Chinese yam composite peptide preparation, preparation method and application of Chinese yam composite peptide preparation in preparation of medicine for strengthening spleen and five internal organs

Abstract

The invention belongs to the technical field of traditional Chinese medicines and biological preparations, and discloses a Chinese yam composite peptide preparation, a preparation method and application of the Chinese yam composite peptide preparation in preparation of medicines for strengthening the spleen and the five internal organs. The Chinese yam composite peptide powder prepared by the invention can improve whole body diseases of rats with spleen deficiency type hyperlipemia through multi-target regulation and control of metabolic pathways, for example, up-regulation of a pentose phosphate pathway, promotion of glutathione metabolism, regulation of nerve-intestine-axis, synergistic restoration of visceral organ functions and the like, and the effect of'synergistic restoration of five internal organs' is achieved from'single spleen tonifying '. In addition, preventive intervention (a protection group) is coordinated with ROR alpha rhythm through early regulation of glutamine metabolism, so that compared with a treatment group, metabolic disorder is more remarkably reversed, multi-organ function repair is realized, body injury caused by hyperlipidemia is systematically improved, and finally, the effects of strengthening the spleen and strengthening the five internal organs are achieved, and the effect of'synergistic repair of the five internal organs' is achieved through regulation and control of metabolic roots.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine and biological agents, and particularly relates to a Chinese yam compound peptide preparation, a preparation method thereof, and application of the Chinese yam compound peptide preparation in preparation of a medicine for invigorating the spleen and strengthening five internal organs. BACKGROUND

[0002] In the field of contemporary biological agent research and development, intervention programs for metabolic disorders of spleen deficiency have long been plagued by systemic deficiencies. Traditional Chinese medicine compounds such as Sijunzi Decoction have the effect of invigorating the spleen, but their mechanisms of action are mostly limited to improving digestive and absorptive functions. Existing probiotic preparations have problems such as inactivation of bacterial cells due to the gastric acid environment, making it difficult to maintain an effective concentration of key metabolites such as short-chain fatty acids in the intestinal tract. More notably, current commercially available peptide supplements generally use an empirical compounding approach, lacking research on synergistic mechanisms with microbial metabolites.

[0003] The existing solid-state fermentation process has obvious defects in the treatment of special substrates such as Chinese yam. First, Chinese patent CN110638035A provides a solid-state fermentation method for blueberry and yam, without detecting the content of short-chain fatty acids before and after fermentation. Chinese patent CN111345348A is a conventional lactic acid bacteria liquid-state fermentation of yam paste and milk, and the short-chain fatty acids are significantly increased after fermentation and mixing with hawthorn jam, but the content of short-chain fatty acids in yam paste before and after fermentation is not detected, which cannot rule out the influence of milk and hawthorn jam on the content of short-chain fatty acids. The postbiotic prepared in Chinese patent CN119999906A can effectively improve glucose and lipid metabolism disorders, relieve liver function damage, and effectively improve intestinal barrier function, but the liquid-state fermentation and fermentation substrate of rice and cereal grains used in the patent are different from those of the present application in terms of fermentation method and fermentation substrate.

[0004] In terms of peptide combination technology, existing products are mostly focused on the regulation of a single functional indicator. For example, the peptide composition prepared in Chinese patent CN117679324A exhibits anti-wrinkle activity and is applied to skin care products, which is different from the application direction and efficacy mechanism of the present application. The KGT-C polypeptide in Chinese patent CN119320429A is only designed for blood lipid abnormalities and does not establish a synergistic action model with the microbial metabolic network, making it difficult to meet the needs of modern preventive medicine for multi-target regulation. The above patents do not find any similarities with the preparation method and preparation process of the Chinese yam powder and peptide composition in the present application.

[0005] Based on the above technical bottleneck, the present application selects Paracaseiclovum IOB413 to direct fermentation of Chinese yam, successfully increases the content of acetic acid and propionic acid in the fermentation product of Chinese yam. More importantly, by establishing the precise compatibility system of post-fermentation bio-fermentation and compound peptides, a "microbial metabolism-host nutrition" bidirectional regulation network is constructed. Animal experiments prove that the composition can not only up-regulate the phosphotriose pathway, but also realize the coordinated repair of multiple organ functions through the regulation of ROR alpha biological rhythm. This systematic intervention strategy from the root of metabolism provides a new idea for the research and development of new biological agents. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art, and provides a Chinese yam compound peptide preparation, a preparation method and its application in preparing drugs for invigorating the spleen and strengthening the five internal organs.

[0007] The technical solution adopted by the present application to solve its technical problems is:

[0008] A Chinese yam compound peptide preparation, the preparation is Chinese yam fermentation post-biofermentation and Chinese yam compound peptide powder.

[0009] The preparation method of the Chinese yam compound peptide preparation as described above comprises the following steps:

[0010] The preparation method of the Chinese yam fermentation post-biofermentation comprises the following steps:

[0011] S1, Paracaseiclovum IOB413 strain activation: inoculate IOB413 strain in the frozen tube into the slope culture medium, and place it in 37±0.5℃ culture, the activation time is 20±2h, the activated colony is white, round, the surface is wet, opaque, the edge is neat, the bacterial body is rod-shaped under the microscope, the individual is uniform and thick, which indicates that the activation is successful;

[0012] S2, take a fresh slope strain and inoculate it in the liquid culture medium containing 2%-4% Chinese yam powder, 36±2℃, close and culture, the culture time is 20±2h, and the seed liquid is obtained;

[0013] S3, inoculate the seed liquid into the Chinese yam fermentation substrate according to the inoculation amount of 5%, 36±2℃, close and culture, the culture time is 24-48h, after fermentation, inactivate at 80-95℃, dry treatment the water content≤10%, then crush to obtain Chinese yam fermentation post-biofermentation;

[0014] The preparation method of the Chinese yam compound peptide powder comprises the following steps:

[0015] The fermented D. japonica bio-substance is compounded with various complex peptides including but not limited to pea peptides, soybean oligopeptides and the like, such as: the fermented D. japonica bio-substance powder, pea peptide powder and soybean oligopeptide are mixed in a mass ratio (1:1:1)~(1:2:2), and are put into a mixing machine, and are mixed at a relative humidity of 45% or less and a temperature of 20~25 DEG C for 20~30 minutes at a rotating speed of 15~25 rpm to obtain the D. japonica complex peptide powder.

[0016] Further, the slant medium uses a modified MRS medium, and the formula is as follows: 4.0~7.0g of proteose peptone, 4.0~7.0g of beef extract, 5.0~7.0g of yeast powder, 13.0~18.0g of glucose, 0.8~1.0mL of Tween 80, 1.5~2.0g of potassium phosphate dibasic heptahydrate, 3.0~4.0g of sodium acetate trihydrate, 1.0~1.2g of triammonium citrate, 0.1~0.2g of magnesium sulfate heptahydrate, 0.03~0.05g of manganese sulfate tetrahydrate, 12~15.0g of agar, pH=6.2±0.2, and the water amount is 1~1.2L.

[0017] Further, the slant medium uses a modified MRS medium, and the formula is as follows: 4.0~7.0g of proteose peptone, 4.0~7.0g of beef extract, 5.0~7.0g of yeast powder, 13.0~18.0g of glucose, 0.8~1.0mL of Tween 80, 1.5~2.0g of potassium phosphate dibasic heptahydrate, 3.0~4.0g of sodium acetate trihydrate, 1.0~1.2g of triammonium citrate, 0.1~0.2g of magnesium sulfate heptahydrate, 0.03~0.05g of manganese sulfate tetrahydrate, pH=6.2±0.2, and the water amount is 1~1.2L.

[0018] Further, the fermented D. japonica substrate is a mixture of D. japonica and water, and the feed liquid ratio g:mL of D. japonica :water is 1:1.5.

[0019] The application of the D. japonica complex peptide preparation as described above in the preparation of a drug for invigorating the spleen and strengthening five internal organs.

[0020] Further, the D. japonica complex peptide powder can improve the spleen deficiency by regulating metabolic pathways through multiple targets, and achieve the effect of repairing five internal organs in coordination with invigorating the spleen.

[0021] Further, the D. japonica complex peptide powder can up-regulate the phosphotriose pathway, promote glutathione metabolism, regulate the nerve-intestine-axis and repair organ functions in coordination.

[0022] The application has the following advantages and positive effects:

[0023] 1. The present application adopts Paracasei IOB413 to ferment Chinese yam by solid state fermentation, through optimizing the fermentation conditions, under the condition of 36±2℃, the fermentation substrate material liquid ratio (Chinese yam: water) is (1~2):(1.5~3.5), and the fermentation is carried out for 24~48h, so that the Chinese yam after fermentation is obtained, so that the content of short-chain fatty acids (acetic acid and propionic acid) in the Chinese yam after fermentation is increased by 16.82 times and the content of propionic acid is increased by 2.46 times compared with the unfermented Chinese yam.

[0024] 2. The present application cooperates the Chinese yam after fermentation with various complex peptides including but not limited to pea peptides, soybean oligopeptides and the like, such as Chinese yam after fermentation, pea peptide powder and soybean oligopeptide according to the mass ratio (1:1:1)~(1:2:2), and finally the Chinese yam complex peptide powder is obtained, forming a two-way regulation system of microbial metabolite superposition host nutrition. The new biological preparation obtained by the directional fermentation of Chinese yam after fermentation and the precise peptide cooperation can be applied to the direction of invigorating the spleen and strengthening the five internal organs.

[0025] 3. The Chinese yam complex peptide powder prepared by the present application can improve the whole body symptoms of the spleen deficiency type hyperlipidemia rats through multi-target point regulation of metabolic pathways, such as up-regulating the phosphotriose pathway, promoting glutathione metabolism, regulating the nerve-intestine-axis and synergistically repairing organ function, etc., achieving the effect of "five internal organs synergistic repair" from "single spleen invigoration". The preventive intervention (protection group) can more significantly reverse metabolic disorders than the treatment group by early regulation of glutamine metabolism and RORα rhythm coordination, realize multi-organ function repair, systemically improve the body damage caused by hyperlipidemia, and finally achieve the effect of invigorating the spleen and strengthening the five internal organs, and achieve the effect of "five internal organs synergistic repair" from metabolic regulation. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is the change graph of acetic acid content before and after fermentation in the present application;

[0027] Figure 2 It is the change graph of propionic acid content before and after fermentation in the present application;

[0028] Figure 3 It is the graph of A for aldosterone (ALD) level in rat serum and B for central natriuretic peptide (ANP) level in rat serum in the present application;

[0029] Figure 4 It is the PAS staining image of five internal organs of rats in each group in the present application; wherein, A is the PAS staining image of rat heart tissue; B is the PAS staining image of rat liver tissue; C is the PAS staining image of rat spleen tissue; D is the PAS staining image of rat lung tissue; E is the PAS staining image of rat kidney tissue;

[0030] Figure 5HE staining images of colon and stomach tissues of rats in each group in the application; wherein, A is the HE staining image of rat colon tissue; B is the HE staining image of rat stomach tissue;

[0031] Figure 6 PCA diagram of serum metabolites of rats in each group in the application;

[0032] Figure 7 Wayne diagram of serum metabolites of rats in each group in the application;

[0033] Figure 8 Cluster heat map of differential metabolites of rat serum of positive control group vs blank group in the application;

[0034] Figure 9 Cluster heat map of differential metabolites of rat serum of treatment group 3 vs blank group in the application;

[0035] Figure 10 Cluster heat map of differential metabolites of rat serum of protection group 3 vs blank group in the application;

[0036] Figure 11 Volcano plot of differential metabolites of rat serum of model group vs blank group in the application;

[0037] Figure 12 Volcano plot of differential metabolites of rat serum of positive control group vs blank group in the application;

[0038] Figure 13 Volcano plot of differential metabolites of rat serum of treatment group 3 vs blank group in the application;

[0039] Figure 14 Volcano plot of differential metabolites of rat serum of protection group 3 vs blank group in the application;

[0040] Figure 15 KEGG pathway analysis diagram of differential metabolites of rat serum in each group in the application; wherein, A is KEGG pathway analysis of model group vs blank group; B is KEGG pathway analysis of positive control group vs blank group; C is KEGG pathway analysis of treatment group 3 vs blank group; D is KEGG pathway analysis of protection group 3 vs blank group. DETAILED DESCRIPTION

[0041] The application will be further described in conjunction with the examples below, which are descriptive rather than limiting, and cannot be used to limit the protection scope of the application.

[0042] The various experimental operations involved in the embodiments are conventional techniques in the art, and the parts not specifically noted herein can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the application date of the present application.

[0043] A Chinese yam compound peptide preparation, which is a Chinese yam post-fermentation probiotic and Chinese yam compound peptide powder.

[0044] A preparation method of the Chinese yam compound peptide preparation as described above, comprising the following steps:

[0045] The preparation method of the Chinese yam post-fermentation probiotic, comprising the following steps:

[0046] S1, activation of Paracasei Lactis IOB413 strain: inoculate IOB413 strain in the frozen tube into the slant medium, and place it into 37±0.5℃ culture, the activation time is 20±2h, the activated colony is white, round, surface is moist, opaque, edge is neat, and the bacterial body is rod-shaped, individual is uniform, and robust, which indicates that the activation is successful;

[0047] S2, take a fresh slant strain and inoculate it into the liquid medium containing 2%-4% Chinese yam powder, 36±2℃, and culture in a closed state, the culture time is 20±2h, and the seed liquid is obtained;

[0048] S3, inoculate the seed liquid into the Chinese yam fermentation substrate according to the inoculation amount of 5%, 36±2℃, and culture in a closed state, the culture time is 24-48h, after fermentation, inactivate at 80-95℃, dry treatment the water content≤10%, then crush, and obtain the Chinese yam post-fermentation probiotic;

[0049] The preparation method of the Chinese yam compound peptide powder, comprising the following steps:

[0050] The Chinese yam post-fermentation probiotic is synergistically matched with various compound peptides including but not limited to pea peptide, soybean oligopeptide, etc., such as Chinese yam post-fermentation probiotic powder, pea peptide powder and soybean oligopeptide, according to the mass ratio (1:1:1)-(1:2:2), put into a mixing machine, mix at 15-25rpm under the condition of relative humidity≤45%, temperature 20-25℃ for 20-30 minutes, and obtain the Chinese yam compound peptide powder.

[0051] Preferably, the slant medium uses a modified MRS medium with the following formulation: 4.0-7.0 g of proteose peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of potassium phosphate dibasic heptahydrate, 3.0-4.0 g of sodium acetate trihydrate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.05 g of manganese sulfate tetrahydrate, 12-15.0 g of agar, pH = 6.2 ± 0.2, and water added to 1-1.2 L.

[0052] Preferably, the liquid medium uses a modified MRS medium with the following formulation: 4.0-7.0 g of proteose peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of potassium phosphate dibasic heptahydrate, 3.0-4.0 g of sodium acetate trihydrate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.05 g of manganese sulfate tetrahydrate, pH = 6.2 ± 0.2, and water added to 1-1.2 L.

[0053] Preferably, the Chinese yam fermentation substrate is a mixture of Chinese yam and water, with a solid-liquid ratio of 1:1.5 (g:mL).

[0054] Use of the Chinese yam compound peptide preparation as described above in the preparation of a drug for invigorating the spleen and strengthening the five zang organs.

[0055] Preferably, the Chinese yam compound peptide powder improves spleen deficiency by regulating metabolic pathways through multiple targets, achieving the effect of simultaneous repair of the five zang organs through single spleen invigoration.

[0056] Preferably, the Chinese yam compound peptide powder can up-regulate the phosphopentose pathway, promote glutathione metabolism, regulate the neural-gut axis, and cooperatively repair organ function.

[0057] Specifically, the preparation and detection are as follows:

[0058] Example 1

[0059] This example is a preparation method of the Chinese yam fermentation postbiotic, and the steps are as follows:

[0060] S1, Paracaseolyticus IOB413 strain activation: inoculate the IOB413 strain in the frozen tube into the slant medium and place it in a 37 ± 0.5℃ incubator for 20 ± 2h of activation time. The activated colonies are white, round, moist, opaque, and have a neat edge. The bacterial cells are rod-shaped, uniform, and robust under microscopic examination, indicating successful activation.

[0061] The slant culture medium used was a modified MRS medium with the following formula: peptone 4.0-7.0 g, beef extract 4.0-7.0 g, yeast powder 5.0-7.0 g, glucose 13.0-18.0 g, Tween 80 0.8-1.0 mL, dipotassium hydrogen phosphate (7H2O) 1.5-2.0 g, sodium acetate (3H2O) 3.0-4.0 g, triammonium citrate 1.0-1.2 g, magnesium sulfate (7H2O) 0.1-0.2 g, manganese sulfate (4H2O) 0.03-0.05 g, agar 12-15.0 g, pH = 6.2±0.2, and the amount of water added was 1-1.2 L.

[0062] S2. Take a loop of fresh slant culture and inoculate it into a liquid culture medium containing 2% to 4% yam powder at a final concentration by mass. Culture it in a sealed container at 36±2°C for 20±2h to obtain the seed solution.

[0063] S3. The seed liquid under the optimal fermentation conditions is inoculated into the Chinese yam fermentation substrate (the Chinese yam fermentation substrate is a mixture of Chinese yam and water, and the Chinese yam: water material-liquid ratio g:mL is 1:1.5) at a volume-mass ratio of 5% (inoculation amount). The mixture is cultured in a closed state at 36±2°C for 24 to 48 hours. After fermentation, the mixture is inactivated at 80 to 95°C, dried to a moisture content of 7%, and then crushed to obtain Chinese yam fermentation biogenics.

[0064] Example 2

[0065] This example tested the content of short-chain fatty acids (acetic acid and propionic acid) in the fermented yam of unfermented yam and yam prepared in Example 1. The results are as follows: Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 It can be seen that the acetic acid content of the fermented yam obtained in Example 1 is increased by 16.82 times, and the propionic acid content is increased by 2.46 times compared with the unfermented yam.

[0066] Example 3

[0067] This embodiment is a method for preparing yam composite peptide powder, and the steps are as follows:

[0068] The Dioscorea opposita fermented probiotic powder, pea peptide powder and soybean oligopeptide prepared in Example 1 were put into a mixer in a mass ratio of 1:1:1, and mixed at a speed of 15-25 rpm for 20-30 minutes under the conditions of relative humidity ≤ 45% and temperature 20-25°C to obtain Dioscorea opposita composite peptide powder.

[0069] Application effect example 1: Study on the effect of Chinese yam composite peptide powder on improving spleen qi deficiency in rats

[0070] 1.1 Experimental Methods

[0071] 1.1.1 Experimental grouping

[0072] After the healthy rats were adaptively fed for 7 days, they were randomly divided into 7 groups: a blank control group, a model group, a positive control group, 3 protection groups, and 3 treatment groups.

[0073] 1.1.2 Establishment of a spleen deficiency and excessive dampness type hyperlipidemia rat model and intervention scheme

[0074] The protection groups were subjected to gavage with the Dioscorea opposita complex peptide powder for 30 days. The protection group 1 (the Dioscorea opposita postbiotic powder prepared in Example 1) was gavaged at a dose of 1.89 g / kg (body weight), the protection group 2 (the complex peptide powder prepared in Example 3) was gavaged at a dose of 1.89 g / kg (body weight), and the protection group 3 (the Dioscorea opposita complex peptide powder prepared in Example 3) was gavaged at a dose of 1.89 g / kg (body weight). The test samples were dissolved in 0.9% normal saline, and then gavaged. The remaining rats were normally fed (normal feed, normal feeding, and 22°C culture).

[0075] After the protection groups were fed for 30 days and the remaining groups were normally fed for 30 days, modeling was started. A 0.2 mg / mL reserpine solution was prepared by diluting reserpine with an appropriate amount of distilled water and acetic acid. Except for the blank group, the remaining groups were subjected to intramuscular injection of the reserpine solution at a dose of 0.2 mg / kg per day. The blank group was subcutaneously injected with the same dose of 0.85% normal saline. The modeling period was 7-10 days. After the modeling was successful, intervention was performed for 2 weeks. The specific intervention methods were as follows: the blank control group, the model group, and the three protection groups were gavaged with 1 mL / kg (body weight) of 0.85% normal saline; the positive control group was gavaged with 0.63 g / kg (body weight) of Shenling Baizhu Powder; the treatment group 1 was gavaged with 1.89 g / kg (body weight) of the Dioscorea opposita postbiotic powder prepared in Example 1; the treatment group 2 was gavaged with 1.89 g / kg (body weight) of the complex peptide powder prepared in Example 3; and the treatment group 3 was gavaged with 1.89 g / kg (body weight) of the Dioscorea opposita complex peptide powder prepared in Example 3. Gavage was performed twice a day, once in the morning and once in the evening.

[0076] 1.2 Index observation

[0077] 1.2.1 Kidney function index determination

[0078] After the serum was obtained, the aldosterone (ALD) and atrial natriuretic peptide (ANP) contents were detected according to the steps in the enzyme-linked immunosorbent assay kit instructions.

[0079] 1.2.2 Organ index

[0080] The organ index of the heart, liver, spleen, lung, and kidney was calculated. For example, the spleen index was calculated as follows: Spleen Index = [Spleen weight (mg) / Body weight (g)].

[0081] Spleen Index = [Spleen weight (mg) / Body weight (g)]

[0082] 1.2.3 Histopathological changes in the colon and stomach

[0083] Diarrhea caused by spleen deficiency can cause microscopic changes in the colon and stomach tissue. In the colon mucosa layer of the spleen deficiency model animals, there are inflammatory cell infiltration, obvious edema and congestion of the mucosa, irregular mucosa layer, and increased goblet cells.

[0084] The rat stomach and colon were placed in 4% paraformaldehyde for 48 hours or more, dehydrated by gradient, paraffin-embedded, and the wax block was placed in ice water, then sectioned (section thickness about 5 μm), the glass slide was taken out and dried, and then stained with HE. After washing for 10 minutes, dehydrate with 95% ethanol for 2 minutes, repeat washing twice, xylene transparency for 5 minutes, repeat washing twice, and seal with neutral gum. Observed under an optical microscope.

[0085] 1.2.4 Histological observation of organs

[0086] After weighing, each organ (heart, liver, spleen, lung, and kidney) was stained with periodic acid-Schiff reagent (PAS) and hematoxylin. Finally, the changes in the tissues of each organ were observed.

[0087] 1.2.5 Metabolic level detection

[0088] Through non-targeted metabolomics technology, the related serum biomarkers of spleen deficiency syndrome rats were studied, and the serum differential metabolites and the main involved metabolic pathways were obtained. The effects of Huaiyiyangcao compound peptide powder on energy metabolism, lipid metabolism, glucose metabolism, and amino acid metabolism in rats with spleen deficiency syndrome were analyzed.

[0089] 1.3 Statistical processing method

[0090] All data were expressed as mean ± standard deviation, and were plotted using GraphPad Prism 8 and Origin 2021 software.

[0091] 1.4 Experimental results

[0092] 1.4.1 Determination of kidney function indicators

[0093] For example, Figure 3Compared with the blank group (ALD content: 5.67 pg / mL, ANP content: 60.50 pg / mL), the ALD content (79.74 pg / mL) in the serum of the model group of rats was significantly increased, and the ANP content (11.17 pg / mL) was significantly reduced, indicating that the metabolism of the kidney of the rats in this group was blocked; compared with the model group, the ALD content in the serum of the protection group and the treatment group of rats was significantly reduced, and the ANP content was significantly increased, and the protection 3 group (ALD content: 22.70 pg / mL, ANP content: 80.50 pg / mL) and the treatment 3 group (ALD content: 27.52 pg / mL, ANP content: 63.50 pg / mL) had the best effect, indicating that the prepared Chinese yam composite peptide powder can effectively regulate kidney metabolism.

[0094] 1.4.2 Organ index

[0095] The five organs (heart, liver, spleen, lung and kidney) of the rats in each group were weighed, and the organ index was calculated, and the results are shown in Table 1. Compared with the blank group, the organ index of the model group of rats increased, indicating that the organs of the rats in this group appeared symptoms such as congestion, edema or hyperplasia; the organ index of the protection 3 group (heart: 3.68±0.57, liver: 36.65±4.68, spleen: 2.13±0.21, lung: 4.39±0.43, kidney: 7.23±0.64) and the treatment 3 group (heart: 3.48±0.40, liver: 33.15±4.26, spleen: 2.48±0.35, lung: 4.63±0.72, kidney: 7.52±0.99) returned to the level consistent with the blank group, indicating that the prepared Chinese yam composite peptide powder can effectively restore the health of the five organs of the spleen deficiency and dampness accumulation rats.

[0096] Compared with the recovery of the organ index (heart: 3.95±0.58, liver: 37.65±1.77, spleen: 2.27±0.39, lung: 4.82±0.71, kidney: 7.59±0.75) of the spleen deficiency and dampness accumulation rats of the Chinese patent CN120227439A Chinese yam composite peptide composition, the effect is more significant.

[0097] Table 1 Organ index statistics table of rats

[0098]

[0099] 1.4.3 PAS staining of each organ tissue

[0100] From Figure 4As shown in the results, there was obvious glycogen deposition in the five internal organs of the rats in the model group, indicating that the five internal organs of the rats in this group were damaged to varying degrees. Compared with the model group, the PAS staining colors of the five internal organs of the rats in the protection group 3 and the treatment group 3 were restored to the level basically consistent with that of the blank group, indicating that the Chinese yam compound peptide powder can effectively promote glycogen metabolism in the five internal organs of rats with spleen deficiency and dampness, and repair the damage to the five internal organs.

[0101] 1.4.4 Pathological morphology of colon and stomach tissues

[0102] like Figure 5 As shown in the results, compared with the model group, the mucosal and inflammatory cell infiltration of the colon and gastric tissues of the rats in the protection group and the treatment group were basically restored to the same level as the blank group, and the protection group 3 and the treatment group 3 had the best effects, indicating that the Chinese yam compound peptide powder can effectively improve the inflammation of the colon and gastric tissues in rats with spleen deficiency and dampness, further repair the damaged mucosal tissue, and restore gastrointestinal motility, and the effect of the protection group is stronger than that of the treatment group.

[0103] 1.4.5 Metabolic level detection

[0104] 1.4.5.1 Principal Component Analysis of QC Samples

[0105] as follows Figure 6 As shown in the PCA model diagram obtained after 7 rounds of cross-validation, the QC samples are closely clustered together, indicating that this experiment has good stability and repeatability.

[0106] 1.4.5.2 Venn diagram of differential metabolites

[0107] according to Figure 7 As shown, metabolite profiles varied significantly and were specific across groups. Comprehensive analysis revealed that inhibition of energy metabolism, such as the pentose phosphate pathway, and detoxification functions, such as cytochrome P450 and glutathione metabolism, was common across all groups. The model group served as a pivotal node (sharing 26 metabolites with multiple groups), and its widespread metabolic inhibition may have exacerbated systemic functional decline. Specific changes in the protection group (nucleotide metabolism inhibition) and the treatment group (sphingolipid signaling inhibition) suggest that their pathological mechanisms may focus on impaired DNA damage repair and imbalanced neural activity regulation, respectively.

[0108] 1.4.5.3 Differential metabolite clustering heat map

[0109] Figure 8 The figure shows that there are significant differences in the expression patterns of metabolites between the blank group and the positive control group. The metabolites listed in the figure are involved in various biological functions, including neurotransmitters, amino acid derivatives, fatty acid derivatives, etc., indicating different regulatory mechanisms in metabolic pathways between the two groups.

[0110] Figure 9The results showed that metabolites such as 3β,7α-dihydroxycholest-5-en-27-oic acid were significantly overexpressed in the treatment group 3 and underexpressed in the blank group, suggesting that the cholesterol metabolic pathway may be activated in the treatment group 3. Anserine and other amino acid derivatives were present at lower levels in the blank group, indicating that the amino acid metabolic pathway is inhibited. Methylone and alaptide were significantly overexpressed in the treatment group 3 and underexpressed in the blank group, suggesting that these metabolites may be involved in specific metabolic regulatory mechanisms. Significant differences in metabolic patterns were observed between the treatment group 3 and the blank group, indicating that metabolic regulatory mechanisms differ under physiological or pathological conditions.

[0111] Figure 10 Metabolites such as pymolidinecarboxaldehyde and 3-(1,3-benzodioxol-5-ylmethylene)-2-oxo- were significantly overexpressed in the blank group and relatively underexpressed in the protection group 3, suggesting that certain metabolic pathways may be activated in the blank group. The elevated expression of fatty acid metabolites such as 9-methoxy-pentadecanoic acid in the blank group may reflect enhanced fatty acid synthesis or degradation pathways. The lower levels of amino acid derivatives such as anserine in the protection group 3 may indicate that amino acid metabolic pathways are inhibited in the blank group. Significant differences in metabolic patterns were observed between the blank and protection groups, suggesting distinct metabolic regulatory mechanisms under physiological or pathological conditions.

[0112] 1.4.5.4 Volcano plot of differential metabolites

[0113] Figure 14 Upregulated metabolites and Figure 11 、 Figure 12 、 Figure 13 Compared with the model / positive control / treatment groups, the expression of the protective group was significantly increased, indicating that the upregulated metabolic pathways in the protection group 3 were also significantly increased compared with the model / positive control / treatment groups.

[0114] 1.4.5.5 KEGG pathway analysis of differential metabolites

[0115] like Figure 15 As shown, KEGG pathway enrichment analysis was performed on the differential metabolites in the serum of rats in each group. Figure 15In A of FIG. 1, the model group showed extensive inhibition of energy metabolism and biosynthetic pathways (e.g., downregulation of pentose phosphate pathway, fatty acid metabolism, amino acid metabolism such as cysteine and methionine, tryptophan metabolism, etc.), and potential weakening of neural transmission and signal regulation functions (e.g., downregulation of serotonergic synapse, neuroactive ligand-receptor interaction). At the same time, the model group also showed signs of impaired detoxification function (e.g., downregulation of cytochrome P450 metabolism). In contrast, the blank group showed specific enhancement of biosynthetic metabolism (e.g., upregulation of pyrimidine metabolism), and possible antioxidant and energy metabolism support (e.g., upregulation of taurine and hypotaurine metabolism).

[0116] Figure 15 In B of FIG. 1, the positive control group showed inhibition of energy metabolism (downregulation of pentose phosphate pathway, fatty acid synthesis) and impaired detoxification function (inhibition of cytochrome P450, ABC transporter); while the blank group showed enhancement of proliferative metabolism (upregulation of choline metabolism, pyrimidine synthesis).

[0117] Figure 15 In C of FIG. 1, the treatment 3 group showed extensive inhibition of energy metabolism and biosynthetic pathways (e.g., downregulation of pentose phosphate pathway, glycine / seryl / threonyl metabolism, glycerophospholipid metabolism), and potential weakening of neural activity and signal transmission (e.g., downregulation of serotonergic synapse, sphingolipid signaling pathway). At the same time, the treatment 3 group also showed signs of impaired detoxification function (e.g., downregulation of cytochrome P450 metabolism). In contrast, the blank group showed enhancement of proliferative and biosynthetic metabolism (e.g., upregulation of choline metabolism, pyrimidine metabolism), and possible enhancement of antioxidant capacity (e.g., upregulation of taurine and hypotaurine metabolism).

[0118] Figure 15 In D of FIG. 1, the protection 3 group showed inhibition of energy metabolism and biosynthesis (downregulation of pentose phosphate pathway, pyrimidine metabolism, purine metabolism, choline metabolism, and amino acid metabolism such as alanine, aspartate, glutamate, arginine, etc.), and impaired detoxification function (downregulation of glutathione metabolism); while the blank group showed specific enhancement of biosynthesis and metabolism (upregulation of taurine and hypotaurine metabolism, pantothenate and coenzyme A biosynthesis, histidine metabolism, etc.), and possible enhancement of neural activity (upregulation of neuroactive ligand-receptor interaction).

[0119] The experimental groups (Y / M / CZ / CB) all showed energy metabolism inhibition (down-regulation of the pentose phosphate pathway as the core) and detoxification dysfunction (inhibition of cytochrome P450 or glutathione metabolism), suggesting that reactive oxygen species (ROS) accumulation and decreased toxin clearance capacity may be common pathological mechanisms. Among them, the model group had the most extensive biosynthesis inhibition, involving comprehensive down-regulation of amino acid and fatty acid metabolism, which may directly weaken cell proliferation and repair capacity; the protection 3 group had the most serious metabolic disorder, with dual inhibition of nucleotide and glutathione metabolism or exacerbation of DNA damage and oxidative stress. The blank group maintained homeostasis through proliferation metabolism enhancement (pyrimidine and choline metabolism) and antioxidant support (taurine / hypotaurine metabolism). These differences may be driven by glutamine metabolism imbalance (affecting NADPH supply and amino acid turnover) and RORα rhythm regulation disorder (leading to temporal disorder of metabolic pathways).

[0120] It can also be seen through comparison that the mixture of the bioferment powder of the Chinese yam after fermentation and the pea peptide powder and the soybean oligopeptide in the present application has a synergistic effect between the two, which can synergistically improve the related performance of the prepared Chinese yam compound peptide powder.

[0121] Although the embodiments of the present application are disclosed for the purpose of illustration, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments.

Claims

1. A method for preparing a composite peptide preparation of Dioscorea opposita, characterized in that: The steps include: The preparation method of the fermented yam postbiotics comprises the following steps: S1. Activation of Lactobacillus paracasei IOB413 strain: Inoculate IOB413 from a cryopreserved tube into a slant culture medium and incubate at 37±0.5°C for 20±2 hours. Activated colonies should be white, round, moist, opaque, and have neat edges. Microscopic examination reveals rod-shaped, uniform, and robust cells, indicating successful activation. S2. Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2% to 4% yam powder at a final concentration by weight. Incubate in a sealed container at 36±2°C for 20±2 hours to obtain the seed solution. S3, inoculating the seed liquid into the Chinese yam fermentation substrate at an inoculum rate of 5%, culturing in a closed state at 36±2°C for 24-48 hours, inactivating at 80-95°C after fermentation, drying to a moisture content of ≤10%, and crushing to obtain Chinese yam fermentation biomass; The preparation method of the Chinese yam composite peptide powder comprises the following steps: The fermented yam and the compound peptide are synergistically mixed and put into a mixer, and mixed at a speed of 15-25 rpm for 20-30 minutes under the conditions of relative humidity ≤ 45% and temperature 20-25°C to obtain the yam compound peptide powder; The Chinese yam composite peptide preparation is Chinese yam fermentation postbiotics and Chinese yam composite peptide powder.

2. The preparation method according to claim 1, wherein: The slant culture medium uses a modified MRS culture medium, which has the following formula: 4.0-7.0 g of peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of dipotassium hydrogen phosphate heptahydrate, 3.0-4.0 g of sodium acetate trihydrate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.05 g of manganese sulfate tetrahydrate, 12-15.0 g of agar, pH=6.2±0.2, and the amount of water added is 1-1.2 L.

3. The preparation method according to claim 1, wherein: The liquid culture medium uses a modified MRS culture medium, which has the following formula: 4.0-7.0 g of peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of dipotassium hydrogen phosphate heptahydrate, 3.0-4.0 g of sodium acetate trihydrate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.05 g of manganese sulfate tetrahydrate, pH=6.2±0.2, and the amount of water added is 1-1.2 L.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The Chinese yam fermentation substrate is a mixture of Chinese yam and water, and the Chinese yam:water material-liquid ratio (g:mL) is 1:1.

5.

5. Use of the Dioscorea opposita composite peptide preparation prepared by the preparation method according to any one of claims 1 to 4 in the preparation of medicines for strengthening the spleen and five internal organs.

6. The use according to claim 5, characterized in that: The Chinese yam composite peptide powder improves spleen deficiency by regulating metabolic pathways through multiple targets, and achieves the effect of coordinated repair of the five internal organs by simply strengthening the spleen.

7. The use according to claim 6, characterized in that: The Chinese yam composite peptide powder can up-regulate the pentose phosphate pathway, promote glutathione metabolism, regulate the nerve-gut axis and coordinately repair organ functions.

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