and at 100 mg / mL, peak broadening and slight tailing occur, reducing the separation efficiency.

[0047] 3.2.4 Effect of elution rate on the separation of acetylcholinesterase inhibitory peptides Two fractions with molecular weights <3 kDa were obtained by ultrafiltration of sheep casein hydrolysate and prepared at concentrations of 80 mg / mL. Pure water was used as the eluent, with a loading volume of 1 mL and elution rates of 0.6 mL / min, 0.8 mL / min, and 1 mL / min. The results are as follows: Figure 12 As shown. By Figure 12 It can be seen that when the elution rate is 1 mL / min, the peak height is moderate, the peak shape is clear, and the interpeak separation is good, which is the optimal flow rate; when the flow rate is 0.6 mL / min and 0.8 mL / min, the elution time is long and the signal is weak.

[0048] 3.2.5. Acetylcholinesterase inhibitory peptide activity of each component under optimal separation conditions of Sephadex G-15 dextran gel. Fraction M1, containing sheep milk casein <3kDa, was eluted using Sephadex G-15 dextran gel under the following conditions: concentration 80mg / mL, pure water as eluent, sample volume 1mL, and elution rate 1mL / min. The resulting gel chromatogram (c) with three peaks is shown in Error! Reference source not found.12. The fractions were collected and named M11, M12, and M13 from left to right. After freeze-drying, they were reconstituted with the <3kDa fraction M1 (from ultrafiltration) to prepare a 15mg / mL solution. The acetylcholinesterase inhibition rate was then measured, and the results are as follows: Figure 13 As shown. By Figure 13It was found that after separation by Sephadex G-15 dextran gel, the inhibition rates of the <3 kDa fractions of sheep casein protein hydrolysate were M12 > M13 > M11 > M1, with M12 exhibiting an AChE inhibition rate of 16.19 ± 0.11%. This indicates that low molecular weight active peptides are mainly concentrated in the intermediate elution peak in Sephadex G-15 dextran gel, making them more readily bound to acetylcholinesterase and exhibiting the highest acetylcholinesterase inhibitory activity.

[0049] 3.3 Preparation of Chromatographic Separation Results The sheep milk casein fraction M12, obtained by Sephadex G-15 gel electrophoresis, was subjected to preparative chromatography separation. The resulting preparative chromatogram and the corresponding elution times of each fraction are shown below. Figure 14 As shown. Sheep milk casein fractions M01-M09 obtained from the preparation chromatography were collected, freeze-dried, and rehydrated to 30 mg / mL. Their cholinesterase inhibitory activity was measured, and the results are shown below. Figure 15 As shown. By Figure 15 It was found that the sheep milk casein component M09 exhibited good cholinesterase inhibition, with an AChE inhibition rate of 86.06 ± 0.04%. This inhibition rate was significantly higher than that obtained by ultrafiltration and dextran gel chromatography, indicating that preparative chromatography is beneficial for the purification of acetylcholinesterase inhibitory peptides.

[0050] 3.4 Results of stability of acetylcholinesterase inhibitory peptide 3.4.1 Effect of pH on the activity of cholinesterase inhibitory peptide M09 Depend on Figure 16 It can be seen that the inhibition rate of M09 against AChE under different pH conditions shows a trend of first increasing and then decreasing with increasing pH, and the inhibition rate changes little at different reaction times under the same pH conditions. Furthermore, M09 exhibits high inhibitory activity against AChE within the pH range of 6.5–10.5, with its inhibition rate varying between 40.18±0.41% and 45.60±0.47%. This indicates that M09 has good stability in near-neutral and alkaline environments; the inhibition rate decreases significantly beyond this range. This suggests that M09 has high inhibitory activity against AChE within the pH range of 6.5–10.5, while its activity decreases significantly under strongly alkaline conditions.

[0051] 3.4.2 Effect of temperature on the activity of cholinesterase inhibitory peptide M09 Depend on Figure 17 It can be seen that within the temperature range of 55~95℃, the inhibition rate of M09 against AChE varies between 50.04±0.09% and 63.80±1.1%. Overall, within the same heat treatment time, the inhibitory activity of M09 against acetylcholinesterase increases with increasing temperature.

[0052] 3.4.3 Effect of simulated gastrointestinal digestion on the activity of acetylcholinesterase inhibitory peptide M09 To investigate the stability of component M09 under simulated gastrointestinal digestion conditions and its changes in cholinesterase inhibitory activity, it was first placed in gastric digestive fluid for 0–120 min, and the cholinesterase inhibitory activity was measured at different time points. Subsequently, after the simulated gastric digestion was completed, it was placed in a simulated intestinal digestion system containing trypsin for 0–4 h, and the acetylcholinesterase inhibition rate was measured at different time points. The results are as follows: Figure 18 As shown. By Figure 18 It was found that during the simulated gastric digestion stage, the inhibition rate of M09 against AChE remained generally between 49.14±0.19% and 52.39±0.44%. The inhibitory activity of M09 against AChE did not change significantly during simulated gastric digestion, indicating that it can maintain a certain level of activity under simulated gastric acid environment and pepsin action. During the simulated intestinal digestion stage, the inhibitory activity of M09 against acetylcholinesterase showed an overall trend of first increasing and then slightly decreasing, with its inhibition rate against AChE varying between 56.35±0.58% and 60.69±0.41%, reaching its highest value at 30 minutes of intestinal digestion. This indicates that under simulated intestinal digestion conditions, the inhibitory activity of M09 against acetylcholinesterase was enhanced.

[0053] 3.5 Identification results of acetylcholinesterase inhibitory peptides Forty-seven peptides were identified from the M09 fraction using HPLC-MS / MS. The molecular weights of the AChE inhibitory peptides in sheep milk casein ranged from 340 Da to 878 Da. These bioactive peptides originated from α. S1 -Casein, α S2 -Casein, lactoferrin, β-casein, osteopontin, and κ-casein. Table 3 shows the amino acid sequences, molecular weights, and sources of the peptides obtained.

[0054] Table 3. Amino acid sequences and origins of peptides identified in component M09. Example 3: Wet preparation of sheep milk powder with memory-enhancing activity The component M09 prepared in Example 2 was subjected to vacuum freeze-drying to obtain acetylcholinesterase inhibitory peptide freeze-dried peptide powder. Component M09 was placed in a freeze dryer (model: FD-1A-50, Shanghai Bilang) and pre-frozen at -40℃ for 4 hours. Then, the vacuum pump was turned on and freeze-dried at 4-6 Pa for 20 hours.

[0055] The lyophilized acetylcholinesterase inhibitor peptide powder was added to sheep milk, along with nutritional fortifiers and prebiotics. The mixture was then added to the sheep milk powder and aseptically mixed to obtain sheep milk powder with memory-enhancing activity. The aseptic mixing was performed using a three-dimensional mixed bacteria at a mixing speed of 10-20 r / min and a mixing time of 15-25 min. The sheep milk powder (calculated per kg of sheep milk powder with memory-improving properties) contains 0.5g-2.0g of acetylcholinesterase inhibitory peptide powder, 1g-5g of phytosterol esters, 0.5g-2.0g of phosphatidylserine, 0.5g-1.5g of N-acetylneuraminic acid, 8g-40g of prebiotics, and 0.3g-1.2g of probiotic powder. The prebiotics are one or more of inulin, isomaltooligosaccharide, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, stachyose, mannose, and chitosan oligosaccharide. The probiotics are one or more of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Lactobacillus fermentum, and Lactobacillus plantarum.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A sheep milk powder with memory-enhancing activity, characterized in that, The milk powder contains a memory-enhancing peptide; the memory-enhancing peptide is an acetylcholinesterase inhibitory peptide, and its amino acid sequence is shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

2. The sheep milk powder according to claim 1, characterized in that, The acetylcholinesterase inhibitory peptide has an acetylcholinesterase inhibition rate of 86.06 ± 0.04%.

3. The method for preparing sheep milk powder according to claim 1, characterized in that, The process includes enzymatic hydrolysis of sheep milk casein using a complex protease, ultrafiltration, gel chromatography separation and purification, and preparation of chromatographic separation and purification and freeze drying to obtain acetylcholinesterase inhibitory peptide powder. Then, the acetylcholinesterase inhibitory peptide powder, nutritional fortifier, prebiotics and probiotics are added to sheep milk powder, and after aseptic mixing, the sheep milk powder with improved memory peptide is obtained. The complex protease includes bromelain and flavor protease; the enzyme activity ratio of bromelain and flavor protease in the complex protease is 4:

1.

4. The preparation method according to claim 3, characterized in that, The enzymatic hydrolysis was performed at a pH of 6.5-7.0, with an enzyme dosage of 8000-10000 U / g; the sheep milk casein concentration was 4wt%-6wt%, the hydrolysis temperature was 50-55℃, and the hydrolysis time was 1-3 hours; the acetylcholinesterase inhibition rate of the sheep milk casein hydrolysate prepared by the enzymatic hydrolysis was 22.15%-33.98%. The ultrafiltration includes the step of collecting components with a molecular weight <3kDa and freeze-drying them to obtain crude acetylcholinesterase inhibitory peptide powder M1.

5. The preparation method according to claim 4, characterized in that, The gel chromatography was performed using Sephadex G-15 dextran gel. During purification, the concentration of the crude peptide powder M1 was 80 mg / mL, the loading volume was 1 mL, the eluent was pure water, the elution rate was 1 mL / min, and the fraction M12 from the second peak was collected.

6. The preparation method according to claim 3, characterized in that, The preparative chromatography was a preparative reversed-phase high-performance liquid chromatography. Mobile phase A was an aqueous solution of acetonitrile containing 0.1% TFA, and mobile phase B was ultrapure water containing 0.1% TFA. A linear gradient elution program was used: 0-45 min, with phase A increasing from 5% to 85%. The injection volume was 0.5 mL, and component M09 was collected and then freeze-dried to obtain the acetylcholinesterase inhibitor lyophilized peptide powder.

7. The preparation method according to claim 6, characterized in that, The amount of lyophilized acetylcholinesterase inhibitory peptide powder added is 0.50g-2.0g / kg sheep milk powder. The nutritional fortifiers include phytosterol esters, phosphatidylserine, and N-acetylneuraminic acid. The amount of phytosterol esters added is 1g-5g / kg sheep milk powder, the amount of phosphatidylserine added is 0.5g-2.0g / kg sheep milk powder, and the amount of N-acetylneuraminic acid added is 0.5g-1.5g / kg sheep milk powder. The amount of prebiotics added is 8g-40g / kg sheep milk powder. The probiotics include one or more of the following: inulin, isomaltooligosaccharide, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, stachyose, mannose, and chitosan oligosaccharide. The amount of probiotics added is 0.3g-1.2g / kg of sheep milk powder. The probiotics are one or more of the following: Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Lactobacillus fermentum, and Lactobacillus plantarum.

8. The preparation method according to claim 4, characterized in that, The aseptic mixing process employs a three-dimensional mixed culture, with a mixing speed of 10-20 r / min and a mixing time of 15-25 min.

9. The application of acetylcholinesterase inhibitory peptide in the preparation of food or pharmaceuticals with memory-improving activity, characterized in that, The amino acid sequences of the acetylcholinesterase inhibitory peptide are shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

10. A product that improves memory activity, characterized in that, The product includes an acetylcholinesterase inhibitory peptide; the amino acid sequence of the acetylcholinesterase inhibitory peptide is shown in SEQ ID NO.1~SEQ ID NO.45, PPQ and PLQ.

Images