Black garlic peptide and application thereof

Abstract

The present application relates to a kind of black garlic peptides and its application, the preparation method of the black garlic peptides is: black garlic is dried and ground, hot water extraction, alkali dissolving acid deposition, enzyme hydrolysis, nanofiltration concentration, decolorization and debittering and freeze-drying etc.Obtain black garlic peptide freeze-dried powder.The total polypeptide content of the black garlic peptide prepared in the present application is 70%~75%, the protein content is 80%~82%, and the peptide segment is mainly concentrated in the interval of 4~9 amino acid residues.The present application provides a kind of standardization preparation method of black garlic peptide, simple operation, controllability is strong, and the obtained polypeptide is high in purity and stable in quality;Black garlic peptides have a positive effect on the defecation behavior of constipated mice, small intestinal propulsion capacity and serum gastrointestinal hormone levels, and have application value in treating or relieving constipation, providing a technical basis for the high-value utilization of black garlic resources and the development of functional foods.It also lays a preliminary experimental basis for subsequent human clinical trials of functional foods or dietary supplements using black garlic peptides as raw materials.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a black garlic peptide and its applications. Background Technology

[0002] Constipation is currently one of the most common chronic digestive system diseases worldwide, with a particularly high incidence among urban populations and the elderly. Constipation not only affects the quality of daily life, but in severe cases can also lead to complications such as hemorrhoids, anal fissures, and intestinal obstruction, and even increase the risk of colorectal cancer. The most common causes of constipation include the following: 1) Dietary reasons: Dehydration-related constipation occurs because the body must absorb more water from its stool to conserve fluids. Stool with low water content is difficult to pass smoothly. Fruits, vegetables, grains, and other fiber-rich foods are natural cleansers for the digestive tract. Insufficient intake of these foods can lead to constipation. A lack of dietary fiber (the undigested portion of food) can cause constipation because fiber helps stool retain water and increase volume, making it easier to pass.

[0003] 2) Medications that slow intestinal motility: The most common medications that slow intestinal motility include opioids, iron salts, and medications with anticholinergic effects (such as many antihistamines and tricyclic antidepressants). Other medications include aluminum hydroxide (common in over-the-counter antacids), bismuth subsalicylate, certain antihypertensive drugs, and many sedatives.

[0004] 3) Causes of defecation disorders: Defecation disorders (difficulty defecating) refer to the rectum's inability to generate sufficient force to propel stool and / or the relaxation and difficulty of the external anal sphincter and surrounding rectal muscle fibers during defecation. Those with defecation difficulties feel the need to defecate but are unable to. Even soft stools are difficult to pass. Patients with irritable bowel syndrome (IBS) may experience IBS-related defecation disorders.

[0005] 4) Causes of IBS: Patients with irritable bowel syndrome (IBS) may experience loose stools, bowel irregularities, or constipation. If IBS is usually accompanied by constipation, it is called constipation-predominant IBS.

[0006] 5) Reasons for laxative abuse: Patients who frequently use laxatives and / or enemas often lose bowel motility without these aids. Constipation leads to more laxative use, which in turn leads to more constipation, creating a vicious cycle.

[0007] However, in many cases, the cause of constipation remains unclear. Traditional treatments for constipation, such as chemical laxatives, stimulant laxatives, and probiotics, can provide short-term relief, but long-term use can lead to side effects such as drug dependence and decreased intestinal sensitivity, and the efficacy is inconsistent in some patients. Therefore, developing naturally derived active ingredients with intestinal regulating functions has become an important direction for research in the prevention and treatment of constipation.

[0008] Black garlic is produced by fermenting fresh garlic under high temperature and humidity. During fermentation, black garlic peptides not only retain the original functional components such as allicin and S-allyl cysteine ​​(SAC), but are also rich in various small-molecule polypeptides. Studies have shown that black garlic peptides possess good antioxidant, anti-inflammatory, immunomodulatory, and gut microbiota-regulating biological activities. These properties are closely related to intestinal peristalsis, intestinal barrier function, and gut microbiota homeostasis. However, to date, there are no clear reports, either domestically or internationally, on the use of black garlic peptide powder to treat or improve constipation. In response to this situation, there is an urgent need to develop a natural intervention product or composition based on black garlic peptides, with controllable processing, suitable for people with constipation, to provide a new approach for the adjunctive treatment of constipation. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a black garlic peptide and its application, in order to solve the problems of insufficient long-term efficacy, easy drug dependence, other side effects, and unstable efficacy of traditional constipation treatments in existing technologies, as well as the lack of application of black garlic peptide powder in the preparation of drugs for treating or improving constipation in related technologies, and to provide an active ingredient that is naturally derived and has intestinal conditioning function.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention relates to a black garlic peptide, which is prepared from black garlic by the following method: 1) Dry grind the black garlic raw material, and then sieve it through a 100-200 mesh screen to obtain black garlic powder; 2) Mix the black garlic powder obtained in step 1) with pure water and perform hot water extraction for 30-60 minutes, then filter and collect the filtrate; 3) Adjust the pH of the filtrate obtained in step 2) to 10.0±0.1 by adding alkaline solution and stirring to dissolve; then add acid solution to adjust the pH to 4.6±0.1, let stand and then centrifuge to collect the protein precipitate; 4) The protein obtained in step 3) is washed and resuspended to obtain the substrate. Then, trypsin is added to the substrate for enzymatic hydrolysis. After enzymatic hydrolysis, nanofiltration, decolorization and debittering, concentration and freeze drying are performed to obtain the black garlic peptide.

[0011] Furthermore, the total polypeptide content of the black garlic peptide is 70%–75%, the protein content is 80%–82%, and the peptide segments are mainly concentrated in the range of 4–9 amino acid residues.

[0012] Preferably, the total polypeptide content of the black garlic peptide is 72%, the protein content is 81%, and the peptide segments are mainly concentrated in the range of 4 to 9 amino acid residues.

[0013] Furthermore, the black garlic peptide contains at least one peptide selected from VVYD, FVVY, LPGK, and YIVF.

[0014] Further, in step 2), the ratio of black garlic powder to pure water is 1:8~12 (g / mL), preferably 1:10 (g / mL).

[0015] Furthermore, in step 2), the processing temperature of the hot water extraction operation is 70-90℃, preferably 80℃.

[0016] Further, in step 2), the extraction time for hot water extraction is 20-40 min, preferably 30 min.

[0017] Further, in step 3), the alkaline solution is a 0.1 mol / L NaOH solution, and the acid solution is a 1 mol / L HCl solution.

[0018] Further, in step 3), the centrifugation conditions are 8000 rpm for 15 min.

[0019] Further, in step 4), the enzymatic hydrolysis temperature is 45-55°C, the amount of enzyme added is 5%-7% of the substrate mass, and the enzymatic hydrolysis time is 3-5 hours; preferably, the enzymatic hydrolysis temperature is 50°C, the amount of enzyme added is 6% of the substrate mass, and the enzymatic hydrolysis time is 5 hours.

[0020] Further, in step 4), the decolorization and debittering process conditions are: temperature 40-60℃, decolorizing agent dosage 1%-2%, time 40-50 minutes, preferably temperature 60℃, decolorizing agent dosage 1%, time 40 minutes.

[0021] Further, in step 4), the freeze-drying process conditions are: temperature -68 to -72°C, time 6 to 8 hours, preferably -70°C pre-freezing for 6 hours, then transferring to a vacuum freeze-drying system (pressure <10 Pa) for primary drying for 24 hours and secondary drying for 4 hours.

[0022] Furthermore, the prepared black garlic peptide has a water content of ≤7%, a light yellow or off-white appearance, and a loose texture with good fluidity.

[0023] The second aspect of the present invention is the use of the black garlic peptide described in the first aspect in the preparation of a medicine for improving constipation.

[0024] Furthermore, the drug is an oral preparation, and the drug is administered by gavage at a dose of 100–600 mg·kg⁻¹.

[0025] Furthermore, the improvement of constipation is achieved by promoting small intestinal propulsion, increasing fecal water content, and regulating serum motilin, gastrin, and nitric oxide levels.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: (1) This invention first establishes a multi-step extraction and hydrolysis process suitable for black garlic protein. The process includes key steps such as raw material pretreatment, hot water purification, protein enrichment, enzymatic hydrolysis, hydrolysis decolorization, and powder drying. Among them, hot water purification is used, and the optimal parameters are determined to be a material-to-liquid ratio of 1:10, a processing time of 30 minutes, and a temperature of 80℃. In the enzymatic hydrolysis stage, trypsin is selected as the hydrolytic enzyme, and the optimal conditions are determined by orthogonal experiments to be a temperature of 50℃, an enzyme addition of 6%, and an enzymatic hydrolysis time of 5 hours. The product is rich in small molecule peptides and has high bioavailability. Subsequently, by optimizing the decolorization process (temperature of 60℃, decolorizing agent of 1%, time of 40 minutes) and the freeze-drying process (-70℃, time of 6 hours), black garlic peptide powder with good sensory quality, uniform structure, and low moisture content is obtained.

[0027] (2) The black garlic peptide extracted in this invention exerts its laxative effect by regulating three factors in the blood: MTL, GAS, and NO. This is different from the mechanism of action of polysaccharides in existing black garlic water extracts, which rely on regulating intestinal flora to improve constipation. Further research on the bioactivity of the black garlic peptide in relieving constipation was conducted in an animal experiment using a mouse constipation model established by compound diphenoxylate. The results showed that medium and high doses of black garlic peptide can significantly improve the defecation behavior of constipated mice, increase the number of fecal particles and wet weight, increase the water content of feces, promote the small intestinal propulsion rate, and effectively regulate the serum levels of motilin (MTL), gastrin (GAS), and nitric oxide (NO). The preliminary findings reveal that its mechanism of action is to regulate the secretion of gastrointestinal hormones and improve intestinal motility.

[0028] In summary, this invention provides a standardized preparation method for black garlic peptides, which is simple to operate, highly controllable, and yields peptides with high purity and stable quality. Furthermore, it clarifies for the first time its application value in treating or relieving constipation, providing a technical foundation for the high-value utilization of black garlic resources and the development of functional foods. Attached Figure Description

[0029] Figure 1 This is a standard curve for determining the polypeptide content in black garlic peptide in one embodiment of the present invention; Figure 2 This is the result of a mouse small intestinal motility test in one embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0031] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0032] Example 1: Preparation and parameter optimization of black garlic peptides This embodiment relates to the preparation and characterization of the black garlic peptide of the present invention. First, the black garlic peptide was extracted from black garlic. Then, multiple experiments were conducted on the extraction process of the black garlic peptide, and the extraction parameters of the black garlic peptide were optimized, as follows: 1. Preparation of black garlic peptides This embodiment provides a process for preparing black garlic peptides from black garlic, specifically including the following steps: Step 1: Dry grinding and pulverizing the raw materials Twenty kg of pretreated black garlic samples were selected and dry-ground using a high-speed grinder. The grinder speed was set to 1500 rpm, and the initial grinding time was 1 minute. After grinding, the powder was sieved through a 100–200 mesh screen, and the powder with the correct particle size was collected. Particles that did not meet the size standard were re-grinded to ensure uniform particle size in the final powder. The entire dry grinding process was controlled within 2 minutes to prevent over-grinding, which could lead to heat accumulation and affect the stability of the active ingredients in the black garlic.

[0033] Step 2: Hot water extraction to remove impurities Black garlic powder was mixed with pure water and stirred for extraction, which promoted the dissolution of soluble impurities such as polysaccharides, inorganic salts, and bitter small molecules. After extraction, a plate and frame filter press at 0.3 MPa was used for solid-liquid separation to obtain a clear and transparent filtrate, removing most of the non-protein impurities.

[0034] Step 3: Alkali dissolution-acid precipitation extraction of protein Slowly add 0.1 mol / L NaOH solution to the obtained filtrate to adjust the pH to 10.0 ± 0.1, and stir at room temperature for 60 min to fully dissolve the protein in the black garlic. Then, slowly add 1 mol / L HCl solution to adjust the pH to 4.6 ± 0.1, causing the protein to aggregate and precipitate at its isoelectric point. After standing for 1 h, collect the protein precipitate by centrifugation at 8000 rpm for 15 min, and discard the supernatant.

[0035] Step 4: Protein washing and resuspending Centrifuge the protein precipitate at 3000 rpm for 10 min, discard the supernatant, add an appropriate amount of deionized water to the precipitate, stir thoroughly, and centrifuge again. Repeat this washing step three times to remove inorganic salts and residual small molecule impurities. Finally, resuspend the protein in PBS buffer at pH 7.0 and sonicate to form a homogeneous protein suspension.

[0036] Step 5: Ultrasound-assisted enzymatic hydrolysis The protein suspension was pretreated with ultrasound at 50 °C with a power of 150 W using an intermittent 5-second on / off cycle for 30 min to loosen the protein structure and enhance enzymatic hydrolysis efficiency. Subsequently, protease was added to the system for enzymatic hydrolysis, and the degree of deamination (DH) was monitored and controlled between 8% and 20%. Once the target degree of hydrolysis was achieved, the enzyme activity was terminated by heating to 80 °C and holding for 10 min.

[0037] Step Six: Nanofiltration Concentration The enzymatic hydrolysate was concentrated using a nanofiltration membrane with a molecular weight cutoff of 10 kDa. The operating pressure was 0.2 MPa, and the throughput was consistently above 20 L / (m²·h). Nanofiltration effectively removed free amino acids, small molecule impurities, and inorganic salts, achieving preliminary enrichment of the target peptides.

[0038] Step 7: Decolorization and debittering with activated carbon Activated carbon is added to the nanofiltration concentrate for reaction, and then the activated carbon is removed by high-speed centrifugation. This step effectively removes melanin, Maillard reaction products, and bitter small molecules, significantly improving the color and flavor of the black garlic peptide product.

[0039] Step 8: Fine-grained grading and concentration Nanofiltration membranes with a molecular weight cutoff of 1–5 kDa were used for further concentration and fractionation to retain the target functional peptides while removing large, unhydrolyzed proteins and very small impurities. The peptide concentration in the system was controlled to reach 15%–20% (w / v) to provide favorable physical conditions for subsequent drying.

[0040] Step 9: Freeze-drying The concentrate was pre-frozen at -70 ℃ for 6 h and then transferred to a vacuum freeze-drying system (pressure <10 Pa) for primary drying for 24 h and secondary drying for 4 h. The resulting black garlic peptide powder had a water content of ≤7%, was light yellow or off-white, had a loose texture and good flowability, and was suitable for storage and product development.

[0041] 2. Orthogonal Experiment Test 1) Hot water extraction for impurity removal experiment Hot water extraction, as a crucial preliminary step in the preparation of water-soluble components from black garlic, significantly impacts the efficiency of subsequent protein extraction and peptide preparation. This experiment used the mass of the water-soluble components as the evaluation index and systematically investigated the effects of three factors on the extraction efficiency: water volume (i.e., the ratio of pure water volume (mL) to the mass of black garlic powder (g) – the following ratios were set: 5, 10, and 15 times), processing time (30 and 60 min), and temperature (60℃, 80℃, and 100℃). The experimental results are shown in Table 1.

[0042] Table 1. Experimental parameters for hot water extraction Temperature has a significant effect on improving the yield of water-soluble substances. As the temperature increases from 60℃ to 80℃, the mass of water-soluble substances gradually increases. However, when the temperature is further increased to 100℃, the increase is limited, and some combinations even show a slight decrease. This indicates that excessively high temperatures may lead to the degradation of some soluble components or affect diffusion efficiency.

[0043] Increasing the amount of water used also helps to improve the yield of water-soluble substances. The extraction yield is highest when the water content is increased by 10 times. Beyond 10 times, the improvement is not obvious or even slightly decreases. It is speculated that excessive dilution may actually affect the extraction efficiency.

[0044] Extending the processing time from 30 min to 60 min had limited effect on improving the yield of water-soluble substances; under most conditions, 30 min was sufficient to extract the main soluble components.

[0045] Comprehensive analysis shows that temperature and water volume are the main factors affecting extraction efficiency, followed by processing time. Under the conditions of this experiment, the combination of 10 times the amount of water, 80℃, and 30 min yielded the highest water-soluble content (3.27 g), and the resulting filtrate was clear and uniform in color, suitable for subsequent processing. Therefore, these conditions are recommended as the optimal process parameters for hot water extraction of black garlic.

[0046] 2) Ultrasound-assisted enzymatic hydrolysis experiment The role of proteases is to hydrolyze peptide bonds in proteins, breaking down large, long-chain proteins into peptides and amino acids of varying lengths. Because different proteases have different enzymatic sites, the same protein, under different enzymes and enzymatic parameters, can produce different polypeptide fragments, resulting in polypeptides with different functional activities. This experiment used different proteases to perform ultrasound-assisted enzymatic hydrolysis of black garlic protein. The proteases used and the experimental results are shown in Table 2.

[0047] Table 2: Information on proteases in ultrasound-assisted enzymatic digestion experiments The trypsin group yielded the highest peptide production, reaching 568.97 mg, significantly higher than other enzyme groups. The peptide production when chymotrypsin was used in combination with trypsin was 437.29 mg, also significantly better than using chymotrypsin alone (255.14 mg) or neutral protease alone (254.68 mg). The papain and bromelain groups had relatively lower peptide production, at 194.68 mg and 171.05 mg, respectively. Besides the differences in yield, the trypsin group showed higher clarity in the reaction solution, with almost no precipitation and stable solution properties; the chymotrypsin and neutral protease groups also exhibited some clarity; while the papain and bromelain groups showed some degree of turbidity, indicating a greater tendency for protein fragment aggregation. Considering both peptide yield and solution stability, trypsin not only has high hydrolysis efficiency but also produces products with excellent properties, making it suitable for the large-scale preparation of black garlic peptides.

[0048] Next, trypsin was used to perform enzymatic hydrolysis under different enzymatic parameters. The specific experimental results are shown in Table 3.

[0049] Table 3: Enzyme to substrate mass ratio information during trypsin hydrolysis Different enzymatic hydrolysis parameters had some impact on the yield of black garlic peptides, but the overall differences were not significant. The peptide content fluctuated between groups from 538.08 mg to 572.12 mg. Comprehensive analysis showed that the influence of each factor on peptide yield was as follows: the enzyme-to-substrate mass ratio and hydrolysis time had a greater impact, while temperature had a relatively smaller impact. Among all experimental groups, group 5 (temperature 50℃, enzyme-to-substrate mass ratio 6%, hydrolysis time 5 h) had the highest peptide yield, reaching 572.12 mg. Other groups with relatively high yields included group 7 (55℃, 5%, 5 h, 551.33 mg) and group 4 (50℃, 5%, 3 h, 550.37 mg). Considering both peptide yield and the economics of the process, the optimal enzymatic hydrolysis parameters were determined to be: temperature 50℃, enzyme-to-substrate mass ratio 6%, and hydrolysis time 5 h.

[0050] 3) Decolorization and debittering experiment The experimental parameters of different decolorization and debittering experiments obviously have an impact on the color and flavor of black garlic peptide products. Next, decolorization and debittering were carried out under different parameters, and the specific experimental results are shown in Table 4.

[0051] Table 4: Experimental parameters for different decolorization and debittering experiments Scoring Instructions: "++" indicates insufficient decolorization, resulting in a dark yellow color; "++" indicates good decolorization, resulting in a light yellow color; "+" indicates sufficient decolorization, resulting in a white color. The decolorization process parameters significantly affected the appearance color of the black garlic polypeptide solution. Based on the combined results of each group of experiments, temperature, decolorizing agent dosage, and decolorization time all had a certain effect on the decolorization effect. The appearance color scoring showed that groups with scores of "+++" included Group 1 (40 ℃, 1%, 20 min), Group 4 (50 ℃, 1%, 20 min), and Group 8 (60 ℃, 2%, 20 min), indicating that the decolorization effect was poor under conditions of lower or moderate temperatures, lower decolorizing agent dosage, and shorter decolorization times. With increasing decolorizing agent dosage and time, some groups (such as Groups 2, 3, 5, 6, and 9) showed good decolorization effects and scored "++", while Group 7 (60 ℃, 1%, 40 min) scored "+", showing the best decolorization effect.

[0052] Under these conditions, the concentration of black allium peptide, as determined by the BCA method, was 559.40 mg / mL, slightly lower than the pre-decolorization concentration of 572.12 mg / mL, with a black allium peptide transfer rate of 97.6%. Therefore, although some peptide loss occurs during the decolorization and debittering process, the overall loss is minimal, and the decolorization effect is significant, effectively improving the appearance quality and added value of the black allium peptide solution. In conclusion, the optimal decolorization process parameters are: temperature 60 ℃, decolorizing agent dosage 1%, and decolorization time 40 min.

[0053] Example 2: Characterization of black garlic peptides This embodiment relates to the characterization of the black garlic peptide prepared by the present invention, including the analysis of peptide chains in the black garlic peptide prepared in Example 1 using LC-MS / MS, and the determination of total protein content and polypeptide content, as detailed below: This embodiment uses the same method for preparing black garlic peptide as in Example 1, and is prepared under the optimal parameters optimized in Example 1, namely: 1) When using hot water method for impurity removal, the optimal parameters are determined to be a material-to-liquid ratio of 1:10, a processing time of 30 minutes, and a temperature of 80°C; 2) In the enzymatic hydrolysis stage, trypsin is selected as the hydrolytic enzyme, with an enzymatic hydrolysis temperature of 50°C, an enzyme addition amount of 6%, and an enzymatic hydrolysis time of 6 hours; 3) The decolorization temperature is 60°C, the decolorizing agent is 1%, and the time is 40 minutes.

[0054] 1. LC-MS / MS analysis The prepared black garlic peptides were analyzed by LC-MS / MS, and after optimization, a total of 482 peptide segments were detected, mainly concentrated in the 4-9 amino acid residue range, of which 346 were tetrapeptides, accounting for 71.8%. Representative short peptides such as VVYD, FVVY, LPGK, and YIVF have strong physiological activity potential, and relevant parameters are detailed in Table 5.

[0055] Table 5: LC-MS / MS analysis results of black garlic peptides 2. Determination of total protein content: The protein content of the prepared black garlic peptides was determined using the Kjeldahl method. The specific operating steps are as follows: 1) Sample Digestion: Accurately weigh approximately 0.2 g (accurate to 0.0001 g) of the black garlic tetrapeptide sample, and record it as m1. Transfer the sample to a Kjeldahl flask, and add 10 g K2SO4 + 0.5 g CuSO4 as a catalyst. Then add 20 mL of concentrated H2SO4 and gently shake to mix. Place the Kjeldahl flask on a digestion furnace and heat over a low flame. After the contents are completely carbonized and foaming stops, increase the heat to maintain a gentle boil until the digestion liquid turns a clear, transparent blue-green color. Continue digestion for another 30-60 minutes to ensure that the nitrogen in the sample is completely converted to ammonium sulfate. Simultaneously, perform a reagent blank test using the same amount of copper sulfate, potassium sulfate, and concentrated sulfuric acid as the treated sample, using the same method.

[0056] 2) Distillation and absorption: After cooling, transfer the digest to a distillation apparatus and rinse the flask with a small amount of water. Add 50 mL of 40% NaOH solution (to make the solution strongly alkaline and release NH3). Pass the distilled ammonia gas into 20 mL of 2% boric acid solution for absorption, and add 2 drops of mixed indicator (the solution turns pink).

[0057] 3) Titration: Titrate with 0.1 mol / L HCl standard solution until the solution changes from blue-green to pale purple. Record the volume of HCl consumed, V1 (mL). Simultaneously perform a blank experiment and record the blank volume, V2.

[0058] Calculate protein content using the following formula: Protein content (%) = 100% × (V1 - V2) × CHCl × 0.01401 × 6.25 / ml; The experiment measured the volume of 0.1 mol / L HCl consumed by the sample. Based on the sample mass and the conversion formula, the protein content was calculated to be 81%.

[0059] 3. Determination of polypeptide content The total polypeptide content was determined by the biuret colorimetric method. BSA standard solutions (0.2–2.0 mg / mL) were prepared. 1 mL of each standard was added to 4 mL of biuret reagent, mixed well, and reacted at room temperature for 30 min. The absorbance (A) was measured at 540 nm, and a standard curve was plotted. Figure 1 As shown.

[0060] The sample processing procedure is as follows: Weigh 10 mg of black garlic peptide powder and dissolve it in 10 mL of water. Centrifuge and take the supernatant. Take 1 mL of the supernatant and participate in the above reaction. Read the absorbance value and substitute it into the standard curve for calculation. The final result is converted into the total peptide percentage content according to the formula.

[0061] After the experimental samples were dissolved in water and centrifuged, the supernatant was taken and its absorbance was measured using the same method. The absorbance was then substituted into the regression equation above to calculate the peptide concentration. The final conversion results showed that the total peptide content in the black garlic peptide powder sample was 72%.

[0062] In summary, LC-MS analysis showed that the product contained 70% to 80% 4-9 peptides, and the total protein content was over 70%. These two sets of data are sufficient to prove that polypeptides are the main component of the extracted product. Therefore, it can be inferred that the active substance that relieves constipation is polypeptide.

[0063] Example 3: The effect of black garlic peptides on improving intestinal function in constipation model mice This embodiment involves the performance verification of the black garlic peptide prepared in Example 1. A mouse constipation model was established using compound diphenoxylate. The effects of black garlic peptide on defecation behavior, small intestinal propulsion function, and serum levels of motilin (MTL), gastrin (GAS), and nitric oxide (NO) were comprehensively evaluated. The mechanism of its action in relieving constipation was preliminarily explored, as follows: 1. Establishment and grouping of a mouse constipation model The animals used in the experiment were SPF-grade Kunming mice, half male and half female, weighing 18-22 g, provided by the Laboratory Animal Center of the Naval Special Medical Center. All mice were acclimatized for 7 days in a clean-grade animal facility before the experiment, with the ambient temperature controlled at 22±2℃, relative humidity at 50%-60%, and 12-hour alternating day and night light. During the experiment, the mice had free access to standard pelleted feed and purified water. All animal experimental procedures followed the animal ethics guidelines of the Naval Special Medical Center.

[0064] In this experiment, mice were randomly divided into two batches for small intestinal propulsion tests and intestinal function improvement indicators. Each batch consisted of six treatment groups: blank control group, model group (diphenoxylate 10 mg / kg), positive control group (diphenoxylate 10 mg / kg + mosapride suspension 1.875 mg / kg), low-dose black garlic peptide group (diphenoxylate 10 mg / kg + 100 mg / kg), medium-dose black garlic peptide group (diphenoxylate 10 mg / kg + 300 mg / kg), and high-dose black garlic peptide group (diphenoxylate 10 mg / kg + 600 mg / kg). Ten mice were in each group.

[0065] All mice were administered the drug via gavage, with a gavage volume of 0.2 ml, for 7 consecutive days. The model group, positive control group, and different doses of black garlic peptide groups were administered compound diphenoxylate (10 mg / kg, i.e., 0.2 ml / 10g) via gavage, while the blank control group received the same dose of distilled water to establish a constipation model. After the last gavage on day 7, all mice were fasted for 16 consecutive hours but allowed free access to water.

[0066] Thirty minutes after the last gavage on day 7, followed by a 16-hour fast, the positive control group and different doses of black garlic peptide groups were administered ink containing 10 mg / kg of compound diphenoxylate (0.2 ml / 10 g). The blank control and model groups were administered the same dose (0.2 ml / 10 g) of ink. Mice were allowed normal food and water intake after gavage. From the start of ink administration, the water intake, body weight, and survival status of each mouse were observed and recorded. The time of the first black feces excretion was recorded, and all black feces were collected within 6 hours. The number of feces and total mass were counted. The collected black feces were immediately weighed (wet weight, M1), then dried in a 105℃ oven for 5 hours. After drying, they were weighed again (dry weight, M2). The fecal moisture content was calculated using the formula (M1-M2) / M1×100%. Fecal characteristics, including color, shape, and moisture, were also observed visually.

[0067] Mice used in the small intestine propulsion experiment were fasted for 16 hours after the last gavage on day 7, but water was allowed. 30 minutes later, all groups of mice were administered ink (formula as before, 0.2 ml / 10 g) by gavage. 25 minutes after gavage, the mice were euthanized by cervical dislocation, and the abdominal cavity was immediately opened. The small intestine from the pylorus to the cecum was completely removed and placed in ice-cold physiological saline. Surface moisture was gently absorbed with filter paper, and the small intestine was laid flat and straightened. The total length of the small intestine and the length from the pylorus to the ink tip were measured with a ruler. The small intestine propulsion rate was calculated using the formula "ink propulsion length / total small intestine length × 100%". Serum MTL, GAS, and NO levels were detected using enzyme-linked immunosorbent assay (ELISA).

[0068] All experimental data are expressed as mean ± standard deviation (x ± s). SPSS 22.0 statistical software was used for analysis, and Prism 9.0 was used for statistical graph generation. One-way ANOVA was used for comparisons between groups, and the LSD method was used for post-experimental comparisons between two groups. A p-value < 0.05 was considered statistically significant. Any abnormalities discovered during the experiment were promptly recorded and investigated to ensure the scientific validity and reliability of the data.

[0069] 2. General observation Before modeling, mice in all groups were in good mental condition, responsive, agile, and had normal food and water intake. Their fur was glossy, and the frequency and consistency of their feces were normal. After modeling with compound diphenoxylate, compared with the control group, the feces of mice in the model group changed from continuous, soft, yellow, elongated strips to dry, hard, brown granular pieces, and the amount of feces decreased. Their mental state and activity level were significantly worse than before, and the luster of their fur decreased. Their food intake generally showed a downward trend. Compared with the model group, the fecal hardening of mice in each treatment group improved, the amount of feces increased to varying degrees, and their mental state gradually improved.

[0070] 3. Effects of black garlic peptides on defecation in constipated mice The defecation patterns of mice in each group are shown in Table 6. Compared with the control group, the time to the first black stool in the model group was significantly prolonged (P<0.001), and the number of stool particles, stool mass, and fecal water content within 6 hours were significantly reduced (P<0.001), indicating that the diarrhea model was successfully established. Medium dose of black garlic peptide (300 mg·kg⁻¹) - ¹), High dose (600 mg·kg) - ¹) The group showed significant improvements compared to the model group in terms of time to first black stool, number of stool particles in 6 hours, stool quality, and stool moisture content (P<0.05, 0.01, 0.001, respectively); while the low-dose black garlic peptide (100 mg·kg) showed significant improvements compared to the model group. - Although the indicators in group ¹) showed some improvement compared to the model group, the difference was not statistically significant. These results indicate that medium- and high-dose black garlic peptides have a significant effect on improving intestinal motility and fecal characteristics in mice.

[0071] Table 6: Effects of black garlic products on mouse defecation ( (X±S, n = 10) 4. Effects of black garlic peptides on small intestinal motility tests.

[0072] Figure 1The study demonstrated the effect of black allium peptide on the small intestinal propulsion rate in mice. Compared with the blank control group, the small intestinal propulsion rate in the model group was significantly decreased (P<0.001). The positive control mosapride group effectively improved and restored the small intestinal propulsion rate (P<0.001); although the low-dose and medium-dose black allium peptide groups showed an upward trend compared with the model group, the differences were not significant; the high-dose black allium peptide group (P<0.05) significantly improved the small intestinal propulsion rate. These results indicate that high-dose black allium peptide can effectively restore small intestinal motility in mice.

[0073] 5. Effects of black garlic peptides on serum MTL, GAS, and NO levels in mice Further experimental results (as shown in Table 7) showed that the levels of MTL and GAS in the serum of mice in the model group decreased, while the level of NO increased; after administration of black garlic peptide, the levels of MTL and GAS in the serum of mice increased (P<0.001, P<0.05), while the level of NO decreased (P<0.01).

[0074] Table 7: Serum MTL, GAS and NO expression in each group ( X±S, n = 7) This study systematically evaluated the effects of different doses of black garlic peptide on improving intestinal function in a mouse constipation model induced by diphenoxylate. Three dosing gradients (low, medium, and high) were used in the animal defecation experiment. The results showed that black garlic peptide had a positive effect on defecation behavior, small intestinal propulsion, and serum gastrointestinal hormone levels in constipated mice, exhibiting a certain dose-dependent effect. In particular, medium and high doses of black garlic peptide (300 and 600 mg / kg) showed significant effects in shortening the defecation latency period, increasing the frequency and weight of defecation, and increasing fecal water content (P<0.05); it also significantly improved small intestinal propulsion rate, suggesting its good ability to promote intestinal peristalsis. Mechanistic studies on serum MTL, GAS, and NO were conducted only using the high-dose black garlic peptide group. The results showed that the effects of black garlic peptide on intestinal proliferating factors such as MTL, GAS, and NO provide preliminary evidence for its mechanism of regulating intestinal function.

[0075] In summary, black garlic peptides demonstrate good anti-constipation potential and can comprehensively improve defecation behavior and intestinal motility. This provides experimental evidence for the functional development of black garlic in the field of intestinal health and lays the foundation for subsequent human clinical trials of functional foods or dietary supplements made from black garlic peptides.

[0076] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A black garlic peptide, characterized in that, The black garlic peptide is prepared from black garlic using the following method: 1) Dry grind the black garlic raw material, and then sieve it through a 100-200 mesh screen to obtain black garlic powder; 2) Mix the black garlic powder obtained in step 1) with pure water and perform hot water extraction for 30-60 minutes, then filter and collect the filtrate; 3) Adjust the pH of the filtrate obtained in step 2) to 10.0±0.1 by adding alkaline solution and stirring to dissolve; then add acid solution to adjust the pH to 4.6±0.1, let stand and then centrifuge to collect the protein precipitate; 4) The protein obtained in step 3) is washed and resuspended to obtain the substrate. Then, trypsin is added to the substrate for enzymatic hydrolysis. After enzymatic hydrolysis, nanofiltration, decolorization and debittering, concentration and freeze drying are performed to obtain the black garlic peptide.

2. The black garlic peptide according to claim 1, characterized in that, The total polypeptide content of the black garlic peptide is 70%–75%, the protein content is 80%–82%, and the peptide segments are mainly concentrated in the range of 4–9 amino acid residues.

3. The black garlic peptide according to claim 2, characterized in that, The black garlic peptide contains at least one peptide selected from VVYD, FVVY, LPGK, and YIVF.

4. The black garlic peptide according to claim 1, characterized in that, In step 2), the ratio of black garlic powder to pure water is 1:8~12 (g / mL), preferably 1:10 (g / mL). And / or, the processing temperature of the hot water extraction operation is 70-90℃, preferably 80℃; the extraction time is 20-40 min, preferably 30 min.

5. The black garlic peptide according to claim 1, characterized in that, In step 3), the alkaline solution is a 0.1 mol / L NaOH solution, and the acid solution is a 1 mol / L HCl solution; And / or, the centrifugation conditions are 8000 rpm for 15 min.

6. The black garlic peptide according to claim 1, characterized in that, In step 4), the enzymatic hydrolysis temperature is 45-55°C, the amount of enzyme added is 5%-7% of the substrate mass, and the enzymatic hydrolysis time is 3-5 hours; preferably, the enzymatic hydrolysis temperature is 50°C, the amount of enzyme added is 6% of the substrate mass, and the enzymatic hydrolysis time is 5 hours. And / or, the decolorization and debittering process conditions are: temperature 40-60℃, decolorizing agent dosage 1%-2%, time 40-50 minutes, preferably temperature 60℃, decolorizing agent dosage 1%, time 40 minutes. And / or, the freeze-drying process conditions are: freeze-drying temperature of -68 to -72°C, and time of 6 to 8 hours.

7. The black garlic peptide according to claim 1, characterized in that, The prepared black garlic peptide has a water content of ≤7%, a light yellow or off-white appearance, and a loose texture with good fluidity.

8. The use of the black garlic peptide according to any one of claims 1 to 7 in the preparation of a medicine for improving constipation.

9. The application according to claim 8, characterized in that, The drug is an oral preparation, and the dosage is 100–600 mg / kg. -1 The dosage was administered via gavage.

10. The application according to claim 8, characterized in that, The improvement in constipation is achieved by promoting small intestinal propulsion, increasing fecal water content, and regulating serum motilin, gastrin, and nitric oxide levels.

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