A method for preparing a fermentation broth having enhanced anti-inflammatory and antioxidant activity and the resulting product

Abstract

The present application belongs to the technical field of biological fermentation, and particularly relates to a preparation method of a fermentation liquor with enhanced anti-inflammatory and anti-oxidation activity and the obtained product. The specific steps of the method are as follows: first, a malus micromalus chung fruit fermentation liquor and a morchella esculenta fermented magnolia officinalis extract are prepared, and then the morchella esculenta fermented magnolia officinalis extract is added into the malus micromalus chung fruit fermentation liquor in a proportion. The present application can reduce the astringency of the malus micromalus chung fruit juice by fermenting the malus micromalus chung fruit juice in advance, and the prepared malus micromalus chung fruit juice has good stability. The present application first provides a traditional Chinese medicine fermented magnolia officinalis malus micromalus chung fruit juice drink with enhanced anti-inflammatory and anti-oxidation activity. By adding a certain concentration of the morchella esculenta fermented magnolia officinalis extract into the malus micromalus chung fruit juice, the anti-inflammatory and anti-oxidation effects of the malus micromalus chung fruit juice are improved under the synergistic effect of the two, and the prepared drink has the effects of food digestion and spleen invigoration, diabetes mellitus and heat relieving, alcohol relieving, and providing a large amount of vitamins, anti-inflammatory and anti-oxidation effects for the human body.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for preparing a fermentation broth with enhanced anti-inflammatory and antioxidant activity, and the resulting product. Technical Background

[0002] Rosa rugosa, also known as mountain king fruit or wood pear, is a perennial deciduous shrub belonging to the genus Rosa in the family Rosaceae. It is mainly distributed in the Yunnan-Guizhou-Sichuan plateau region of my country, with Guizhou Province having the richest resources and highest annual yield. Rosa rugosa is a plant used for both food and medicine; its fruit is rich in various active ingredients, including vitamin C, superoxide dismutase, peroxidase, polyphenols, flavonoids, polysaccharides, triterpenes, amino acids, and trace elements. Among these, the polyphenol content is as high as 15.93 mg / g. -1 The average flavonoid content is approximately 6.8 mg / g. -1 These substances mainly include gallocatechin, quercetin, kaempferol, and their derivatives. These substances possess a variety of pharmacological activities, giving prickly pear high value in both food and clinical medicine, and promising application prospects. It is also a research hotspot in phytochemistry.

[0003] Morel mushrooms ( Morchella esculenta Morel mushrooms (L.) are named for their honeycomb-like cap, resembling a sheep's stomach. There are 28 species of wild morels worldwide, with 20 species found in my country. Morels are recognized globally as a rare and precious higher fungus, primarily growing in neutral to slightly alkaline humus-rich soils, abundant among broad-leaved trees such as paulownia, apple, pear, elm, and poplar, as well as sparse reed beds. Morels 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.

[0004] Magnolia officinalis, named for its thick bark and woody texture, is primarily cultivated in mountainous areas with a long growing season. It is a Class II protected rare plant in China, mainly distributed in Hubei, Sichuan, Zhejiang, and Jiangxi provinces, with the "Sichuan Magnolia officinalis" variety considered superior. Modern research has revealed various chemical components in Magnolia officinalis, including lignans, alkaloids, and volatile oils. Its main active ingredients possess pharmacological effects such as anti-tumor, anti-inflammatory, gastrointestinal regulation, and liver protection. Among these, Magnolia officinalis exhibits significant anti-tumor activity, inhibiting malignant tumors of the liver, colon, pancreas, and lungs. However, as a traditional Chinese medicine, Magnolia officinalis is primarily used in prescriptions as a decoction. In recent years, research on its water-soluble components has gradually emerged, leading to the discovery of new glycosides and alkaloids.

[0005] In the prior art, the taste of the roxburgh rose juice itself is sour and astringent, and the taste of the related drinks prepared from the roxburgh rose juice is poor; and there is no related record of preparing a drink from roxburgh rose and magnolia officinalis fermentation liquor. SUMMARY

[0006] In view of the problems in the prior art, the application provides a preparation method of a fermentation liquor with enhanced anti-inflammatory and antioxidant activity.

[0007] The application further provides the fermentation liquor obtained by the preparation method.

[0008] The application adopts the technical scheme as follows to achieve the above object:

[0009] The application provides a preparation method of a fermentation liquor with enhanced anti-inflammatory and antioxidant activity, comprising the following steps:

[0010] (1) Clean the roxburgh rose, and beat the pulp to obtain roxburgh rose pulp; add Aspergillus oryzae and pectinase into the roxburgh rose, and ferment at room temperature to obtain fermentation slurry; after enzyme inactivation of the fermentation slurry, squeeze and filter to obtain roxburgh rose fermentation liquor;

[0011] (2) Crush the magnolia officinalis, and sieve to obtain magnolia officinalis powder;

[0012] (3) Add PDA liquid medium into the magnolia officinalis powder, and stir while adding until the magnolia officinalis powder is neither loose nor in a lump, but in a state of being tightly held into a lump and being dispersed when released, to obtain magnolia officinalis culture medium;

[0013] (4) High-pressure steam sterilize the magnolia officinalis culture medium in a sealed state;

[0014] (5) Ferment the magnolia officinalis: clamp the morchella and insert it into the bottom of the magnolia officinalis culture medium, and stir uniformly; after sealing, seal and ferment the culture medium to obtain the fermentation product of the morchella fermented magnolia officinalis;

[0015] (6) Soak the dried fermentation product in 70% ethanol (V / V), filter with gauze, and centrifuge at 8000 r / min to obtain supernatant; perform rotary evaporation on the obtained supernatant, and then perform freeze-drying with a freeze-drying machine; after grinding, obtain the powder-like morchella fermented magnolia officinalis extract;

[0016] (7) Add the morchella fermented magnolia officinalis extract into the roxburgh rose fermentation liquor, and stir uniformly.

[0017] Further, in step (1), the addition amount of the pectinase accounts for 0.1% of the weight of the roxburgh rose; and the addition amount of the Aspergillus oryzae accounts for 0.05% of the weight of the roxburgh rose.

[0018] Further, in step (1), the fermentation time is 10-12 h.

[0019] Further, in step (2), the Magnolia officinalis powder is passed through a 60 mesh sieve.

[0020] Further, in step (5), the Morchella is inoculated into the Magnolia officinalis culture medium in an amount of 5-6%.

[0021] Further, in step (5), the fermentation culture is cultured in a 20 DEG C incubator for 30 days.

[0022] Further, in step (6), the volume ratio of the fermented product to 70% ethanol is 1:10.

[0023] Further, in step (6), the soaking is carried out at room temperature for 24 hours.

[0024] Further, in step (7), the content of the Morchella fermented Magnolia officinalis extract in the Rosa roxburghii fermentation liquid is 0.15 g per L.

[0025] The application also provides a Magnolia officinalis Rosa roxburghii juice beverage prepared by the above preparation method.

[0026] The Morchella used in the application is inoculated into PDA solid culture medium in an amount of 5%, and then cultured in a 20 DEG C constant temperature incubator for 2-3 days.

[0027] The Morchella strain used in the application is commercially available, and its apparent characteristics are as follows: in the early stage, the Morchella mycelium is white or light yellow, and has luster; the mycelium tip is multi-fingered or dendritic; in the middle stage, the colony is formed on the culture medium, the older mycelium in the center of the colony secretes pigment, so that the mycelium is brownish yellow; when the mycelium is aging, the colony turns from dark brown to black brown, and the luster disappears. The enzyme activity of the pectinase used in the application is 10000 u / g.

[0028] The application has the following beneficial effects:

[0029] (1) The application can reduce the astringency of the Rosa roxburghii juice by fermenting the juice, and the prepared Rosa roxburghii juice has good stability.

[0030] (2) The application provides a traditional Chinese medicine fermented Magnolia officinalis Rosa roxburghii juice beverage with enhanced anti-inflammatory and antioxidant activities for the first time. By adding a certain concentration of Morchella fermented Magnolia officinalis extract into the Rosa roxburghii juice, the anti-inflammatory and antioxidant effects of the juice are improved under the synergistic action of the two, and the prepared beverage has the effects of food digestion and spleen invigoration, diabetes and heat relief, alcohol relief, and provides a large amount of vitamins, anti-inflammatory and antioxidant effects for the human body. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The proliferation inhibition of different concentrations of fermented crude extracts on RAW264.7 cells;

[0032] Figure 2 Effects of Magnolia officinalis extract and Morchella officinalis fermented Magnolia officinalis extract on DPPH free radicals. Specific implementation method

[0033] In order to make the purpose, technical means and beneficial effects of the present application more clear and explicit, the present application is further described in detail below in combination with the drawings and examples. However, the content of the present application is not limited to the scope described in the examples.

[0034] Example 1 Preparation of Morchella officinalis fermented Magnolia officinalis extract

[0035] 1. Activation of Morchella officinalis

[0036] Morchella officinalis is a fungus of the Morchellaceae family and the Morchella genus. The culture medium used is composed of 200 g of potatoes, 20 g of glucose and 15-20 g of agar per L of culture medium. The prepared PDA solid culture medium is sterilized (115°C, 30 min) and then cooled to about 50°C, and then transferred to a clean bench to pour the plates. After the solid culture medium in the plates is formed, the Morchella strain is inoculated (5%) in the center of the plates and placed in a constant temperature incubator at 20°C for 2 days to obtain activated Morchella officinalis.

[0037] 2. Fermentation of Magnolia officinalis

[0038] (1) Grind Magnolia officinalis: Grind Magnolia officinalis and sieve it through a 60-mesh sieve to obtain Magnolia officinalis powder.

[0039] (2) Add a certain amount of Magnolia officinalis powder to the previously prepared PDA liquid medium (1:10) while stirring to obtain a uniform mixture. Add distilled water as needed until the Magnolia officinalis powder is neither loose nor clumped, but forms a clump that can be broken apart.

[0040] (3) Put the prepared Magnolia officinalis into a box, add a plastic sealing film, cover and seal, and then place it in a sterilization pot for high-pressure steam sterilization at 115°C for 30 min.

[0041] (4) Ferment the Magnolia officinalis: Take a proper amount of Morchella officinalis and inoculate it into the Magnolia officinalis culture medium (inoculation amount 5%) as much as possible at the bottom of the culture medium, stir well, cover with a plastic sealing film, cover and seal; cultivate in a 20°C incubator for 30 days, and then dry to constant weight to obtain dried fermented product.

[0042] 3. Extraction

[0043] Take the dried fermented product, soak it in 10 times the volume of 70% ethanol (V / V) at room temperature for 48 h, and then centrifuge to obtain the supernatant. Perform rotary evaporation on the obtained supernatant, freeze-dry it using a freeze-drying machine, and then grind it to obtain a powder-like Morchella officinalis fermented Magnolia officinalis extract that is easy to preserve.

[0044] Example 2 Determination of Anti-inflammatory Ability of Morinda officinalis How Extract Fermented by Morchella

[0045] 1. Cell recovery

[0046] The frozen RAW264.7 cells were removed and transferred to a constant temperature water bath at 37°C, shaken to melt. Centrifuged at 1000 rpm for 5 min, discarded the supernatant, resuspended in DMEM complete medium, inverted microscope observed the cell density. Transferred to the incubator at 37°C, 5% CO2 culture, observed the cell growth.

[0047] 2. Cell culture and subculture

[0048] The culture medium was changed every 2-3 days, and the cells were subcultured when the cell density reached 70-90%. The old culture medium was discarded, washed twice with PBS, and an appropriate amount of trypsin was added for digestion for 1-2 min. The digestion was terminated with complete medium containing serum, mixed uniformly by blowing, centrifuged at 1000 rpm for 5 min, resuspended in fresh culture medium, 1:2 aliquoted, cultured at 37°C, 5% CO2 in the incubator, and the cell growth was observed.

[0049] 3. Cell survival rate determination

[0050] (1) Recover and culture the above cells.

[0051] (2) 2.0-3.0 x 10 5 / ml cell density was inoculated into 96-well plates and cultured for 24 h.

[0052] (3) The crude extract prepared by diluting with 20% ethanol was prepared into sample solution containing different concentrations of crude extract.

[0053] (4) After overnight culture, Morchella officinalis How extract sample solution (directly using 70% soaked Morinda officinalis powder for 48 h) with concentrations of 0 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, and unfermented Morinda officinalis extract sample solution were added to each well, 5 μl per well, 6 replicate wells for each concentration, and normal control was set up, also with 6 replicate wells, cultured for 12-24 h.

[0054] (5) 10 μL CCK-8 was added to each well and incubated for 2 h.

[0055] (6) After incubation, place on a shaker for 10 min, after shaking, place on an enzyme marker, detect OD value at 450 nm, calculate cell survival rate according to the following formula.

[0056] Cell viability (%) = [(OD value of sample well - OD value of zeroing well) / (OD value of control well - OD value of zeroing well)] × 100%.

[0057] Experimental results are as follows Figure 1 As shown, within the range of 50-200 μg / mL, both fermented and unfermented Magnolia officinalis extracts promoted the proliferation of RAW264.7 cells, and enhanced macrophage activity in a dose-dependent manner, with the former showing a more pronounced promoting effect than the latter. The optimal effect was observed at a concentration of 150 μg / mL, resulting in a 15% increase in survival rate compared to treatment with the unfermented Magnolia officinalis extract.

[0058] Example 3: Determination of the antioxidant capacity of morel mushroom fermented Magnolia officinalis extract

[0059] Prepare 10 -4 A 1 mol / L DPPH solution was mixed with 150 μg / mL of unfermented Magnolia officinalis extract and morel-fermented Magnolia officinalis extract to form the test group; an equal volume of DPPH solution of the same concentration was mixed with an equal volume of deionized water to form the blank group; an equal volume of 20% ethanol was mixed with an equal volume of the above-mentioned test samples of different concentrations to form the control group. After reacting the test solutions for 30 min, the absorbance at 517 nm was measured, and the DPPH free radical scavenging rate was calculated, where DPPH free radical scavenging rate = (A... h +A i -A j ) / A h ×100%

[0060] A h This indicates the absorbance value at 517 nm in the control well;

[0061] A i This indicates the absorbance value at 517 nm in the control well;

[0062] A j This indicates the absorbance value measured at 517 nm in the well.

[0063] Depend on Figure 2 It is known that both Magnolia officinalis extract and morel-fermented Magnolia officinalis extract can scavenge DPPH free radicals, with morel-fermented Magnolia officinalis extract showing better effect and stronger antioxidant capacity.

[0064] Example 4: Preparation of Prickly Pear Juice

[0065] After cleaning the prickly pear, it is pulped to obtain prickly pear pulp. 0.05% Aspergillus oryzae and 0.1% pectinase by weight of the prickly pear are added to the prickly pear and fermented at room temperature for 12 hours to obtain fermented slurry. After inactivating the enzymes in the fermented slurry, it is squeezed and filtered to obtain prickly pear fermentation liquid.

[0066] Respectively, in the addition of different amounts of Aspergillus oryzae and pectinase in the fermentation of the Malus robusta juice, the effect of the astringency of the fermented juice was evaluated by tasting, and the score was given by 10 points.

[0067] Table 1

[0068]

[0069] The evaluation results are shown in Table 1. When Aspergillus oryzae and pectinase were added at the same time, the astringency of the prepared Malus robusta fermented juice was the lowest. When Aspergillus oryzae was added alone, the prepared Malus robusta fermented juice had a special bitter taste. When pectinase was added alone, although the astringency was appropriately reduced, the Malus robusta juice had a special smell. When Aspergillus oryzae and pectinase were used for fermentation, there was no such smell, and the astringency was better.

[0070] Example 5

[0071] The content of the Morchella-fermented Magnolia officinalis extract in each L of the Malus robusta fermented juice was 0.15 g, and the Malus robusta drink was obtained.

[0072] The antioxidant capacity of the drink prepared in Example 5 was determined, and the specific results are shown in Table 2.

[0073] Table 2

[0074]

[0075] The above specific embodiments do not constitute a limitation on the protection scope of the present application. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modification, equivalent replacement, and improvement made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a fermentation broth for enhancing the efficacy of anti-inflammatory and antioxidant activities, characterized by, It comprises the following steps: (1) clean the Rosa roxburghii Tratt, beat the pulp to get the pulp, add Aspergillus oryzae and pectinase to the pulp, ferment at room temperature to get the fermented pulp, squeeze and filter the fermented pulp after enzyme inactivation to get the fermented liquid of Rosa roxburghii Tratt; (2) crush Magnolia officinalis, sieve to get Magnolia officinalis powder; (3) add PDA liquid medium to the Magnolia officinalis powder, stir while adding, until the Magnolia officinalis powder becomes neither loose nor clustered, but tight and loose, to get the Magnolia officinalis culture medium; (4) sterilize the Magnolia officinalis culture medium under high-pressure steam in a sealed state; (5) ferment the Magnolia officinalis: clamp Morchella into the bottom of the Magnolia officinalis culture medium, stir evenly, seal and ferment to get the fermented product of Morchella fermented Magnolia officinalis; (6) soak the dried fermented product in 70% ethanol (V / V), filter with gauze, centrifuge at 8000 r / min to get the supernatant, rotary evaporate the supernatant, freeze-dry the rotary evaporated product, grind again to get the powder of Morchella fermented Magnolia officinalis extract; (7) add the Morchella fermented Magnolia officinalis extract to the fermented liquid of Rosa roxburghii Tratt, stir evenly; In step (1), the fermentation time is 10-12 h; In step (5), the fermentation culture is cultured in a 20℃ incubator for 30 d; In step (6), the volume ratio of the fermented product to 70% ethanol is 1:

10.

2. The production method according to claim 1, characterized by, In step (1), the addition amount of pectinase accounts for 0.1% of the weight of Rosa roxburghii Tratt; the addition amount of Aspergillus oryzae accounts for 0.05% of the weight of Rosa roxburghii Tratt.

3. The preparation method according to claim 1, characterized in that, In step (2), the Magnolia officinalis powder is sieved through a 60-mesh sieve.

4. The method of claim 1, wherein, In step (5), the amount of Morchella added to the Magnolia officinalis culture medium is 5-6%.

5. The preparation method according to claim 1, characterized in that, In step (6), the soaking is at room temperature for 24 h.

6. The method of any one of claims 1-5, wherein, In step (7), the content of Morchella fermented Magnolia officinalis extract in each L of the fermented liquid of Rosa roxburghii Tratt is 0.15 g.

7. A Magnolia officinalis Rosa roxburghii juice beverage prepared by the method of any one of claims 1-6.

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