Method for fermenting mulberry leaves by using dominant strains

Abstract

The invention belongs to the field of mulberry leaf fermentation, and particularly relates to a method for fermenting mulberry leaves by using a dominant strain, which comprises the following steps: carrying out steam fixation on fresh mulberry leaves, adding a glucose solution, carrying out inoculated fermentation by using lactobacillus rhamnosus, and carrying out aerobic fermentation in a 37 DEG C constant-temperature environment with the oxygen introduction amount of 12% and the fermentation time of 6 hours; after fermentation is finished, the fermented mulberry leaves are dried. According to the mulberry leaves obtained through fermentation, the total acid is larger than or equal to 0.755, the OD600 is larger than or equal to 0.081, the pH value is larger than or equal to 5.47, and the fermented mulberry leaves are good in fragrance and taste. The fermentation product of the mulberry leaves fermented by adopting the specific strain lactobacillus rhamnosus has obvious inhibitory activity on tumor cells, especially liver cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of mulberry leaf fermentation, and particularly relates to a method for fermenting mulberry leaves with dominant strains. Background Art

[0002] Fresh mulberry leaves are highly nutritious and rich in various nutrients such as amino acids, crude protein, and vitamins. However, mulberry leaves contain 8% - 12% crude fiber and 8% - 12% crude ash, which seriously affect the taste. Moreover, fresh mulberry leaves contain antinutritional factors phytic acid and tannins. Phytic acid will combine with mineral elements and vitamins to form complex precipitates, which is not conducive to the absorption and utilization of the body. Tannins combine with digestive enzymes such as amylase and trypsin in the body to reduce the utilization of nutrients. Fresh mulberry leaves contain 75% - 90% water, with a large water content, and are extremely prone to spoilage and difficult to preserve. If sugar substances are added during the silage process of mulberry leaves to degrade the relevant enzymes of fiber, the crude fiber content of mulberry leaves can be reduced and the palatability can be improved. If fermented with microorganisms, the pH of silage mulberry leaves decreases significantly, the lactic acid content of mulberry leaves increases, which improves the digestion of the body and is conducive to the absorption of nutrients. However, because the fermentation of mulberry leaves has not been industrialized yet, there are still some problems. The control of fermentation molds is not good, resulting in mold contamination and toxin overstandard.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for fermenting mulberry leaves with dominant strains to solve the above problems.

[0005] A method for fermenting mulberry leaves with dominant strains includes the following steps:

[0006] Step 1: Select fresh mulberry leaves without diseases and insect bites, wash, drain, cut into wide strips, and steam for fixation; add a 5% glucose solution by 4% of the mulberry leaf feeding amount, and supplement sterile pure water according to the material-liquid ratio of 1:6;

[0007] Step 2: Dissolve Lactobacillus rhamnosus stored at 2 - 6°C in a low-temperature refrigerator, evenly coat it on a solid medium for cultivation; put it into a constant temperature incubator for activation to obtain an activated strain;

[0008] Step 3: Dilute the activated strain 1000 times with physiological saline, inoculate it on the mulberry leaves, and shake well; ferment aerobically in a constant temperature environment at 37°C, with an oxygen supply of 12%, and the fermentation time is 6h;

[0009] Step 4: After the fermentation is completed, dry the fermented mulberry leaves.

[0010] Further improvement: In step 1, the steam fixation time is 3 minutes.

[0011] For further improvement, in step 2, the method for preparing the solid medium is as follows:

[0012] Weigh the yeast extract peptone dextrose agar medium powder into a bottle, add pure water, and the mass-to-volume ratio of the yeast extract peptone dextrose agar medium to pure water is 4.9:100; heat and stir until it dissolves to a transparent state, seal the bottle mouth, and perform high-pressure sterilization for 20 minutes at a pressure of 0.11 - 0.14 MPa. After the sterilization is completed and cooled, the solid medium is obtained.

[0013] For further improvement, in step 4, the drying temperature is 60 °C.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. For the mulberry leaves fermented by the present invention, the total acid ≥ 0.755, OD600 ≥ 0.081, pH value ≥ 5.47, and the fermented mulberry leaves have a good aroma and excellent taste.

[0016] 2. The fermentation products of the mulberry leaves fermented with the specific strain Lactobacillus rhamnosus have obvious inhibitory activity against tumor cells, especially liver cancer cells.

[0017] 3. The fermentation method of the present invention is easy to operate, and the fermentation molds are easy to control, and it will not cause mold contamination and excessive toxin levels. Description of the Drawings

[0018] Figure 1 is a physical photo of the fermented mulberry leaves;

[0019] Figure 2 is a column chart of the activities of tumor cells in each group;

[0020] Figure 3 is a flow cytometry scatter plot of apoptosis in Control Group 1;

[0021] Figure 4 is a flow cytometry scatter plot of apoptosis in Control Group 2;

[0022] Figure 5 is a flow cytometry scatter plot of apoptosis in Control Group 3;

[0023] Figure 6 is a flow cytometry scatter plot of apoptosis in the Fermented Mulberry Leaf Group 1;

[0024] Figure 7 is a flow cytometry scatter plot of apoptosis in the Fermented Mulberry Leaf Group 2;

[0025] Figure 8 is a flow cytometry scatter plot of apoptosis in the Fermented Mulberry Leaf Group 3;

[0026] Figure 9It is the flow cytometry scatter plot of cell apoptosis in Group 1 of fresh mulberry leaves;

[0027] Figure 10 It is the flow cytometry scatter plot of cell apoptosis in Group 2 of fresh mulberry leaves;

[0028] Figure 11 The flow cytometry scatter plot of cell apoptosis in Group 3 of fresh mulberry leaves;

[0029] Figure 12 It shows the scratch healing of tumor cells in each group;

[0030] Figure 13 It is the fluorescence image of the cell ROS experiment in Blank Group 1;

[0031] Figure 14 It is the fluorescence image of the cell ROS experiment in Group 1 of fresh mulberry leaves;

[0032] Figure 15 It is the fluorescence image of the cell ROS experiment in Group 1 of fermented mulberry leaves;

[0033] Figure 16 It is the relative expression abundance of mRNA;

[0034] Figure 17 It is the expression levels of Bax and Bcl-2 proteins in cells of each group;

[0035] Figure 18 It is the differential analysis between Lactobacillus rhamnosus and three other strains;

[0036] Figure 19 It is the bar chart of COG pathway enrichment analysis;

[0037] Figure 20 It is the bar chart of KO pathway enrichment analysis;

[0038] Figure 21 It is the active binding protein map of Pregnanediol 3-O-glucuronide and Bcl-2 receptor protein;

[0039] Figure 22 It is the active binding protein map of Apigenin 7-[rhamnosyl-(1->2)-galacturonide] and Bcl-2 receptor protein;

[0040] Figure 23 It is the active binding protein map of Asperuloside acid and Bcl-2 receptor protein;

[0041] Figure 24 It is the active binding protein map of Kaempferol 3-O-(6-malonyl-glucoside) and Bcl-2 receptor protein. Detailed implementation mode

[0042] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0043] Example 1

[0044] 1. Preparation of solid medium: Weigh 4.9 grams of YPD (yeast extract peptone dextrose agar medium) powder into a conical flask, add 100 ml of pure water, heat, and stir with a glass rod until it dissolves to a transparent state. Seal the mouth of the conical flask with absorbent cotton, and sterilize it under high pressure for 20 minutes at a pressure of 0.11 - 0.14 MPa. After sterilization, wait for it to cool, and then take it to the sterile operation table for operation.

[0045] 2. Preparation before operation: Sterilize the required glass instruments under high pressure for about 30 minutes at a pressure of 0.11 - 0.14 MPa for 17 minutes.

[0046] 3. Mulberry leaf treatment: Select fresh mulberry leaves without diseases and insect bites, wash them, drain the clear water, cut them into 2 - cm wide strips, steam for 3 minutes, and immediately put them into a sterilized (sterilized for 17 minutes) conical flask. Add a 5% glucose solution by 4% of the mulberry leaf feeding amount, and supplement sterile pure water according to the solid - liquid ratio of 1:6.

[0047] 4. Strain activation: Dissolve the strain (Lactobacillus rhamnosus) stored at low temperature of 2 - 6 °C, evenly coat it on the solid medium, smear it evenly with a spreading rod, and culture it. Put it into a constant - temperature incubator in time for activation to obtain a fully activated strain.

[0048] 5. Inoculation and fermentation: Dilute the activated bacteria with physiological saline to 1000 times for inoculation. Inoculate it onto the mulberry leaves and shake well. Ferment aerobically at a constant temperature of 37 °C for 6 hours, and the oxygen supply amount is 12%.

[0049] 6. Measurement of values: Before measurement, pour the fermented bacterial liquid into a sterile centrifuge tube, centrifuge at 3000 r / min for 10 minutes, take out the supernatant, and measure the total acidity, pH value, and OD value.

[0050] 7. Drying: Spread the mulberry leaf samples at different fermentation times on a tray and put them into a constant - temperature drying oven for drying. The drying conditions are 60 °C, and store them sealed at room temperature for later use. See the physical picture of the fermented mulberry leaves in Figure 1 .

[0051] 8. Quality evaluation of fermented mulberry leaves: Using UPLC - Q / TOF - MS to detect the chemical components in mulberry leaves, it is found that the fermented mulberry leaves have new active components, and these components have anti - cancer activity.

[0052] Example 2

[0053] Technical indicators

[0054] (1) The addition amount of 5% glucose solution by mass fraction is 4%, the temperature is 37 °C, and the fermentation is carried out for 24 h. At this time, the pH is the lowest, the total acid content is the highest, and the OD600 value and the sensory score are also relatively high.

[0055] (2) The total acid content and pH value can evaluate the fermentation effect of Lactobacillus rhamnosus, and OD600 can evaluate the growth and reproduction status of fungi. Therefore, the three indicators of total acid, OD600, and pH value are used as technical indicators. The sensory evaluation is composed of a nine-member professional evaluation team to conduct a sensory evaluation of the aroma, taste, and color of fermented mulberry leaves.

[0056] Total acid ≥ 0.755, OD600 ≥ 0.081, pH value ≥ 5.47, sensory score ≥ 70.

[0057] (3) Cell CCK-8 assay (Here, human hepatocellular carcinoma cells, abbreviated as Hep G2, are used)

[0058] Preliminary preparation: Weigh 0.250 mg of the fermented mulberry leaf sample. Add 2.5 mL of sterile water and dissolve it thoroughly. Filter it with a 0.22 μm filter membrane to prepare a 100 mg / L stock solution. Prepare a 10 mg / L working solution with the culture medium. Inoculate the cell suspension (100 μL / well, 10 4 ~10 5 cells) in a 96-well plate. Place the culture plate in an incubator for pre-incubation for 24 h (37 °C, 5% CO2). Prepare a new CCK-8 working solution: Dilute the 100X CCK-8 stock solution with the culture medium to 1X for use, and prepare it freshly before use. Remove the culture medium from the 96-well plate, and add 110 μL of CCK-8 solution to each well. Incubate the culture plate in the incubator for 1 - 4 hours. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability (%) = (A value of the experimental well - A value of the control well) / (A value of the blank well - A value of the control well) × 100%.

[0059] Table 1 Tumor inhibition results of fermented mulberry leaves by single strain combined with compound strains

[0060]

[0061] In Table 1, compared with Example 1, all the fermented mulberry leaf samples are only different in strains.

[0062] It can be seen from Table 1 that the tumor inhibition effect of fermentated mulberry leaves by Lactobacillus brevis, Lactobacillus casei, etc. is general.

[0063] Example 3

[0064] 3.1 Tumor cell viability characterization test

[0065] 1. Take 50 g of fermented mulberry leaf powder, pass it through an 80-mesh sieve, add 60% ethanol for reflux extraction for 2 hours according to a solid-liquid ratio of 1:20, filter, and freeze-dry the filtrate after rotary evaporation to obtain the fermented mulberry leaf extract.

[0066] 2. Take 50 g of fresh mulberry leaf powder, pass it through an 80-mesh sieve, add 60% ethanol for reflux extraction for 2 hours according to a solid-liquid ratio of 1:20, filter, and freeze-dry the filtrate after rotary evaporation to obtain the fresh mulberry leaf extract.

[0067] 3. Weigh 0.250 mg of the fermented mulberry leaf extract and 0.250 mg of the fresh mulberry leaf extract. Add 2.5 mL of sterile water to dissolve them fully, filter using a 0.22-μm filter membrane, and prepare a 100 mg / L stock solution. Prepare working solutions of 0 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, and 20 mg / L using the culture medium.

[0068] 4. Inoculate the cell suspension (100 μL / well, 10^4 - 10^5 cells) in a 96-well plate. Place the culture plate in an incubator for pre-incubation for 24 h (37 °C, 5% CO2).

[0069] 5. Treat the cells in groups: fermented mulberry leaf extract at 0 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L; control group (fresh mulberry leaf extract) at 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L. Incubate for 12 h, 24 h, 48 h, and 75 h.

[0070] 6. Prepare a new CCK-8 working solution: Dilute the 100X CCK-8 stock solution with the culture medium to 1X for use, and prepare it fresh as needed.

[0071] 7. Remove the culture medium from the 96-well plate, and add 110 μL of CCK-8 solution to each well (note that no bubbles should be generated in the wells as they will affect the OD value reading).

[0072] 8. Incubate the culture plate in the incubator for 1 - 4 hours.

[0073] 9. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0074] 10. Tumor cell viability (%) = (A value of experimental well - A value of control well) / (A value of blank well - A value of control well) * 100%.

[0075] 3.2. Single-factor experimental design

[0076] (1). Selection of glucose addition amount: The addition amounts of a 5% (mass fraction) glucose solution are 2%, 4%, 8%, 16%, and 20% respectively, the fermentation temperature is 37 °C, the oxygen supply is 10%, the inoculum addition amount is 10%, and the fermentation time is 24 h.

[0077] (2) Determination of fermentation temperature: The fermentation temperatures are 30°C, 32°C, 35°C, 37°C, and 40°C respectively. The addition amount of the glucose solution is 4%, the oxygen supply amount is 10%, the inoculant addition amount is 10%, and the fermentation time is 24 h.

[0078] (3) Selection of oxygen supply amount: The oxygen supply amounts are 6%, 12%, 18%, 20%, and 24% respectively. The addition amount of the glucose solution is 4%, the fermentation temperature is 37°C, the inoculant addition amount is 10%, and the fermentation time is 24 h.

[0079] (4) Inoculant addition amount: They are 5%, 10%, 15%, 20%, and 25% respectively. The addition amount of the glucose solution is 4%, the oxygen supply amount is 10%, the fermentation temperature is 37°C, and the fermentation time is 24 h.

[0080] (5) Fermentation time: They are 6 h, 12 h, 24 h, 32 h, and 48 h respectively. The addition amount of the glucose solution is 4%, the fermentation temperature is 37°C, the oxygen supply amount is 10%, and the inoculant addition amount is 10%.

[0081] The test results are shown in Tables 2 - 6:

[0082] Table 2

[0083] Addition amount of glucose solution (%) Tumor cell viability (%) 2％ 20.5074 4％ 11.7506 8％ 21.3645 16％ 23.6667 20％ 63.5491

[0084] Table 3

[0085] Fermentation temperature (°C) Tumor cell viability (%) 30 30.6818 33 24.5311 35 21.0084 37 21.2020 40 27.848

[0086] Table 4

[0087] Oxygen supply amount (%) Tumor cell viability (%) 6％ 34.4402 12％ 30.2434 18％ 28.5853 20％ 29.6650 24％ 30.7970

[0088] Table 5

[0089]

[0090]

[0091] Table 6

[0092] Fermentation time (h) Tumor cell viability (%) 6 29.6650 12 30.1517 24 34.7757 32 45.1311 48 45.6769

[0093] A large inoculation amount will cause the rapid reproduction of bacteria, generating excessive metabolic wastes; a small inoculation amount will cause the slow growth and reproduction of bacteria, increasing the fermentation cycle; an appropriate inoculation amount can not only effectively shorten the culture cycle, obtain a high cell biomass, but also obtain more enzymes and improve the activity of fermentation products.

[0094] Continuously increasing the glucose addition amount is likely to cause an imbalance in the carbon-nitrogen ratio in the substrate, creating a high osmotic pressure environment and leading to the early death of lactic acid bacteria.

[0095] The fermentation temperature is 33 °C, 35 °C or 37 °C.

[0096] After a large number of experiments, it was found in the present invention that: when the fermentation is completed, the sour taste is strong, but continuous fermentation leads to the aging of the strains, and after the activity of lactic acid bacteria decreases, it is easy to be infected by miscellaneous bacteria to produce peculiar smells or even mildew and rot smells.

[0097] Example 4

[0098] Taking the anti-tumor activity of the fermented mulberry leaf extract as the evaluation index, experiments were carried out with the addition amount of glucose (2%, 4%, 8%) at a mass fraction of 5%, fermentation temperature (33 °C, 35 °C, 37 °C), oxygen ventilation amount (12%, 18%, 20%), inoculant addition amount (5%, 10%, 15%), and fermentation time (6 h, 12 h, 24 h) as fermentation parameters, and the optimal parameter combination of Lactobacillus rhamnosus inoculated on mulberry leaves was determined. The results are shown in Table 7:

[0099] Table 7 Orthogonal experiment detection results of Lactobacillus rhamnosus fermenting mulberry leaves

[0100]

[0101]

[0102] Taking the tumor cell activity as the index, the range R D > R E > R B > R C > R A , the influence degrees of the five factors on tumor cells in this experiment are in turn: D > E > B > C > A. That is, the addition amount of the glucose solution with a mass fraction of 5% is 4%, the fermentation temperature is 37 °C, the oxygen ventilation amount is 12%, the inoculant addition amount is 10%, and the fermentation time is 6 h. Under these conditions, the inhibition rate of tumor cells is relatively high, and the fermentation time is short and the glucose addition amount is small, which can save time and material costs and has high application value in actual production.

[0103] Example 5

[0104] Cell viability detection

[0105] Hep G2 human liver cancer cells were resuscitated and passaged, and cells with good growth and in the logarithmic growth phase were selected to detect the growth inhibitory effect of the fermented mulberry leaf extract (0, 2, 2.5, 5, 10, 20 mg / ml) on tumor cells to determine sensitive tumor cells.

[0106] Cell CCK-8 was used to detect cell viability

[0107] Weigh 0.250 mg of fermented mulberry leaf extract and 0.250 mg of fresh mulberry leaf extract. Add 1 mL of sterile water to dissolve them completely, and filter through a 0.22 μm filter membrane to prepare a 2.5 mg / L working solution. Inoculate the cell suspension (100 μL / well, 10 4 ~10 5 cells) into a 96-well plate. Place the culture plate in an incubator for pre-incubation for 24 h (37 °C, 5% CO2). Dilute the 100X CCK-8 stock solution with the culture medium to 1X for use. Remove the culture medium from the 96-well plate, add 110 μL of CCK-8 solution to each well, and incubate for 1 - 4 hours. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability (%) = (A value of the experimental well - A value of the control well) / (A value of the blank well - A value of the control well) × 100%.

[0108] Flow cytometry for detecting cell apoptosis

[0109] Inoculate the cell suspension into a 6-well plate and culture for 24 h. After centrifuging the suspended cells (centrifuge at 2000 rpm for 5 min), collect the cells and wash them twice with PBS. Collect 1 - 5 × 10 5 cells. Then add 500 μL of Binding Buffer to resuspend the cells, add 5 μL of AnnexinV-FITC, mix well, and then add 5 μL of Propidium Iodide. React at room temperature in the dark for 5 - 15 min. Detect using a flow cytometer with an excitation wavelength Ex = 488 nm and an emission wavelength Em = 530 nm. The green fluorescence of AnnexinV-FITC is detected through the FITC channel (FL1), and the red fluorescence of PI is detected through the FL3 channel.

[0110] Flow cytometry for detecting cell cycle

[0111] Inoculate the cell suspension into a 6-well plate and pre-incubate in an incubator for 24 h (37 °C, 5% CO2). After staining, detect using a flow cytometer with an excitation wavelength Ex = 488 nm and an emission wavelength Em = 530 nm. The red fluorescence of PI (flow cytometry Ex = 488 nm, Em ≥ 630 nm) is detected through the FL3 channel.

[0112] Cell scratch assay

[0113] Inoculate 10 5 cells into a 6-well plate, add 2 mL of culture medium, and culture until the cells cover the well plate. Use a 10 μL pipette tip to make a cross-shaped scratch in the well plate, replace the culture medium with a new one, and take a photo to record the cell scratch. Treat the cells with 2.5 mg / L of fermented mulberry leaf extract and 2.5 mg / L of fresh mulberry leaf extract for 48 h. Replace the culture medium with a new one, take a photo, and record the cell scratch.

[0114] Detection of the content of immunosuppressive factors secreted by liver tumor cells

[0115] The contents of L-1β-ELK1270, IL-4-ELK1152, IL-6-ELK1156, IL-10-ELK1142, and TNF-a-ELK1190 in the supernatant of the culture medium of liver cancer cells were detected by ELISA.

[0116] The expression levels of BCL-2 and BAX mRNA in cells of each group were detected by qPCR.

[0117] Liver tumor cells in the logarithmic growth phase were selected, and total RNA of liver cancer cells was extracted using TRI Reagent, reverse transcribed into cDNA by a reverse transcription kit, and then subjected to PCR amplification.

[0118] The expression levels of Bax and Bcl-2 proteins in cells of each group were detected by Western blotting.

[0119] Protein concentration was determined by BCA method: 5 mg / ml standard bovine serum albumin was added to the standard wells of a 96-well plate. The sample was diluted to a certain concentration with the standard diluent and 20 μl was added to the 96-well plate. An appropriate amount of BCA working solution was prepared by mixing 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), thoroughly mixed, 200 μL of the BCA working solution was added to each well, and incubated at 37 °C for 30 min. The absorbance value at 562 nm was measured.

[0120] The results were analyzed

[0121] 1. Determination of the concentrations of fermented mulberry leaves and raw mulberry leaf extracts

[0122] In the range of 2 - 20 mg / L, the fermented mulberry leaf extract had a certain inhibitory effect on the growth of human liver cancer cells, and the inhibitory effect increased in a concentration-dependent manner. When the concentration of 2.5 mg / L was selected, the inhibitory effect on liver cancer cells was used as the cell activity as Figure 2 shown.

[0123] 2. Detection of the apoptosis rate of liver tumor cells

[0124] The fermented mulberry leaf extract had the effect of inducing apoptosis of liver cancer cells, as shown in the fluorescence signal data table 8 and Figures 3 - 11 shown. The Control group refers to the blank control group (liver cancer cells cultured normally); among them, in Figures 3 - 11 , LL is the proportion of tumor live cells, LR is the proportion of early apoptotic tumor cells, UR is the proportion of late apoptotic cells, and UL is the proportion of dead cells. From the above, it can be seen that the fermented mulberry leaf extract has significant activity.

[0125] Table 8 Apoptosis rate of tumor cells

[0126] Grouping Live cells Early apoptosis Late apoptosis Dead cells Apoptosis rate Control Group 1 94.04 3.70 1.08 1.18 4.78 Control Group 2 94.13 3.56 1.04 1.27 4.6 Control Group 3 94.45 3.32 0.98 1.25 4.3 Fermented mulberry leaf Group 1 70.39 7.59 16.16 5.85 23.75 Fermented mulberry leaf Group 2 72.57 7.21 14.00 6.22 21.21 Fermented mulberry leaf Group 3 72.71 7.76 13.41 6.11 21.17 Fresh mulberry leaf Group 1 90.84 3.71 3.72 1.73 7.43 Fresh mulberry leaf Group 2 91.40 3.52 3.26 1.82 6.78 Fresh mulberry leaf Group 3 91.84 3.29 2.99 1.89 6.28

[0127] 3. Flow cytometry for cell cycle detection

[0128] The SPSS results showed that there were significant differences in the G2 phase of tumor cells between the control group and the fresh mulberry leaf group, and the results are shown in Table 9. The fermented mulberry leaf extract acts on the cells in the G2 phase, causing them to die or stop dividing, thus reducing the number of cells, indicating that the fermented mulberry leaf extract has a positive effect on inhibiting tumor cells.

[0129] Table 9 Statistical table of the effects of fresh mulberry leaf and fermented mulberry leaf extracts on the tumor cell growth cycle

[0130]

[0131]

[0132] In Table 9, the Control group is calculated using the average value of the data in Control groups 1 - 3, and the fermented mulberry leaf group is calculated using the average value of the data in fermented mulberry leaf groups 1 - 3.

[0133] 4. Scratch healing rate of tumor cells in each group

[0134] Compared with the blank group (hepatoma cells cultured normally) and the fresh mulberry leaf group (powder of fresh mulberry leaves, extracted with 50% ethanol, concentrated, rotary evaporated, purified by macroporous resin, and finally freeze - dried), the scratch healing rate of tumor cells was significantly reduced (P < 0.01), as shown in Figure 12 . This indicates that the interaction between tumor cells and the extracellular matrix is weakened, resulting in a decrease in cell migration ability.

[0135] 5. Detection of the content of immunosuppressive factors secreted by tumor cells, and the results are shown in Table 10:

[0136] Table 10 Effects of mulberry leaf extracts on the secretion of immunosuppressive factors by tumor cells

[0137]

[0138] In Table 10, the blank group refers to the group of hepatoma cells cultured normally. The fresh mulberry leaf group is the powder of fresh mulberry leaves, extracted with 50% ethanol, concentrated, rotary evaporated, purified by macroporous resin, and finally freeze - dried. The fermented mulberry leaf group is obtained by steaming fresh mulberry leaves with steam and then fermenting with Lactobacillus rhamnosus and drying (refer to Example 1), and the extraction and purification process is the same as that of the fresh mulberry leaf group. The concentrations of the fresh mulberry leaf group and the fermented mulberry leaf group acting on tumor cells are both 2.5 mg / L.

[0139] The autocrine IL-4 factor in tumor tissues directly acts on tumor tissues, inducing cachexia and neovascularization, etc., which indirectly promotes the growth of tumor cells; the autocrine IL-10 factor in tumor tissues promotes tumor growth, inhibits programmed cell death, and promotes tumor metastasis. Both inhibit the activation of tumor cells, thereby reducing the release of inflammatory mediators and playing an important role in the anti-inflammatory response.

[0140] 6. Detection results of ROS in tumor cells

[0141] As Figures 13 - 15 shown, compared with blank group 1 (hepatoma cells cultured normally without any extract treatment) and raw mulberry leaf group 1, the fluorescence intensity of cells in the fermented mulberry leaf group 1 was significantly higher than that of the control group and the raw mulberry leaf group. The ROS level in tumor cells of the fermented mulberry leaf extract increased, causing damage to them or leading to their oxidation.

[0142] 7. Expression levels of BCL-2 and BAX mRNA in small tumor cells of each group

[0143] Compared with the control group (hepatoma cells cultured normally without any extract treatment) and the raw mulberry leaf group (calculated from the average value of the data in raw mulberry leaf groups 1-3), the expression abundance of BCL-2 in the fermented mulberry leaf group (calculated from the average value of the data in fermented mulberry leaf groups 1-3) was significantly reduced, while the expression abundance of BAX was significantly increased, as shown in Table 11 and Figure 16 shown.

[0144] Table 11 Statistical table of fluorescence quantitative PCR results

[0145]

[0146] 8. Expression levels of Bax and Bcl-2 proteins in cells of each group

[0147] See Figure 17 , compared with the control group (hepatoma cells cultured normally without any extract treatment), the expression levels of Bcl-2 in both the raw mulberry leaf and fermented mulberry leaf extract groups decreased, and the expression level of BAX was relatively high. There was a significant difference in the expression level in the fermented mulberry leaf group (P<0.01), indicating that the tumor cells treated with the fermented mulberry leaf extract were more prone to apoptosis.

[0148] Overexpression of BAX and targeted downregulation of the expression of Bcl-2 inhibit the proliferation, migration, and invasion abilities of tumor cells, indicating that the fermented mulberry leaf extract has the potential to be used as a biomarker for the clinical diagnosis and treatment of pre-hepatic tumor cells.

[0149] Example 6

[0150] Mulberry leaves are fermented with Lactobacillus delbrueckii, and its metabolites consist of 26 types; mulberry leaves are fermented with Lactobacillus brevis, and its metabolites consist of 28 types; mulberry leaves are fermented with Lactobacillus casei, and its metabolites consist of 21 types; mulberry leaves are fermented with Lactobacillus rhamnosus, and its metabolites consist of 40 types; the types and quantities of metabolites of Lactobacillus rhamnosus fermented mulberry leaves are higher than those of other strains.

[0151] Transcriptomics experimental results

[0152] Since the active ingredients of Lactobacillus rhamnosus fermented mulberry leaves are higher than those of other strains fermented mulberry leaves, therefore, Lactobacillus rhamnosus and the other three strains are compared to explore the genes that may be involved in metabolism in the strains. First, differential expression analysis is performed on Lactobacillus rhamnosus and the other three groups of strains by Deseq2, and then the differential genes are visually analyzed with a volcano plot. The results are shown in Figure 18 .

[0153] From Figure 18 it can be seen that compared with the other three strains, the expressions of yhcF, uidA (B9H01_RS05640), pyrG, alr-2, treS, adhE, AaMFS54, EF-P, ulaC, pdhB, and mgrA of Lactobacillus rhamnosus are significantly up-regulated. In order to further understand which metabolic pathways these differentially expressed genes are involved in, the KOBAS database is used to perform metabolic pathway enrichment analysis of COG and KO respectively.

[0154] As Figure 19 , 20 shown, Beta-galactosidase and beta-glucuronidase are two important hydrolases, and the metabolic pathways they participate in have multiple functions in organisms. β-Galactosidase mainly catalyzes the hydrolysis of β-galactoside bonds. For example, it decomposes lactose into glucose and galactose, which is also the reason why the glycoside components of Lactobacillus rhamnosus fermented mulberry leaves are higher than those of other strains fermented. β-Glucuronidase hydrolyzes β-glucuronide bonds and assists in releasing glucuronide conjugates. The production of aldehyde glycosides in the metabolites of Lactobacillus rhamnosus fermented mulberry leaves comes from this.

[0155] YqhD belongs to the iron-dependent alcohol dehydrogenase family, and its core function is similar to that of class III ADH (glutathione-dependent formaldehyde dehydrogenase), and it may be involved in formaldehyde metabolism. By catalyzing the binding of formaldehyde and glutathione to generate S-hydroxymethylglutathione, and then oxidizing it to formic acid, it helps to remove toxic formaldehyde in cells, and this function helps to improve the effect of inhibiting the activity of tumor cells.

[0156] Transketolase is the key enzyme of the pentose phosphate pathway (PPP). It converts 5-carbon sugars and 4-carbon sugars into 6-carbon sugars through the transketol reaction, and undergoes a hydroxyl reaction with alcohols or phenols to form glycosides. Therefore, this enzyme provides substrates for the synthesis of glycosides and is the key enzyme for the metabolites of Lactobacillus rhamnosus fermented mulberry leaves.

[0157] Figures 21 - 24 It is the binding diagram of pregnanediol 3-O-glucuronide, apigenin 7-[rhamnosyl-(1->2)-galacturonide], asperuloside acid, kaempferol 3-O-(6-malonyl-glucoside) and the tumor receptor protein Bcl-2. Bcl-2 (B-cell lymphoma / leukemia-2 gene) is an important anti-apoptotic protein and belongs to the Bcl-2 family members. It participates in the occurrence, development and treatment resistance of cancer by regulating apoptosis. For example, in cancer cells, the overexpression of Bcl-2 can resist apoptosis induced by chemotherapy, radiotherapy, etc., resulting in the escape of tumor cells from death and the generation of treatment resistance. The metabolites pregnanediol 3-O-glucuronide, apigenin 7-[rhamnosyl-(1->2)-galacturonide], asperuloside acid, kaempferol 3-O-(6-malonyl-glucoside) in Lactobacillus rhamnosus fermented mulberry leaves act on the Bcl-2 target of the human body and down-regulate the expression of Bcl-2, thereby playing an anti-tumor role.

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for fermenting mulberry leaves with a dominant strain, characterized in that: It includes the following steps: Step 1: Select fresh mulberry leaves, wash them, drain them, steam them for fixation, and add a nutrient solution; Step 2: Culture and activate the Lactobacillus rhamnosus stored in the refrigerator to obtain an activated strain; Step 3: Dilute the activated strain and inoculate it onto the mulberry leaves for aerobic fermentation; Step 4: After the fermentation ends, dry the fermented mulberry leaves.

2. The method for fermenting mulberry leaves with a dominant strain according to claim 1, characterized in that: In Step 1, the steam fixation time is 3 min.

3. The method for fermenting mulberry leaves with a dominant strain according to claim 1, characterized in that: In Step 2, the steps for culturing and activating the Lactobacillus rhamnosus stored in the refrigerator are as follows: Dissolve the Lactobacillus rhamnosus stored at 2-6°C in the refrigerator, evenly coat it on a solid medium for culturing; place it in a constant temperature incubator for activation to obtain an activated strain.

4. A method for fermenting mulberry leaves with dominant strains according to claim 3, characterized in that: In Step 2, the preparation method of the solid medium is as follows: Weigh the yeast extract peptone dextrose agar medium powder into a bottle, add pure water, and the mass-volume ratio of the yeast extract peptone dextrose agar medium to pure water is 4.9:100; heat and stir until it dissolves to a transparent state, seal the bottle mouth, and sterilize it under high pressure for 20 minutes at a pressure of 0.11-0.14 MPa. After the sterilization ends and it cools down, the solid medium is obtained.

5. The method for fermenting mulberry leaves with a dominant strain according to claim 1, characterized in that: In Step 4, the drying temperature is 60°C.

6. A method for fermenting mulberry leaves with dominant bacteria according to claim 1, characterized in that: In Step 1, the nutrient solution is a glucose solution. Add a 5% glucose solution by 4% of the mulberry leaf input amount, and supplement sterile pure water according to the material-liquid ratio of 1:

6.

7. A method for fermenting mulberry leaves with dominant strains according to claim 1, characterized in that: In Step 3, the dilution of the activated strain is to dilute the activated strain 1000 times with physiological saline.

8. A method for fermenting mulberry leaves with dominant bacteria strains according to claim 1, characterized in that: In Step 3, during the aerobic fermentation of the mulberry leaves, the fermentation temperature is a constant temperature environment of 37°C, the oxygen supply amount is 12%, and the fermentation time is 6 h.

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