A kind of Chinese yam composite peptide preparation, preparation method and its application in the preparation of spleen-strengthening and five-organ-strengthening Chinese medicine

Abstract

The application belongs to the technical field of traditional Chinese medicine and biological preparation, and discloses a Chinese yam compound peptide preparation, a preparation method and application of the Chinese yam compound peptide preparation in preparation of a medicine for invigorating the spleen and strengthening five internal organs. The preparation is a Chinese yam bioferment after fermentation and Chinese yam compound peptide powder. The Chinese yam compound peptide powder prepared by the application improves the whole body symptoms of a rat with hyperlipidemia of the spleen deficiency type through multi-target regulation of metabolic pathways, such as up-regulation of the phosphopentose pathway, promotion of glutathione metabolism, regulation of the nerve-intestine-axis and synergistic repair of organ functions, so as to achieve the effect of 'five internal organs synergistic repair' from'single spleen invigoration'. Preventive intervention (the protection group) reverses metabolic disorders more significantly than the treatment group by early regulation of glutamine metabolism and ROR alpha rhythm coordination, realizes multi-organ function repair, systematically improves the body damage caused by hyperlipidemia, and finally achieves the effect of invigorating the spleen and strengthening five internal organs, so as to achieve the effect of 'five internal organs synergistic repair' from metabolic regulation.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and biological agents, and in particular to a compound peptide preparation of Chinese yam, its preparation method and its application in the preparation of drugs for strengthening the spleen and five internal organs. Background Technology

[0002] In the field of contemporary biopharmaceutical research and development, intervention programs for spleen-deficiency-type metabolic disorders have long faced a systemic deficiency. While traditional Chinese medicine formulas such as Sijunzi Tang (Four Gentlemen Decoction) have spleen-strengthening effects, their mechanisms of action are mostly limited to improving digestive and absorptive functions. Current probiotic preparations suffer from problems such as bacterial inactivation due to the acidic environment of the stomach, making it difficult to maintain effective concentrations of key metabolites such as short-chain fatty acids in the intestines. More importantly, currently commercially available peptide supplements generally employ empirical formulation methods, lacking research on the synergistic mechanisms of action with microbial metabolites.

[0003] Existing solid-state fermentation processes exhibit significant shortcomings in handling special substrates such as Chinese yam. Firstly, Chinese patent CN110638035A provides a solid-state fermentation method for blueberry yam, but it does not compare the short-chain fatty acid content before and after fermentation. Secondly, Chinese patent CN111345348A involves conventional lactic acid bacteria liquid fermentation of yam pulp and milk; after fermentation, mixing with hawthorn sauce significantly increases the short-chain fatty acid content, but it does not compare the short-chain fatty acid content in the yam pulp before and after fermentation, making it impossible to rule out the influence of milk and hawthorn sauce on the short-chain fatty acid content. Thirdly, the post-biotic prepared in Chinese patent CN119999906A can effectively improve glucose and lipid metabolism disorders, alleviate liver damage, and effectively improve intestinal barrier function; however, it uses liquid fermentation and rice-like grains as the fermentation substrate, which differs from the present invention in both fermentation method and substrate.

[0004] In the field of peptide combination technology, existing products mostly focus on regulating single functional indicators. For example, the peptide composition prepared in Chinese patent CN117679324A exhibits anti-wrinkle activity and is used in skincare products, which differs from the application direction and mechanism of action of this invention. The KGT-C peptide in Chinese patent CN119320429A is designed only for dyslipidemia and fails to establish a synergistic model with the microbial metabolic network, making it difficult to meet the needs of modern preventive medicine for multi-target regulation. No similarities were found between the above patents and the preparation methods and processes of the Chinese yam powder and peptide composition in this invention.

[0005] Addressing the aforementioned technical bottlenecks, this invention utilizes *Lactobacillus paracasei* IOB413 for targeted fermentation of Chinese yam, successfully significantly increasing the acetic acid and propionic acid content in the fermentation products. More importantly, by establishing a precise formulation system of post-fermentation biotics and complex peptides, a bidirectional regulatory network of "microbial metabolism-host nutrition" is constructed. Animal experiments have confirmed that this composition not only upregulates the pentose phosphate pathway but also achieves synergistic repair of multiple organ functions by regulating RORα circadian rhythms. This systematic intervention strategy, addressing the root causes of metabolism, provides a novel approach for the development of new biological agents. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Chinese yam compound peptide preparation, a preparation method thereof, and its application in the preparation of drugs for strengthening the spleen and five internal organs.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] A compound peptide preparation of Chinese yam, wherein the preparation is a fermented Chinese yam extract and a compound peptide powder of Chinese yam.

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

[0010] The preparation method of the glycogen after fermentation of Chinese yam includes the following steps:

[0011] S1. Activation of Lactobacillus paracasei strain IOB413: Inoculate the IOB413 strain from the cryopreservation tube into an agar slant culture medium and incubate at 37±0.5℃ for 20±2h. Activated colonies are white, round, moist, opaque, and have neat edges. Microscopic examination shows that the bacteria are rod-shaped, uniform, and robust, indicating successful activation.

[0012] S2. Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2%–4% Chinese yam powder at a final mass concentration. Incubate at 36±2℃ in a sealed container for 20±2 hours to obtain the seed culture.

[0013] S3. Inoculate the seed liquid into the fermentation substrate of Chinese yam at an inoculation rate of 5%, and incubate in a sealed environment at 36±2℃ for 24 to 48 hours. After fermentation, inactivate the seed liquid at 80 to 95℃, dry it until the moisture content is ≤10%, and then crush it to obtain the fermented yam biogen.

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

[0015] The glycogen produced by fermentation of Chinese yam is synergistically combined with various complex peptides, including but not limited to pea peptides and soybean oligopeptides. For example, the glycogen powder produced by fermentation of Chinese yam, pea peptide powder and soybean oligopeptides are put into a mixer at a mass ratio of (1:1:1) to (1:2:2). Under the conditions of relative humidity ≤45% and temperature 20~25℃, the mixture is mixed at a speed of 15~25rpm for 20~30 minutes to obtain Chinese yam complex peptide powder.

[0016] Furthermore, the slant culture medium used is 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 heptahydrate 1.5–2.0 g, sodium acetate trihydrate 3.0–4.0 g, triammonium citrate 1.0–1.2 g, magnesium sulfate heptahydrate 0.1–0.2 g, manganese sulfate tetrahydrate 0.03–0.05 g, agar 12–15.0 g, pH = 6.2 ± 0.2, and water volume of 1–1.2 L.

[0017] Furthermore, the liquid culture medium used is 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 heptahydrate 1.5–2.0 g, sodium acetate trihydrate 3.0–4.0 g, triammonium citrate 1.0–1.2 g, magnesium sulfate heptahydrate 0.1–0.2 g, manganese sulfate tetrahydrate 0.03–0.05 g, pH=6.2±0.2, and water volume of 1–1.2 L.

[0018] Furthermore, the fermentation substrate of the Chinese yam is a mixture of Chinese yam and water, and the ratio of Chinese yam to water in g:mL is 1:1.5.

[0019] The above-mentioned application of the Chinese yam compound peptide preparation in the preparation of drugs for strengthening the spleen and five internal organs.

[0020] Furthermore, the Chinese yam compound peptide powder improves spleen deficiency by regulating metabolic pathways through multiple targets, achieving the effect of synergistic repair of the five internal organs by simply strengthening the spleen.

[0021] Furthermore, the Chinese yam compound peptide powder can upregulate the pentose phosphate pathway, promote glutathione metabolism, regulate the neuro-gut axis, and synergistically repair organ function.

[0022] The advantages and positive effects of this invention are as follows:

[0023] 1. This invention uses Lactobacillus paracasei IOB413 for solid-state fermentation of Chinese yam. By optimizing the fermentation conditions, at 36±2℃, the ratio of fermentation substrate to liquid (Chinese yam: water) is (1~2):(1.5~3.5), and the fermentation time is 24~48h. The fermented Chinese yam produces a glycogenase, which increases the content of short-chain fatty acids (acetic acid and propionic acid) by 16.82 times and the content of propionic acid by 2.46 times compared with unfermented Chinese yam.

[0024] 2. This invention synergistically combines fermented yam biogenic agents with various complex peptides, including but not limited to pea peptides and soybean oligopeptides. For example, fermented yam biogenic agent powder, pea peptide powder, and soybean oligopeptides are mixed in a mass ratio of (1:1:1) to (1:2:2) to obtain yam complex peptide powder, forming a bidirectional regulatory system of microbial metabolites superimposed on host nutrition. The novel biological agents obtained by the directional fermentation of yam biogenic agents and precise peptide formulation of this invention can be applied to the treatment of spleen deficiency and strengthening of the five internal organs.

[0025] 3. The compound peptide powder of Dioscorea opposita prepared in this invention improves the systemic symptoms of spleen-deficiency type hyperlipidemic rats by regulating metabolic pathways through multiple targets, such as upregulating the pentose phosphate pathway, promoting glutathione metabolism, regulating the neuro-gut axis, and synergistically repairing organ function, thereby achieving the effect of "synergistic repair of the five internal organs" from "single spleen strengthening". Moreover, the preventive intervention (protective group) significantly reversed metabolic disorders more significantly than the treatment group by regulating glutamine metabolism and coordinating RORα rhythm in the early stage, achieving multi-organ function repair, systematically improving the body damage caused by hyperlipidemia, and ultimately achieving the effect of strengthening the spleen and the five internal organs, thereby achieving the effect of "synergistic repair of the five internal organs" by regulating the metabolic root cause. Attached Figure Description

[0026] Figure 1 This is a graph showing the change in acetic acid content before and after fermentation in this invention;

[0027] Figure 2 This is a graph showing the change in propionic acid content before and after fermentation in this invention;

[0028] Figure 3 In this invention, A represents the level of aldosterone (ALD) in rat serum, and B represents the level of central natriuretic peptide (ANP) in rat serum.

[0029] Figure 4 These are PAS-stained images of the five internal organs of rats in each group of rats in this invention; wherein, A is a PAS-stained image of rat heart tissue; B is a PAS-stained image of rat liver tissue; C is a PAS-stained image of rat spleen tissue; D is a PAS-stained image of rat lung tissue; and E is a PAS-stained image of rat kidney tissue.

[0030] Figure 5Images of HE-stained colon and stomach tissues from rats in each group of this invention; wherein, A is an HE-stained image of rat colon tissue; B is an HE-stained image of rat stomach tissue;

[0031] Figure 6 This is a PCA diagram of serum metabolites in rats from each group in this invention;

[0032] Figure 7 This is a Venn diagram of serum metabolites from each group of rats in this invention;

[0033] Figure 8 This is a heatmap of differential metabolite clustering in the serum of rats in the positive control group and the blank group in this invention.

[0034] Figure 9 This is a heatmap of differential metabolite clustering in the serum of rats in the three treatment groups versus the blank group in this invention;

[0035] Figure 10 This is a heatmap of differential metabolite clustering in the serum of rats in the three protected groups versus the blank group in this invention;

[0036] Figure 11 This is a volcano diagram of differentially expressed metabolites in the serum of rats in the model group versus the blank group in this invention.

[0037] Figure 12 This is a volcano diagram of differentially expressed metabolites in the serum of rats in the positive control group and the blank group in this invention.

[0038] Figure 13 This is a volcano diagram of differential metabolites in the serum of rats in the three treatment groups versus the blank group in this invention;

[0039] Figure 14 This is a volcano diagram of differential metabolites in the serum of rats in the three protected groups versus the blank group in this invention;

[0040] Figure 15 The diagram shows the KEGG pathway analysis of differentially metabolized rat serum in each group in this invention; where A represents the KEGG pathway analysis of the model group vs. the blank group; B represents the KEGG pathway analysis of the positive control group vs. the blank group; C represents the KEGG pathway analysis of the three treatment groups vs. the blank group; and D represents the KEGG pathway analysis of the three protection groups vs. the blank group. Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0042] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0043] A compound peptide preparation of Chinese yam, wherein the preparation is a fermented Chinese yam extract and a compound peptide powder of Chinese yam.

[0044] The preparation method of the Chinese yam compound peptide preparation as described above includes the following steps:

[0045] The preparation method of the glycogen after fermentation of Chinese yam includes the following steps:

[0046] S1. Activation of Lactobacillus paracasei strain IOB413: Inoculate the IOB413 strain from the cryopreservation tube into an agar slant culture medium and incubate at 37±0.5℃ for 20±2h. Activated colonies are white, round, moist, opaque, and have neat edges. Microscopic examination shows that the bacteria are rod-shaped, uniform, and robust, indicating successful activation.

[0047] S2. Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2%–4% Chinese yam powder at a final mass concentration. Incubate at 36±2℃ in a sealed container for 20±2 hours to obtain the seed culture.

[0048] S3. Inoculate the seed liquid into the fermentation substrate of Chinese yam at an inoculation rate of 5%, and incubate in a sealed environment at 36±2℃ for 24 to 48 hours. After fermentation, inactivate the seed liquid at 80 to 95℃, dry it until the moisture content is ≤10%, and then crush it to obtain the fermented yam biogen.

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

[0050] The glycogen produced by fermentation of Chinese yam is synergistically combined with various complex peptides, including but not limited to pea peptides and soybean oligopeptides. For example, the glycogen powder produced by fermentation of Chinese yam, pea peptide powder and soybean oligopeptides are put into a mixer at a mass ratio of (1:1:1) to (1:2:2). Under the conditions of relative humidity ≤45% and temperature 20~25℃, the mixture is mixed at a speed of 15~25rpm for 20~30 minutes to obtain Chinese yam complex peptide powder.

[0051] Preferably, the slant culture medium is 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 heptahydrate 1.5–2.0 g, sodium acetate trihydrate 3.0–4.0 g, triammonium citrate 1.0–1.2 g, magnesium sulfate heptahydrate 0.1–0.2 g, manganese sulfate tetrahydrate 0.03–0.05 g, agar 12–15.0 g, pH = 6.2 ± 0.2, and water volume of 1–1.2 L.

[0052] Preferably, the liquid culture medium is a modified MRS medium with the following formula: 4.0–7.0 g peptone, 4.0–7.0 g beef extract, 5.0–7.0 g yeast extract, 13.0–18.0 g glucose, 0.8–1.0 mL Tween 80, 1.5–2.0 g dipotassium hydrogen phosphate heptahydrate, 3.0–4.0 g sodium acetate trihydrate, 1.0–1.2 g triammonium citrate, 0.1–0.2 g magnesium sulfate heptahydrate, 0.03–0.05 g manganese sulfate tetrahydrate, pH = 6.2 ± 0.2, and a water volume of 1–1.2 L.

[0053] Preferably, the fermentation substrate of Chinese yam is a mixture of Chinese yam and water, and the ratio of Chinese yam to water in g:mL is 1:1.5.

[0054] The above-mentioned application of the Chinese yam compound peptide preparation in the preparation of drugs for strengthening the spleen and five internal organs.

[0055] Preferably, the Chinese yam compound peptide powder improves spleen deficiency by regulating metabolic pathways through multiple targets, thereby achieving the effect of synergistic repair of the five internal organs by simply strengthening the spleen.

[0056] Preferably, the Chinese yam compound peptide powder can upregulate the pentose phosphate pathway, promote glutathione metabolism, regulate the neuro-gut axis, and synergistically repair organ function.

[0057] Specifically, the relevant preparation and testing methods are as follows:

[0058] Example 1

[0059] This embodiment describes a method for preparing glycogen after fermentation of Chinese yam, and the steps are as follows:

[0060] S1. Activation of Lactobacillus paracasei strain IOB413: Inoculate the IOB413 strain from the cryopreservation tube into an agar slant and incubate at 37±0.5℃ for 20±2h. Activated colonies are white, round, moist, opaque, and have neat edges. Microscopic examination shows that the bacteria are rod-shaped, uniform, and robust, 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 extract 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 water volume of 1–1.2 L.

[0062] S2. Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2%–4% Chinese yam powder at a final mass concentration. Incubate at 36±2℃ in a sealed container for 20±2 hours to obtain the seed culture.

[0063] S3. Inoculate the seed liquid with optimal fermentation conditions at a volume-to-mass ratio of 5% (inoculation amount) into the Huai yam fermentation substrate (the Huai yam fermentation substrate is a mixture of Huai yam and water, with a material-to-liquid ratio of Huai yam:water of 1:1.5 g:mL). Incubate at 36±2℃ in a sealed container for 24–48 hours. After fermentation, inactivate the fermented product at 80–95℃, dry it to a moisture content of 7%, and then pulverize it to obtain the Huai yam fermented biogen.

[0064] Example 2

[0065] This embodiment tested the content of short-chain fatty acids (acetic acid and propionic acid) in the glycogen after fermentation of unfermented Chinese yam and Chinese yam prepared in Example 1. The results are as follows: Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 As can be seen from the results, the fermented yam obtained in Example 1 had 16.82 times higher acetic acid content and 2.46 times higher propionic acid content compared to the unfermented yam.

[0066] Example 3

[0067] This embodiment describes a method for preparing compound peptide powder from Chinese yam, and the steps are as follows:

[0068] The fermented yam powder, pea peptide powder, and soybean oligopeptide obtained in Example 1 were added to a mixer at a mass ratio of 1:1:1. The mixture was stirred for 20 to 30 minutes at a speed of 15 to 25 rpm under conditions of relative humidity ≤45% and temperature 20 to 25℃ to obtain yam compound peptide powder.

[0069] Application Effect Example 1: Study on the Improvement of Spleen Qi Deficiency Syndrome in Rats by Chinese Yam Compound Peptide Powder

[0070] 1.1 Experimental Methods

[0071] 1.1.1 Experimental Grouping

[0072] After 7 days of adaptive feeding, healthy rats were randomly divided into 7 groups: blank control group, model group, positive control group, protection groups 1, 2, and 3, and treatment groups 1, 2, and 3.

[0073] 1.1.2 Establishment and intervention protocol of a rat model of hyperlipidemia with spleen deficiency and dampness accumulation

[0074] The protection groups were administered Huai yam compound peptide powder by gavage for 30 days. The gavage dose for protection group 1 (Huai yam post-regenerative powder prepared in Example 1) was 1.89 g / kg (body weight), the gavage dose for protection group 2 (compound peptide: pea peptide and soybean oligopeptide mass ratio 1:1) was 1.89 g / kg (body weight), and the gavage dose for protection group 3 (Huai yam compound peptide powder prepared in Example 3 of this invention) was 1.89 g / kg (body weight). All test samples were dissolved in 0.9% physiological saline before gavage. The remaining rats were fed normally (normal feed, normal feeding, cultured at 22℃).

[0075] After 30 days of feeding as described above in the protective group, and 30 days of normal feeding in the other groups, modeling was initiated. Reserpine was diluted with an appropriate amount of distilled water and acetic acid to prepare a 0.2 mg / mL reserpine solution. Except for the control group, rats in the other groups were administered the solution intramuscularly at a dose of 0.2 mg / kg daily. The control group received a subcutaneous injection of the same dose of 0.85% saline. The modeling period was 7–10 days. After successful modeling, intervention was initiated for 2 weeks. Specific intervention methods included: gavage administration of 1 mL / kg (body weight) of 0.85% saline to the control group, model group, and three protective groups; and gavage administration of 0.63 mg / kg (body weight) of 0.63% saline to the positive control group. The following treatments were administered: Ginseng and Atractylodes Macrocephala Powder (1.89 g / kg body weight); Chinese Yam Post-natal Essence Powder (prepared in Example 1, administered via gavage to Group 1); Composite peptide powder obtained by mixing pea peptides and soybean oligopeptides in a 1:1 mass ratio (1.89 g / kg body weight / gavage to Group 2, administered via gavage to Group 3); and Chinese Yam Composite Peptide Powder (prepared in Example 3 of this invention, administered via gavage to Group 3, administered via gavage to Group 3). All treatments were administered twice daily, once in the morning and once in the evening.

[0076] 1.2 Indicator Observation

[0077] 1.2.1 Measurement of renal function indicators

[0078] After obtaining serum, the levels of aldosterone (ALD) and atrial natriuretic peptide (ANP) were detected according to the instructions of the enzyme-linked immunosorbent assay kit.

[0079] 1.2.2 Organ Index

[0080] The organ indices for the five internal organs—heart, liver, spleen, lungs, and kidneys—are calculated using the spleen as an example; the formulas for calculating the indices of the other organs are the same.

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

[0082] 1.2.3 Pathological morphology of colon and stomach tissues

[0083] Diarrhea caused by spleen deficiency can cause microscopic changes in the colon and stomach tissues. In animals with spleen deficiency, the colonic mucosa shows inflammatory cell infiltration, significant mucosal edema and congestion, irregular mucosal layer, and increased goblet cells.

[0084] Rats' stomachs and colons were placed in 4% paraformaldehyde for more than 48 hours for graded dehydration, then embedded in paraffin. The paraffin blocks were placed in ice water, followed by sectioning (approximately 5 μm thick). The slides were then removed and dried to remove surface moisture. The slides were stained with hematoxylin and eosin (HE), washed for 10 min, dehydrated in 95% ethanol for 2 min (repeated twice), cleared in xylene for 5 min (repeated twice), mounted with neutral resin, and 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 iodic 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] We used non-targeted metabolomics to study the relevant serum biomarkers of rats with spleen deficiency syndrome, obtained the differentially expressed serum metabolites and the main metabolic pathways involved, and analyzed the effects of Chinese yam compound peptide powder on energy metabolism, lipid metabolism, glucose metabolism and amino acid metabolism in rats with spleen deficiency syndrome.

[0089] 1.3 Data Statistical Processing Methods

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

[0091] 1.4 Experimental Results

[0092] 1.4.1 Measurement of renal function indicators

[0093] like Figure 3As shown, compared with the blank group (ALD content: 5.67 pg / mL, ANP content: 60.50 pg / mL), the serum ALD content (79.74 pg / mL) of the model group rats was significantly increased, and the ANP content (11.17 pg / mL) was significantly decreased, indicating that the renal metabolism of the rats in this group was impaired. Compared with the model group, the serum ALD content of the protection group and the treatment group rats was significantly decreased, and the ANP content was significantly increased. The protection group 3 (ALD content: 22.70 pg / mL, ANP content: 80.50 pg / mL) and the treatment group 3 (ALD content: 27.52 pg / mL, ANP content: 63.50 pg / mL) showed the best effects, indicating that the Chinese yam compound peptide powder prepared in this invention can effectively regulate renal metabolism.

[0094] 1.4.2 Organ Index

[0095] The organs (heart, liver, spleen, lung, and kidney) of rats in each group were weighed, and the organ indices were calculated. The results are shown in Table 1. Compared with the blank group, the organ indices of rats in the model group increased, indicating that the organs of rats in this group showed symptoms such as congestion, edema, or hyperplasia and hypertrophy. The organ indices of rats in the protection group 3 (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 group 3 (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) recovered to the same level as the blank group, indicating that the Chinese yam compound peptide powder prepared in this invention can effectively restore the health of the five organs of rats with spleen deficiency and dampness.

[0096] Compared with the results of the Chinese patent CN120227439A on the recovery of organ indices (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) in rats with spleen deficiency and dampness, the effect is more significant.

[0097] Table 1. Statistical table of organ indices in rats

[0098]

[0099] 1.4.3 PAS staining of various organ tissues

[0100] Depend on Figure 4As shown, there was obvious glycogen deposition in the five internal organs of the model group rats, 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 color of the five internal organs of the protected group 3 and the treated group 3 rats recovered to the same level as 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, compared with the model group, the colonic and gastric tissue mucosa and inflammatory cell infiltration of rats in the protection group and treatment group were basically restored to the same level as the blank group. The protection group 3 and treatment group 3 showed the best effects, indicating that the Chinese yam compound peptide powder can effectively improve the inflammation of the colonic and gastric tissues of rats with spleen deficiency and dampness, further repair the damaged mucosal tissue, restore gastrointestinal motility, and the protection group has a stronger effect than 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 cycles of cross-validation, the QC samples are closely clustered together, indicating that the experiment has good stability and repeatability.

[0106] 1.4.5.2 Venn diagram of differential metabolites

[0107] according to Figure 7 As shown, significant and specific differences in metabolites exist among different groups. Comprehensive analysis reveals that inhibition of energy metabolism, such as the pentose phosphate pathway, and detoxification functions, such as cytochrome P450 and glutathione metabolism, are common across groups. The model group, acting as a pivotal node (sharing 26 metabolites with multiple groups), may exacerbate systemic functional decline due to its widespread metabolic inhibition. The specific changes observed in the protective group 3 (nucleotide metabolism inhibition) and the treatment group 3 (sphingolipid signaling inhibition) suggest that their pathological mechanisms may focus on impaired DNA damage repair and imbalanced regulation of neural activity, respectively.

[0108] 1.4.5.3 Clustering Heatmap of Differential Metabolites

[0109] Figure 8 The study showed significant differences in metabolite expression patterns between the blank control group and the positive control group. The metabolites listed in the figure involve multiple biological functions, including neurotransmitters, amino acid derivatives, and fatty acid derivatives, 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 treatment group 3, but underexpressed in the control group, suggesting that the cholesterol metabolism pathway may be activated in treatment group 3. Anserine and other amino acid derivatives were present at lower levels in the control group, indicating that the amino acid metabolism pathway was inhibited. Methylone and Alaptide were significantly overexpressed in treatment group 3, but underexpressed in the control group, suggesting that these metabolites may be involved in specific metabolic regulatory mechanisms. The significant differences in metabolic patterns between treatment group 3 and the control group indicate different metabolic regulatory mechanisms under physiological or pathological conditions.

[0111] Figure 10 The results showed that metabolites such as Pymolidinecarboxaldehyde and 3-(1,3-benzodioxol-5-ylmethylene)-2-oxo- were significantly overexpressed in the control group but relatively underexpressed in the protection group 3, suggesting that certain metabolic pathways may be activated in the control group. The increased levels of fatty acid metabolites such as 9-Methoxy-pentadecanoic acid in the control 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 were inhibited in the control group. The significant differences in metabolic patterns between the control group and the protection group 3 suggest different metabolic regulatory mechanisms under physiological or pathological conditions.

[0112] 1.4.5.4 Volcano Plot of Differential Metabolites

[0113] Figure 14 Upregulation of metabolites and Figure 11 , Figure 12 , Figure 13 Compared to the model / positive control / treatment groups, the upregulation of metabolic pathways in the protective group 3 was significantly increased, indicating that the upregulation of metabolic pathways in the protective group 3 was also significantly increased compared to the model / positive control / treatment groups 3.

[0114] 1.4.5.5 Analysis of the differentially metabolized KEGG pathway

[0115] like Figure 15 As shown, KEGG pathway enrichment analysis was performed on differentially metabolites in the serum of rats from each group. Figure 15In group A, the model group exhibited widespread inhibition of energy metabolism and biosynthetic pathways (e.g., downregulation of the pentose phosphate pathway, fatty acid metabolism, amino acid metabolism such as cysteine ​​and methionine, and tryptophan metabolism), as well as potential weakening of neurotransmission and signal regulation functions (e.g., downregulation of serotonergic synapses and neuroactive ligand-receptor interactions). Simultaneously, the model group also showed signs of impaired detoxification function (e.g., downregulation of cytochrome P450 metabolism). In contrast, the control group showed enhanced specific biosynthetic metabolism (e.g., upregulation of pyrimidine metabolism), and potential support for antioxidant and energy metabolism (e.g., upregulation of taurine and alpha-taurine metabolism).

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

[0117] Figure 15 In group C, treatment group 3 showed widespread inhibition of energy metabolism and biosynthetic pathways (e.g., downregulation of the pentose phosphate pathway, glycine / serine / threonine metabolism, and glycerophospholipid metabolism), as well as potential impairment of neural activity and signal transduction (e.g., downregulation of serotonergic synapses and sphingolipid signaling pathways). Simultaneously, treatment group 3 also showed signs of impaired detoxification function (e.g., downregulation of cytochrome P450 metabolism). In contrast, the control group showed enhanced proliferation and biosynthetic metabolism (e.g., upregulation of choline metabolism and pyrimidine metabolism), as well as potential enhanced antioxidant capacity (e.g., upregulation of taurine and alpha-taurine metabolism).

[0118] Figure 15 In group D, the protection group 3 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, aspartic acid, glutamic acid, arginine, etc.), as well as impaired detoxification function (downregulation of glutathione metabolism); while the blank group showed enhancement of specific biosynthesis and metabolism (upregulation of taurine and taurine metabolism, pantothenic acid and coenzyme A biosynthesis, histidine metabolism, etc.), as well as possible enhancement of neuroactive activity (upregulation of neuroactive ligand-receptor interaction).

[0119] All experimental groups (Y / M / CZ / CB) exhibited inhibition of energy metabolism (primarily due to downregulation of the pentose phosphate pathway) and impaired detoxification function (inhibition of cytochrome P450 or glutathione metabolism), suggesting that the accumulation of reactive oxygen species (ROS) and decreased toxin clearance may be common pathological mechanisms. Among them, the model group showed the most extensive inhibition of biosynthesis, involving comprehensive downregulation of amino acid and fatty acid metabolism, which may directly weaken cell proliferation and repair capabilities. The three protective groups showed the most severe metabolic disorders, with dual inhibition of nucleotide and glutathione metabolism, potentially exacerbating DNA damage and oxidative stress. The control group maintained homeostasis through enhanced proliferation metabolism (pyrimidine and choline metabolism) and antioxidant support (taurine / hypoturine metabolism). These differences may be driven by a combination of glutamine metabolic imbalance (affecting NADPH supply and amino acid turnover) and RORα rhythm dysregulation (leading to temporal disruption of metabolic pathways).

[0120] The comparison also shows that the mixture of fermented Chinese yam gluten powder, pea peptide powder and soybean oligopeptide in this invention has a synergistic effect, which can synergistically improve the relevant properties of the prepared Chinese yam composite peptide powder.

[0121] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a compound peptide preparation of Dioscorea opposita, characterized in that: The preparation is a fermented yam extract and a compound peptide powder of yam; The preparation includes the following steps: The preparation method of the glycogen after fermentation of Chinese yam includes the following steps: S1. Activation of Lactobacillus paracasei strain IOB413: Inoculate the IOB413 strain from the cryopreservation tube into an agar slant culture medium and incubate at 37±0.5℃ for 20±2h. Activated colonies are white, round, moist, opaque, and have neat edges. Microscopic examination shows that the bacteria are rod-shaped, uniform, and robust, indicating successful activation. S2. Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2% to 4% Chinese yam powder. Incubate at 36±2℃ in a sealed container for 20±2 hours to obtain the seed culture. S3. Inoculate the seed liquid into the fermentation substrate of Chinese yam at an inoculation rate of 5%, and incubate in a closed environment at 36±2℃ for 24-48 hours. After fermentation, inactivate the seed liquid at 80-95℃, dry it to a moisture content of ≤10%, and pulverize it to obtain the glycogen after fermentation of Chinese yam. The slant culture medium used is 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 heptahydrate 1.5–2.0 g, sodium acetate trihydrate 3.0–4.0 g, triammonium citrate 1.0–1.2 g, magnesium sulfate heptahydrate 0.1–0.2 g, manganese sulfate tetrahydrate 0.03–0.05 g, agar 12–15.0 g, pH = 6.2 ± 0.2, and water volume of 1–1.2 L. The liquid culture medium used is 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 heptahydrate 1.5–2.0 g, sodium acetate trihydrate 3.0–4.0 g, triammonium citrate 1.0–1.2 g, magnesium sulfate heptahydrate 0.1–0.2 g, manganese sulfate tetrahydrate 0.03–0.05 g, pH = 6.2 ± 0.2, and water volume of 1–1.2 L. The preparation method of the Chinese yam compound peptide powder includes the following steps: After fermentation, the raw material powder of Chinese yam, pea peptide powder and soybean oligopeptide are added to a mixer in a mass ratio of 1:1:1 to 1:2:

2. The mixture is stirred for 20 to 30 minutes at a speed of 15 to 25 rpm under conditions of relative humidity ≤45% and temperature 20 to 25℃ to obtain Chinese yam compound peptide powder.

2. The preparation method according to claim 1, characterized in that: The fermentation substrate of Huai yam is a mixture of Huai yam and water, and the ratio of Huai yam to water in g:mL is 1:1.5.

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