A multifunctional yam composite peptide composition, preparation method and application in strengthening the heart

Abstract

The present invention belongs to the field of functional food technology and discloses a multifunctional Chinese yam composite peptide composition, a preparation method, and its application in cardiotonic treatment. The preparation method of the multifunctional Chinese yam composite peptide composition comprises the following steps: preparing pea protein powder; preparing pea peptide powder A; and preparing the multifunctional Chinese yam composite peptide composition. The present invention wet-grafts highly active pea peptide powder with Chinese yam powder. The polysaccharides in the Chinese yam effectively neutralize the bitterness of the pea peptides and increase their hydrolysis rate to 94.32% at pH 2.0-3.0. Simultaneously, the composition significantly enhances its antioxidant capacity and gastric mucosal protection ability, but also increases its hygroscopicity, with a water content of 8.79%.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides and functional foods, in particular to a multifunctional yam composite peptide composition, a preparation method and an application thereof in strengthening the heart. Background Art

[0002] The spleen governs transportation and transformation, serving as the central link in energy metabolism throughout the body. Its physiological structure is the hub for the transport of essence, water, and moisture, responsible for the body's entire food metabolism. Its anatomical structure is represented by the liver, the largest digestive organ in modern medicine. Its substance exchange system is responsible for digestion, absorption, and excretion, encompassing the digestive system and metabolism. Spleen deficiency and dampness often coexist, acting as a causal link. Spleen deficiency, Qi deficiency, and excess dampness are all common physical conditions or health issues in Traditional Chinese Medicine.

[0003] Chinese yam has a sweet and mild nature and enters the spleen, lung, and kidney meridians. It has the benefits of invigorating qi and nourishing yin, tonifying the spleen, lungs, and kidneys, consolidating essence, and stopping leukorrhea. It can be used for spleen and stomach deficiency. Its balanced qi and yin-tonifying properties and astringent properties make it suitable for spleen deficiency, poor appetite, fatigue, loose stools, leukorrhea in women, and indigestion in children. Clinically, it is commonly used for spleen deficiency, poor appetite, chronic diarrhea, wheezing and coughing due to lung deficiency, spermatorrhea due to kidney deficiency, leukorrhea, frequent urination, deficiency-heat and thirst, and neurasthenia.

[0004] Pea peptide is a protein hydrolyzate obtained by the action of protease on pea protein and then undergoing special treatment. As a new type of pea deep-processing product and nutritional supplement with higher quality than pea protein, it has shown attractive development and application prospects in the fields of food, medicine, daily chemical industry, etc.

[0005] Because the rich fiber content in yam is difficult to fully digest and absorb in a short period of time, excessive consumption of yam may cause bloating or worsen constipation in those with weak digestive systems or those prone to constipation. Pea peptides, small molecule peptides extracted from pea protein through enzymatic hydrolysis, are not only easily digested and absorbed but also have higher bioavailability, entering the bloodstream more quickly and providing nutrients to the body quickly, making them particularly suitable for post-exercise recovery. They also have additional health benefits such as antioxidant, anti-inflammatory, and gut microbiome regulation. Compared to traditional proteins, pea peptides place less strain on the digestive system during absorption, making them suitable for those with weak gastrointestinal function. Therefore, wet-grafting pea peptides onto Chinese yam to produce a polymer is being considered to improve digestion and absorption issues experienced by patients with spleen deficiency. This treatment also significantly enhances its antioxidant and gastric mucosal protective properties. However, this treatment increases the polymer's hygroscopicity and the risk of allergic reactions to acetylcholine in Chinese yam.

[0006] The neurotransmitter acetylcholine in Chinese yam is responsible for allergic reactions, causing symptoms such as autonomic dysfunction and vasodilation, as well as symptoms such as generalized itching, decreased heart rate, decreased blood pressure, palpitations, flushing, sweating, nausea, vomiting, abdominal pain, diarrhea, bronchoconstriction, and bronchial asthma. Existing research indicates that Lactobacillus johnsonii can prevent allergic reactions, regulate allergic constitutions, and reduce bacterial and viral infections. Furthermore, Lactobacillus johnsonii stabilizes intestinal flora and stimulates mucosal immune responses, effectively treating acute diarrhea, abdominal distension, and irritable bowel syndrome. Therefore, the innovative use of Lactobacillus johnsonii IOB 801 to ferment the grafted copolymerization product of Chinese yam and pea peptides can reduce the risk of Chinese yam allergy in patients and investigate the effects of the fermented Chinese yam peptide complex on the five internal organs in patients with spleen deficiency. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a multifunctional Dioscorea opposita composite peptide composition, a preparation method and an application thereof in strengthening the heart.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A method for preparing a multifunctional yam composite peptide composition comprises the following steps:

[0010] Preparation of pea protein powder;

[0011] Preparation of pea peptide powder A;

[0012] Preparation of multifunctional yam composite peptide composition:

[0013] Add pea peptide powder A and Chinese yam powder at a mass ratio of 3:5 to an appropriate amount of 3 mol / L phosphate buffer solution, mix thoroughly, and adjust the pH of the mixture to 7.0 ± 0.2 using 20% ​​edible sodium hydroxide solution or hydrochloric acid solution;

[0014] The mixture was placed in a hot water bath at 87±2°C to react while magnetically stirring at 35±5 r / min for 3±0.5 h.

[0015] After the reaction is complete, the mixture is quickly placed in ice water to cool down to stop the reaction. The cooled mixture is inoculated with Lactobacillus johnsonii IOB 801 seed liquid at a rate of 1%, cultured at 37°C, with the pH maintained at 6.0-6.5, at a rotation speed of 45±5r / min, and cultured for 8±2h. After the fermentation liquid is concentrated, it is spray-dried to obtain a multifunctional Chinese yam composite peptide composition.

[0016] Furthermore, the preparation method of pea protein powder is specifically as follows:

[0017] Pea pretreatment: Wash, screen and peel fresh peas, soak them in water for 12 hours with a solid-liquid ratio of 1:30. After soaking, grind them into a homogenate.

[0018] Initial separation: sieve the homogenized slurry to separate the slurry residue and keep the slurry for later use;

[0019] Alkaline extraction: add edible sodium hydroxide solution with a mass concentration of 20% to the slurry, adjust the pH to 8.0, the extraction temperature is 35°C, the extraction time is 40 minutes, after the extraction is completed, centrifuge at 4000 rpm for 30 minutes, and collect the supernatant;

[0020] Acid precipitation separation: The supernatant was concentrated, cooled, and then a 20% hydrochloric acid solution was added to adjust the pH of the concentrate to 4.0. The concentrate was precipitated for 30 minutes and centrifuged at 4000 rpm for 30 minutes to obtain pea protein precipitate.

[0021] Spray drying: Add edible sodium hydroxide solution with a mass concentration of 20%, adjust the pH to 7.0, neutralize the pea protein precipitate, and spray dry to obtain pea protein powder.

[0022] Furthermore, the preparation method of pea peptide powder A is specifically as follows:

[0023] Prepare pea protein liquid: add pea protein powder to pure water at a mass ratio of pea protein powder to pure water of 1:10, and stir for 30 minutes to obtain pea protein liquid;

[0024] Preparation of compound protease: neutral protease, alkaline protease and flavor protease were compounded in a mass ratio of 1.0:0.2:0.8 to prepare compound protease system A;

[0025] Hydrolysis of compound protease system A: using pea protein liquid as substrate, add compound protease system A with a final mass concentration of 1.0%~1.5% and stir, perform enzymatic hydrolysis at 52°C for 5-6 hours, and after the enzymatic hydrolysis is completed, inactivate the enzyme at 90°C to obtain pea peptide hydrolyzate;

[0026] Membrane filtration: The obtained pea peptide hydrolysate was centrifuged at 6000 r / min for 8 min, the supernatant was collected, and filtered through a membrane to obtain the pea peptide collection solution;

[0027] Freeze drying: The pea peptide collection liquid is pre-frozen and then vacuum freeze-dried to obtain pea peptide powder A.

[0028] Furthermore, the Lactobacillus johnsonii 10B 801 is a strain of Lactobacillus johnsonii screened from fermented kimchi.

[0029] Furthermore, the Lactobacillus johnsonii 10B 801 is named: Lactobacillus johnsonii 10B 801, the classification name is: Lactobacillus johnsonii, the preservation number is: CGMCC No.16824, the preservation date is: November 26, 2018, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0030] Furthermore, the preparation method of the Lactobacillus johnsonii 10B 801 seed liquid is as follows:

[0031] Strain activation: Activate the frozen Lactobacillus johnsonii IOB 801 three times on MRS agar plates, pick a single colony and inoculate it into MRS liquid medium, culture it at 37°C for 10±2h, then transfer it to MRS liquid medium at a 1% inoculum volume and culture it at 37°C for 10±2h as seed liquid.

[0032] The multifunctional Dioscorea opposita composite peptide composition is prepared by the above-mentioned preparation method.

[0033] The multifunctional Dioscorea opposita composite peptide composition is used in the preparation of cardiotonic drugs.

[0034] The use of the multifunctional Dioscorea opposita composite peptide composition as described above in the preparation of a drug for restoring cardiac function of spleen deficiency and dampness excess type.

[0035] Furthermore, the multifunctional Chinese yam composite peptide composition can significantly improve the weight loss of rats with spleen deficiency and dampness, i.e., the recovery rate is increased by 60%, loose stools, i.e., the stool moisture content is reduced from 65.54% to 37.40%, and abnormal organ indexes, i.e., the spleen index is restored from 3.23 to 2.64.

[0036] The advantages and positive effects achieved by the present invention are:

[0037] 1. This invention develops a highly efficient enzymatic hydrolysis system by combining neutral protease, alkaline protease, and flavor protease. The three enzymes work synergistically to significantly improve the enzymatic hydrolysis efficiency of pea protein, achieving a peptide content of ≥81.72% while reducing the production of bitter peptides. This overcomes the low peptide yield and poor taste of traditional single-enzyme processes.

[0038] 2. The present invention wet-grafts and copolymerizes highly active pea peptide powder with Chinese yam powder. The polysaccharide in Chinese yam can effectively neutralize the bitterness of pea peptide and increase its hydrolysis rate to 94.32% under pH 2.0-3.0 conditions. At the same time, its antioxidant capacity and gastric mucosal protection ability are significantly improved, but its hygroscopicity increases, and the water content is 8.79%.

[0039] 3. The present invention has shown through experiments that the Dioscorea opposita composite peptide grafted with Lactobacillus johnsonii 10B 801 is fermented to obtain a Dioscorea opposita composite peptide composition, which significantly reduces the allergenicity of Dioscorea opposita, and at the same time reduces the water content to 5.31%, reducing the hygroscopicity; wherein the saponins, allantoin and other ingredients act synergistically with the small molecule active peptides of pea peptides to target and regulate metabolic disorders, inflammatory responses and multiple organ functions related to spleen deficiency, achieving the comprehensive therapeutic effect of "tonifying the spleen and removing dampness, and strengthening the five internal organs", among which the effect on strengthening cardiac function is the best.

[0040] A Chinese yam peptide combination significantly improved weight loss (recovery rate increased by 60%), loose stools (stool moisture decreased from 65.54% to 37.40%), and abnormal organ indices (spleen index restored from 3.23 to 2.64) in rats with spleen deficiency and dampness. It also prevented and alleviated cardiovascular and cerebrovascular diseases. Its multi-system synergistic intervention mechanism was validated by regulating serum inflammatory factors and endocrine metabolic indicators. This approach fills a gap in the existing technology for integrating functional peptide compounding with traditional Chinese medicine theory, providing a new strategy for nutritional intervention in spleen deficiency-related diseases.

[0041] 4. The present invention uses a combination of multiple proteases in different ratios to hydrolyze pea protein, significantly increasing the peptide content of pea peptides. Then, through wet graft copolymerization of pea peptides with Chinese yam powder, the taste of pea peptides is improved, the digestion problem of Chinese yam powder is solved, and the antioxidant activity and gastric mucosal protection ability are improved.

[0042] Pea peptide and Chinese yam powder polymer were fermented using Lactobacillus johnsonii IOB 801, and the resulting Chinese yam composite peptide composition significantly reduced the allergenicity and hygroscopicity of Chinese yam powder; finally, animal experiments verified that the Chinese yam composite peptide composition obtained after fermentation with Lactobacillus johnsonii IOB 801 was unexpectedly able to significantly restore the heart function of rats with spleen deficiency and dampness, thereby promoting the health of other organs and improving symptoms such as mental depression and inflammation, endocrine disorders, and decreased organ function caused by spleen deficiency in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph showing changes in rat body weight in the present invention;

[0044] Figure 2 is a graph showing the nitric oxide level in the blood of rats in the present invention;

[0045] Figure 3 is a graph showing the level of endothelin-1 (ET-1) in the blood of rats in the present invention;

[0046] Figure 4 is a graph showing the level of triiodothyronine (T3) in the blood of rats in the present invention;

[0047] Figure 5is a graph showing the level of tetraiodothyronine (T4) in the blood of rats in the present invention;

[0048] Figure 6 This is a graph of rat motilin (MTL) levels in the present invention;

[0049] Figure 7 is a graph of gastrin (GAS) levels in rats in the present invention;

[0050] Figure 8 This is a graph showing the level of interleukin-6 (IL-6), an inflammatory factor, in the serum of rats in the present invention;

[0051] Figure 9 This is a graph showing the level of inflammatory factor tumor necrosis gene α (TNF-α) in rat serum in the present invention;

[0052] Figure 10 This is a graph showing the level of the inflammatory factor transforming growth factor β (TGF-β) in the serum of rats in the present invention;

[0053] Figure 11 is a graph showing the level of aldosterone (ALD) in rat serum in the present invention;

[0054] Figure 12 is a graph of the level of atrial natriuretic peptide (ANP) in rat serum in the present invention;

[0055] Figure 13 This is a comparison diagram of periodic acid-Schiff (PAS) staining of rat heart tissue in the present invention;

[0056] Figure 14 This is a comparison chart of the clearance rate of diphenylpicrylphenylhydrazine (DPPH) in Example 4 of the present invention;

[0057] Figure 15 This is a comparison chart of superoxide anion radical scavenging rates in Example 4 of the present invention;

[0058] Figure 16 These are HE staining images of gastric mucosal tissue sections of mice in each group in Example 5 of the present invention.

[0059] A Lactobacillus johnsonii (Lactobacillus johnsonii) IOB 801 screened from fermented kimchi, its name is: Lactobacillus johnsonii IOB 801, the classification name is: Lactobacillus johnsonii, the preservation number is: CGMCC No.16824, the preservation date is: November 26, 2018, the preservation unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0061] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0062] A method for preparing a multifunctional yam composite peptide composition comprises the following steps:

[0063] Preparation of pea protein powder;

[0064] Preparation of pea peptide powder A;

[0065] Preparation of multifunctional yam composite peptide composition:

[0066] Add pea peptide powder A and Chinese yam powder at a mass ratio of 3:5 to an appropriate amount of 3 mol / L phosphate buffer solution, mix thoroughly, and adjust the pH of the mixture to 7.0 ± 0.2 using 20% ​​edible sodium hydroxide solution or hydrochloric acid solution;

[0067] The mixture was placed in a hot water bath at 87±2°C to react while magnetically stirring at 35±5 r / min for 3±0.5 h.

[0068] After the reaction is complete, the mixture is quickly placed in ice water to cool down to stop the reaction. The cooled mixture is inoculated with Lactobacillus johnsonii IOB 801 seed liquid at a rate of 1%, cultured at 37°C, with the pH maintained at 6.0-6.5, at a rotation speed of 45±5r / min, and cultured for 8±2h. After the fermentation liquid is concentrated, it is spray-dried to obtain a multifunctional Chinese yam composite peptide composition.

[0069] Preferably, the preparation method of pea protein powder is as follows:

[0070] Pea pretreatment: Wash, screen and peel fresh peas, soak them in water for 12 hours with a solid-liquid ratio of 1:30. After soaking, grind them into a homogenate.

[0071] Initial separation: sieve the homogenized slurry to separate the slurry residue and keep the slurry for later use;

[0072] Alkaline extraction: add edible sodium hydroxide solution with a mass concentration of 20% to the slurry, adjust the pH to 8.0, the extraction temperature is 35°C, the extraction time is 40 minutes, after the extraction is completed, centrifuge at 4000 rpm for 30 minutes, and collect the supernatant;

[0073] Acid precipitation separation: The supernatant was concentrated, cooled, and then a 20% hydrochloric acid solution was added to adjust the pH of the concentrate to 4.0. The concentrate was precipitated for 30 minutes and centrifuged at 4000 rpm for 30 minutes to obtain pea protein precipitate.

[0074] Spray drying: Add edible sodium hydroxide solution with a mass concentration of 20%, adjust the pH to 7.0, neutralize the pea protein precipitate, and spray dry to obtain pea protein powder.

[0075] Preferably, the preparation method of pea peptide powder A is as follows:

[0076] Prepare pea protein liquid: add pea protein powder to pure water at a mass ratio of pea protein powder to pure water of 1:10, and stir for 30 minutes to obtain pea protein liquid;

[0077] Preparation of compound protease: neutral protease, alkaline protease and flavor protease were compounded in a mass ratio of 1.0:0.2:0.8 to prepare compound protease system A;

[0078] Hydrolysis of compound protease system A: using pea protein liquid as substrate, add compound protease system A with a final mass concentration of 1.0%~1.5% and stir, perform enzymatic hydrolysis at 52°C for 5-6 hours, and after the enzymatic hydrolysis is completed, inactivate the enzyme at 90°C to obtain pea peptide hydrolyzate;

[0079] Membrane filtration: The obtained pea peptide hydrolysate was centrifuged at 6000 r / min for 8 min, the supernatant was collected, and filtered through a membrane to obtain the pea peptide collection solution;

[0080] Freeze drying: The pea peptide collection liquid is pre-frozen and then vacuum freeze-dried to obtain pea peptide powder A.

[0081] Preferably, the Lactobacillus johnsonii 10B 801 is a strain of Lactobacillus johnsonii screened from fermented kimchi.

[0082] Preferably, the Lactobacillus johnsonii 10B 801 is named: Lactobacillus johnsonii 10B 801, the classification name is: Lactobacillus johnsonii, the preservation number is: CGMCC No.16824, the preservation date is: November 26, 2018, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0083] Preferably, the preparation method of the Lactobacillus johnsonii 10B 801 seed liquid is:

[0084] Strain activation: Activate the frozen Lactobacillus johnsonii IOB 801 three times on MRS agar plates, pick a single colony and inoculate it into MRS liquid medium, culture it at 37°C for 10±2h, then transfer it to MRS liquid medium at a 1% inoculum volume and culture it at 37°C for 10±2h as seed liquid.

[0085] The multifunctional Dioscorea opposita composite peptide composition is prepared by the above-mentioned preparation method.

[0086] The multifunctional Dioscorea opposita composite peptide composition is used in the preparation of cardiotonic drugs.

[0087] The use of the multifunctional Dioscorea opposita composite peptide composition as described above in the preparation of a drug for restoring cardiac function of spleen deficiency and dampness excess type.

[0088] Preferably, the multifunctional Chinese yam composite peptide composition can significantly improve the weight loss of rats with spleen deficiency and dampness, i.e., the recovery rate is increased by 60%, loose stools, i.e., the stool moisture content is reduced from 65.54% to 37.40%, and abnormal organ indexes, i.e., the spleen index is restored from 3.23 to 2.64.

[0089] Specifically, the relevant preparation and detection are as follows:

[0090] Example 1

[0091] This embodiment provides a method for preparing pea protein powder, comprising the following steps:

[0092] S1. Pea pretreatment: fresh peas were washed, screened and peeled, and soaked in water for 12 h with a solid-liquid ratio of g:mL of 1:30. After soaking, the peas were crushed and ground into a homogenate.

[0093] S2. Preliminary separation: sieve the homogenized slurry to separate the slurry residue and keep the slurry for later use.

[0094] S3. Alkaline extraction: add edible sodium hydroxide solution with a mass concentration of 20% to the slurry, adjust the pH to 8.0, the extraction temperature is 35°C, the extraction time is 40 minutes, after the extraction is completed, centrifuge at 4000r / min for 30 minutes, and take the supernatant.

[0095] S4. Acid precipitation separation: The supernatant was concentrated, cooled, and then a 20% hydrochloric acid solution was added to adjust the pH of the concentrate to 4.0. The concentrate was precipitated for 30 minutes and centrifuged at 4000 rpm for 30 minutes to obtain a pea protein precipitate.

[0096] S5. Spray drying: Add edible sodium hydroxide solution with a mass concentration of 20%, adjust the pH to 7.0, neutralize the pea protein precipitate, and spray dry to obtain pea protein powder (the measured protein content is 81.37%).

[0097] Example 2

[0098] This embodiment is a method for preparing a composite protease, comprising the following steps:

[0099] S1. Prepare pea protein liquid: add the pea protein powder prepared in Example 1 to pure water at a mass ratio of pea protein powder to pure water of 1:10, and stir for 30 minutes to obtain a pea protein liquid.

[0100] S2. Preparation of compound protease:

[0101] Neutral protease, alkaline protease and flavor protease were compounded in a mass ratio of 1.0:0.2:0.8 to prepare compound protease system A.

[0102] S3. Hydrolysis of complex protease system A

[0103] Pea protein liquid was used as the substrate, and a composite protease system A with a final mass concentration of 1.0% to 1.5% was added and stirred. Enzymatic hydrolysis was carried out at 52°C for 5-6 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 90°C to obtain a pea peptide enzymatic hydrolyzate.

[0104] S4, membrane filtration

[0105] The obtained pea peptide enzymatic hydrolyzate was centrifuged at 6000 r / min for 8 min, the supernatant was collected, and the supernatant was filtered through a membrane to obtain a pea peptide collection solution.

[0106] S5. Freeze drying

[0107] The pea peptide collection liquid was pre-frozen and then vacuum freeze-dried to obtain pea peptide powder A, wherein the pea peptide product recovery rate obtained by enzymatic hydrolysis with the composite protease system A was 72.14%, the protein content (dry basis) was 87.56%, and the peptide content was 81.72%.

[0108] Example 3

[0109] This embodiment is a method for preparing a lyophilized powder of a composite peptide polymer of Dioscorea opposita, comprising the following steps:

[0110] S1. The pea peptide powder A and Chinese yam powder prepared in Example 2 were added to an appropriate amount of 3 mol / L phosphate buffer solution at a mass ratio of 3:5, mixed thoroughly, and the pH of the mixture was adjusted to 7.0±0.2 using edible sodium hydroxide solution or hydrochloric acid solution with a mass concentration of 20%.

[0111] S2. Place the mixed solution in a hot water bath at 87±2°C to react while magnetically stirring at a speed of 35±5 r / min for a reaction time of 3±0.5 h.

[0112] S3. After the reaction is complete, the mixture is quickly placed in ice water to cool down to stop the reaction. The cooled mixed solution is vacuum filtered and the filtrate is placed in a freeze dryer for freeze drying.

[0113] S4. Freeze-drying yields a lyophilized powder of the Chinese yam composite peptide polymer. The graft copolymerization results in a polymer that neutralizes the bitterness of the pea peptide, offering an excellent flavor and a hydrolysis rate of 94.32% at pH 2.0-3.0. However, the polymer exhibits increased hygroscopicity, with a water content of 8.79%.

[0114] Example 4

[0115] The in vitro antioxidant capacity determination of the yam composite peptide polymer includes the following steps:

[0116] S1. Comparative Example Preparation of Dioscorea opposita composite peptide powder: The multifunctional peptide, pea peptide powder A, and Dioscorea opposita powder were mixed at a mass ratio of 3:5 to obtain Dioscorea opposita composite peptide powder.

[0117] S2. Using vitamin C as a positive control, the antioxidant capacity of the yam composite peptide polymer prepared in Example 3 was tested to see if it was improved compared with the yam composite peptide powder prepared in S1. The clearance rates of two key antioxidant indicators, diphenylpicrylphenylhydrazine (DPPH) and superoxide anion, were tested respectively. The results are as follows: Figure 14 and Figure 15 shown.

[0118] Depend on Figure 14 and Figure 15 As shown in the figure, with the increase of the concentration of Chinese yam composite peptide powder and Chinese yam composite peptide polymer, the scavenging ability of DPPH free radicals and superoxide anions also increased; when the concentration of Chinese yam composite peptide powder was 8 mg / mL, the DPPH scavenging rate was 46.23%, while when the concentration of Chinese yam composite peptide polymer was 8 mg / mL, the DPPH scavenging rate was close to 100%; when the concentration of Chinese yam composite peptide powder was 16 mg / mL, the superoxide anion scavenging rate was 39.30%, while when the concentration of Chinese yam composite peptide polymer was 16 mg / mL, the superoxide anion scavenging rate was 71.71%; this shows that the scavenging ability of the polymer for DPPH free radicals and superoxide anions is significantly stronger than that of Chinese yam composite peptide powder.

[0119] Example 5

[0120] The determination of the gastric mucosal protective ability of the yam composite peptide polymer includes the following steps:

[0121] S1. Thirty-two healthy male Kunming mice were randomly divided into a blank group, a model group, a Chinese yam complex peptide powder group, and a Chinese yam complex peptide polymer group, with 8 mice in each group.

[0122] The S2, blank, and model groups were gavaged daily with normal saline (10 ml / kg). The Dioscorea opposita peptide powder and Dioscorea opposita peptide polymer groups were gavaged daily with the same dose (20 mg / kg) of Dioscorea opposita peptide powder and Dioscorea opposita peptide polymer. This gavage was continued for 14 consecutive days. Thirty minutes after the last dose on the 14th day, all mice except the blank group were gavaged simultaneously with 50% ethanol solution (10 ml / kg) every 12 hours for a total of six times.

[0123] S3. After the last oral administration, all mice were fasted for 12 hours and then killed. The stomach tissues were taken and HE sections were stained to observe the damage of gastric mucosa. The results are as follows: Figure 16 shown.

[0124] Depend on Figure 16 It can be seen that Chinese yam complex peptide powder and Chinese yam complex peptide polymer have obvious protective effects on alcohol-induced gastric mucosal damage in mice; and the protective effect of the polymer on the gastric mucosa of mice is significantly stronger than that of Chinese yam complex peptide powder.

[0125] Example 6

[0126] This embodiment is a method for preparing a composite peptide composition of Dioscorea opposita, comprising the following steps:

[0127] S1. Strain activation: Activate the frozen Lactobacillus johnsonii 10B 801 three times on MRS agar plates, pick a single colony and inoculate it into MRS liquid medium, culture it at 37°C for 10±2 hours, then transfer it to MRS liquid medium at a 1% inoculum volume and culture it at 37°C for 10±2 hours as seed liquid.

[0128] S2. Inoculate the strain at a 1% inoculum size into the cooled mixed solution obtained in step S3 of Example 3, incubate at 37° C., maintain the pH at 6.0-6.5, rotate at 45±5 r / min, and incubate for 8±2 h.

[0129] S3. After the fermentation liquid is concentrated, it is spray-dried to obtain a Chinese yam composite peptide composition. After allergy test, the Chinese yam composite peptide composition prepared by this method significantly reduced the allergenicity, and the water content was also reduced to 5.31%, reducing the hygroscopicity.

[0130] Among them, the Lactobacillus johnsonii 10B 801 is a strain of Lactobacillus johnsonii 10B 801 screened from fermented kimchi, its name is: Lactobacillus johnsonii 10B 801, its classification name is: Lactobacillus johnsonii, its preservation number is: CGMCC No.16824, the preservation date is: November 26, 2018, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0131] The screening method of the Lactobacillus johnsonii IOB801 comprises the following steps:

[0132] Take 1 mL of kimchi water from a resident's home in Tianjin and put it into a test tube containing 9 mL of sterile saline (mass concentration is 0.9%). After thorough mixing, take 1 mL of water and put it into a test tube containing 9 mL of saline. Then, dilute it to 10% by gradient. -5 100 μL of each sample solution was spread onto MRS agar medium and incubated at 37°C for 48 hours. Two replicates were made for each gradient. Colonies of varying morphology, size, and color were selected from the plates, repeatedly streaked and purified, and then incubated in a 37°C incubator for 48 hours. This process was repeated until pure colonies appeared. Finally, the obtained strains were sequenced for the 16S rDNA gene, then numbered and stored.

[0133] Example 7

[0134] This example is a study on the effect of a composite peptide composition of Chinese yam on improving spleen deficiency syndrome in rats, including:

[0135] 1.1 Experimental Materials

[0136] 1.1.1 Experimental animals

[0137] Specific pathogen-free (SPF) healthy male Sprague-Dawley rats were purchased from Sbefor (Beijing, China) under license number SCXK (Beijing) 2024-0001. They were 6-7 weeks old and weighed (180 ± 20) g. Rats had free access to water during the experiment. The room temperature was 20-26°C, the relative humidity was 40%-70%, and the light cycle was 12 h: lights on from 7:00 AM to 7:00 PM, and dark from 7:00 PM to 7:00 AM. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0138] 1.2 Experimental methods

[0139] 1.2.1 Experimental Grouping

[0140] After 7 days of adaptive feeding, the healthy rats were randomly divided into 7 groups (8 rats in each group): blank control group, model group, positive control group, protection group 1, protection group 2, treatment group 1, and treatment group 2.

[0141] 1.2.2 Establishment of the rat model of spleen deficiency and dampness excess type hyperlipidemia and intervention plan

[0142] The protection group was gavaged with the test samples for 30 days. The gavage dose of protection group 1 (the Dioscorea opposita composite peptide polymer prepared in Example 3) was 1.89 g / kg (body weight), and the gavage dose of protection group 2 (the Dioscorea opposita composite peptide composition prepared in Example 6 of the present invention) was 1.89 g / kg (body weight). The test samples were all dissolved in 0.9% saline at a mass concentration of 1 mL / kg (body weight) and then gavaged. The remaining rats were raised normally (ordinary feed, normal eating, and cultured at 22°C).

[0143] After 30 days of feeding, modeling began. Reserpine was diluted with distilled water and acetic acid to prepare a 0.2 mg / mL reserpine solution. Except for the blank group, all rats in the remaining groups were administered intramuscularly at a dose of 0.2 mg / kg per day. The blank group was subcutaneously injected with 0.85% saline at the same mass concentration. The modeling period was 7-10 days. Scoring was performed according to Table 2. A score of 3 for each symptom indicated a successful modeling. After the modeling was completed, intervention was performed for a period of 2 weeks. The specific intervention methods are as follows:

[0144] Table 1 Intervention treatment operation table for each group

[0145]

[0146] Note: Treatment group 1 was orally administered with the Dioscorea opposita composite peptide polymer prepared in Example 3, and treatment group 2 was orally administered with the Dioscorea opposita composite peptide composition prepared in Example 6. The test samples were dissolved in 0.9% saline at a mass concentration of 1 mL / kg (body weight) and then orally administered.

[0147] Table 2 Rats' general condition score sheet

[0148]

[0149] 1.3 Indicator Observation

[0150] 1.3.1 Weight Index

[0151] Adaptive feeding began 7 days later and lasted until the end of the experiment. The body weight and changes of the rats in each group were recorded every 7 days.

[0152] 1.3.2 Defecation

[0153] After completing all the operations according to Section 1.2.2 of Example 7, the rats in each group were fasted for 12 hours, with the wet and dry weights of stool recorded, and the stool humidity was calculated.

[0154] Stool moisture (%) = [stool wet mass (g) - stool dry mass (g)] / stool wet mass (g) × 100%

[0155] 1.3.3 Behavioral experiments

[0156] 1.3.3.1 Open field test

[0157] After completing the procedures in 1.3.2, conduct the open field test. In a quiet environment, place the rat in the center of the bottom of the open field chamber. A camera, positioned 2 m above a 1 m × 1 m square on the bottom, simultaneously records and measures the rat's movements. Record and analyze the percentage of time spent in the central area, the number of squares walked, the number of times the rats stand, and the distance they move within the open field.

[0158] 1.3.3.2 Forced swimming test

[0159] After completing the open-field test described in 1.3.3.1, a forced swim test was performed. A transparent container was filled with room-temperature water approximately 30 cm high. Twenty-four hours before the actual experiment, each rat underwent 10 minutes of adaptive swimming training. During the actual experiment, each rat underwent a 6-minute swimming test, which was recorded in full. The video was digitally analyzed using Etho Vision software, and the cumulative immobility time (2–6 minutes) was calculated. The criterion for this was immobility of all four limbs or only slight movement of the hind limbs.

[0160] 1.3.4 Effects on blood rheology and vascular endothelial cell function

[0161] After completing the behavioral experiment in 1.3.3, blood was collected from the eyeballs of all rats, and then all rats were killed. The levels of bioactive molecules nitric oxide (NO) and endothelin-1 (ET-1) in the rat serum were determined strictly according to the instructions of the kit.

[0162] 1.3.5 Detection of endocrine system related indicators

[0163] Place the whole blood sample obtained in step 1.3.4 at room temperature for 2 hours or at 4°C overnight, then centrifuge at 3000 rpm for 15 minutes. Take the supernatant and test the contents of tetraiodothyronine (T4) and triiodothyronine (T3) according to the kit instructions.

[0164] 1.3.6 Detection of gastrointestinal motility recovery in rats

[0165] The rat serum obtained in step 1.3.4 was tested for motilin (MTL) and blood gastrin (GAS) levels according to the instructions of the enzyme-linked immunosorbent assay kit.

[0166] 1.3.7 Serum inflammatory factor determination

[0167] The rat serum obtained in step 1.3.4 was assayed for changes in the levels of pro-inflammatory factors interleukin-6 (IL-6), tumor necrosis gene α (TNF-α), and anti-inflammatory factor transforming growth factor β (TGF-β) according to the instructions of the enzyme-linked immunosorbent assay kit.

[0168] 1.3.8 Determination of renal function indicators

[0169] The rat serum obtained in step 1.3.4 was assayed for aldosterone (ALD) and atrial natriuretic peptide (ANP) levels according to the instructions of the enzyme-linked immunosorbent assay kit.

[0170] 1.3.9 Organ Index

[0171] In step 1.3.4, after sacrificing the rats in each group, the five internal organs (heart, liver, spleen, lungs, and kidneys) were removed and the organ indices of the heart, liver, spleen, lungs, and kidneys were calculated. The spleen is used as an example; the calculation formulas for the other organ indices are the same.

[0172] Spleen Index = [rat spleen weight (mg) / rat body weight (g)]

[0173] 1.3.10 Cardiac Histological Observation

[0174] After weighing all rat organs as described in 1.3.9, the hearts were fixed in Bouin's solution for 2 days, then fixed in 70% ethanol (refrigerated at 4°C) until use. Heart tissue blocks were fixed in Bouin's solution, dehydrated with ethanol and n-butanol, and embedded in methacrylate resin. A 20 μm-thick section was cut from the embedded block and stained with periodic acid-Schiff reagent (PAS) and hematoxylin. Finally, changes in the heart tissue were observed.

[0175] 1.4 Data statistical processing methods

[0176] All data were expressed as mean ± standard deviation and plotted using GraphPad Prism 8 and Origin 2021 software.

[0177] 1.5 Experimental Results

[0178] 1.5.1 Changes in rat body weight

[0179] Adaptive feeding began 7 days later and continued until the end of the experiment. The weight of each group of rats was recorded every 7 days, and the weight changes were recorded. The results are as follows: Figure 1 As shown, during the modeling period, except for the blank group, the weight of rats in other groups decreased. After 14 days of intervention with normal saline, the weight of rats in the two protection groups recovered significantly; however, after 14 days of intervention with normal saline, the weight of rats in the model group did not recover significantly; after 14 days of intervention with normal saline, the weight of rats in the positive control group recovered significantly; after 14 days of intervention with the Chinese yam composite peptide, the weight loss of rats in the two treatment groups was also significantly improved; this shows that the two Chinese yam composite peptides can prevent and treat the weight loss of rats caused by spleen deficiency and dampness to a certain extent.

[0180] 1.5.2 Defecation

[0181] After 2 weeks of intervention, the stool wet weight and dry weight of each group of rats were recorded within 12 hours, and the stool humidity was calculated. The results are shown in Table 5. Compared with the blank group (stool humidity 34.09%), the stool humidity of the model group rats (65.54%) was significantly increased; compared with the model group, the stool humidity of the protection group (stool humidity 37.21% in protection group 1 and 19.32% in protection group 2) and the treatment group (stool humidity 53.45% in treatment group 1 and 37.40% in treatment group 2) rats was significantly reduced and returned to normal levels, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can effectively improve the symptoms of loose stools in rats with spleen deficiency and dampness, and the effect is significantly better than the Dioscorea opposita composite peptide polymer prepared in Example 3.

[0182] Table 5 Statistics of rat feces weight and humidity

[0183]

[0184] 1.5.3 Behavioral Experiments

[0185] 1.5.3.1 Mine Experiment

[0186] In the open field test, the model group rats spent less time in the central area within 10 minutes compared to the normal group, indicating that spleen deficiency and dampness excess can cause a decrease in the rats' exploratory and autonomous activity. The prevention and treatment effects of the protection group 2 and treatment group 2 on depression and anxiety caused by spleen deficiency and dampness excess in rats were stronger than those of the protection group 1 and treatment group 1, with more significant improvements in the rats' activity and exploratory behavior, as shown in Table 3.

[0187] Table 3 Statistics of central area activity time in rats

[0188]

[0189] 1.5.3.2 Forced swimming test

[0190] In the forced swimming experiment, the model group spent significantly more time immobile on the water surface than the normal group, indicating that spleen deficiency and dampness can cause depressive behaviors such as fatigue and decreased mobility in rats. Compared with the model group, the protection group 2 and treatment group 2 spent significantly less time immobilized in the water, significantly preventing and alleviating negative behaviors such as fatigue and decreased mobility caused by spleen deficiency and dampness, and improving the rats' desire to survive and autonomous activity. The effects were significantly stronger than those in the protection group 1 and treatment group 1, as shown in Table 4.

[0191] Table 4 Statistics of rat swimming immobility time

[0192]

[0193] 1.5.4 Effects on Hemorheology and Endothelial Cell Function

[0194] Endothelin-1 (ET-1) is the most potent vasoconstrictor, while nitric oxide (NO) is a vasodilator. The balance of this pair of vasoactive substances is important for maintaining normal vascular tone and hemodynamics. Figure 2-Figure 3 It can be seen that compared with the blank group, the NO level in the model group was significantly decreased, and the ET-1 level was significantly increased, indicating that the NO / ET-1 imbalance in the model group rats was related to the occurrence of cardiovascular and cerebrovascular diseases; compared with the model group, the NO level in the protection group 2 and the treatment group 2 was significantly increased, and the ET-1 level was significantly decreased, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can significantly prevent and restore the NO / ET-1 imbalance in rats caused by spleen deficiency and dampness, and can prevent and treat the occurrence of cardiovascular and cerebrovascular diseases, and the effect is significantly better than the Dioscorea opposita composite peptide polymer prepared in Example 3.

[0195] 1.5.5 Detection of endocrine system related indicators

[0196] The thyroid gland is the largest endocrine gland in the human body. Its main active substances are triiodothyronine (T3) and tetraiodothyronine (T4). Figure 4-Figure 5 It can be seen that the T3 and T4 levels in the model group were significantly decreased compared with the blank control group, indicating that the metabolic level of the rats in the model group was reduced; compared with the model group, the T3 and T4 levels in the protection group 2 and the treatment group 2 were significantly increased, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can prevent and restore the endocrine metabolic level of rats caused by spleen deficiency and dampness, and improve the basal metabolic rate, and the effect is significantly better than the Dioscorea opposita composite peptide powder prepared in Example 3.

[0197] 1.5.6 Detection of gastrointestinal motility recovery in rats

[0198] Motilin (MTL) and gastrin (GAS) are hormones normally secreted by the gastrointestinal tract, which play an important regulatory role in gastrointestinal motility. Figure 6-Figure 7 As shown, compared with the blank group, the MLT and GAS levels of the model group were significantly reduced, indicating that the gastrointestinal motility of the rats with spleen deficiency and dampness was significantly decreased; the MLT and GAS levels of the protection group 2 and the treatment group 2 were increased to varying degrees compared with the model group, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can restore the gastrointestinal motility of rats with spleen deficiency and dampness, and the effect is significantly better than the Dioscorea opposita composite peptide polymer prepared in Example 3.

[0199] 1.5.7 Determination of serum inflammatory factors

[0200] The occurrence and development of spleen deficiency syndrome is closely related to inflammatory factors, such as Figures 8-10 As shown in the results, compared with the blank group, the levels of proinflammatory factors IL-6 and TNF-α in the serum of the rats in the model group were significantly increased, and the level of anti-inflammatory factor TGF-β was significantly decreased, indicating that spleen deficiency and dampness can induce inflammatory response in rats; compared with the model group, the levels of proinflammatory factors IL-6 and TNF-α in the serum of the rats in the protection group 2 and the treatment group 2 were significantly decreased, and the level of anti-inflammatory factor TGF-β was significantly increased, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can prevent and alleviate inflammation in rats with spleen deficiency and dampness, and the effect is significantly better than the Dioscorea opposita composite peptide polymer prepared in Example 3.

[0201] 1.5.8 Determination of renal function indicators

[0202] ALD is an important mineralocorticoid secreted by the zona glomerulosa of the adrenal cortex, which has the physiological function of regulating sodium and potassium metabolism and extracellular fluid volume; the main function of ANP is to promote the excretion of sodium and water by the kidneys. Figure 11-12 As shown in the results, compared with the blank group, the ALD content in the serum of the rats in the model group was significantly increased, and the ANP content was significantly decreased, indicating that the renal metabolism of the rats in this group was blocked; compared with the model group, the ALD content in the serum of the rats in the protection group 2 and the treatment group 2 was significantly decreased, and the ANP content was significantly increased, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can effectively regulate renal metabolism, and the effect is significantly better than the Dioscorea opposita composite peptide polymer prepared in Example 3.

[0203] 1.5.9 Organ Index

[0204] The five internal organs (heart, liver, spleen, lungs, and kidneys) of the rats in each group were weighed, and organ indices were calculated. The results are shown in Table 6. Compared with the blank group, the organ indices of the rats in the model group were all increased, indicating that these rats showed symptoms such as congestion, edema, or hypertrophy of the organs. Compared with the model group, the organ indices of the rats in the protection group 2 and the treatment group 2 returned to the same level as the blank group. This shows that the Dioscorea opposita composite peptide composition prepared by the present invention can effectively restore the health of the five internal organs of rats with spleen deficiency and dampness, and the effect is significantly better than that of the Dioscorea opposita composite peptide polymer prepared in Example 3. As shown in Table 6.

[0205] Table 6 Statistics of rat organ index

[0206]

[0207] 1.5.10 Cardiac Histological Observation

[0208] Depend on Figure 13 As shown in the table, compared with the blank group, there was obvious glycogen deposition in the heart tissue of the rats in the model group, indicating that the heart tissue of the rats in this group was damaged. Compared with the model group, the heart color of the rats in the protection group 2 and the treatment group 2 was restored to the same level as that of the blank group, indicating that the Dioscorea opposita composite peptide composition prepared by the present invention can effectively promote glycogen metabolism in the heart tissue of rats with spleen deficiency and dampness, restore heart health, and the effect is significantly better than that of the Dioscorea opposita composite peptide polymer prepared in Example 3. Combined with Table 6, it is shown that the Dioscorea opposita composite peptide composition can promote the recovery of other organs by restoring and strengthening heart health.

[0209] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a multifunctional yam composite peptide composition, characterized in that: The steps include: Preparation of pea protein powder; Preparation of pea peptide powder A; Preparation of multifunctional yam composite peptide composition: Add pea peptide powder A and Chinese yam powder at a mass ratio of 3:5 to an appropriate amount of 3 mol / L phosphate buffer solution, mix thoroughly, and adjust the pH of the mixture to 7.0 ± 0.2 using 20% ​​edible sodium hydroxide solution or hydrochloric acid solution; The mixture was placed in a hot water bath at 87±2°C to react while magnetically stirring at 35±5 r / min for 3±0.5 h. After the reaction is complete, the mixture is quickly placed in ice water to cool down to stop the reaction. The cooled mixture is inoculated with Lactobacillus johnsonii IOB 801 seed liquid at a rate of 1%, and cultured at 37°C, with the pH maintained at 6.0-6.5, at a rotation speed of 45±5 r / min, for 8±2 hours. After the fermentation liquid is concentrated, it is spray-dried to obtain a multifunctional Chinese yam composite peptide composition. The preparation method of pea peptide powder A is specifically as follows: Prepare pea protein liquid: add pea protein powder to pure water at a mass ratio of pea protein powder to pure water of 1:10, and stir for 30 minutes to obtain pea protein liquid; Preparation of compound protease: neutral protease, alkaline protease and flavor protease were compounded in a mass ratio of 1.0:0.2:0.8 to prepare compound protease system A; Hydrolysis of compound protease system A: using pea protein liquid as substrate, add compound protease system A with a final mass concentration of 1.0%~1.5% and stir, perform enzymatic hydrolysis at 52°C for 5-6 hours, and after the enzymatic hydrolysis is completed, inactivate the enzyme at 90°C to obtain pea peptide hydrolyzate; Membrane filtration: The obtained pea peptide hydrolysate was centrifuged at 6000 r / min for 8 min, the supernatant was collected, and filtered through a membrane to obtain the pea peptide collection solution; Freeze drying: The pea peptide collection solution is pre-frozen and then vacuum freeze-dried to obtain pea peptide powder A; The Lactobacillus johnsonii 10B 801 is named as Lactobacillus johnsonii 10B 801 and its classification name is Lactobacillus johnsonii Lactobacillus johnsonii , the deposit number is: CGMCC No.16824, the deposit date is: November 26, 2018, the deposit unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing the multifunctional Dioscorea opposita composite peptide composition according to claim 1, wherein: The preparation method of pea protein powder is specifically as follows: Pea pretreatment: Wash, screen and peel fresh peas, soak them in water for 12 hours with a solid-liquid ratio of 1:

30. After soaking, grind them into a homogenate. Initial separation: sieve the homogenized slurry to separate the slurry residue and keep the slurry for later use; Alkaline extraction: add edible sodium hydroxide solution with a mass concentration of 20% to the slurry, adjust the pH to 8.0, the extraction temperature is 35°C, the extraction time is 40 minutes, after the extraction is completed, centrifuge at 4000 rpm for 30 minutes, and collect the supernatant; Acid precipitation separation: The supernatant was concentrated, cooled, and then a 20% hydrochloric acid solution was added to adjust the pH of the concentrate to 4.

0. The concentrate was precipitated for 30 minutes and centrifuged at 4000 rpm for 30 minutes to obtain pea protein precipitate. Spray drying: Add edible sodium hydroxide solution with a mass concentration of 20%, adjust the pH to 7.0, neutralize the pea protein precipitate, and spray dry to obtain pea protein powder.

3. The method for preparing the multifunctional Dioscorea opposita composite peptide composition according to claim 1 or 2, characterized in that: The preparation method of the Lactobacillus johnsonii 10B 801 seed liquid is: Strain activation: Activate the frozen Lactobacillus johnsonii IOB 801 three times on MRS agar plates, pick a single colony and inoculate it into MRS liquid medium, culture it at 37°C for 10±2h, then transfer it to MRS liquid medium at a 1% inoculum volume and culture it at 37°C for 10±2h as seed liquid.

4. The multifunctional Dioscorea opposita composite peptide composition prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the multifunctional Dioscorea opposita composite peptide composition according to claim 4 in preparing a drug for restoring cardiac function in patients with spleen deficiency and dampness excess.

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