A soy dregs solid fermentation preparation and its application in treating or preventing sarcopenia

Abstract

The present invention discloses a bean dregs solid-state fermentation preparation and its application in treating or preventing sarcopenia, relates to the field of bean dregs processing, wherein the bean dregs solid-state fermentation preparation is based on Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 as fermentation strains, bean dregs as fermentation substrate, first by solid-state fermentation, and then by extraction. The present invention establishes a myotube atrophy model by inducing mouse myoblast C2C12 damage with TNF‑α in vitro, explores the effect of the bean dregs solid-state fermentation preparation on C2C12 cells, and studies the effect of the bean dregs solid-state fermentation preparation on LPS-induced muscle atrophy mice in vivo. The results show that the bean dregs solid-state fermentation preparation has the potential to delay aging, prevent muscle atrophy and weakness associated with aging, and treat sarcopenia, providing a reference for exploring the potential application of plant active ingredients in preventing or treating muscle function.

Description

Technical Field

[0001] The present invention relates to the field of bean dregs processing, and in particular to a bean dregs solid-state fermentation preparation and its application in treating or preventing sarcopenia. Background Art

[0002] Sarcopenia, also known as muscle loss or muscle attenuation, is a progressive, age-related, systemic skeletal muscle disease that occurs frequently in the elderly. It is characterized by decreased physical fitness and a gradual loss of muscle mass, strength, and function, leading to difficulty in daily activities, an increased risk of falls and fractures, and even a shortened life expectancy. The risk of sarcopenia increases with age and is a leading cause of death and disability in the elderly.

[0003] Studies have shown that the occurrence of sarcopenia is affected by the interaction of multiple factors such as genetics and the environment, and its occurrence and progression are related to multiple mechanisms related to the imbalance of skeletal muscle protein synthesis and degradation, neuromuscular integrity and muscle fat content. The pathophysiological mechanism of sarcopenia is quite complex, and it is a syndrome involving the dysregulation of multiple signaling pathways. Sarcopenia is related to changes in skeletal muscle physiology and cellular mechanisms, including metabolic, cellular, vascular and inflammatory levels. Specifically, possible causative factors of sarcopenia include malnutrition, lack of exercise, chronic inflammation, oxidative stress damage, mitochondrial dysfunction, age-related hormonal changes and weakened neuromuscular function. Intervention measures for sarcopenia mainly consider exercise intervention, nutritional support and drug treatment.

[0004] Regarding drug treatment, currently, there is a lack of suitable specific medications for sarcopenia. Hormone replacement and its adjuvant therapy are commonly used clinically to prevent or treat skeletal muscle atrophy, but this approach needs improvement due to significant side effects. Studies have shown that testosterone replacement therapy can improve muscle strength and mass, reduce fat content, and significantly reduce side effects. Drugs such as growth hormones, β-adrenergic receptor agonists, and angiotensin-converting inhibitors are commonly used in the treatment of sarcopenia, but their clinical efficacy and safety require further study. Overall, domestic research on drug treatments for sarcopenia is still in the exploratory stage. Therefore, it is particularly important to explore new effective functional ingredients for the prevention or treatment of sarcopenia.

[0005] Current research shows that many plant active ingredients with antioxidant and anti-inflammatory properties have shown good intervention effects on the occurrence and progression of sarcopenia. There are a large number of animal experiments and in vitro experiments on sarcopenia and they show good research prospects, but this is not enough to directly prove that these plant active ingredients can effectively prevent or treat the loss of muscle mass and muscle function. In order to further verify the potential of plant active ingredients in preventing sarcopenia, more relevant research is needed. At present, the number of studies published on the potential application of herbal medicines in preventing or treating muscle function is relatively small, and relevant scholars still need to continue their efforts to continuously explore effective methods for treating sarcopenia. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a soy dregs solid fermentation preparation and its application in treating or preventing sarcopenia.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides a bean dregs solid-state fermentation preparation, which is obtained by solid-state fermentation and extraction using Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 as fermentation strains and bean dregs as a fermentation substrate.

[0009] As a further optimization scheme of the present invention, the Lactobacillus paracasei LMX1 has a deposit number of CCTCC M20211073 and was deposited in the China Center for Type Culture Collection on August 24, 2021;

[0010] The preservation number of the Pediococcus acidilactici IFJ1 is CCTCCM2022828, and it was deposited in the China Center for Type Culture Collection on June 8, 2022.

[0011] As a further optimized solution of the present invention, the volume ratio of the bacterial liquid of Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 is (1-3):(1-3), and more preferably 1:1.

[0012] As a further optimized solution of the present invention, the method for preparing the okara solid-state fermentation preparation comprises the following steps:

[0013] (1) Dry bean dregs are added to sterile water to prepare a solid fermentation medium, and then Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 are cultured and activated to prepare seed liquids, and the seed liquids are mixed to prepare a fermentation liquid;

[0014] (2) inoculating the fermentation liquid obtained in step (1) into a solid-state fermentation medium for solid-state fermentation, and obtaining a solid-state fermentation product of bean dregs after the solid-state fermentation is completed;

[0015] (3) Using distilled water as the extracting liquid, the bean dregs solid fermentation product obtained in step (2) is extracted to obtain the bean dregs solid fermentation preparation, and the extraction process is further preferably such that the extraction material-liquid ratio is 1:12, the extraction temperature is 50° C., and the extraction time is 30 min.

[0016] As a further optimized solution of the present invention, the initial water content of the solid-state fermentation medium is 40-80%, the inoculation amount of the fermentation liquid is 2-10% of the mass of the solid-state fermentation medium, the solid-state fermentation temperature is 24-32°C, and the fermentation time is 3-7 days.

[0017] The initial water content of the solid-state fermentation medium is more preferably 70%, the inoculation amount of the fermentation liquid is more preferably 2% of the mass of the solid-state fermentation medium, the solid-state fermentation temperature is more preferably 28° C., and the fermentation time is more preferably 6 days.

[0018] The second aspect of the present invention also provides a use of any of the above-mentioned solid-state fermentation preparations of okara in the preparation of a drug for treating or preventing sarcopenia.

[0019] The beneficial effects of the present invention are:

[0020] The present invention obtains a bean dregs solid-state fermentation preparation rich in oligopeptides by solid-state fermentation of bean dregs with a mixture of high-aminopeptidase-producing Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1. Furthermore, the present invention uses TNF-α to induce mouse myoblast C2C12 damage in vitro to establish a myotube atrophy model, explores the effect of the bean dregs solid-state fermentation preparation on C2C12 cells, and studies the effect of the bean dregs solid-state fermentation preparation on LPS-induced muscle atrophy mice in vivo. The results indicate that the bean dregs solid-state fermentation preparation can promote the proliferation of C2C12 cells, improve the inhibitory effect of TNF-α on cell migration ability, reduce the ratio of cells in the G0 / G1 phase of the cell cycle, increase the ratio of cells in the G2 / M phase, improve the cell cycle arrest caused by TNF-α, and improve the promoting effect of TNF-α on cell aging.

[0021] In addition, solid-state fermentation preparations of soy dregs can also promote the differentiation of C2C12 cells into myotubes, increase the number, length and diameter of myotubes, upregulate the expression of early differentiation markers MyoD1 and myogenesis marker MyoG, and downregulate the expression of muscle atrophy markers MuRF and Atrogin-1. Furthermore, it can improve the weight loss of LPS-stimulated mice, improve the exercise capacity of LPS-stimulated mice, and increase the weight of the calf muscles of the hind limbs of mice.

[0022] Based on the above, it can be concluded that solid-state fermentation preparations of soybean dregs have the potential to delay aging, prevent aging-related muscle atrophy and weakness, and treat sarcopenia, providing a reference for exploring the potential application of plant active ingredients in preventing or treating muscle function. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of different concentrations of okara solid-state fermentation preparations on C2C12 cell proliferation;

[0024] Figure 2 The effects of different concentrations of okara solid-state fermentation preparations on C2C12 cell migration;

[0025] Figure 3 The effect of different concentrations of okara solid fermentation preparations on the migration of C2C12 cells induced by 20 ng / mL TNF-α;

[0026] Figure 4 The effect of different concentrations of okara solid fermentation preparations on the cell cycle of C2C12 cells induced by 20 ng / mL TNF-α;

[0027] Figure 5 β-galactosidase staining was used to observe the effects of different concentrations of okara solid-state fermentation preparations on TNF-α-induced C2C12 cell senescence;

[0028] Figure 6 The effect of different concentrations of solid-state fermentation preparations of soybean dregs on myotube differentiation of C2C12 cells without TNF-α induction;

[0029] Figure 7 The effect of different concentrations of solid-state fermentation preparations of soybean dregs on the myotube differentiation of C2C12 cells induced by TNF-α;

[0030] Figure 8 qPCR was used to detect the mRNA expression levels of relevant myogenesis and muscle atrophy markers;

[0031] Figure 9a To detect the protein expression of MyoD1, a marker of early myotube differentiation;

[0032] Figure 9b To detect the protein expression of myogenesis marker MyoG;

[0033] Figure 9c To detect the protein expression of MuRF1, a muscle atrophy marker;

[0034] Figure 9d To detect the protein expression of Atrogin-1, a muscle atrophy marker;

[0035] Figure 10 Effects of different concentrations of solid-state fermentation preparations of soybean dregs on the body weight of mice stimulated with LPS;

[0036] Figure 11 The effects of different concentrations of okara solid-state fermentation preparations on the grasping force of mice stimulated by LPS;

[0037] Figure 12 The effects of different concentrations of solid fermentation preparations of soybean dregs on the time mice spend on the rod stimulated by LPS;

[0038] Figure 13 Effects of different concentrations of solid-state fermentation preparations of soybean dregs on the calf muscle weight of mice stimulated with LPS;

[0039] Figure 14 HE staining images of mouse muscle tissue (A: control group - normal saline; B: model group - lipopolysaccharide-induced muscle atrophy; C: treatment group 1-100 mg / kg soy dregs solid-state fermentation preparation; D: treatment group 2-200 mg / kg soy dregs solid-state fermentation preparation; E: treatment group 3-300 mg / kg soy dregs solid-state fermentation preparation);

[0040] Figure 15 The effect of different initial fermentation water contents on the concentration of okara oligopeptides in okara solid-state fermentation preparations;

[0041] Figure 16 The effect of different fermentation times on the concentration of okara oligopeptides in okara solid-state fermentation preparations;

[0042] Figure 17 The effect of different fermentation temperatures on the concentration of okara oligopeptides in okara solid-state fermentation preparations;

[0043] Figure 18 The effect of different LMX1 and IFJ1 inoculation amounts on the concentration of okara oligopeptides in okara solid-state fermentation preparations;

[0044] Figure 19 The effect of different LMX1 and IFJ1 bacterial liquid ratios on the concentration of okara oligopeptides in okara solid-state fermentation preparations. DETAILED DESCRIPTION

[0045] The present invention is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0046] 1. Reagents and Materials

[0047] Unless otherwise specified, the reagents and materials used in this example were commercially available.

[0048] The preservation number of Lactobacillus paracasei LMX1 is CCTCCM20211073, and it was deposited in the China Center for Type Culture Collection on August 24, 2021; (the patent "CN117286047A A composite fermentation agent and its application in the production of raw tofu" discloses the isolation, screening, identification and cultural characteristics of this strain).

[0049] The Pediococcus acidilactici IFJ1 has a deposit number of CCTCCM2022828 and was deposited in the China Center for Type Culture Collection on June 8, 2022; (the article "Li Dongqi. Isolation and Identification of Aminopeptidase-Producing Bacteria in Northern Anhui Bean Paste and Research on Its Fermentation Effect [D]" discloses the isolation, screening, identification and cultural characteristics of the strain).

[0050] MRS broth liquid culture medium: 10 g peptone, 8 g beef powder, 4 g yeast powder, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween 80, dissolved in 1 L distilled water and sterilized at 121°C for 15 min.

[0051] Mouse myoblasts: C2C12 cells were obtained from the Institute of Geriatrics, the First Affiliated Hospital, University of Science and Technology of China.

[0052] Mice: C57BL / 6, 6–8 weeks old, were purchased from Henan Sikebes Biotechnology Co., Ltd. The animals involved in this invention were housed and managed in strict accordance with the Regulations on the Administration of Laboratory Animals and the guidelines established by the Experimental Animal Ethics Committee of Anhui Agricultural University. All animal experimental procedures were carried out in strict accordance with the standards of the Ethics Committee of Anhui Agricultural University.

[0053] DMEM complete medium: DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0054] Differentiation medium: DMEM high glucose medium containing 2% horse serum and 1% penicillin-streptomycin.

[0055] Alkaline copper test solution: Solution A: Accurately weigh 10 g of NaOH and 50 g of Na2CO3, add them to 400 mL of pure water, stir and dissolve for later use; Solution B: Accurately weigh 0.5 g of potassium tartrate and dissolve it in 50 mL of pure water, and accurately weigh 0.25 g of copper sulfate and dissolve it in 30 mL of pure water; transfer Solution A and Solution B to a 500 mL volumetric flask, add pure water to dissolve and dilute to 500 mL.

[0056] 2. Methods

[0057] Unless otherwise specified, the following methods are conventional methods known to those skilled in the art.

[0058] 1. Preparation of solid-state fermentation preparation of soybean dregs

[0059] 1.1. Preparation of fermentation substrate

[0060] The soy dregs used in this invention are fresh soy dregs produced as a byproduct during the soy milk production process at Anhui Dafu Food Co., Ltd. The fresh soy dregs are sterilized in an autoclave at 121°C for 15 minutes, placed on a baking tray, and dried in an oven at 55°C for 48 hours, with frequent stirring. The dried soy dregs are then refrigerated in a freezer at 4°C.

[0061] 1.2 Preparation of fermentation broth

[0062] (1) Activation of IFJ1 and LMX1 strains

[0063] The IFJ1 and LMX1 glycerol strains stored in the laboratory were inoculated into 75 mL of MRS broth liquid culture medium under sterile conditions and cultured in a 37°C incubator for 48 h.

[0064] Under sterile conditions, the bacterial solution was diluted, and then the appropriate gradient of diluted bacterial solution (10 -6 , 10 -7 , 10 -8 ) 30 μL and spread it on the solidified solid culture medium, spread it back and forth several times, and place it upside down in a 37°C incubator for 24 hours. Observe the spreading results, select single colonies with good growth status and obvious morphology, inoculate them on the solid culture medium, continue to streak purification for 3 generations, and determine whether the streaked colonies are IFJ1 or LMX1 based on colony morphology and Gram staining.

[0065] (2) Preparation of IFJ1 and LMX1 seed solutions

[0066] Use a sterile inoculating loop to select a single colony with good phenotype from the above-identified IFJ1 and LMX1, and inoculate them into MRS broth liquid culture medium respectively. Incubate them in a 37°C incubator for 48 hours to obtain IFJ1 and LMX1 seed liquid.

[0067] (3) Mix the IFJ1 seed solution and the LMX1 seed solution in a volume ratio of 1:1 to obtain a fermentation solution.

[0068] 1.3 Solid-state fermentation of soybean dregs

[0069] First, take 200 g of the dry bean dregs prepared in step 1.2, add sterile water to prepare a solid fermentation medium with an initial moisture content of 70%, and then inoculate the fermentation liquid prepared in step 1.2 into the solid fermentation medium for solid fermentation. The inoculation amount of the fermentation liquid is 2% of the mass of the solid fermentation medium, the solid fermentation temperature is 28°C, the fermentation time is 6 days, and the bean dregs solid fermentation product is obtained after the solid fermentation is completed.

[0070] 1.4 Extraction of solid fermentation products from soybean dregs

[0071] The okara solid-state fermentation product was extracted with distilled water at a material-to-liquid ratio of 1:12, an extraction temperature of 50°C, and an extraction time of 30 minutes to obtain an okara solid-state fermentation preparation (hereinafter referred to as DZFJZJ). DZFJZJ was freeze-dried to obtain DZFJZJ freeze-dried powder, which was stored in a refrigerator at -20°C.

[0072] 2. Exploring the biological functions of DZFJZJ

[0073] 2.1 Effects of DZFJZJ on C2C12 cell senescence

[0074] 2.1.1 Myotube differentiation of C2C12 cells

[0075] After recovery, culture, and passage, C2C12 cells in the logarithmic growth phase were collected and inoculated into 6-well culture plates with DMEM complete medium and placed in an incubator at 37°C and 5% CO2. When the cells grew to 80%, the medium was removed and replaced with differentiation medium to induce differentiation. The differentiation medium was replaced every two days. From the third day on, a small amount of myotube formation could be observed. Thereafter, the myotubes became thicker and more numerous. After the fifth day of differentiation, they could be used for subsequent experiments.

[0076] 2.1.2. CCK-8 assay to determine the effect of DZFJZJ on C2C12 cell proliferation

[0077] (1) Remove cells from the incubator and observe them under an inverted microscope. Prepare a cell suspension of C2C12 cells in good condition with a cell fusion of about 80%. Count the cells and evenly seed 5,000 cells per well in a 96-well plate. Place the plate in an incubator and culture for 24 hours.

[0078] (2) The next day, the original culture medium was aspirated and different concentrations of DZFJZJ were added and cultured for another 96 h. The concentrations of DZFJZJ were set to 0, 100, 200, 400, 800, and 1600 μg / mL, which were prepared by diluting 120 mg / mL DZFJZJ lyophilized powder stock solution (120 mg DZFJZJ lyophilized powder dissolved in 1 mL PBS solution and filtered and sterilized after dissolution). The medium was changed every day.

[0079] (3) After 0 h, 24 h, 48 h, 72 h, and 96 h, the old culture medium in the 96-well plate was discarded, and 100 μL of CCK-8 detection reagent (CCK-8: complete culture medium = 1:10) was added to each well, followed by incubation at 37 °C in the dark for 1 h.

[0080] (4) After 1 hour, the 96-well plate was taken out and the color depth was observed by naked eyes to preliminarily judge the experimental results. Then, the effect of DZFJZJ on cell activity was detected using a microplate reader. The detection wavelength was set to 450nm, and the OD value of each well was obtained. The survival rate of C2C12 cells after treatment with different concentrations of DZFJZJ was calculated. Cell proliferation activity (%) = (OD 实验 -OD 空白 ) / (OD 对照 -OD 空白 ), and each group of experiments was performed 3 times.

[0081] The results are as follows Figure 1 As shown in the figure, with the increase of time, the ability of DZFJZJ to promote the proliferation of C2C12 cells gradually increased. Compared with the control group, different concentrations of DZFJZJ can promote the proliferation of C2C12 cells, and the effect of 800μg / mL DZFJZJ is more significant.

[0082] 2.1.3 Cell scratch assay to detect the effect of DZFJZJ on the migration ability of C2C12 cells

[0083] (1) Take out C2C12 cells in good condition with a cell confluence of about 80% from the cell culture incubator, digest and collect them, prepare them into a cell suspension, and count the cells. 5 The cells were seeded in a 6-well plate, and the cells in the 6-well plate were shaken by a cross-cross method. The 6-well plate was then placed in a cell culture incubator and cultured for 24 h.

[0084] (2) The next day, the 6-well plate was taken into the clean bench for operation, the old culture medium was aspirated and discarded, the culture plate was covered and inverted, and five straight lines were drawn on the back of the plate with a marker and a ruler as marks. Then, a 200 μL pipette tip was used to draw vertical lines along the marks of the black marker. The 6-well plate was taken into the clean bench for operation, the old culture medium was aspirated and discarded, the culture plate was covered and inverted, and five straight lines were drawn on the back of the plate with a marker and a ruler as marks. Then, a 200 μL pipette tip was used to draw vertical lines along the marks of the black marker. The cell surface was rinsed with PBS to remove cells detached due to scratches. Then, complete culture medium containing 0, 100, 200, 400, 800 and 1600 μg / mL DZFJZJ was added respectively. The cell culture plate was placed under an inverted microscope for photography and recording, and the initial scratch width was recorded.

[0085] (3) After 24 h, the cell culture plate was taken to the clean bench for operation. The old culture medium was first removed, and the cells were washed with PBS. The culture plate was then placed under a microscope to observe the cell migration of the groups treated with different concentrations of DZFJZJ and take photos to record the results. The scratch healing rate of each group of cells was analyzed and counted.

[0086] Scratch healing rate = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0087] The results are as follows Figure 2 As shown in the figure, compared with the blank control group, different concentrations of DZFJZJ could significantly enhance the migration ability of C2C12 cells.

[0088] Furthermore, at 0 h, the cells were scratched and recorded, and then treated with 20 ng / mL TNF-α and different concentrations of DZFJZJ for 24 h, and the width of the cell scratch was recorded again. The results are shown in Figure 2. Figure 3 As shown in the results, TNF-α treatment can reduce the migration rate of C2C12 cells, but the addition of different concentrations of DZFJZJ can improve the inhibitory effect of TNF-α on cell migration ability to varying degrees. Therefore, DZFJZJ has the effect of promoting the migration of C2C12 cells.

[0089] 2.1.4 Flow cytometry analysis of the effect of DZFJZJ on the C2C12 cell cycle

[0090] C2C12 cells in good condition with a cell confluence of about 80% were taken out from the cell culture incubator for digestion and collection, and then prepared into a cell suspension. The cells were counted and 2×10 cells were used per well. 5 Cells were seeded in a 6-well plate, shaken to mix the cells, and then placed in a cell culture incubator for 24 hours. The experiment included a blank control group and six groups treated with different concentrations of DZFJZJ.

[0091] After 24 hours, the blank control group was treated with complete DMEM medium, while the DZFJZJ-treated groups were treated with various concentrations of prepared DZFJZJ solutions and incubated in an incubator. After 24 hours, the original culture medium was aspirated, and the blank control group was treated with complete DMEM medium. The other groups were treated with 20 ng / mL TNF-α solution and incubated in an incubator. After 4 hours, the original culture medium was aspirated, and the cells were rinsed 1-2 times with PBS. The cells were trypsinized and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed once with PBS and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were added with 70% ethanol (pre-cooled), mixed by pipetting, and incubated at 4°C overnight. The cells were centrifuged at 1000 rpm for 5 minutes, the ethanol was discarded, and the cells were washed once with PBS and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the appropriate amount of PI working solution was added, mixed, and incubated at 37°C. After 30 minutes, the cells were centrifuged at 1000 rpm for 5 minutes. Discard the supernatant, rinse once with an appropriate amount of PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of PBS to resuspend the cells, and detect using a flow cytometer.

[0092] The results are as follows Figure 4 As shown in the figure, compared with 20 ng / mL TNF-α, DZFJZJ significantly reduced the proportion of cells in the G0 / G1 phase of the cell cycle and significantly increased the proportion of cells in the G2 / M phase, indicating that DZFJZJ can partially improve the cell cycle arrest caused by TNF-α.

[0093] 2.1.5. β-Galactosidase staining to detect the effect of DZFJZJ on the senescence process of C2C12 cells

[0094] C2C12 cells in good condition with a cell confluence of 80% were prepared at a concentration of 1.5×10 4 Cell suspension was prepared at a concentration of 1.5×10 cells / mL per well. 4 Cells were seeded in a 24-well plate and cultured in an incubator. After 24 hours, the original culture medium was removed and washed once with PBS, and then DMEM complete medium was added. Different drug treatments were performed according to the experimental groups and the cells were cultured in an incubator for 4 hours. The liquid in the wells was removed, and the cells were washed once with PBS, and then the fixative was added and fixed at room temperature. After 15 minutes, the fixative was removed and the cells were rinsed 3 times with PBS, each time for 3 minutes. The staining working solution was added and incubated at 37°C overnight. The staining working solution was prepared according to Table 1:

[0095] Table 1. Preparation of β-galactosidase staining working solution

[0096]

[0097] Discard the staining working solution, rinse with PBS 1-2 times, add appropriate amount of PBS, and observe the staining under an ordinary optical microscope.

[0098] The results are as follows Figure 5 Compared with the control group, 20 ng / mL TNF-α significantly increased the number of senescent C2C12 cells. Treatment with different concentrations of DZFJZJ could improve the promoting effect of TNF-α on cell senescence to varying degrees, and 800 μg / mL DZFJZJ had the best effect. Therefore, DZFJZJ has the effect of delaying cell senescence.

[0099] 2.2 Effect of DZFJZJ on the content of inflammatory factor TNF-α in C2C12 cells

[0100] 2.2.1 Effect of DZFJZJ on TNF-α-induced C2C12 cell myotube atrophy

[0101] 2.2.1.1 Effects of DZFJZJ on the diameter of C2C12 cell myotubes

[0102] To verify the effect of DZFJZJ on the diameter of C2C12 cell myotubes, differentiation medium was added to C2C12 cells after the confluence reached 80% and cultured for 4 days. Then, different concentrations of DZFJZJ (0, 100, 200, 400, 800, and 1600 μg / mL) were added for 2 days. The diameter of myotubes was analyzed by immunofluorescence observation and Image J software. Figure 6 As shown in the figure, compared with the control group, with the increase of DZFJZJ concentration, the diameter of myotube cells also increased. The myotube cells in the G3 group were the thickest and the myotube structure was compact, which significantly increased the diameter of normal myotube cells. With the continued increase of concentration, the diameter of myotube cells began to shorten. Therefore, it is believed that the concentration of 800μg / mL DZFJZJ has a positive effect on myotube differentiation.

[0103] Furthermore, after the confluence of C2C12 cells reached 80%, differentiation medium was added and cultured for 4 days, and then 20 ng / mL TNF-α and different concentrations of DZFJZJ (0, 100, 200, 400, 800 and 1600 μg / mL) were added for 2 days. The myotube diameter was observed by immunofluorescence staining and analyzed using Image J software. Figure 7As shown, compared with the control, mature C2C12 myotubes treated with TNF-α exhibited significant myotube atrophy, clustered nuclei, and a loose structure with cavitation, narrow diameter, and poor myotube quality. However, treatment with DZFJZJ significantly reduced myotube atrophy with increasing concentrations, resulting in a more compact structure and improved quality, with a significant increase in myotube diameter. The T20+G3 group exhibited the best cell condition, indicating that 800 μg / mL DZFJZJ significantly ameliorated the TNF-α-induced inhibition of myotube differentiation.

[0104] 2.2.1.2 Effects of DZFJZJ on related mRNA expression in C2C12 cells

[0105] Fluorescence quantitative PCR was used to detect the mRNA expression levels of MyoD1, a marker of early differentiation of myotubes, MyoG, a marker of myogenesis, and MuRF1 and Atrogin-1, markers of muscle atrophy. The specific steps are as follows:

[0106] (1) Extract RNA: Digest the cells with trypsin and collect them by centrifugation, discarding the supernatant. Extract RNA according to the instructions of the Cell RNA Rapid Extraction Kit. Add 500uL Lysis Buffer to the cell pellet, mix well by pipetting, and let it stand for 1 minute. Add the mixture to the adsorption column, centrifuge at 12000rpm for 30 seconds, and discard the filtrate. Add 500uL Washing Buffer D2 to the adsorption column, centrifuge at 12000rpm for 30 seconds, and discard the filtrate. Repeat once. Place the adsorption column in an empty collection tube, centrifuge at 12000rpm for 30 seconds to remove the rinse solution, remove the adsorption column and place it in a clean RNase Free centrifuge tube. Add 25-50uL RNase Free H2O to the middle of the adsorption membrane, let it stand at room temperature for 1 minute, and centrifuge at 1000rpm for 1 minute. Use NanoDrop to measure the RNA concentration.

[0107] (2) Reverse transcription to synthesize cDNA: Take 1 μg of total RNA, 4 μL of 5×Uni All-In-One SuperMix for qPCR, and 1 μL of gDNA Remover and add them to an RNase-free PCR tube. Then add RNase-free water to 20 μL, mix gently and centrifuge briefly to perform reverse transcription to synthesize cDNA. Incubate at 42°C for 15 min, continue incubation at 85°C for 10 min, heat at 95°C for 5 s, and store the obtained cDNA at -80°C for later use.

[0108] (3) RT-qPCR: The cDNA obtained by reverse transcription was subjected to RT-qPCR using the following reaction system and reaction conditions. Primer sequences were designed using PrimerBank. The RT-qPCR reaction system was prepared by adding reagents according to Table 2:

[0109] Table 2. Reaction system for RT-qPCR detection of gene expression

[0110]

[0111] Table 3. Gene names and GeneIDs

[0112]

[0113]

[0114] Table 4. Primer names and sequences

[0115]

[0116] RT-qPCR reaction conditions are shown in Table 5:

[0117] Table 5. RT-qPCR reaction conditions

[0118]

[0119] like Figure 8 As shown, 20 ng / mL TNF-α inhibited the mRNA expression of MyoD1, a marker of early myotube differentiation, and MyoG, a marker of myogenesis. Treatment with high concentrations of DZFJZJ ameliorated the inhibitory effect of TNF-α on MyoD1 and MyoG mRNA expression. Compared with the control group, 20 ng / mL TNF-α significantly increased the mRNA expression of MuRF1, a marker of muscle atrophy. With the exception of 1600 μg / mL DZFJZJ, treatment with different concentrations of DZFJZJ inhibited the TNF-α-stimulated effect on MuRF1 mRNA expression, with 400 μg / mL DZFJZJ having a more pronounced effect than other DZFJZJ concentrations. 20 ng / mL TNF-α significantly increased the mRNA expression of Atrogin-1, a marker of muscle atrophy. Treatment with different concentrations of DZFJZJ significantly reduced Atrogin-1 mRNA expression.

[0120] 2.2.1.3 Effects of DZFJZJ on the expression levels of related proteins in C2C12 cells

[0121] Key proteins associated with myotube cell status include MyoD1, an early differentiation marker for myotubes, MyoG, a myogenic marker, and MuRF1 and Atrogin-1, markers for muscle atrophy. Western blot was used to detect the expression of these proteins. The specific steps are as follows:

[0122] (1) Extract protein from cells: After the drug-treated cells were rinsed once with PBS, 200 μL 1× SDS-PAGE protein loading buffer, 2 μL protease inhibitor PMSF and 2 μL Cocktail were added to lyse the cells. The cells were scraped with a cell scraper and aspirated into a 1.5 mL EP tube. The protein was denatured by heating in a 97°C metal bath for 10 min. The EP tube was removed and immediately placed on ice. After the sample temperature dropped, it was ready for loading or stored in a -80°C refrigerator.

[0123] (2) Preparation of polyacrylamide gel: Take out two glass plates used for making gel, check whether the glass plates are damaged or chipped (if damaged or chipped, replace with new glass plates), wash twice with ultrapure water and dry. Then align and clamp the glass plates with the cover plate and place them horizontally on the gel rack. Add an appropriate amount of double distilled water to the glass plate and let it stand for 10-15 minutes to observe whether there is leakage. If there is no leakage, pour out the double distilled water and turn the glass plate upside down to dry the residual water. Take a clean 50mL centrifuge tube and prepare the lower layer of gel according to the solution volume listed in Table 6:

[0124] Table 6. Components of the lower gel layer of SDS-PAGE gel

[0125]

[0126] After adding TEMED and mixing thoroughly, slowly add the prepared lower layer glue along the edge of the glass plate within 10 minutes, avoiding the formation of bubbles. Add 10mL of the mixture to each glass plate. Once the liquid level is level, add 1mL of isopropyl alcohol and let it sit for 20-30 minutes. After the lower layer glue solidifies, pour out the isopropyl alcohol and invert the glass plate to dry any remaining isopropyl alcohol. In another clean 50mL centrifuge tube, prepare the upper layer glue according to the solution volumes listed in Table 7:

[0127] Table 7. Components of the top layer of SDS-PAGE gel

[0128]

[0129]

[0130] After adding TEMED and mixing thoroughly, slowly fill the prepared top layer of glue along the edge of the glass plate within 10 minutes, avoiding the formation of bubbles. Slowly insert a clean, dry 15-hole comb into the top layer of glue along the edge of the glass plate. If bubbles form beneath the comb, remove it, rinse it with deionized water, and reinsert the top layer of glue. Let the prepared glue plate rest for 20-30 minutes. Remove the glass plate, soak it in deionized water, and refrigerate at 4°C until ready to use.

[0131] (3) Sample loading and electrophoresis: Add equal amounts of protein sample and Maker to the gel placed in the electrophoresis tank and perform electrophoresis. Take a 1L measuring cylinder and rinse it with double distilled water. Add 18.8g glycine, 3.0g Tris powder and 1.0g SDS, dilute to 1L with double distilled water, put in a magnetic rotor, and stir the solution thoroughly. Take out the prepared SDS-PAGE gel, put it into the electrophoresis tank and fix it. Add electrophoresis fluid and ensure that the liquid level inside and outside the electrophoresis tank is clear to prevent leakage. Slowly pull out the comb, add 5μL protein sample to each well, and add 3.5μL protein marker to the reserved well. Connect the matching power supply and start electrophoresis at a constant voltage of 80V. After the protein sample reaches the lower layer of gel, continue electrophoresis at a constant voltage of 120V until it stops around the bottom of the gel.

[0132] (4) Transfer: Remove the PAGE gel and cut the PAGE gel according to the molecular weight of the target protein and the internal reference. At the same time, cut the PVDF membrane to the same size as the PAGE gel. Soak the PVDF membrane in formaldehyde for about 10 seconds before transfer to activate the PVDF membrane. Prepare the transfer tank by assembling the transfer clamp in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge from the negative electrode to the positive electrode. The transfer clamp must be clamped tightly and cannot be misaligned or have bubbles. Use a wet transfer system and transfer at a constant current of 300mA for 35-40 minutes.

[0133] (5) Blocking: Take out the transferred membrane and observe whether the transfer is complete (observe according to the pre-stained color protein mark), rinse twice with TBST, place the transferred PVDF membrane in blocking solution, and block at room temperature for 1 hour. The blocking solution is protein-free rapid blocking solution (1×). After blocking, wash three times with TBST.

[0134] (6) Antibody incubation: Cut the bands according to the molecular weight of the internal reference protein and the target protein, then add the bands to the diluted primary antibody incubation tubes and incubate overnight at 4°C on a shaker. Then wash with TBST three times, add the secondary antibody at a ratio of 1:4000, incubate on a shaker for 1 hour, discard the secondary antibody, and wash three times with TBST.

[0135] (7) Exposure and photography. Mix solution A and solution B in the chemiluminescent substrate kit in a ratio of 1:1 to prepare 2 mL of developer solution. Pour the developer solution dropwise onto the parallel position of the protein on the PVDF membrane. Let it sit for about 20 seconds. Place the PVDF membrane into the chemiluminescent detection instrument and take a photo. The exposure time depends on the intensity of the fluorescence signal. Adjust the exposure time to achieve the best exposure result. Repeat 3 times and take a photo to save.

[0136] (8) Analysis of protein expression: Image J software was used to perform statistical analysis on the grayscale values ​​of protein bands.

[0137] The results are shown in Figure 9. Compared with the control group, 20 ng / mL TNF-α reduced the expression of MyoD1, an early myotube differentiation marker, and MyoG, a myogenic marker. Low-concentration DZFJZJ treatment reduced the expression of MyoD1 and MyoG proteins. However, increasing DZFJZJ concentration gradually increased MyoD1 expression, but without significant differences, while MyoG expression significantly increased. 20 ng / mL TNF-α significantly increased the expression of MuRF1 and Atrogin-1, muscle atrophy markers. Increasing DZFJZJ concentration gradually decreased MuRF1 and Atrogin-1 expression, and 800 μg / mL and 1600 μg / mL DZFJZJ significantly decreased MuRF1 expression. Therefore, DZFJZJ can increase the expression of myogenic markers while decreasing the expression of muscle atrophy markers, thereby ameliorating TNF-α-induced C2C12 myotube atrophy.

[0138] 2.3 Effect of DZFJZJ on LPS-induced skeletal muscle atrophy in mice

[0139] 2.3.1 Effect of DZFJZJ on LPS-induced body weight in mice

[0140] Six- to eight-week-old C57BL / 6 male mice were randomly divided into five groups (n=8 per group): control group (normal saline); LPS group (2 mg / kg); LPS (2 mg / kg) + DZFJZJ group (100 mg / kg); LPS (2 mg / kg) + DZFJZJ group (200 mg / kg); and LPS (2 mg / kg) + DZFJZJ group (300 mg / kg). LPS and DZFJZJ were intraperitoneally injected daily and the mice were weighed.

[0141] The results are as follows Figure 10 As shown, the weight of mice in the control group remained stable over time. However, the weight of the LPS-treated group and the 100mg / kg and 300mg / kg DZFJZJ-treated groups initially decreased after LPS injection, then increased and stabilized after 12 days, remaining significantly lower than that of the control group. The 200mg / kg DZFJZJ group showed no significant difference from the control group starting on day 10, with the mice's weight essentially recovering. The weight gain of mice treated with 200mg / kg DZFJZJ was significantly greater than that of the LPS group, with statistically significant differences observed on days 10 and 13. This suggests that DZFJZJ can alleviate the weight loss of mice induced by LPS.

[0142] 2.3.2 Effects of DZFJZJ on LPS-induced locomotor activity in mice

[0143] The grasping strength of mice was measured before administration, on the 7th day and on the 14th day after administration. Before administration, the grasping strength of mice in each group was equivalent, with no significant difference; on the 7th day after administration, the grasping strength of the LPS group and the DZFJZJ group decreased compared with the control group, and the grasping strength of the 200mg / kgDZFJZJ group increased significantly compared with the LPS group; on the 14th day after administration, the grasping strength of the LPS group decreased compared with the control group; the grasping strength of the DZFJZJ group increased compared with the LPS group, with statistical differences, and no significant difference compared with the control group. The grasping strength of mice in the same group at different times was compared. Except for the control group, the grasping strength of mice in the LPS and DZFJZJ treatment groups showed a trend of first decreasing and then increasing. The grasping strength of mice in the DZFJZJ treatment group could return to the level before administration at 14 days. The grasping strength of mice stimulated by LPS was improved ( Figure 11 ).

[0144] The rod-spent time of mice in the rotarod test was measured before administration, on the 7th day, and on the 14th day. Before administration, the rod-spent time of mice in each group was comparable, with no significant difference. On the 7th day, the rod-spent time of both the LPS and DZFJZJ-treated groups decreased compared with the control group, with a significant difference in the 200mg / kg DZFJZJ group. On the 14th day, the rod-spent time of the LPS and 100mg / kg DZFJZJ groups decreased significantly compared with the control group, while the 200mg / kg DZFJZJ group showed no significant difference compared with the control group. The rod-spent time of the 100mg / kg and 200mg / kg DZFJZJ groups increased compared with the LPS group, with a statistical difference in the 200mg / kg DZFJZJ group. When the rod-spent time of mice in the same group was compared at different times, the rod-spent time of mice in the LPS and DZFJZJ-treated groups, except for the control group, showed a trend of first decreasing and then increasing. The rod time of LPS-stimulated mice was also improved ( Figure 12 ).

[0145] In summary, DZFJZJ can improve the motor ability of LPS-stimulated mice.

[0146] 2.3.3 Effect of DZFJZJ on LPS-induced muscle mass in mice

[0147] To evaluate skeletal muscle atrophy, the hind limb calf weights of mice were measured before the end of the experiment. Figure 13 It can be seen that the calf muscle weight of the hind limbs of mice injected with LPS was lower than that of the control group. The muscle weight of mice increased after injection of DZFJZJ. The 200 mg / kg DZFJZJ group was able to significantly increase the calf muscle weight of the hind limbs of mice compared with the LPS-treated group, and there was no statistical difference compared with the control group.

[0148] 2.3.4 Effects of DZFJZJ on LPS-induced myofiber structure in mice

[0149] On day 14, after dosing and behavioral testing, mice rested overnight. The next day, they were anesthetized and their eyes were removed for blood collection. Serum was collected by centrifugation at 12,000 rpm for 20 minutes and stored at -80°C for subsequent experiments. Calf muscle tissue was then removed from both limbs and weighed. The left muscle tissue was used for protein-related experiments and stored at -80°C. The right muscle tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E to measure muscle fiber number and cross-sectional area.

[0150] Depend on Figure 14 The results showed that the control group showed mild inflammatory cell infiltration in the interstitial myofibers, without other pathological changes such as muscle fiber atrophy, degeneration, or necrosis. The LPS group showed moderate inflammatory cell infiltration in the interstitial myofibers of the calf muscle tissue, with muscle atrophy, partial muscle fiber atrophy, moderate degeneration, and necrosis. After adding different concentrations of DZFJZJ, the interstitial inflammatory cell infiltration of the myofibers in mice improved from moderate to mild infiltration, and the muscles became more regularly arranged, with the muscle fibers more tightly arranged, and some samples showed only mild atrophy.

[0151] 2.4 Conclusion

[0152] The present invention obtains oligopeptide-rich DZFJZJ by solid-state fermentation of bean dregs mixed with high-aminopeptidase-producing Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1, and then extracting the solid-state fermentation product with water. Furthermore, the present invention establishes a myotube atrophy model by inducing mouse myoblast C2C12 damage with TNF-α in vitro, explores the effect of DZFJZJ on C2C12 cells, and studies the effect of DZFJZJ on LPS-induced muscle atrophy mice in vivo. The above experiments verify that, first, DZFJZJ can promote the proliferation of C2C12 cells, improve the inhibitory effect of TNF-α on cell migration ability, reduce the ratio of cells in the G0 / G1 phase of the cell cycle, increase the ratio of cells in the G2 / M phase, improve the cell cycle arrest caused by TNF-α, and also improve the promoting effect of TNF-α on cell aging.

[0153] Secondly, DZFJZJ can also promote the differentiation of C2C12 cells into myotubes, increase the number, length and diameter of myotubes, upregulate the expression of MyoD1 and MyoG, and downregulate the expression of MuRF and Atrogin-1. Furthermore, it can improve the weight loss of LPS-stimulated mice, improve the exercise ability of LPS-stimulated mice, and increase the weight of the calf muscles of the hind limbs of mice.

[0154] Based on the above, it can be concluded that DZFJZ has the potential to delay aging, prevent aging-related muscle atrophy and weakness, and treat sarcopenia.

[0155] 3. Process optimization of DZFJZJ preparation by mixed bacteria solid-state fermentation of soybean dregs

[0156] Soybean dregs contain 10-20% protein on a dry basis, and their amino acid composition is essentially the same as that of soybeans. Numerous studies have shown that soy protein can produce short peptide chains through the action of microorganisms, enzymes, or other biochemical reactions. These short peptides, known as soy peptides, possess biological functions such as antioxidant, anti-cancer, anti-hypertensive, intestinal barrier protection, immune regulation, and mineral absorption and utilization promotion. The present invention first utilizes a mixture of high-aminopeptidase-producing Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 to solid-state ferment soy dregs to obtain a solid-state fermentation product. This solid-state fermentation product is then subjected to water extraction to obtain DZFJZJ, which is rich in dregs oligopeptides. Considering that the concentration of dregs oligopeptides in DZFJZJ may be a factor potentially affecting the aforementioned efficacy of DZFJZJ, the present invention uses the concentration of dregs oligopeptides in DZFJZJ as an indicator to further optimize the process conditions for preparing DZFJZJ from mixed solid-state fermentation of dregs.

[0157] The experimental design is as follows:

[0158] Single-factor experiments were conducted on the preparation of soybean dregs oligopeptides by solid-state fermentation of IFJ1 and LMX1, and single-factor optimization experiments were carried out at five levels for five factors, namely initial moisture content, fermentation liquid inoculation amount, fermentation temperature, fermentation time, and fermentation liquid ratio.

[0159] 3.1 Effect of different initial moisture contents on the concentration of okara oligopeptides

[0160] Accurately weigh 200 g of dregs into a clean sealed bag, add 6% seed liquid, take the initial moisture content as the independent variable, control the fermentation time 6d, fermentation temperature 28℃, bacteria-liquid ratio 1:1 and other factors unchanged, analyze the effects of initial moisture content of 40%, 50%, 60%, 70% and 80% on the concentration of dregs oligopeptides in DZFJZJ.

[0161] 3.2 Effect of different inoculation amounts on the concentration of okara oligopeptides

[0162] Accurately weigh 200 g of dregs into a clean sealed bag, add 70% sterile water, take the inoculation amount as the independent variable, keep the fermentation time 6d, fermentation temperature 28℃, bacterial liquid ratio 1:1 and other factors unchanged, analyze the effects of inoculation amounts of 2%, 4%, 6%, 8% and 10% on the concentration of dregs oligopeptides in DZFJZJ.

[0163] 3.3 Effects of different fermentation temperatures on the concentration of okara oligopeptides

[0164] Accurately weigh 200 g of dregs into a clean sealed bag, add 70% sterile water, take fermentation temperature as the independent variable, keep the fermentation time 6d, inoculation size 6%, bacterial liquid ratio 1:1 and other factors unchanged, analyze the effects of fermentation temperatures of 24℃, 26℃, 28℃, 30℃ and 32℃ on the concentration of dregs oligopeptides in DZFJZJ.

[0165] 3.4 Effects of different fermentation times on the concentration of okara oligopeptides

[0166] Accurately weigh 200 g of dregs into a clean sealed bag, add 70% sterile water, take fermentation time as the independent variable, keep the inoculation amount 6%, fermentation temperature 28℃, bacterial liquid ratio 1:1 and other factors unchanged, analyze the effect of fermentation time of 3d, 4d, 5d, 6d and 7d on the concentration of dregs oligopeptides in DZFJZJ.

[0167] 3.5 Effects of different bacterial liquid ratios on the concentration of okara oligopeptides

[0168] Accurately weigh 200 g of okara into a clean sealed bag, add 70% sterile water, take the bacterial solution ratio as the independent variable, keep the fermentation time 6d, inoculation size 6%, fermentation temperature 28℃ and other factors unchanged, analyze the effects of using IFJ1 bacterial solution alone, LMX1 bacterial solution and mixed use, the IFJ1:LMX1 bacterial solution ratio of 1:1, 1:2, 2:1, 2:3, 3:2 on the concentration of okara oligopeptides.

[0169] 3.6 Determination of Okara Oligopeptide Concentration in DZFJZJ

[0170] The concentration of okara oligopeptides was determined by the Folin-phenol method. The specific steps are as follows:

[0171] (1) Preparation of standard solution

[0172] An appropriate amount of bovine serum albumin standard (Beijing Wokai Biotechnology) was accurately weighed, dissolved in pure water, and diluted to a standard solution containing 0.3 mg of bovine serum albumin per 1 mL of water.

[0173] (2) Drawing of standard curve

[0174] Take 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 0.3 mg / mL bovine serum albumin solution and place them in stoppered test tubes respectively. Add pure water to 1.0 mL, add 1.0 mL of alkaline copper test solution to each tube, mix immediately, and let it stand at room temperature for 10 minutes. Add 4.0 mL of folin phenol (Beijing Wokai Biotechnology) test solution to each tube, mix immediately, and let it stand at room temperature for 30 minutes. Measure the absorbance at a wavelength of 650 nm. At the same time, use the tube with 0.0 mL of standard solution as a blank.

[0175] With the concentration of the standard solution as the horizontal axis and the corresponding absorbance as the vertical axis, the regression equation is y=0.6126x+0.0196, (R 2 =0.997), indicating a good linear relationship with the absorbance in the concentration range of 0 mg / mL to 0.18 mg / mL.

[0176] (3) Determination of okara oligopeptide concentration in the DZFJZJ sample to be tested

[0177] Dilute DZFJZJ to an appropriate concentration, accurately pipette 1.0 mL of the sample to be tested, measure the absorbance according to the method in (2), substitute it into the regression equation, and calculate the concentration of okara oligopeptides in DZFJZJ.

[0178] Here are the results:

[0179] When the initial water content was 70%, the concentration of okara oligopeptides in DZFJZJ reached a maximum value of 13.07 mg / mL; when the initial water content increased to 80%, the concentration of okara oligopeptides decreased. Compared with the initial water content of 60% and 80%, the concentration of okara oligopeptides increased by 36.73% and 12.32% respectively when the initial water content was 70% (P < 0.05). Therefore, the appropriate initial water content for the solid-state fermentation of LMX1 and IFJ1 to prepare okara is 70% ( Figure 15 ).

[0180] When the fermentation time was 6 days, the concentration of okara oligopeptides reached a maximum value of 15.25 mg / mL; when the fermentation time increased to 7 days, the concentration of okara oligopeptides decreased. Compared with the fermentation time of 5 days and 7 days, the concentration of okara oligopeptides increased by 24.33% and 15.15% at 6 days (P < 0.05), respectively. Therefore, the optimal fermentation time for LMX1 and IFJ1 solid-state fermentation to prepare okara oligopeptides is 6 days ( Figure 16 ).

[0181] When the fermentation temperature was 28℃, the oligopeptide concentration reached a maximum of 12.75mg / mL; when the fermentation temperature increased to 30℃, the concentration of okara oligopeptides decreased. Compared with the fermentation temperatures of 26℃ and 30℃, the concentration of okara oligopeptides increased by 9% and 45.1% respectively at the fermentation temperature of 28℃ (P<0.05). Therefore, the suitable fermentation temperature for LMX1 and IFJ1 solid-state fermentation to prepare okara oligopeptides is 28℃ ( Figure 17 ).

[0182] When the inoculation amount of LMX1 and IFJ1 was 2%-10%, the concentration of okara oligopeptides decreased with the increase of inoculation amount. When the inoculation amount of LMX1 and IFJ1 was 2%, the concentration of okara oligopeptides reached the highest value of 18.71 mg / mL; therefore, the appropriate inoculation amount for the preparation of okara oligopeptides by solid-state fermentation of LMX1 and IFJ1 was 2% ( Figure 18 ).

[0183] When the ratio of LMX1 and IFJ1 bacterial liquid was 1:1, the concentration of okara oligopeptides in DZFJZJ reached the highest value of 13.07 mg / mL, which was 1.6 times and 2.0 times that of the fermentation of Lactobacillus paracasei LMX1 and Lactobacillus paracasei LMX1, respectively. Therefore, the suitable strain ratio for the solid-state fermentation of Lactobacillus paracasei LMX1 and Lactobacillus paracasei IFJ1 to prepare DZFJZJ is 1:1 ( Figure 19 ).

[0184] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A soy dregs solid-state fermentation preparation for preparing a drug for treating or preventing sarcopenia, characterized in that: The bean dregs solid-state fermentation preparation is based on Lactobacillus paracasei ( Lacticaseibacillus paracasei ) LMX1 and Pediococcus acidilactici ( Pediococcus acidilactici ) IFJ1 is used as the fermentation strain, and bean dregs are used as the fermentation substrate, first by solid-state fermentation and then by extraction. The Lactobacillus paracasei LMX1 has a deposition number of CCTCC M20211073 and was deposited in the China Center for Type Culture Collection on August 24, 2021; The Pediococcus acidilactici IFJ1 has a deposit number of CCTCC M2022828 and was deposited in the China Center for Type Culture Collection on June 8, 2022; The preparation method of the bean dregs solid-state fermentation preparation comprises the following steps: (1) Dry bean dregs were added to sterile water to prepare a solid fermentation medium, and then Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 were cultured and activated to prepare seed liquids, and the seed liquids were mixed to prepare a fermentation liquid; (2) inoculating the fermentation liquid obtained in step (1) into a solid-state fermentation medium for solid-state fermentation, and obtaining a solid-state fermentation product of bean dregs after the solid-state fermentation is completed; (3) Using distilled water as an extracting liquid, the okara solid-state fermentation product obtained in step (2) is extracted to obtain the okara solid-state fermentation preparation.

2. The soy dregs solid-state fermentation preparation for preparing a drug for treating or preventing sarcopenia according to claim 1, characterized in that: The bacterial liquid volume ratio of the Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 is (1-3): (1-3).

3. The soy dregs solid-state fermentation preparation for preparing a drug for treating or preventing sarcopenia according to claim 1, characterized in that: The initial water content of the solid-state fermentation medium is 40-80%, the inoculation amount of the fermentation liquid is 2-10% of the mass of the solid-state fermentation medium, the solid-state fermentation temperature is 24-32° C., and the fermentation time is 3-7 days.

4. Use of the okara solid-state fermentation preparation according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing sarcopenia.

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