Bean dreg solid-state fermentation preparation and application thereof in treating or preventing sarcopenia

Abstract

The invention discloses a bean dreg solid state fermentation preparation and application thereof in treating or preventing sarcopenia, and relates to the field of bean dreg processing, the bean dreg solid state fermentation preparation is obtained by taking lactobacillus paracasei LMX1 and pediococcus acidilactici IFJ1 as fermentation strains, taking bean dregs as a fermentation substrate, firstly performing solid state fermentation and then performing extraction. A myotubular atrophy model is established by inducing mouse myoblast C2C12 injury with TNF-alpha in vitro, the effect of the bean dreg solid-state fermentation preparation on C2C12 cells is explored, the influence of the bean dreg solid-state fermentation preparation on LPS-induced muscular atrophy mice is researched in vivo, and the result shows that the bean dreg solid-state fermentation preparation has potential effects of delaying senescence, promoting growth of muscular atrophy, preventing and treating muscular atrophy. The composition has the effects of preventing muscle atrophy and weakness related to aging and treating sarcopenia, and reference is provided for exploring potential application of plant active ingredients in the aspect of preventing or treating muscle functions.

Description

Technical Field

[0001] The present invention relates to the field of okara processing, and particularly to a solid-state fermentation preparation of okara and its application in the treatment or prevention of sarcopenia. Background Art

[0002] Sarcopenia, also known as muscle wasting disease or muscle atrophy, is a progressive systemic skeletal muscle disease related to age, which mostly occurs in the elderly population. The characteristics of this disease are the decline of physical fitness, the gradual loss of muscle mass, strength and function, resulting in difficulties in daily activities, increasing the risk of falls and fractures, and even shortening the life expectancy of the elderly. The risk of sarcopenia gradually intensifies with age and is one of the main causes of death and disability in the elderly.

[0003] Research shows that the occurrence of sarcopenia is affected by multiple factors such as genetics and environment, and its appearance and progression are related to multiple mechanisms related to the imbalance between skeletal muscle protein synthesis and degradation, neuromuscular integrity and muscle fat content. The pathophysiological mechanism of sarcopenia is quite complex and is a syndrome involving the dysregulation of multiple signaling pathways. Sarcopenia is related to the changes in skeletal muscle physiology and cellular mechanisms, and these changes include multiple aspects such as metabolism, cells, blood vessels and inflammation levels. Specifically, the possible pathogenic factors of sarcopenia include malnutrition, lack of exercise, chronic inflammation, oxidative stress injury, mitochondrial dysfunction, age-related hormone changes and weakened neuromuscular function, etc. For the intervention measures of sarcopenia, they mainly consider exercise intervention, nutritional support and drug treatment.

[0004] Regarding the intervention measure of drug treatment, currently, there is a lack of suitable specific drugs for sarcopenia. The common strategies for the prevention or treatment of skeletal muscle atrophy in clinical practice are hormone replacement and its adjuvant therapies, but they need to be improved due to the relatively large side effects of hormone therapy. Some 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 enzyme inhibitors are commonly used in the treatment of sarcopenia, but the clinical application effects and safety still need to be further studied. Generally speaking, the research on drug treatment of sarcopenia in China is still in the exploratory stage. Therefore, it is particularly important to explore new effective functional components for the prevention or treatment of sarcopenia.

[0005] Current research shows that many plant active ingredients with antioxidant and anti-inflammatory properties exhibit good intervention effects on the occurrence and progression of sarcopenia. There are numerous animal experiments and in vitro experiments on sarcopenia, showing good research prospects. However, this is not sufficient to directly prove that these plant active ingredients can effectively prevent or treat the loss of muscle mass and muscle function. To further verify the potential of plant active ingredients in preventing sarcopenia, more relevant research needs to be carried out. Currently, the number of published studies on the potential application of plant drugs 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 object of the present invention is to overcome the deficiencies of the prior art and provide a solid-state fermentation preparation of soybean residue and its application in the treatment or prevention of sarcopenia.

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

[0008] In the first aspect of the present invention, a solid-state fermentation preparation of soybean residue is provided. The solid-state fermentation preparation of soybean residue uses Lacticaseibacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 as fermentation strains, and soybean residue as a fermentation substrate. It is first subjected to solid-state fermentation and then obtained through extraction.

[0009] As a further optimized scheme of the present invention, the preservation number of Lacticaseibacillus paracasei LMX1 is CCTCC M20211073, and it was preserved at the China Center for Type Culture Collection on August 24, 2021;

[0010] The preservation number of Pediococcus acidilactici IFJ1 is CCTCC M2022828, and it was preserved at the China Center for Type Culture Collection on June 8, 2022.

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

[0012] As a further optimized scheme of the present invention, the preparation method of the solid-state fermentation preparation of soybean residue includes the following steps:

[0013] (1) Add dried okara to sterile water to prepare a solid-state fermentation medium. Subsequently, Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 are respectively cultured and activated to obtain seed solutions, and the seed solutions are mixed to obtain a fermentation broth.

[0014] (2) Inoculate the fermentation broth obtained in step (1) into the solid-state fermentation medium for solid-state fermentation. After the solid-state fermentation is completed, okara solid-state fermented product is obtained.

[0015] (3) Use distilled water as the extraction solution to extract the okara solid-state fermented product obtained in step (2) to obtain the okara solid-state fermentation preparation. The extraction process is further preferably that the ratio of extraction material to liquid is 1:12, the extraction temperature is 50 °C, and the extraction time is 30 min.

[0016] As a further optimization scheme of the present invention, the initial water content of the solid-state fermentation medium is 40 - 80%, the inoculation amount of the fermentation broth 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 d.

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

[0018] In the second aspect of the present invention, there is also provided an application of the okara solid-state fermentation preparation as described in any one of the above in the preparation of a drug for treating or preventing sarcopenia.

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

[0020] The present invention obtains an okara solid-state fermentation preparation rich in oligopeptides by mixing and solid-state fermenting okara with Lactobacillus paracasei LMX1 with high aminopeptidase productivity and Pediococcus acidilactici IFJ1. Further, the present invention establishes a myotube atrophy model by inducing C2C12 mouse myoblast injury with TNF-α in vitro, explores the effect of the okara solid-state fermentation preparation on C2C12 cells, and studies the effect of the okara solid-state fermentation preparation on LPS-induced muscle atrophy mice in vivo. The results indicate that the okara solid-state fermentation preparation can promote the proliferation of C2C12 cells, improve the inhibitory effect of TNF-α on cell migration ability, reduce the proportion of cells in the G0 / G1 phase of the cell cycle, increase the proportion of cells in the G2 / M phase, improve the cell cycle arrest caused by TNF-α, and can also improve the promoting effect of TNF-α on cell senescence.

[0021] In addition, the solid-state fermentation preparation of soybean dregs can also promote the differentiation of C2C12 cells into myotubes, increase the number, length, and diameter of myotubes, up-regulate the expression of early differentiation marker MyoD1 and myogenesis marker MyoG, and down-regulate the expression of muscle atrophy markers MuRF and Atrogin-1. Further, it can improve the weight loss of LPS-stimulated mice, improve the motor ability of LPS-stimulated mice, and increase the weight of the calf muscles in the hind limbs of mice.

[0022] Based on the above, it is concluded that the solid-state fermentation preparation of soybean dregs has 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. Description of the Drawings

[0023] Figure 1 Shows the effects of solid-state fermentation preparations of soybean dregs at different concentrations on the proliferation of C2C12 cells;

[0024] Figure 2 Shows the effects of solid-state fermentation preparations of soybean dregs at different concentrations on the migration of C2C12 cells;

[0025] Figure 3 Shows the effects of solid-state fermentation preparations of soybean dregs at different concentrations on the migration of C2C12 cells induced by 20 ng / mL TNF-α;

[0026] Figure 4 Shows the effects of solid-state fermentation preparations of soybean dregs at different concentrations on the cell cycle of C2C12 cells induced by 20 ng / mL TNF-α;

[0027] Figure 5 Shows the effects of solid-state fermentation preparations of soybean dregs at different concentrations on the senescence of C2C12 cells induced by TNF-α observed by β-galactosidase staining;

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

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

[0030] Figure 8 Shows the mRNA expression levels of related myogenesis and muscle atrophy markers detected by qPCR;

[0031] Figure 9a Shows the protein expression level of the early differentiation marker MyoD1 of myotubes detected;

[0032] Figure 9b Shows the protein expression level of the myogenesis marker MyoG detected;

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

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

[0035] Figure 10 The effect of solid-state fermentation preparations of okara at different concentrations on the body weight of LPS-stimulated mice;

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

[0037] Figure 12 The effect of solid-state fermentation preparations of okara at different concentrations on the time of mice on the rod under LPS stimulation;

[0038] Figure 13 The effect of solid-state fermentation preparations of okara at different concentrations on the calf muscle weight of LPS-stimulated mice;

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

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

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

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

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

[0044] Figure 19 The effect of different ratios of LMX1 and IFJ1 bacterial solutions on the okara oligopeptide concentration in the solid-state fermentation preparation of okara. Specific implementation manners

[0045] The present invention is further described in 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. Technicians in this field 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 are commercially available products;

[0048] The preservation number of Lactobacillus paracasei LMX1 is CCTCCM20211073, and it was preserved 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 Study on Its Fermentation Effect [D]" discloses the isolation, screening, identification and cultural characteristics of the strain).

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

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

[0052] Mice: C57BL / 6, 6-8 weeks old, purchased from Henan Sikebes Biotechnology Co., Ltd. The animals involved in the present invention were raised and managed in strict accordance with the "Regulations on the Management of Experimental Animals" and the guidelines formulated by the Experimental Animal Ethics Committee of Anhui Agricultural University, and all animal experimental operations were strictly carried out in accordance with the specifications 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 to 400 mL of pure water, stir and dissolve for standby; Solution B: Accurately weigh 0.5 g of potassium sodium tartrate and dissolve it in 50 mL of pure water, 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 make up the volume to 500 mL.

[0056] II. Methods

[0057] Unless otherwise specified, the methods used below are all 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 soybean dregs used in the present invention are fresh soybean dregs, which are by-products generated during the production of soybean milk by Anhui Dafu Food Co., Ltd. The fresh soybean dregs are sterilized in an autoclave at 121 °C for 15 min, then placed on a baking tray and dried in an oven at 55 °C for 48 h. During this period, the soybean dregs need to be frequently turned. The dried soybean dregs are stored in a 4 °C ice storage.

[0061] 1.2 Preparation of fermentation bacterial liquid

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

[0063] The glycerol bacteria of IFJ1 and LMX1 preserved in the laboratory are respectively inoculated into 75 mL of MRS broth liquid medium under sterile conditions and cultured statically in an incubator at 37 °C for 48 h;

[0064] Under sterile conditions, the bacterial liquid is diluted, and then a sterilized pipette tip is used to accurately aspirate 30 μL of the appropriate gradient of diluted bacterial liquid (10 -6 、10 -7 、10 -8 ) and spread it on the solidified solid medium. Spread it back and forth several times, invert it and place it in an incubator at 37 °C for 24 h. Observe the spreading result, select the single colonies with good growth state and obvious morphology, inoculate them on the solid medium, and continue to streak and purify for 3 generations. According to the colonies after streaking, identify whether they are IFJ1 and LMX1 by colony morphology and Gram staining.

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

[0066] Using a sterile inoculation loop, pick one single colony with a better phenotype from the above-determined IFJ1 and LMX1 respectively, and inoculate them into MRS broth liquid medium. Incubate statically in an incubator at 37°C for 48 h to obtain the IFJ1 and LMX1 seed solutions.

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

[0068] 1.3 Solid-state fermentation of soybean residue

[0069] First, take 200 g of the dry soybean residue prepared in step 1.2, add sterile water to prepare a solid-state fermentation medium with an initial water content of 70%. Subsequently, inoculate the fermentation bacterial solution prepared in step 1.2 into the solid-state fermentation medium for solid-state fermentation. The inoculation amount of the fermentation bacterial solution is 2% of the mass of the solid-state fermentation medium. The solid-state fermentation temperature is 28°C, and the fermentation time is 6 d. After the solid-state fermentation is completed, a soybean residue solid-state fermentation product is obtained.

[0070] 1.4 Extraction of the soybean residue solid-state fermentation product

[0071] Using distilled water as the extraction solution, extract the soybean residue solid-state fermentation product under the conditions of an extraction material-liquid ratio of 1:12, an extraction temperature of 50°C, and an extraction time of 30 min to obtain a soybean residue solid-state fermentation preparation (hereinafter referred to as DZFJZJ). Perform freeze-drying treatment on DZFJZJ to obtain DZFJZJ freeze-dried powder, and store the DZFJZJ freeze-dried powder in a -20°C refrigerator.

[0072] 2 Exploration of the biological functions of DZFJZJ

[0073] 2.1 Effect of DZFJZJ on the senescence of C2C12 cells

[0074] 2.1.1 Myotube differentiation of C2C12 cells

[0075] After resuscitating, culturing, and subculturing C2C12 cells, collect the C2C12 cells in the logarithmic growth phase and inoculate them into a 6-well culture plate with DMEM complete medium. Place them in an incubator at 37°C and 5% CO2. When the cells grow to 80%, remove the medium and replace it with a differentiation medium for induced differentiation. Replace the differentiation medium every two days. From the third day, a small amount of myotubes can be observed to form. Thereafter, the myotubes become thicker and the number increases. Differentiation can be used for subsequent experiments on the fifth day.

[0076] 2.1.2 Determination of the effect of DZFJZJ on the proliferation of C2C12 cells by the CCK-8 method

[0077] (1) Take out the cells from the incubator and place them under an inverted microscope to observe the cells. Select C2C12 cells with good condition and about 80% cell fusion to prepare a cell suspension. Count the cells and evenly inoculate 5000 cells per well in a 96-well plate, then place it in the incubator for 24 h.

[0078] (2) The next day, aspirate the original culture medium, add different concentrations of DZFJZJ and continue to culture for 96 h. The concentrations of DZFJZJ are set to 0, 100, 200, 400, 800, and 1600 μg / mL, which are respectively diluted and prepared from a stock solution of DZFJZJ freeze-dried powder with a concentration of 120 mg / mL (120 mg of DZFJZJ freeze-dried powder is dissolved in 1 mL of PBS solution, and after dissolution, it is filtered and sterilized). Change the medium every day.

[0079] (3) After 0 h, 24 h, 48 h, 72 h, and 96 h respectively, aspirate the old medium in the 96-well plate, add 100 μL of CCK-8 detection reagent (CCK-8: complete medium = 1:10) to each well, and then incubate at 37 °C in the dark for 1 h.

[0080] (4) After 1 h, take out the 96-well plate, first visually observe the color depth to preliminarily judge the experimental results, and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the effect of DZFJZJ on cell viability. The detection wavelength is set to 450 nm to obtain the OD value of each well, and calculate the survival rate of C2C12 cells after treatment with different concentrations of DZFJZJ. Cell proliferation activity (%) = (OD 实验 - OD 空白 ) / (OD 对照 - OD 空白 ), and each experiment is carried out 3 times.

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

[0082] 2.1.3. Detect the effect of DZFJZJ on the migration ability of C2C12 cells by cell scratch assay

[0083] (1) Take out C2C12 cells with good condition and about 80% cell confluence from the cell culture incubator, digest and collect them, prepare a cell suspension, count the cells, and inoculate 2×10 5 cells per well in a 6-well plate. Shake the cells in the 6-well plate by the cross-cross method, and then place the 6-well plate in the cell culture incubator for 24 h.

[0084] (2) The next day, take the 6-well plate into the laminar flow hood for operation. Aspirate and discard the old medium, cover the culture plate and invert it. Use a marker pen and a ruler to draw five straight lines on the back of the plate as marks. Then use a 200 μL pipette tip to make vertical scratches along the marks of the black marker pen. Take the 6-well plate into the laminar flow hood for operation. Aspirate and discard the old medium, cover the culture plate and invert it. Use a marker pen and a ruler to draw five straight lines on the back of the plate as marks. Then use a 200 μL pipette tip to make vertical scratches along the marks of the black marker pen. Rinse the cell surface with PBS to remove the cells detached due to scratching. Then add complete medium containing 0, 100, 200, 400, 800, and 1600 μg / mL DZFJZJ respectively. Place the cell culture plate under an inverted microscope for photographing and record the initial scratch width.

[0085] (3) After 24 h, take the cell culture plate to the laminar flow hood for operation. First, remove the old medium, then wash the cells with PBS, and then place the culture plate under the microscope to observe the cell migration of different concentration DZFJZJ treatment groups and take pictures for record. Analyze and statistically calculate the scratch healing rate of each group of cells.

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

[0087] The results are as Figure 2 shown. Compared with the blank control group, different concentrations of DZFJZJ can significantly improve the migration ability of C2C12 cells.

[0088] Furthermore, at 0 h, make scratches on the cells and record them. Then treat the cells with 20 ng / mL TNF-α and different concentrations of DZFJZJ for 24 h, and record the width of the cell scratches again. The results are as Figure 3 shown. Treatment with TNF-α 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. Detection of the effect of DZFJZJ on the cell cycle of C2C12 cells by flow cytometry

[0090] Take out the C2C12 cells with good condition and about 80% cell confluence from the cell incubator for digestion and collection, prepare a cell suspension, and perform cell counting. Inoculate 2×10 5 cells per well in a 6-well plate, and shake the cells in the 6-well plate by the cross method. Then place the 6-well plate in the cell incubator for 24 h. The experiment has 6 groups including a blank control and different concentration DZFJZJ treatments.

[0091] After 24 h, the blank control group was added with DMEM complete medium, and the DZFJZJ treatment group was added with the prepared DZFJZJ solution at different concentrations and cultured in an incubator. After 24 h, the original culture medium was aspirated, the blank control group was added with DMEM complete medium, and the other groups were added with 20 ng / mL TNF-α culture medium and cultured in an incubator. After 4 h, the original culture medium was aspirated, rinsed 1-2 times with PBS, the cells were digested with trypsin, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, washed once with PBS, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, 70% ethanol (pre-cooled) was added, pipetted and mixed evenly, and placed at 4 °C overnight. Centrifuged at 1000 rpm for 5 min, the ethanol was discarded, the cells were washed once with PBS, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, an appropriate amount of PI working solution was added, mixed evenly, and incubated at 37 °C. After 30 min, centrifuged at 1000 rpm for 5 min. The supernatant was discarded, rinsed once with an appropriate amount of PBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, an appropriate amount of PBS was added to resuspend the cells, and the cells were detected by flow cytometry.

[0092] The results are as Figure 4 shown. 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. It is shown that DZFJZJ can partially improve the cell cycle arrest caused by TNF-α.

[0093] 2.1.5 Detection of the effect of DZFJZJ on the senescence process of C2C12 cells by β-galactosidase staining

[0094] Take C2C12 cells in good condition with a cell confluence of 80% and prepare a cell suspension with a concentration of 1.5×10 4 cells / mL. Inoculate 1.5×10 4 cells per well in a 24-well plate and culture in an incubator. After 24 h, aspirate the original culture medium, wash once with PBS, and then add DMEM complete medium. Perform different drug addition treatments according to the experimental grouping and culture in an incubator for 4 h. Aspirate the liquid in the wells, wash once with PBS, and then add the fixing solution for fixation at room temperature. After 15 min, aspirate the fixing solution, rinse 3 times with PBS, 3 min each time. Add the staining working solution and incubate overnight at 37 °C. Prepare the staining working solution 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 an appropriate amount of PBS, and observe the staining under an ordinary optical microscope.

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

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

[0100] 2.2.1. Effects of DZFJZJ on myotube atrophy induced by TNF-α in C2C12 cells

[0101] 2.2.1.1. Effects of DZFJZJ on the myotube diameter of C2C12 cells

[0102] To verify the effect of DZFJZJ on the myotube diameter of C2C12 cells, after the C2C12 cell fusion rate reached 80%, add differentiation medium and culture for 4 days, and then add different concentrations of DZFJZJ (0, 100, 200, 400, 800, and 1600 μg / mL) and treat for 2 days. Observe the myotube diameter by immunofluorescence and analyze it with Image J software. As Figure 6 shown, compared with the control group, as the concentration of DZFJZJ increased, the myotube cell diameter also increased. The myotube cells in group G3 were the thickest, and the myotube structure was tight, significantly increasing the diameter of normal myotube cells. As the concentration continued to increase, the myotube cell diameter began to shorten. Therefore, it is considered that the concentration of 800 μg / mL DZFJZJ has a positive effect on myotube differentiation.

[0103] Furthermore, after the C2C12 cell fusion rate reached 80%, add differentiation medium and culture for 4 days, and then add 20 ng / mL TNF-α and different concentrations of DZFJZJ (0, 100, 200, 400, 800, and 1600 μg / mL) and treat for 2 days. Observe the myotube diameter by immunofluorescence staining and analyze it with Image J software. As Figure 7As shown, compared with the control, the myotubes of C2C12 mature myotubes treated with TNF-α showed obvious atrophy, with nuclei aggregating into clusters, and the myotube structure was very loose, showing a bubble-like cavity. The myotube diameter was narrow and the myotube quality was poor. After treatment with DZFJZJ, as the concentration increased, the degree of myotube atrophy decreased significantly, the myotube structure was tighter, the quality was better, and the myotube diameter also increased significantly. Moreover, the cell state in the T20+G3 group was the best, that is, 800 μg / mL DZFJZJ had a better inhibitory effect on TNF-α-induced inhibition of myotube differentiation.

[0104] 2.2.1.2, Effects of DZFJZJ on the expression of related mRNAs in C2C12 cells

[0105] The mRNA expression levels of the myotube early differentiation marker MyoD1, the myogenesis marker MyoG, and the muscle atrophy markers MuRF1 and Atrogin-1 were detected by fluorescence quantitative PCR technology. The specific steps are as follows:

[0106] (1) RNA extraction: Digest the cells with trypsin and collect by centrifugation. Discard the supernatant. Extract RNA according to the steps in the cell RNA rapid extraction kit manual. Add 500 μL of Lysis Buffer to the cell pellet, pipette and mix well, and let stand for 1 min. Add the mixture to the adsorption column, centrifuge at 12,000 rpm for 30 s and discard the filtrate. Add 500 μL of WashingBuffer D2 to the adsorption column, centrifuge at 12,000 rpm for 30 s and discard the filtrate, and repeat once. Then place the adsorption column in an empty collection tube, centrifuge at 12,000 rpm for 30 s to remove the washing solution, take out the adsorption column and place it in a clean RNase Free centrifuge tube. Add 25 - 50 μL of RNase Free H2O to the middle position of the adsorption membrane, let stand at room temperature for 1 min, and centrifuge at 1,000 rpm for 1 min. Measure the RNA concentration using NanoDrop.

[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 a RNase-free PCR tube. Then make up to 20 μL with RNase-free Water, gently mix and centrifuge briefly to perform reverse transcription to synthesize cDNA. Incubate at 42 °C for 15 min, continue to incubate 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: Perform RT-qPCR on the cDNA obtained by reverse transcription according to the following reaction system and reaction conditions. Design primer sequences using PrimerBank. For the reaction system of RT-qPCR, add reagents according to Table 2:

[0109] Table 2. Reaction system for detecting gene expression levels by RT-qPCR

[0110]

[0111] Table 3. Gene names and GeneIDs

[0112]

[0113]

[0114] Table 4. Primer names and sequences

[0115]

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

[0117] Table 5. Reaction conditions for RT-qPCR

[0118]

[0119] As Figure 8 shown, 20 ng / mL TNF-α inhibited the mRNA expression of the early myotube differentiation marker MyoD1 and the myogenesis marker MyoG. Treatment with high concentrations of DZFJZJ could improve the inhibitory effect of TNF-α on the mRNA expression of MyoD1 and MyoG. Compared with the control group, 20 ng / mL TNF-α significantly increased the mRNA expression of the muscle atrophy marker MuRF1. Except for 1600 μg / mL DZFJZJ, treatment with different concentrations of DZFJZJ could inhibit the promoting effect of TNF-α on the mRNA expression of MuRF1, and the effect of 400 μg / mL DZFJZJ was better than that of other DZFJZJ concentrations. 20 ng / mL TNF-α significantly increased the mRNA expression of the muscle atrophy marker Atrogin-1, and treatment with different concentrations of DZFJZJ could significantly reduce the mRNA expression of Atrogin-1.

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

[0121] The key proteins related to the myotube cell state are the early myotube differentiation marker MyoD1, the myogenesis marker MyoG, and the muscle atrophy markers MuRF1 and Atrogin-1. The expression levels of related proteins were detected by Western blot, and the specific steps are as follows:

[0122] (1) Protein extraction from cells: After rinsing the drug-treated cells once with PBS, add 200 μL of 1× SDS-PAGE protein loading buffer, 2 μL of protease inhibitor PMSF, and 2 μL of Cocktail to lyse the cells. Scrape the cells with a cell scraper and aspirate them into a 1.5 mL EP tube. Heat the tube in a 97 °C metal bath for 10 min to denature the proteins. Take out the EP tube and immediately place it on ice. After the sample temperature drops, prepare for loading or store it in a -80 °C refrigerator.

[0123] (2) Preparation of polyacrylamide gel: Take out two glass plates for gel preparation and check if there are any cracks or chips (if there are, replace with new glass plates). Wash them twice with ultrapure water and let them dry. Then align and clamp the glass plates with the cover plate and place them horizontally on the gel preparation rack. Add an appropriate amount of double-distilled water to the glass plates and let it stand for 10 - 15 min to check for leakage. If there is no leakage, pour out the double-distilled water and invert the glass plates to dry the remaining water. Take a clean 50 mL centrifuge tube and prepare the lower layer gel according to the solution volumes listed in Table 6:

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

[0125]

[0126] After adding TEMED and mixing well, within 10 min, slowly add the prepared lower layer gel solution along the edge of the glass plate, preventing the formation of bubbles. Add 10 mL of the mixture to each glass plate. After the liquid level is level, add 1 mL of isopropanol and let it stand for 20 - 30 min. After the lower layer gel solidifies, pour out the isopropanol and invert the glass plates to dry the remaining isopropanol. Then take another clean 50 mL centrifuge tube and prepare the upper layer gel according to the solution volumes listed in Table 7:

[0127] Table 7. Components required for the upper layer of SDS-PAGE gel

[0128]

[0129]

[0130] After adding TEMED and mixing well, within 10 min, slowly fill the prepared upper layer gel solution along the edge of the glass plate to the brim, preventing the formation of bubbles. Slowly insert the washed and dried 15-well comb along the upper edge of the glass plate into the upper layer gel. If bubbles form under the comb, pull out the comb, rinse it with double-distilled water, and then re-insert it into the upper layer gel. Let the prepared gel plate stand for 20 - 30 min, remove the glass plate, soak it in double-distilled water, and store it in a 4 °C refrigerator for later use.

[0131] (3) Loading and Electrophoresis: Add an equal amount of protein sample and Maker to the gel placed in the electrophoresis tank and perform electrophoresis. Take a 1L graduated cylinder, rinse it thoroughly with double-distilled water. Add 18.8g of glycine, 3.0g of Tris powder, and 1.0g of SDS, make up the volume to 1L with double-distilled water, put in a magnetic rotor, and stir the solution thoroughly. Take out the prepared SDS-PAGE gel, place it in the electrophoresis tank and fix it. Add electrophoresis buffer, and ensure that the liquid levels in the inner and outer electrophoresis tanks are flat to prevent leakage. Slowly pull out the comb, add 5 μL of protein sample to each well, and add 3.5 μL of protein Marker to the reserved well. Connect the supporting power supply and start electrophoresis at a constant voltage of 80V. After the protein sample reaches the lower 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, then cut the PAGE gel according to the molecular weight sizes of the target protein and the internal reference, and at the same time cut the PVDF membrane to the same size as the PAGE gel. Soak the PVDF membrane in formaldehyde for about 10s before transfer to activate the PVDF membrane. Prepare the transfer cassette sandwich in the order of sponge - filter paper - gel - PVDF membrane - filter paper - sponge from the negative pole to the positive pole, and the transfer cassette needs to be clamped tightly without misalignment or air bubbles. Use a wet transfer system and transfer at a constant current of 300mA for 35 - 40min.

[0133] (5) Blocking: Take out the transferred membrane and observe whether the transfer is complete (observed according to the pre-stained colored protein Mark), wash it 2 times with TBST, place the transferred PVDF membrane in the blocking solution, and block it at room temperature for 1h. The blocking solution is a protein-free rapid blocking solution (1×). After blocking, wash it 3 times with TBST.

[0134] (6) Antibody Incubation: Cut the strips according to the molecular weight sizes of the internal reference protein and the target protein, then add the strips to the diluted primary antibody incubation tubes respectively, and incubate overnight at 4°C on a shaker. Then wash it 3 times with TBST, add the secondary antibody at a ratio of 1:4000, incubate at room temperature on a shaker for 1h, pour out the secondary antibody, and then add TBST to wash it 3 times.

[0135] (7) Exposure and Photographing. Mix solution A and solution B in the chemiluminescence substrate kit in a ratio of 1:1 to prepare 2mL of developing solution. Drop the prepared developing solution drop by drop onto the parallel position of the protein on the PVDF membrane, let it stand and react for about 20s, put the PVDF membrane into the chemiluminescence detection instrument to take a photo and image. The exposure time depends on the fluorescence signal intensity. Adjust the exposure time to achieve the best exposure result, repeat 3 times and save the photos.

[0136] (8) Analyze Protein Expression Level: Use Image J software to statistically analyze the gray values of protein bands.

[0137] The results are shown in Figure 9. Compared with the control group, 20 ng / mL TNF-α decreased the expression levels of the early myotube differentiation marker MyoD1 and the myogenesis marker MyoG proteins; when treated with low concentrations of DZFJZJZJ, the expression levels of MyoD1 and MyoG proteins were decreased, but as the concentration of DZFJZJZJ increased, the expression level of MyoD1 protein gradually increased, but there was no significant difference, while the expression level of MyoG protein increased significantly. 20 ng / mL TNF-α significantly increased the expression levels of the muscle atrophy markers MuRF1 and Atrogin-1 proteins; as the concentration of DZFJZJZJ increased, the expression levels of MuRF1 and Atrogin-1 proteins gradually decreased, and 800 μg / mL and 1600 μg / mL DZFJZJZJ significantly decreased the expression level of MuRF1 protein. Therefore, DZFJZJZJ can increase the expression levels of myogenesis markers and, at the same time, decrease the expression levels of muscle atrophy markers, so as to improve TNF-α-induced C2C12 myotube atrophy.

[0138] 2.3. Effects of DZFJZJZJ on LPS-induced skeletal muscle atrophy in mice

[0139] 2.3.1. Effects of DZFJZJZJ on the body weight of LPS-induced mice

[0140] Male C57BL / 6 mice aged 6 - 8 weeks were randomly divided into 5 groups (8 mice in each group): control group (normal saline); LPS group (2 mg / kg); LPS (2 mg / kg) + DZFJZJZJ group (100 mg / kg); LPS (2 mg / kg) + DZFJZJZJ group (200 mg / kg); LPS (2 mg / kg) + DZFJZJZJ group (300 mg / kg). LPS and DZFJZJZJ were intraperitoneally injected daily and the mice were weighed.

[0141] The results are as Figure 10 shown. As time increased, the body weight of the mice in the control group was basically stable. The body weights of the LPS treatment group and the 100 mg / kg and 300 mg / kg DZFJZJZJ treatment groups decreased first and then increased after LPS injection and were basically stable after 12 days, significantly lower than the control group. The 200 mg / kg DZFJZJZJ group had no significant difference compared with the control group starting from the 10th day, and the body weight of the mice basically recovered. The amplitude of body weight increase in the mice treated with 200 mg / kg DZFJZJZJ was significantly higher than that in the LPS group, and there were statistical differences on the 10th and 13th days. This indicates that DZFJZJZJ has a certain improvement effect on the body weight loss of LPS-stimulated mice.

[0142] 2.3.2. Effects of DZFJZJZJ on the motor ability of LPS-induced mice

[0143] The grip strength of mice was measured before administration, on the 7th day and the 14th day after administration. Before administration, the grip strength of mice in each group was comparable, with no significant difference; on the 7th day after administration, the grip strength of the LPS group and the DZFJZJ group decreased compared with that of the control group, and the grip strength of the 200 mg / kg DZFJZJ group increased significantly compared with that of the LPS group; on the 14th day after administration, the grip strength of the LPS group decreased compared with that of the control group; the grip strength of the DZFJZJ group increased compared with that of the LPS group, showing a statistical difference, and there was no significant difference compared with the control group. When comparing the grip strength of mice in the same group at different times, except for the control group, the grip strength of mice in the LPS and DZFJZJ treatment groups showed a trend of first decreasing and then increasing, and the grip strength of mice in the DZFJZJ treatment group could recover to the level before administration at 14 d. The grip strength of LPS-stimulated mice was improved( Figure 11 ).

[0144] The time on the rod of the rotarod test of mice was measured before administration, on the 7th day and the 14th day after administration. Before administration, the time on the rod of mice in each group was comparable, with no significant difference; on the 7th day after administration, the time on the rod of the LPS group and the DZFJZJ treatment group decreased compared with that of the control group, and there was a significant difference in the 200 mg / kg DZFJZJ group; on the 14th day after administration, the time on the rod of the LPS group and the 100 mg / kg DZFJZJ group decreased significantly compared with that of the control group, there was no significant difference in the 200 mg / kg DZFJZJ group compared with that of the control group, the time on the rod of the 100 mg / kg and 200 mg / kg DZFJZJ groups increased compared with that of the LPS group, and there was a statistical difference in the 200 mg / kg DZFJZJ group. When comparing the time on the rod of mice in the same group at different times, except for the control group, the time on the rod of mice in the LPS and DZFJZJ treatment groups showed a trend of first decreasing and then increasing. The time on the rod 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. Effects of DZFJZJ on muscle mass of LPS-induced mice

[0147] To evaluate skeletal muscle atrophy, the weight of the hindlimb calves of mice was measured before the end of the experiment. From Figure 13 it can be seen that the weight of the hindlimb calf muscles of mice injected with LPS was lower than that of the control group, the muscle weight of mice increased after injecting DZFJZJ, and the 200 mg / kg DZFJZJ group could significantly increase the weight of the hindlimb calf muscles of mice compared with the LPS treatment group, and there was no statistical difference compared with the control group.

[0148] 2.3.4. Effects of DZFJZJ on muscle fiber structure of LPS-induced mice

[0149] After the administration on the 14th day and the completion of the behavioral tests, the mice were allowed to rest overnight. The next day, the mice were anesthetized, and their eyes were first removed to collect blood. After centrifugation at 12,000 rpm for 20 min, the serum was collected and stored at -80 °C for subsequent experiments. Subsequently, the left and right calf muscle tissues of the mice were taken out 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 fixative, then embedded in paraffin, stained with H&E, and the number and cross-sectional area of muscle fibers were measured.

[0150] It was Figure 14 found that there was mild infiltration of inflammatory cells in the interstitial space of muscle fibers in the muscle control group of mice, and no other pathological changes such as muscle fiber atrophy, degeneration or necrosis were observed. In the LPS group, moderate infiltration of inflammatory cells was seen in the interstitial space of muscle fibers in the calf muscle tissue, and muscle atrophy, partial muscle fiber atrophy, moderate degeneration and necrosis occurred; the infiltration of inflammatory cells in the interstitial space of muscle fibers in mice after adding different concentrations of DZFJZJ improved, changing from moderate to mild infiltration, the arrangement between muscles became more regular, the arrangement between muscle fibers became closer, and only mild atrophy was observed in some samples.

[0151] 2.4, Conclusion

[0152] In the present invention, okara was mixed and solid-state fermented by Lactobacillus paracasei LMX1 with high ammonia peptidase activity and Pediococcus acidilactici IFJ1, and then the solid-state fermented product was subjected to water extraction to obtain DZFJZJ rich in oligopeptides. Further, in vitro, a myotube atrophy model was established by inducing damage to mouse myoblast C2C12 cells with TNF-α to explore the effect of DZFJZJ on C2C12 cells, and in vivo, the effect of DZFJZJ on LPS-induced muscle atrophy in mice was studied. It can be known from the above experimental verification that, firstly, DZFJZJ can promote the proliferation of C2C12 cells, improve the inhibitory effect of TNF-α on cell migration ability, reduce the proportion of cells in the G0 / G1 phase of the cell cycle, increase the proportion of cells in the G2 / M phase, improve the cell cycle arrest caused by TNF-α, and can also improve the promoting effect of TNF-α on cell senescence.

[0153] Secondly, DZFJZJ can also promote the differentiation of C2C12 cells into myotubes, increase the number, length and diameter of myotubes, up-regulate the expression of MyoD1 and MyoG, and down-regulate the expression of MuRF and Atrogin-1. Further, it can improve the weight loss of mice stimulated by LPS, improve the motor ability of mice stimulated by LPS, and increase the weight of the calf muscle in the hind limbs of mice.

[0154] Based on the above, it is 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 solid-state fermentation of soybean dregs with mixed bacteria

[0156] The dry basis of soybean dregs contains 10-20% protein, and its amino acid composition is basically consistent with that of soybeans. Many studies have shown that soybean protein can produce short peptide chains through the action of microorganisms or enzymes, or other biochemical reactions. That is, soybean peptides, which have biological functions such as anti-oxidation, anti-cancer, anti-hypertension, protection of intestinal barrier, enhancement of immune regulation, and promotion of mineral absorption and utilization. The present invention first uses high-yield aminopeptidase Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 to mix solid-state fermentation of soybean dregs to obtain solid-state fermentation products, and then water extracts the solid-state fermentation products to obtain DZFJZJ rich in soybean dregs oligopeptides. Considering that the soybean dregs oligopeptide concentration in DZFJZJ may be a factor that potentially affects the above-mentioned efficacy of DZFJZJ, the present invention uses the soybean dregs oligopeptide concentration in DZFJZJ as an indicator to further optimize the process conditions for preparing DZFJZJ by solid-state fermentation of soybean dregs by mixed bacteria.

[0157] The experimental design is as follows:

[0158] Single-factor experiments were conducted on the preparation of okara 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 water content, fermentation liquid inoculation amount, fermentation temperature, fermentation time, and fermentation liquid ratio.

[0159] 3.1 Effect of different initial water 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 of 6d, fermentation temperature of 28℃, bacteria-liquid ratio of 1:1 and other factors unchanged, and analyze the effects of initial moisture contents 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, control the fermentation time of 6d, fermentation temperature of 28℃, bacteria-liquid ratio of 1:1 and other factors unchanged, and 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 soybean dregs into a clean sealed bag, add 70% sterile water, take the fermentation temperature as the independent variable, keep the fermentation time at 6 d, the inoculation amount at 6%, the bacterial liquid ratio at 1:1 and other factors unchanged, and analyze the effects of fermentation temperatures of 24 °C, 26 °C, 28 °C, 30 °C, and 32 °C on the concentration of soybean dreg oligopeptides in DZFJZJ respectively.

[0165] 3.4. Effects of Different Fermentation Times on the Concentration of Soybean Dreg Oligopeptides

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

[0167] 3.5. Effects of Different Bacterial Liquid Ratios on the Concentration of Soybean Dreg Oligopeptides

[0168] Accurately weigh 200 g of soybean dregs into a clean sealed bag, add 70% sterile water, take the bacterial liquid ratio as the independent variable, keep the fermentation time at 6 d, the inoculation amount at 6%, the fermentation temperature at 28 °C and other factors unchanged, and analyze the effects of using IFJ1 bacterial liquid alone, LMX1 bacterial liquid, and when used in combination, with the IFJ1:LMX1 bacterial liquid ratios of 1:1, 1:2, 2:1, 2:3, and 3:2 on the concentration of soybean dreg oligopeptides respectively.

[0169] 3.6. Determination of the Concentration of Soybean Dreg Oligopeptides in DZFJZJ

[0170] The Folin-Ciocalteu method is used to determine the concentration of soybean dreg oligopeptides, and the specific steps are as follows:

[0171] (1) Preparation of the Standard Solution

[0172] Accurately weigh an appropriate amount of bovine serum albumin standard (Beijing Vokai Biotechnology), dissolve it in pure water, and dilute it to a standard solution containing 0.3 mg of bovine serum albumin per 1 mL of water.

[0173] (2) Plotting of the Standard Curve

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

[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 that it has 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 DZFJZJ Samples

[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] The results are as follows:

[0179] When the initial water content was 70%, the concentration of okara oligopeptides in DZFJZJ reached the highest 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% (P < 0.05) when the initial water content was 70%, respectively. 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 the 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% respectively when the fermentation time was 6 days (P < 0.05). Therefore, the appropriate 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 the maximum value of 12.75mg / mL; when the fermentation temperature increased to 30℃, the concentration of okara oligopeptide decreased. Compared with the fermentation temperatures of 26℃ and 30℃, the concentration of okara oligopeptide increased by 9% and 45.1% respectively when the fermentation temperature was 28℃ (P<0.05). Therefore, the suitable fermentation temperature for preparing okara oligopeptide by solid-state fermentation of LMX1 and IFJ1 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 maximum value of 18.71 mg / mL; therefore, the appropriate inoculation amount for preparing okara oligopeptides by solid-state fermentation of LMX1 and IFJ1 was 2% (Figure 18 )。

[0183] When the proportion of the bacterial suspensions of LMX1 and IFJ1 is 1:1, the concentration of okara oligopeptides in DZFJZJ reaches the highest value of 13.07 mg / mL, which is 1.6 times and 2.0 times that of the fermentation by Lactobacillus pentosus IFJ1 and Lactobacillus paracasei LMX1 respectively. Therefore, the suitable strain ratio for the preparation of DZFJZJ by the mixed solid-state fermentation of Lactobacillus paracasei LMX1 and Lactobacillus pentosus IFJ1 is 1:1( Figure 19 )。

[0184] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A bean dregs solid fermentation preparation, characterized in that: The bean dregs solid fermentation preparation uses Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 as fermentation strains and bean dregs as fermentation substrate, and is firstly solid fermented and then extracted.

2. The okara solid fermentation preparation according to claim 1, characterized in that: 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; The preservation number of the Pediococcus acidilactici IFJ1 is CCTCC M2022828, and it was deposited in the China Center for Type Culture Collection on June 8, 2022.

3. The solid-state fermentation preparation of soybean dregs according to claim 1, characterized in that: The bacterial liquid volume ratio of the Lactobacillus paracasei LMX1 and the Pediococcus acidilactici IFJ1 is (1-3): (1-3).

4. The okara solid-state fermentation preparation according to claim 1, characterized in that: The preparation method of the bean dregs solid-state fermentation preparation comprises the following steps: (1) adding sterile water to dry bean dregs to prepare a solid fermentation medium, then culturing and activating Lactobacillus paracasei LMX1 and Pediococcus acidilactici IFJ1 to prepare seed liquids, and mixing the seed liquids to prepare a fermentation liquid; (2) inoculating the fermented bacterial 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 extract, extracting the okara solid-state fermentation product obtained in step (2) to obtain the okara solid-state fermentation preparation.

5. The bean curd dregs solid fermentation preparation according to claim 4, 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.

6. Use of the okara solid-state fermentation preparation according to any one of claims 1 to 5 in the preparation of a drug for treating or preventing sarcopenia.

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