A method for preparing a functional food for enhancing the ability of immune cells based on fermented magnolia officinalis

Abstract

This invention belongs to the field of health functional foods, specifically relating to a method for preparing functional foods that enhance the ability of immune cells based on fermented Magnolia officinalis. This invention uses Magnolia officinalis as a fermentation substrate, adding a small amount of water chestnut powder to the culture medium. This prevents spoilage during fermentation, greatly improving the success rate. Furthermore, the raw materials are simple, readily available, and inexpensive, enhancing the nutritional content of the fermentation product. The fermented food produced using this product has no off-flavors. This fermentation product promotes the proliferation and differentiation of Naïve T cells and also promotes the release of immune cytokines. The Magnolia officinalis fermentation product prepared by this invention has the effect of enhancing immunity, providing a scientific basis for the study of morel-fermented Magnolia officinalis extract as a fermented food and its effects on the immune system.

Description

Technical Field

[0001] This invention belongs to the field of health functional foods, specifically relating to a method for preparing functional foods that enhance the ability of immune cells based on fermented Magnolia officinalis. Technical Background

[0002] Morel mushrooms (scientific name: *Morchella esculenta* (L.) Pers.) belong to the family Morchicaceae and the genus *Morchella*. The cap is nearly spherical, oval to elliptical, reaching up to 10 cm in height, with a blunt, rounded apex and pitted surfaces resembling a sheep's stomach. Morels are distributed worldwide and are used for both food and medicinal purposes. They are rich in various nutrients, including protein, amino acids, vitamins, fatty acids, as well as abundant polysaccharides, trace elements, dietary fiber, and minerals. Morels possess multiple health benefits, such as boosting immunity, reducing inflammation, killing bacteria, fighting viruses, inhibiting tumor growth, and lowering blood lipids. They can also alleviate the side effects of chemotherapy in cancer patients and prevent atherosclerosis, making them suitable for all ages.

[0003] Modern research has discovered that Magnolia officinalis contains various types of chemical components, including volatile oils, alkaloids, and lignans. Its main active ingredients possess pharmacological effects such as anti-tumor, anti-inflammatory, gastrointestinal regulation, and liver protection. However, as a traditional Chinese medicine, Magnolia officinalis is mostly used in prescriptions by decoction. In recent years, research on the water-soluble components of Magnolia officinalis has gradually emerged, and new glycosides, alkaloids, and other components have been discovered.

[0004] Cells involved in or related to the immune response are collectively referred to as immune cells. These mainly include lymphocytes, monocytes / macrophages, dendritic cells, mast cells, and granulocytes. They are crucial components of the body's immune system, defending against pathogen invasion, building immune defenses, clearing foreign substances, and repairing damaged tissues, thus fundamentally ensuring human health. Currently, there is no record of using the fermentation products of Magnolia officinalis to prepare related functional foods. Summary of the Invention

[0005] To address the market gap in existing technologies, this invention provides a method for preparing functional foods that enhance the ability of immune cells based on fermented Magnolia officinalis. This method utilizes morel mushrooms to ferment Magnolia officinalis to prepare corresponding extracts. When the concentration of the morel mushroom fermented Magnolia officinalis extract used is 20-40 μM, it can effectively promote the proliferation and differentiation of immune cells (Niave T cells) into CD4+ T cells. When the concentration of the morel mushroom fermented Magnolia officinalis extract is 20-40 μM, it can promote the release of the immune cell cytokine IFN-γ.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0007] This invention provides a method for preparing functional foods that enhance the ability of immune cells based on fermented Magnolia officinalis, comprising the following steps:

[0008] (1) Take morel strains, pick out mycelia and inoculate them in PDA medium, and incubate at constant temperature inverted position to obtain activated morel strains;

[0009] (2) Add Magnolia officinalis powder to PDA medium containing water chestnut powder while stirring. Stir until the dry powder is neither clumped nor loose, and can be tightly clumped but dispersed when released. This is Magnolia officinalis medium. Seal and then autoclave.

[0010] (3) Morel mushrooms were inoculated into the bottom of Magnolia officinalis culture medium, stirred evenly, and then brewer's yeast was added. After sealing with a sealing film, fermentation culture was carried out, and then dried to constant weight to obtain dried fermented product.

[0011] (4) Take the dried fermented material and soak it in gradient ethanol, combine the soaking liquids, centrifuge, take the supernatant and put it into a rotary evaporator for rotary evaporation, freeze dry it in a freeze dryer, and then grind it to obtain powdered morel fermented magnolia bark extract as the main raw material for fermented food.

[0012] Furthermore, in step (1), the inoculation amount of the mycelium in the PDA medium is 5%; the isothermal inverted culture is cultured at 20°C for 3 days.

[0013] Furthermore, in step (2), the formula of the PDA culture medium containing water chestnut powder is as follows: 200g potato, 45g water chestnut powder, 20g glucose, 20g agar, 5g peptone, 3g potassium dihydrogen phosphate, 15g magnesium sulfate, 1000ml water, and natural pH.

[0014] The water chestnut powder used in this invention is prepared by the following steps: freshly cleaned water chestnuts are directly mashed, then an appropriate amount of acetic acid is added, and after stirring evenly, calcium bicarbonate is added. After stirring for a period of time, the mixture is freeze-dried and ground into powder.

[0015] The amount of acetic acid added is 5% of the weight of the water chestnut; the amount of calcium bicarbonate added is 3% of the weight of the water chestnut.

[0016] Furthermore, in step (2), the mass ratio of the Magnolia officinalis powder to the PDA culture medium containing water chestnut powder is 1:10.

[0017] Furthermore, in step (3), the amount of morel mushrooms added is 10%; the amount of brewer's yeast added accounts for 2-3% of the magnolia bark culture medium.

[0018] Furthermore, in step (3), the fermentation culture is carried out in an incubator at 20°C for 15 days.

[0019] Furthermore, in step (4), the specific gradient of the gradient ethanol is 35% ethanol (V / V) - 50% ethanol (V / V) - 70% ethanol (V / V); the mass ratio of the fermentation product to ethanol is 1:10; the extraction is carried out at room temperature for 8 hours under each gradient.

[0020] In the preparation of the culture medium in this invention, the high-pressure steam sterilization conditions are 115℃ for 30 minutes;

[0021] The preparation process of the Magnolia officinalis powder used in this invention is as follows: Magnolia officinalis is pulverized using a pulverizer and then passed through a 60-mesh sieve.

[0022] The fermented Magnolia officinalis extract prepared by this invention can effectively promote the proliferation of immune cells when the concentration is 5-40 μM; preferably, the proliferation-promoting effect is better at 10-20 μM; the fermented Magnolia officinalis extract can effectively promote the differentiation of immune cells when the concentration is 20-40 μM; the fermented Magnolia officinalis extract can effectively promote the release of the immune cell cytokine IFN-γ when the concentration is 20-40 μM.

[0023] The morel mushrooms used in this invention are commercially available. Their appearance is as follows: In the early stage, the morel mycelium is glossy white or pale yellow, and the tips of the mycelium are branched in a dendritic manner; in the middle stage, colonies are formed on the culture medium, and the older mycelium in the center of the colony secretes pigment, making the mycelium brownish-yellow; when the mycelium ages, the colony changes from dark brown to dirty brown, and the luster disappears.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. This invention uses morel mushrooms as the fermentation strain and adds brewer's yeast to prepare a fermentation product that can promote the immune function of the immune system.

[0026] 2. The fermentation method provided by this invention can improve fermentation efficiency, produce richer nutrients during fermentation, and convert macromolecules into smaller molecules that are easier to absorb, thereby enhancing the effect on immune cells.

[0027] 3. This invention uses Magnolia officinalis as a fermentation substrate, adding a small amount of water chestnut powder to the culture medium. This prevents spoilage during fermentation, greatly improving the success rate. The raw materials are simple, readily available, and inexpensive, enhancing the nutritional content of the fermentation product. Fermented foods made from this product have no off-flavors. This fermentation product promotes the proliferation and differentiation of Naïve T cells and also promotes the release of immune cytokines. The Magnolia officinalis fermentation product prepared by this invention has the effect of enhancing immunity, providing a scientific basis for the study of morel-fermented Magnolia officinalis extract as a fermented food and its effects on the immune system. Attached Figure Description

[0028] Figure 1 The effect of different concentrations of fermented Magnolia officinalis extract on the proliferation capacity of Naïve T cells;

[0029] Figure 2 The effect of different concentrations of fermented Magnolia officinalis extract on the differentiation capacity of CD4+ T cells;

[0030] Figure 3 The effect of different concentrations of fermented Magnolia officinalis extract on the release of the immune cytokine IFN-γ. Detailed Implementation

[0031] To make the objectives, technical means, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the scope of this invention is not limited to the embodiments described.

[0032] Example 1

[0033] 1. Activation of morel mushrooms

[0034] (1) Prepare PDA comprehensive culture medium: Peel the potato, weigh 200g, cut into small pieces, add ultrapure water and cook over low heat until thickened, filter with gauze, add other culture medium components in Table 1 PDA comprehensive culture medium, and make up to 1L with ultrapure water.

[0035] Table 1

[0036]

[0037] (2) After sterilizing the prepared PDA comprehensive culture medium by high pressure steam (115℃, 30min), place it in a clean bench, disinfect it with ultraviolet light for 10min and avoid light for 5min, then pour it into a plate. After the culture medium solidifies, take morel strain, pick the mycelium and inoculate it in the center of the plate, and place it in a constant temperature incubator at 20℃ and invert it for 3 days to obtain activated morel.

[0038] 2. Fermentation of Magnolia officinalis

[0039] (1) Crushing Magnolia officinalis: Crush Magnolia officinalis with a crusher, pass it through a 60-mesh sieve, and obtain Magnolia officinalis powder after screening.

[0040] (2) Add a certain amount of Magnolia officinalis powder to PDA liquid culture medium (200g potato, 45g water chestnut powder, 20g glucose, 20g agar, 5g peptone, 3g potassium dihydrogen phosphate, 15g magnesium sulfate, 1L ultrapure water, pH natural) at a ratio of 1:10. Stir while adding the powder until it is evenly mixed. You can add an appropriate amount of distilled water until the Magnolia officinalis powder is neither clumped nor loose, and it is in a state where it is tightly clumped but dispersed when loosened.

[0041] (3) Put the prepared Magnolia officinalis into a box, seal it with sealing film and cover it, and put it in an autoclave at 115°C for 30 minutes for high-pressure steam sterilization.

[0042] (4) Fermentation of Magnolia officinalis: Select morel mushrooms of appropriate size and inoculate them into Magnolia officinalis culture medium (inoculation amount 10%), inoculate them into the bottom of Magnolia officinalis as much as possible, stir evenly, add brewer's yeast accounting for 3% of the mass of Magnolia officinalis culture medium, seal with sealing film, cover and seal; culture in an incubator at 20℃ for 15 days, dry in a vacuum drying oven to constant weight, and obtain dried fermented product.

[0043] 3. Extraction:

[0044] The dried fermented material was extracted with 10 times its volume of 35%-50%-70% graded ethanol (V / V), each grade for 8 hours. The extracts were combined, centrifuged, and the supernatant was placed in a rotary evaporator for rotary evaporation. After rotary evaporation, it was freeze-dried and then ground to obtain a powdered, easily preserved Magnolia officinalis extract.

[0045] 4. Drug preparation

[0046] (1) Weigh out the fermented Magnolia officinalis extract, add 25% ethanol to each, and sonicate until the extract is fully dissolved. Prepare drug concentrations of 5μM, 10μM, 20μM, 30μM, 40μM and 80μM, and store in a refrigerator at 4℃ for later use.

[0047] 5. Cell Acquisition: Extraction of primary naïve mouse T cells

[0048] 4-8 week old female C57BL / 6 mice were selected. Spleens were aseptically removed in a clean bench and placed in FBS-containing culture medium. The spleens were then placed on a 200-mesh cell sieve, and a single-cell suspension was prepared by grinding the spleen with the soft tip of a syringe. These single-cell suspensions were centrifuged at 1200 rpm for 5 min. After removing the supernatant, the cells were resuspended in 3 ml of erythrocyte lysis buffer and lysed on ice for 3 min. Lysis was then terminated by adding 5-10 times the volume of PBS. The cells were centrifuged at 400 g for 5 min. After centrifugation, the supernatant was removed, and the cells were resuspended in 1 ml of RPMI-1640 medium containing 10% FBS. MouseNaïve T cells were then sorted using an immunomagnetic assay kit provided by the manufacturer and cultured in 96-well plates.

[0049] 6. Cell viability assay

[0050] (1) 2.0~3.0×10 5 Cells at the above-mentioned cell density of / ml were seeded into 96-well plates.

[0051] (2) The fermented product prepared by diluting with 20% ethanol was used to make fermented Magnolia officinalis products of different concentrations.

[0052] (3) Add 5 μl of fermented Magnolia officinalis extract of different concentrations to each well to make experimental groups with different concentration gradients of 5 μM, 10 μM, 20 μM, 30 μM, 40 μM and 80 μM. Each concentration has 6 replicates. At the same time, a normal control group is set up, that is, the group with only PBS added, also with 6 replicates. The mixture is cultured for 24 h.

[0053] (4) Add 10 μl of CCK-8 to each well and incubate for 4 h.

[0054] (5) After incubation, place the cell on a shaker and shake for 10 minutes. After shaking, place the cell on a microplate reader and detect its OD value at 450 nm. Calculate the cell viability using the following formula.

[0055] Cell viability (%) = [(OD value of sample well - OD value of zeroing well) / (OD value of control well - OD value of zeroing well)] × 100%.

[0056] 7. The ability of T cells to differentiate into CD4+ T cells

[0057] (1) 2.0~3.0×10 5 Cells at the above-mentioned cell density of / ml were seeded into 96-well plates.

[0058] (2) The fermented product was diluted with 20% ethanol to prepare fermented magnolia officinalis fermented product solutions of different concentrations.

[0059] (3) Add 5 μl of fermented Magnolia officinalis extract of different concentrations to each well to make experimental groups with different concentration gradients of 10 μM, 20 μM and 40 μM. Each concentration has 3 replicates. At the same time, a control group is set up, i.e. the group with only PBS added, also with 3 replicates. Culture for 24 h.

[0060] (4) After gently blowing the liquid in the 96-well plate, aspirate the cells and add them to the corresponding centrifuge tubes. Wash the cells twice by centrifugation with PBS.

[0061] (5) Remove the supernatant and resuspend the precipitate in an appropriate amount of PBS.

[0062] (6) Add FITC anti-mouse CD4 flow cytometry antibody and PE / Cyanine7 anti-mouse CD3e flow cytometry antibody and mix well.

[0063] (7) After incubation, add an appropriate amount of PBS and centrifuge to wash the staining solution. Centrifuge at 300-400g for 5 minutes, and wash twice. Then place in an ice bath in the dark until ready for instrumental analysis.

[0064] 8. Detection of the release of the immune factor IFN-γ

[0065] (1) 2.0~3.0×10 5 Cells at the above-mentioned cell density of / ml were seeded into 96-well plates.

[0066] (2) The fermented product was diluted with 20% ethanol to prepare fermented magnolia officinalis fermented product solutions of different concentrations.

[0067] (3) Add 5 μl of fermented Magnolia officinalis extract of different concentrations to each well to make experimental groups with different concentration gradients of 10 μM, 20 μM, 30 μM and 40 μM. Each concentration has 3 replicates. At the same time, a control group is set up, that is, the group with only PBS added, also with 3 replicates. Culture for 24 h.

[0068] (4) The release of IFN-γ in the cell culture supernatant was determined according to the instructions of the MouseIFN-gamma ELISA Kit.

[0069] Application Example 1: Effect of fermented Magnolia officinalis extract on Naïve T cell proliferation.

[0070] like Figure 1 As shown, within the 10-40 μM range, fermented Magnolia officinalis extract promoted the proliferation of Naïve T cells, with the best effect observed at a concentration of 10-20 μM. Simultaneously, the experiment revealed that when only Morchella esculenta was used for fermentation without the addition of Saccharomyces cerevisiae, the fermentation products obtained at the same concentration gradient all exhibited reduced effects on Naïve T cell proliferation. For example, at 10 μM, fermentation with only Morchella esculenta reduced the proliferation effect by approximately 0.75%. Furthermore, when no water chestnut powder was added to the Magnolia officinalis culture medium during fermentation, the fermentation products obtained at the same concentration gradient all exhibited reduced effects on Naïve T cell proliferation. For example, at 10 μM, the fermentation product obtained without the addition of water chestnut powder showed a reduction in proliferation effect of approximately 0.28%.

[0071] Application Example 2: Effect of fermented Magnolia officinalis extract on T cell differentiation into CD4+ T cells.

[0072] like Figure 2 As shown, fermented Magnolia officinalis extract promoted the differentiation of CD4+ T cells to some extent in the range of 20-40 μM, and the differentiation-promoting ability of CD4+ T cells was the highest at 40 μM.

[0073] Application Example 3: Effect of fermented Magnolia officinalis extract on the release of immune cell cytokine IFN-γ.

[0074] like Figure 3As shown, within the range of 10-40 μM, fermented Magnolia officinalis extract can promote the release of the immune factor IFN-γ to a certain extent in a concentration-dependent manner, which is beneficial to the enhancement of immune regulation.

[0075] In summary, the extract obtained by fermenting Magnolia officinalis with Morel mushrooms and Saccharomyces cerevisiae in this invention, when used as a fermented food, can effectively promote the differentiation and proliferation of T lymphocytes and enhance immunomodulatory capabilities. As a control, we found that the Morel mushroom-fermented Magnolia officinalis extract at a concentration of 80 μM exhibited a cytotoxic effect on T cells. Therefore, when preparing the concentration, we selected a gradient below 40 μM to maintain the beneficial effects on lymphocyte differentiation and the release of immune factors while preventing toxic effects on T lymphocytes. This also provides valuable reference for formulating novel fermented foods in the future. Furthermore, the Morel mushroom-fermented Magnolia officinalis extract described in this invention can provide a basis for developing novel drugs that enhance immune function in the biopharmaceutical field.

[0076] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for preparing functional foods that enhance the ability of immune cells based on fermented Magnolia officinalis, characterized in that, Includes the following steps: (1) Take morel strains, pick out mycelia and inoculate them in PDA medium, and incubate at constant temperature inverted position to obtain activated morel strains; (2) Add Magnolia officinalis powder to PDA medium containing water chestnut powder while stirring. Stir until the dry powder is neither clumped nor loose, and can be tightly clumped but dispersed when released. This is Magnolia officinalis medium. Seal and then autoclave. (3) Morel mushrooms were inoculated into the bottom of Magnolia officinalis culture medium, stirred evenly, and then brewer's yeast was added. After sealing with a sealing film, fermentation culture was carried out, and then dried to constant weight to obtain dried fermented product. (4) Take the dried fermented material and soak it in gradient ethanol, combine the soaking liquids, centrifuge, take the supernatant and put it into a rotary evaporator for rotary evaporation, freeze dry it in a freeze dryer, and then grind it to obtain powdered morel mushroom fermented magnolia bark extract as the main raw material for fermented food. The water chestnut powder is prepared by the following steps: freshly cleaned water chestnuts are directly mashed, then an appropriate amount of acetic acid is added, stirred evenly, calcium bicarbonate is added, stirred for a period of time, freeze-dried, and then ground into powder. The amount of acetic acid added is 5% of the weight of the water chestnuts; the amount of calcium bicarbonate added is 3% of the weight of the water chestnuts. In step (2), the mass ratio of the magnolia bark powder to the PDA culture medium containing water chestnut powder is 1:10; In step (3), the amount of morel mushrooms added is 10%; the amount of brewer's yeast added accounts for 2-3% of the magnolia bark culture medium.

2. The method according to claim 1, characterized in that, In step (1), the inoculation amount of the mycelium in the PDA medium is 5%; the isothermal inverted culture is cultured at 20℃ for 3 days.

3. The method according to claim 1, characterized in that, In step (3), the fermentation culture is carried out in an incubator at 20°C for 15 days.

4. The method according to any one of claims 1-3, characterized in that, In step (4), the specific gradient of the gradient ethanol is 35% ethanol (V / V) - 50% ethanol (V / V) - 70% ethanol (V / V); the mass ratio of the fermentation product to ethanol is 1:10; the soaking is 8 hours of room temperature extraction under each gradient.

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