Ginseng peptide and its preparation method and application

Abstract

The present invention relates to a ginseng peptide and a preparation method and application thereof, belonging to the field of biotechnology. The present invention provides a ginseng peptide, wherein the ginseng peptide comprises a characteristic peptide having an amino acid sequence of at least one of LY, MM, LR, AP, LAR, RLDFR, SALAFR, LLLLGH and LLLLLHG. The present invention obtains ginseng peptides by treating fermented ginseng residue, and identifies the characteristic peptides containing the amino acid sequences of LY, MM, LR, AP, LAR, RLDFR, SALAFR, LLLLGH and LLLLLHG in the ginseng peptides by HPLC-MS / MS. The present invention confirms through experiments that ginseng peptides and fermented ginseng hydrolysates containing ginseng peptides have excellent efficacy in inhibiting the differentiation of bone marrow mononuclear cells into osteoclasts, can improve hormone-induced osteoporosis, and can be applied to products for improving osteoporosis and its related symptoms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a ginseng peptide and a preparation method and application thereof. Background Art

[0002] Ginseng (Panax ginseng CA Meyer), a perennial herbaceous plant of the genus Panax L. in the Araliaceae family, is also known as the "King of Herbs" and has a long history of use. It is one of the most valuable medicinal plants in traditional medicine. Since its inclusion in the list of new resource foods in 2012, its applications have continued to expand, and demand has increased. Ginseng processing techniques are becoming increasingly diverse. In addition to traditional decoction, industrial extraction and microbial fermentation methods all produce large amounts of ginseng residue. Currently, ginseng residue is primarily used as organic fertilizer and animal feed, or directly discharged into the environment. This resource utilization rate is not maximized, and there is still a lack of high-value-added, high-return ginseng residue recycling methods.

[0003] Osteoporosis is a common bone metabolic disease characterized by pathological changes such as decreased bone mass and abnormal bone microarchitecture, ultimately leading to thinning, brittle bones, and increased risk of fracture. It is predominantly seen in middle-aged and elderly individuals. The disease is caused by a phenomenon in which bone resorption exceeds bone formation. This imbalance can be triggered by factors such as hormonal changes, malnutrition, lack of exercise, smoking, excessive alcohol consumption, and drug use. Early symptoms are often asymptomatic, and complications such as fractures or physical deformities are not detected, severely impacting quality of life. Treatment strategies involve promoting bone formation and inhibiting bone resorption. Commonly used drugs include calcium supplements, vitamin D, growth hormone, glucocorticoid antagonists, calmodulin, bisphosphonates, estrogen, and RANKL inhibitors. These drugs are often used in combination to enhance efficacy, but long-term use can cause side effects such as hypercalcemia, vascular sclerosis, kidney stones, diabetes, joint pain, gastrointestinal discomfort, and muscular dystrophy. Therefore, the development of new, safe, and effective treatment strategies is crucial for patients with osteoporosis. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a ginseng peptide and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a ginseng peptide, comprising a characteristic peptide having an amino acid sequence of at least one of Leu-Tyr, Met-Met, Leu-Arg, Leu-Ala-Arg, Ser-Ala-Leu-Ala-Phe-Arg, Arg-Leu-Asp-Phe-Arg, Leu-Leu-Leu-Leu-Gly-His, Leu-Leu-Leu-Leu-Leu-Gly-His and Ala-Pro.

[0007] The invention obtains ginseng peptides by treating fermented ginseng residue, and identifies the ginseng peptides as containing characteristic peptides with amino acid sequences of LY, MM, LR, AP, LAR, RLDFR, SALAFR, LLLLGH and LLLLLHG through HPLC-MS / MS.

[0008] As a preferred embodiment of the ginseng peptide of the present invention, the ginseng peptide includes a characteristic peptide with an amino acid sequence of Leu-Tyr, a characteristic peptide of Met-Met, a characteristic peptide of Leu-Arg, a characteristic peptide of Leu-Ala-Arg, a characteristic peptide of Ser-Ala-Leu-Ala-Phe-Arg, a characteristic peptide of Arg-Leu-Asp-Phe-Arg, a characteristic peptide of Leu-Leu-Leu-Leu-Gly-His, a characteristic peptide of Leu-Leu-Leu-Leu-Leu-His-Gly and a characteristic peptide of Ala-Pro.

[0009] In a second aspect, the present invention provides a fermented ginseng hydrolyzate comprising the above-mentioned ginseng peptide.

[0010] As a preferred embodiment of the fermented ginseng hydrolyzate of the present invention, the mass ratio of the characteristic peptide Leu-Tyr, characteristic peptide Met-Met, characteristic peptide Leu-Arg, characteristic peptide Leu-Ala-Arg, characteristic peptide Ser-Ala-Leu-Ala-Phe-Arg, characteristic peptide Arg-Leu-Asp-Phe-Arg, characteristic peptide Leu-Leu-Leu-Leu-Gly-His, characteristic peptide Leu-Leu-Leu-Leu-Leu-His-Gly and characteristic peptide Ala-Pro of ginseng peptide in the fermented ginseng hydrolyzate is Leu-Tyr:Met-Met. et:Leu-Arg:Leu-Ala-Arg:Ser-Ala-Leu-Ala-Phe-Arg:Arg-Leu-Asp-Phe-Arg:Leu-Leu-Leu-Leu-Gly-His:Leu-Leu-Leu-Leu-Leu -Gly-His: Ala-Pro=(49.6-52.1): (6.3-7.6): (7.5-8.8): (1.5-1.8): (18.1-19.5): (2.6-3.1): (1.4-1.6): (1.2-1.5): (7.8-8.0).

[0011] As a preferred embodiment of the fermented ginseng hydrolyzate of the present invention, the characteristic peptide Leu-Tyr, characteristic peptide Met-Met, characteristic peptide Leu-Arg, characteristic peptide Leu-Ala-Arg, characteristic peptide Ser-Ala-Leu-Ala-Phe-Arg, characteristic peptide Arg-Leu-Asp-Phe-Arg, characteristic peptide Leu-Leu-Leu-Leu-Gly-His, characteristic peptide Leu-Leu-Leu-Leu-Leu-His-Gly and The mass ratio of the characteristic peptide Ala-Pro is Leu-Tyr: Met-Met: Leu-Arg: Leu-Ala-Arg: Ser-Ala-Leu-Ala-Phe-Arg: Arg-Leu-Asp-Phe-Arg: L eu-Leu-Leu-Leu-Gly-His: Leu-Leu-Leu-Leu-Leu-Gly-His: Ala-Pro=49.6:7.6:8.8:1.5:19.5:2.6:1.4:1.2:7.8.

[0012] In a third aspect, the present invention provides a method for preparing the fermented ginseng hydrolyzate, comprising the following steps:

[0013] (1) Mixing the fermented ginseng residue with water, adjusting the pH to 7.0-8.0, extracting at 45-55°C for 30-40 minutes, adding protease, bathing at 45-55°C for 4-5 hours, and centrifuging to obtain an enzymatic hydrolyzate;

[0014] (2) Drying the enzymatic hydrolyzate obtained in step (2) to obtain a fermented ginseng hydrolyzate containing ginseng peptides.

[0015] The present invention extracts and enzymatically hydrolyzes fermented ginseng residue to obtain fermented ginseng hydrolyzate, and concentrates and dries the fermented ginseng hydrolyzate to prepare fermented ginseng hydrolyzate containing ginseng peptides. In vivo and in vitro experiments have verified that the fermented ginseng hydrolyzate has excellent efficacy in inhibiting the differentiation of bone marrow mononuclear cells into osteoclasts, and can improve hormone-induced osteoporosis.

[0016] As a preferred embodiment of the preparation method of the present invention, in step (1), the ratio of the fermented ginseng residue to water is fermented ginseng residue: water = 1 g: (25-30) mL.

[0017] As a preferred embodiment of the preparation method of the present invention, in step (2), the type of protease includes at least one of papain, trypsin and alkaline protease.

[0018] As a preferred embodiment of the preparation method of the present invention, in step (2), the amount of the protease added is 0.75-0.80 mg of protease per 1 g of fermented ginseng residue, and the concentration of the protease is 1350-1400 U / mg.

[0019] As a preferred embodiment of the preparation method of the present invention, in step (1), the fermented ginseng residue is obtained by centrifuging and filtering ginseng after microbial fermentation.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned ginseng peptides or fermented ginseng hydrolysates in the preparation of products for improving osteoporosis and its complications.

[0021] In vivo and in vitro experiments have confirmed that the ginseng peptides and fermented ginseng hydrolysates provided by the present invention can significantly increase resistance to osteoporosis.

[0022] In a fifth aspect, the present invention provides the use of the above-mentioned ginseng peptides or fermented ginseng hydrolysates in the preparation of products for preventing osteoporosis and its complications.

[0023] As a preferred embodiment of the application of the present invention, the products include medicines, foods, health products, cosmetics and biological products.

[0024] As a preferred embodiment of the application of the present invention, the dosage of the fermented ginseng hydrolyzate is 250-1000 mg per 1 kg body weight of the organism.

[0025] As a preferred embodiment of the application of the present invention, the osteoporosis includes primary osteoporosis, secondary osteoporosis, idiopathic osteoporosis and regional osteoporosis. The primary osteoporosis is postmenopausal osteoporosis and senile osteoporosis; the secondary osteoporosis is osteoporosis caused by endocrine diseases (hyperparathyroidism, Cushing's syndrome, diabetes), chronic diseases (chronic kidney disease, liver disease, chronic obstructive pulmonary disease), drug use (glucocorticoids, antiepileptic drugs, anticoagulants), and lifestyle (long-term alcoholism, smoking, malnutrition, lack of exercise); the idiopathic osteoporosis is of unknown cause, usually occurring in young people or children, with no clear secondary cause; and the regional osteoporosis is caused by local ischemia, trauma, infection or nervous system disease.

[0026] As a preferred embodiment of the application of the present invention, the ginseng peptides and fermented ginseng hydrolyzate have the effect of improving osteoporosis by relieving bone pain and back pain, correcting hunchback and body posture changes, enhancing exercise motivation, and preventing fractures and disabilities.

[0027] As a preferred embodiment of the application of the present invention, the improvement effects of the ginseng peptides and fermented ginseng hydrolyzate on osteoporosis are manifested in the following aspects: increasing bone density, reducing trabecular spaces, increasing the number of trabecular connections, reducing trabecular fractures, and improving bone mineralization.

[0028] As a preferred embodiment of the application of the present invention, the improvement effect of the ginseng peptide and fermented ginseng hydrolyzate on osteoporosis is also reflected in inhibiting the differentiation of osteoclasts and reducing the levels of osteoclast markers, including but not limited to serum calcium, serum phosphorus, serum 25-hydroxyvitamin D, serum alkaline phosphatase, and serum PTH.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention obtains ginseng peptides by treating fermented ginseng residue, and identifies the ginseng peptides as containing characteristic peptides with amino acid sequences of LY, MM, LR, AP, LAR, RLDFR, SALAFR, LLLLGH and LLLLLHG by HPLC-MS / MS.

[0031] 2. The present invention has experimentally confirmed that ginseng peptides and fermented ginseng hydrolyzates containing ginseng peptides have excellent efficacy in inhibiting the differentiation of bone marrow mononuclear cells into osteoclasts, can improve hormone-induced osteoporosis, and have the advantages of being safe, long-term, and having no side effects. They can be used in products that improve osteoporosis and its related symptoms, and have broad application prospects and economic value.

[0032] 3. The ginseng peptides and fermented ginseng hydrolysates provided by the present invention have a small molecular weight and are easily absorbed, and have relatively few application restrictions on elderly osteoporosis patients with poor gastrointestinal function.

[0033] 4. The fermented ginseng hydrolyzate of the present invention is derived from ginseng residue after microbial fermentation, which can improve the utilization rate of ginseng and reduce resource waste and environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the liquid-to-mass BPC diagram of the fermented ginseng hydrolyzate in Example 1 of the present invention;

[0035] Figure 2 This is the secondary mass spectrum of the LY peptide segment in Example 1 of the present invention;

[0036] Figure 3 This is the secondary mass spectrum of the MM peptide segment in Example 1 of the present invention;

[0037] Figure 4 This is the secondary mass spectrum of the LR peptide segment in Example 1 of the present invention;

[0038] Figure 5 This is the secondary mass spectrum of the LAR peptide segment in Example 1 of the present invention;

[0039] Figure 6 This is the secondary mass spectrum of the SALAFR peptide segment in Example 1 of the present invention;

[0040] Figure 7 This is the secondary mass spectrum of the RLDFR peptide segment in Example 1 of the present invention;

[0041] Figure 8 This is the secondary mass spectrum of the LLLLGH peptide segment in Example 1 of the present invention;

[0042] Figure 9 This is the secondary mass spectrum of the LLLLLHG peptide segment in Example 1 of the present invention;

[0043] Figure 10 This is the secondary mass spectrum of the AP peptide segment in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0045] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.

[0046] The ginseng species used in the ginseng peptides and fermented ginseng hydrolysates of the present invention are not limited. The fermented ginseng residue used in the following examples and comparative examples is prepared by subjecting ginseng to microbial fermentation according to the method of Example 1 in the technology disclosed in CN 116334147 A, followed by centrifugal filtration after the fermentation is completed.

[0047] SPF female C57BL / 6 mice were purchased from Guangdong Medical Laboratory Animal Center (SCXK (Yue) 2022-0002).

[0048] The techniques not described in detail in the following examples, comparative examples and effect examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0049] In the following examples and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine ​​(Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), and "T" refers to threonine (Thr).

[0050] In the following examples, comparative examples and effect examples, LY refers to the Leu-Tyr characteristic peptide, MM refers to the Met-Met characteristic peptide, LR refers to the Leu-Arg characteristic peptide, LAR refers to the Leu-Ala-Arg characteristic peptide, SALAFR refers to the Ser-Ala-Leu-Ala-Phe-Arg characteristic, RLDFR refers to the Arg-Leu-Asp-Phe-Arg characteristic peptide, LLLLGH refers to the Leu-Leu-Leu-Leu-Gly-His characteristic peptide, LLLLLHG refers to the Leu-Leu-Leu-Leu-Leu-His-Gly characteristic peptide, and AP refers to the Ala-Pro characteristic peptide. The amino acid sequences of the above characteristic peptides are shown in SEQ ID NO.1-9.

[0051] Example 1

[0052] This embodiment provides a fermented ginseng hydrolyzate and a preparation method thereof, the preparation method comprising the following steps:

[0053] S1. Mix the fermented ginseng residue with water at a solid-liquid ratio of 1 g:30 mL, adjust the pH to 8.0 with 2 M NaOH solution, extract at 50°C for 30 min, add 0.00075 g of trypsin (concentration of 1350 U / mg) per 1 g of fermented ginseng residue, hydrolyze at 50°C for 5 h, incubate in a water bath at 85°C for 30 min, cool to room temperature, and centrifuge at 10,000 rpm for 10 min. Collect the supernatant to obtain the enzymatic hydrolyzate;

[0054] S2. The enzymatic hydrolyzate obtained in step S1 was concentrated under reduced pressure at 65° C. and −0.09 MPa, and freeze-dried to obtain the fermented ginseng hydrolyzate.

[0055] Effect Example 1

[0056] The ginseng obtained in Example 1, the fermented ginseng residue, and the fermented ginseng hydrolyzate were characterized as follows:

[0057] 1. Determination of ginsenosides

[0058] Determination of ginsenosides in ginseng, fermented ginseng residue and fermented ginseng peptides by HPLC. Waters UPLC liquid chromatograph, chromatographic column: Waters XBridge BEH Shield C18 2.1mm×100mm×1.7μm, column temperature: 40℃, detection wavelength: 203nm, flow rate: 0.6mL / min, injection volume: 2μL, mobile phase composed of water (A) and acetonitrile (B), elution method: 0-0.5min 15% B, 40.5-14.5min 15-30% B, 14.5-15.5min 30-32% B, 15.5-18.5min 32-38% B, 18.5-24.0min 38-43% B, 24.0-27.0min 43.0-55.0% B, 27.0-31.0min, 55% B, 31.0-35.0min, 55-70% B, 35.0-38.0min, 70-90% B.

[0059] Preparation of reference solution: Weigh 10.35 mg of ginsenoside Rg1, 10.25 mg of ginsenoside Re, 10.08 mg of ginsenoside Rb1, 10.07 mg of ginsenoside Rd, 10.70 mg of ginsenoside Rc, and 10.02 mg of ginsenoside Rb2 respectively, add appropriate amount of methanol to dissolve them, make the volume to 5 mL, and shake well. Add appropriate amount of methanol to dissolve 3.16 mg of ginsenoside Rf, make the volume to 1.5 mL, and shake well. Accurately pipette 1 mL of the above solution into a 20 mL volumetric flask, dilute to the mark with methanol and shake well to obtain a reference solution with a ginsenoside Rg1 concentration of 103.5 μg / mL, a ginsenoside Re concentration of 102.5 μg / mL, a ginsenoside Rb1 concentration of 100.8 μg / mL, a ginsenoside Rd concentration of 100.7 μg / mL, a ginsenoside Rf concentration of 105.35 μg / mL, a ginsenoside Rc concentration of 107.0 μg / mL, and a ginsenoside Rb2 concentration of 100.2 μg / mL.

[0060] Preparation of sample solution: Take about 1g each of ginseng powder (passed through No. 4 sieve), fermented ginseng residue, and fermented ginseng peptide, accurately weigh, place in a 20mL volumetric flask, add appropriate amount of methanol, and treat with ultrasound (500W, 37kHz) for 30min. Take out, leave overnight, and ultrasound (500W, 37kHz) for 30min. After cooling, add methanol to the scale, shake well, filter with a 0.22μm organic filter membrane, and take the filtrate.

[0061] Table 1. Ginsenoside detection results

[0062]

[0063] As shown in Table 1, the ginsenoside content in the fermented ginseng hydrolysate is much lower than that in the ginseng before fermentation. The total saponin content is only 25.8% of that before fermentation, and ginsenosides Rg1 and Re are not detected. This indicates that after fermentation, the ginsenosides are almost transferred to the fermentation liquid, while the ginsenoside content in the fermented ginseng residue and fermented ginseng hydrolysate is very low.

[0064] 2. Determination of amino acid composition in fermented ginseng hydrolysate.

[0065] The amino acid content of the fermented ginseng hydrolyzate prepared in Example 1 was analyzed with reference to the national safety standard GB 5009.124-2016 for the determination of amino acids in food. An S7130 fully automatic amino acid analyzer (SYKAM, Germany) was used, using a Cation Separation Column LCA K06 / Na, 150 mm × 4.6 mm, and an Ammonia Filtration Column LCA K04 / Na, 100 mm × 4.6 mm. The results are shown in Table 2. The assay principle involves hydrolysis of protein in food by hydrochloric acid to form free amino acids. After separation on the ion exchange column of the amino acid analyzer, the free amino acids react with a ninhydrin solution to produce a color reaction. Detection is performed at the maximum absorption peak wavelengths of 570 nm and 440 nm. The amino acid composition of the fermented ginseng peptides is determined based on retention time and peak area.

[0066] Table 2 Amino acid content of fermented ginseng hydrolysate

[0067]

[0068] As shown in Table 2, aspartic acid, arginine, etc. are the main amino acids in the fermented ginseng hydrolysate, followed by glutamic acid and leucine, indicating that the content of acidic amino acids is relatively high. The fermented ginseng residue is acidic under natural conditions, and the alkaline dissolution method can effectively extract protein from the fermented ginseng residue.

[0069] 3. The peptide sequence of the fermented ginseng hydrolysate obtained in Example 1 was identified.

[0070] The amino acid sequence of the fermented ginseng hydrolyzate prepared in Example 1 was identified using the HPLC-MS / MS method. The specific detection method is as follows:

[0071] The samples were separated and detected using an X500 LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometer (AB SCIEX, USA). The liquid chromatography and mass spectrometry control software used was SCIEX OS2.0 integrated version (AB SCIEX, USA); the chromatographic column used was 1×100 mm HSS T3 (1.8 μm, Waters, USA).

[0072] The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B), and the elution method was: 0-4.00 min 5.0% B, 4.00-6.00 min 5.0-10.0% B, 6.00-30.00 min 10.0-40.0% B, 30.00-34.00 min 40.0-90.0% B, 34.00-40.00 min 90% B, 40.00-42.00 min 90.0-5.0% B, 42.00-52.00 min 5.0% B, flow rate 0.05 mL / min, injection volume 1 μL, column temperature 40°C, the percentages of the above mobile phases are volume percentages, volume of mobile phase B / total volume of mobile phase A and mobile phase B.

[0073] Mass spectrometry detection method: scan cycle 0.642s, ESI ion source temperature 500℃, positive ion mode, spray voltage 5500V, TOF primary scan range 100-1200Da, secondary scan range 50-1200Da, working mode IDA, maximum number of candidate ions 4, dynamic exclusion turned on, and other parameters used the proteomics method default values. Note that the dead volume of the instrument should be minimized before use. The identification results are shown in Figure 1-10 and Table 3.

[0074] Table 3 Identification results of characteristic peptide sequences in fermented ginseng hydrolysate

[0075]

[0076]

[0077] like Figure 1-10 As shown in Table 3, the present invention uses HPLC-MS / MS technology to identify 9 characteristic amino acid sequences from the fermented ginseng hydrolyzate, including 4 dipeptides (LY, MM, LR, AP), 1 tripeptide (LAR), 1 pentapeptide (RLDFR), 2 hexapeptides (SALAFR, LLLLGH), and 1 7-peptide (LLLLLHG), which can be used as quality control standards for the enzymatically fermented ginseng peptides.

[0078] Effect Example 2

[0079] Tartrate-resistant acid phosphatase (TRAP) is present in many cells, but it abnormally aggregates during bone resorption and is secreted by osteoclasts. It participates in the degradation and remodeling of bone matrix and is used as a marker to assess bone resorption activity. Based on this, this effect example aims to explore the effect of the fermented ginseng hydrolyzate obtained in Example 1 on osteoclast differentiation. The specific scheme is as follows:

[0080] Bone marrow mononuclear cells were isolated from the femur and tibia of 1-month-old C57BL / 6 mice and plated in 24-well plates at a rate of 1 × 10 5 Each well was cultured overnight and the culture medium was replaced with differentiation medium to induce differentiation of bone marrow mononuclear cells into osteoclasts. The differentiation medium consisted of α-MEM medium containing 10% fetal bovine serum and supplemented with M-CSF (25 ng / mL) and RANKL (25 ng / mL). While replacing the differentiation medium, different concentrations of the fermented ginseng hydrolyzate prepared in Example 1 (12.5, 25, 50, and 100 μg / mL) were added, and alendronic acid (1 μM) was used as a positive control drug. The differentiation medium was replaced on the 4th day, and TRAP staining was performed on the 7th day. The osteoclasts were observed and counted under a microscope according to their characteristics. The statistical results are shown in Table 4. The characteristics of osteoclasts are: cells with more than 3 nuclei and TRAP positive cells.

[0081] Table 4 The number of osteoclasts in different treatment groups

[0082]

[0083]

[0084] As shown in Table 3, different concentrations of fermented ginseng hydrolyzate all have the effect of significantly reducing osteoclasts, and when the concentration is 100 μg / mL, the effect of fermented ginseng hydrolyzate is better than that of the positive drug alendronic acid, indicating that the fermented ginseng hydrolyzate of the present invention has the effect of inhibiting the differentiation of bone marrow mononuclear cells into osteoclasts.

[0085] Effect Example 3

[0086] Ovariectomized mice (OVX mice) are a classic animal model for studying postmenopausal osteoporosis. The ovaries of mice are surgically removed to simulate the hormonal changes in women after menopause, especially the significant decrease in estrogen levels. This hormonal change leads to increased bone resorption and reduced bone formation, which in turn leads to decreased bone density and degradation of bone microstructure, ultimately leading to the occurrence of osteoporosis. On this basis, this effect example constructs an OVX mouse model to evaluate the efficacy of the fermented ginseng hydrolyzate obtained in Example 1 on osteoporosis. The specific scheme is as follows:

[0087] Six-week-old SPF-grade C57BL / 6 female mice were purchased from the Guangdong Medical Laboratory Animal Center (SCXK (Yue) 2022-0002). The ambient temperature was 20-25°C, the humidity was 40%-60%, and the light intensity was 12 h per day (7:00-19:00). They had free access to food and water. Experiments were performed when they were 3 months old.

[0088] The OVX model was established as follows: Mice were fasted from food but not water for 12 hours before ovariectomy. Mice were anesthetized with isoflurane and immobilized in a supine position. The abdominal skin of the surgical area was disinfected with 75% alcohol. A longitudinal incision of approximately 0.5 cm was made through the skin and muscle to expose the pink ovaries. The uterus and ovaries were ligated at their junction, and the ovaries were removed. The muscles and skin were sutured layer by layer, and the mice were returned to a clean home cage. For sham surgery, only a ventral incision was made, and the ovaries were not removed. Following surgery, all mice received intraperitoneal penicillin injections every other day to prevent infection. The home cages were changed daily, and subsequent experiments were performed one week later.

[0089] Ovariectomized mice were randomly divided into an OVX model group, a low-dose fermented ginseng hydrolyzate (250 mg / kg) group, a medium-dose fermented ginseng hydrolyzate (500 mg / kg) group, a high-dose fermented ginseng hydrolyzate (1000 mg / kg) group, and a 17β-estradiol-positive drug group (10 μg / kg), with 5 mice in each group. Sham-operated mice served as a normal control group. Enzymatically hydrolyzed fermented ginseng peptides were administered orally, and 17β-estradiol was administered subcutaneously once daily for 2 months. After the last dose, the mice were fasted for 12 hours and euthanized. The right hind limbs of the mice were scanned to measure bone density and trabecular number. The results are shown in Table 5.

[0090] Table 5 Bone density and trabecular number of mice in different treatment groups

[0091]

[0092] As shown in Table 4, compared with the normal group (sham operation), the bone density and trabecular number of the right hind limbs of the OVX model group mice were significantly reduced, indicating that the osteoporosis model was successfully constructed. In contrast, the fermented ginseng hydrolyzate can significantly improve the bone density and trabecular number reduced by removing the ovaries. At a dose of 1000 mg / kg, its effect is better than that of the positive drug 17β-estradiol. The above results suggest that the fermented ginseng hydrolyzate provided by the present invention can resist osteoporosis caused by hormonal changes and has a certain bone tissue protection effect.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fermented ginseng hydrolyzate, characterized in that: The invention comprises a ginseng peptide, wherein the ginseng peptide comprises a characteristic peptide of the amino acid sequence of Leu-Tyr, a characteristic peptide of Met-Met, a characteristic peptide of Leu-Arg, a characteristic peptide of Leu-Ala-Arg, a characteristic peptide of Ser-Ala-Leu-Ala-Phe-Arg, a characteristic peptide of Arg-Leu-Asp-Phe-Arg, a characteristic peptide of Leu-Leu-Leu-Leu-Gly-His, a characteristic peptide of Leu-Leu-Leu-Leu-Leu-His-Gly, and a characteristic peptide of Ala-Pro; The fermented ginseng hydrolyzate is prepared by the following steps: (1) Mix the fermented ginseng residue with water, adjust the pH to 7.0-8.0, extract at 45-55°C for 30-40 min, add trypsin, bathe in 45-55°C water for 4-5 h, and centrifuge to obtain the enzymatic hydrolyzate; (2) drying the enzymatic hydrolyzate obtained in step (1) to obtain a fermented ginseng hydrolyzate containing ginseng peptides; In step (1), the method for preparing the fermented ginseng residue comprises the following steps: (a) 200 g of raw sun-dried ginseng was pulverized using a high-speed pulverizer, passed through a 10-mesh sieve, soaked in 90-100° C. hot water with a material-liquid ratio of 1:20 for 12 h, and then pulverized using a colloid mill. 1.0% by weight of pectinase was added for enzymatic hydrolysis to obtain a ginseng fermentation matrix. The enzymatic hydrolysis temperature was 47° C. ± 5° C., and the enzymatic hydrolysis time was 3 h. (b) Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMRs broth respectively and cultured in a constant temperature incubator at 37°C ± 1°C for 24 h. The two bacterial cultures were then inoculated into 500 mL of culture medium at a ratio of 1:1 with 2% of each culture medium and cultured for 24 h at 37°C ± 1°C. The culture medium contained 10 7 cfu / mL to 10 9 cfu / mL; wherein the culture medium comprises 2% fructooligosaccharides, 1.25% wheat protein hydrolyzate powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80 and the remainder purified water; the Lactobacillus paracasei B04WI2501 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.62875; the Lactobacillus paracasei B16NY2107 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.62874; (c) adding calcium carbonate to the ginseng fermentation matrix prepared in step (a) to adjust the pH to 6.0, adding 5% by weight of white sugar to the ginseng fermentation matrix, heating to 90-100° C., and keeping the temperature for 3 hours to obtain a fermentation liquid; (d) The fermentation liquid obtained in step (c) was transferred to a fermentation tank, stirred and cooled with circulating cooling water. After the liquid was cooled to 37°C, the strain obtained in step (b) was inoculated into a 5L fermentation tank at a 1% inoculation rate. The fermentation temperature was 30°C and the rotation speed was 50 rpm. The fermentation was carried out at a constant temperature for 14 days. During the fermentation process, the activity of the strain in the fermentation liquid was maintained at 10 7 cfu / mL to 10 9 cfu / mL; after the fermentation, centrifugal filtration was performed with the centrifugal parameters of 3000 rpm for 10 min, and the mixture was passed through an 80-mesh vibrating sieve to obtain the fermented ginseng residue.

2. The fermented ginseng hydrolyzate according to claim 1, wherein The mass ratios of the characteristic peptide Leu-Tyr, characteristic peptide Met-Met, characteristic peptide Leu-Arg, characteristic peptide Leu-Ala-Arg, characteristic peptide Ser-Ala-Leu-Ala-Phe-Arg, characteristic peptide Arg-Leu-Asp-Phe-Arg, characteristic peptide Leu-Leu-Leu-Leu-Gly-His, characteristic peptide Leu-Leu-Leu-Leu-Leu-His-Gly and characteristic peptide Ala-Pro of the ginseng peptides in the fermented ginseng hydrolyzate are Leu-Tyr:Met-Met:Leu-Arg: Leu-Ala-Arg: Ser-Ala-Leu-Ala-Phe-Arg: Arg-Leu-Asp-Phe-Arg: Leu-Leu-Leu-Leu-Gly-His: Leu-Leu-Leu-Leu-Leu-His-G ly: Ala-Pro= (49.6-52.1): (6.3-7.6): (7.5-8.8): (1.5-1.8): (18.1-19.5): (2.6-3.1): (1.4-1.6): (1.2-1.5): (7.8-8.0).

3. The method for preparing the fermented ginseng hydrolyzate according to claim 1, wherein: The following steps are involved: (1) Mix the fermented ginseng residue with water, adjust the pH to 7.0-8.0, extract at 45-55°C for 30-40 min, add trypsin, bathe in 45-55°C water for 4-5 h, and centrifuge to obtain an enzymatic hydrolyzate; (2) drying the enzymatic hydrolyzate obtained in step (1) to obtain a fermented ginseng hydrolyzate containing ginseng peptides; In step (1), the method for preparing the fermented ginseng residue comprises the following steps: (a) 200 g of raw sun-dried ginseng was pulverized using a high-speed pulverizer, passed through a 10-mesh sieve, soaked in 90-100° C. hot water with a material-liquid ratio of 1:20 for 12 h, and then pulverized using a colloid mill. 1.0% by weight of pectinase was added for enzymatic hydrolysis to obtain a ginseng fermentation matrix. The enzymatic hydrolysis temperature was 47° C. ± 5° C., and the enzymatic hydrolysis time was 3 h. (b) Lactobacillus paracasei B04WI2501 and Lactobacillus paracasei B16NY2107 were inoculated into 100 mL of LMRs broth respectively and cultured in a constant temperature incubator at 37°C ± 1°C for 24 h. The two bacterial cultures were then inoculated into 500 mL of culture medium at a ratio of 1:1 with 2% of each culture medium and cultured for 24 h at 37°C ± 1°C. The culture medium contained 10 7 cfu / mL to 10 9 cfu / mL; wherein the culture medium comprises 2% fructooligosaccharides, 1.25% wheat protein hydrolyzate powder, 0.1% magnesium sulfate, 5.0% sodium acetate, 2.0% potassium dihydrogen phosphate, 1.0% Tween 80 and the remainder purified water; the Lactobacillus paracasei B04WI2501 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.62875; the Lactobacillus paracasei B16NY2107 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.62874; (c) adding calcium carbonate to the ginseng fermentation matrix prepared in step (a) to adjust the pH to 6.0, adding 5% by weight of white sugar to the ginseng fermentation matrix, heating to 90-100° C., and keeping the temperature for 3 hours to obtain a fermentation liquid; (d) The fermentation liquid obtained in step (c) was transferred to a fermentation tank, stirred and cooled with circulating cooling water. After the liquid was cooled to 37°C, the strain obtained in step (b) was inoculated into a 5L fermentation tank at a 1% inoculation rate. The fermentation temperature was 30°C and the rotation speed was 50 rpm. The fermentation was carried out at a constant temperature for 14 days. During the fermentation process, the activity of the strain in the fermentation liquid was maintained at 10 7 cfu / mL to 10 9 cfu / mL; after the fermentation, centrifugal filtration was performed with the centrifugal parameters of 3000 rpm for 10 min, and the mixture was passed through an 80-mesh vibrating sieve to obtain the fermented ginseng residue.

4. The preparation method according to claim 3, wherein In step (1), the ratio of the fermented ginseng residue to water is fermented ginseng residue: water = 1 g: (25-30) mL.

5. The preparation method according to claim 3, wherein In step (1), the amount of trypsin added is 0.75-0.80 mg of trypsin per 1 g of fermented ginseng residue, and the concentration of trypsin is 1350-1400 U / mg.

6. Use of the fermented ginseng hydrolyzate according to claim 1 in the preparation of a medicine for improving postmenopausal osteoporosis.

7. Use of the fermented ginseng hydrolyzate according to claim 1 in the preparation of a medicine for preventing postmenopausal osteoporosis.

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