Fermented genus daphne juice and its production method and application

Abstract

The present invention discloses a fermented radix dahurica juice and a production method thereof, belonging to the field of microbial fermentation. The present invention uses Bacillus coagulans CGMCC 1.3220 to ferment the radix dahurica juice, thereby increasing the contents of flavonoids, polyphenols, saponins, and polysaccharides in the radix dahurica juice to 887 mg / L, 594 mg / L, 765 mg / L, and 41 g / L, respectively. These contents are 19.3%, 14%, 17.1%, and 28.1% higher than those of unfermented radix dahurica juice, respectively. The fermented radix dahurica juice supernatant has a good tyrosinase inhibition rate and antioxidant capacity. The present invention uses microbial fermentation of the radix dahurica juice, further increasing the content of active ingredients therein and expanding the application of radix dahurica.

Description

Technical Field

[0001] The invention relates to fermented genus daphne juice and a production method and application thereof, belonging to the field of microbial fermentation. Background Art

[0002] Daphne radix is ​​a traditional Chinese medicinal herb that primarily grows at altitudes above 3,500 meters, primarily in Qujing, Kunming, and Lhasa in Tibet. Its roots contain a variety of active ingredients, including polysaccharides, glucosinolates, saponins, polyphenols, and flavonoids. The Four Medical Classics, a jewel of Tibetan medicine, describes it as resembling a round radish, small and sweet, with a high density. Its sweet flavor, warm properties, and the ability to clear heat and detoxify, as well as nourish and increase oxygen, make it of great medicinal and research value in Traditional Chinese Medicine.

[0003] The existing use of radix daphne is limited to the food field, including beverages and health products, and its specific functions involve anti-hypoxia, anti-fatigue, lipid-lowering, etc. For example, the prior art use of radix daphne total flavonoids to prepare hypoglycemic drugs CN102293868B discloses a method for preparing total flavonoids from radix daphne, and shows the function of lowering blood sugar in mice; a solid beverage that can quickly increase human blood oxygen saturation and its preparation process CN113519660A discloses a health product that improves hypoxia tolerance; a cordyceps and red yeast rice enzyme beverage and its preparation method CN102018259B discloses the use of radix daphne and red yeast rice to prepare an enzyme beverage; a processing method of radix daphne leisure food CN103110070B discloses a method for preparing crisp chips using radix daphne; a method for making health wine by co-fermenting Tibetan special resources with highland barley CN107034103B discloses a method for producing health wine using daphne, which contains high levels of sterols and alkaloids.

[0004] It can be found that the existing preparation of radix daphne is limited to isolating or utilizing the active ingredients contained in radix daphne and using them in the preparation of food or health products. However, the antioxidant properties of the active ingredients contained in radix daphne are not fully utilized, which further limits the utilization of radix daphne.

[0005] Bacillus coagulans is taxonomically classified as a member of the genus Bacillus. Its cells are rod-shaped, Gram-positive, with terminal spores and no flagella. It can break down sugars to produce L-lactic acid, making it a homolactic fermenter. As an edible probiotic, Bacillus coagulans is widely used in fermented foods. Furthermore, as an edible probiotic, Bacillus coagulans helps maintain intestinal microecological balance and supports a healthy digestive system. In the context of modern biotechnology and microbiology, in-depth research on Bacillus coagulans is expected to reveal more information about its metabolic pathways and fermentation products, which will also help to better understand and utilize this microbial resource.

[0006] Based on the current status of existing technologies, attempts to expand the application of radix guianensis and improve its utilization value have extremely high practical value and economic benefits. Summary of the Invention

[0007] The inventors have discovered that Bacillus coagulans CGMCC 1.3220 can ferment radix dahurica juice and increase the content of polyphenols, flavonoids, saponins, and other substances in the juice. The resulting fermented radix dahurica juice supernatant has excellent tyrosinase inhibition and antioxidant capacity. This invention utilizes microbial fermentation of radix dahurica juice to increase the content of active ingredients, further expanding the application of radix dahurica.

[0008] The first object of the present invention is to provide a fermentation clear liquid of Dahurian radix juice, which is obtained by fermenting Dahurian radix juice with Bacillus coagulans CGMCC 1.3220.

[0009] In one embodiment, the fermentation is carried out by inoculating the genus daphne juice with Bacillus coagulans CGMCC 1.3220 and then fermenting it at a temperature of 35-40° C. for 5-9 days.

[0010] The invention also provides a composition containing the fermented clear liquid of genus daphne juice.

[0011] The second object of the present invention is a method for producing daphne daphne juice fermentation clear liquid, comprising: squeezing daphne daphne juice to obtain daphne daphne juice, fermenting the daphne juice with Bacillus coagulans CGMCC 1.3220 for 5 to 9 days, filtering and removing residue after fermentation to obtain daphne daphne juice fermentation clear liquid.

[0012] In one embodiment, the genus Veneris of the present invention is produced in Tibet or Qinghai.

[0013] In one embodiment, the seed liquid of Bacillus coagulans CGMCC 1.3220 is inoculated into the genus daphne juice at an inoculum amount of 1-5% v / v.

[0014] In one embodiment, Bacillus coagulans CGMCC 1.3220 is inoculated into genus daphne juice and fermented for 5 to 9 days.

[0015] In one embodiment, Bacillus coagulans CGMCC 1.3220 is inoculated into genus daphne juice and then cultured at 35-40° C. and 100 rpm.

[0016] The third object of the present invention is to provide the use of the fermented clear solution of genus daphne juice in the preparation of whitening and antioxidant products.

[0017] The present invention also provides the use of the composition in preparing whitening and anti-oxidation products.

[0018] In one embodiment, the product is a food, a health product, a medicine, or a skin topical product.

[0019] In one embodiment, the product has at least one of the following functions:

[0020] (a) Inhibit tyrosinase;

[0021] (b) Antioxidant.

[0022] Beneficial effects

[0023] (1) The present invention prepares a fermented liquid of daphne radix juice by fermenting daphne radix juice with Bacillus coagulans CGMCC 1.3220, thereby increasing the contents of flavonoids, polyphenols, and saponins in the daphne radix juice to 887 mg / L, 594 mg / L, and 765 mg / L, respectively, which are 19.3%, 14%, and 17.1% higher than those of unfermented daphne radix juice, respectively;

[0024] (2) The fermented liquid of the genus dahurica juice of the present invention has a total acid content of 5.64 g / L, which is 2.5 times higher than that of the unfermented genus dahurica juice; and the polysaccharide content is 28.1% higher than that of the unfermented genus dahurica juice;

[0025] (3) The fermented liquid of the genus dahurica juice of the present invention has a good tyrosinase inhibition rate of 98.7%, which is 82.8% higher than that of the unfermented genus dahurica juice;

[0026] (4) The fermented clear liquid of the genus dahurica juice of the present invention has good hydroxyl radical scavenging rates and DPPH radical scavenging rates, reaching 64.4% and 83.6% respectively, which are increased by 51.7% and 66.3% respectively compared with the unfermented genus dahurica juice. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0028] Use bacteria:

[0029] Bacillus coagulans CGMCC 1.3220, Lactobacillus plantarum CGMCC 1.1856, Pediococcus acidilactici CGMCC 1.4, Lactococcus lactis CGMCC 1.2030, and Lactobacillus fermentum CGMCC 1.15608 were purchased from China General Microorganism Culture Collection.

[0030] Example 1: Preparation of fermented genus angelica juice

[0031] (1) Preparation of genus daphne juice

[0032] Buy cilantro produced in Tibet, take 1 kg, wash and cut into small pieces, put it in a blender and crush it, take it out and set aside.

[0033] (2) Seed solution preparation

[0034] Bacillus coagulans, Lactobacillus plantarum, Pediococcus acidilactici, Lactococcus lactis, and Lactobacillus fermentum were scraped from a solid MRS culture medium plate and inoculated into a shaking flask containing 10 mL of seed culture medium, respectively. The culture temperature was 37° C., the shaking speed was 150 rpm, and the culture was carried out for 12 hours to obtain seed liquids of different strains. The formula (w / v) of the seed culture medium included 1000 mL of deionized water, 3% glucose, 1% beef powder, 1% yeast powder, 0.1% potassium dihydrogen phosphate, and 0.05% anhydrous magnesium sulfate.

[0035] (3) Inoculation and fermentation

[0036] The seed liquid obtained in step (2) was inoculated into the genus dahurica juice prepared in step (1) at an inoculum amount of 5% v / v, and fermented continuously at 37° C. and 100 rpm for 7 days to obtain products of genus dahurica juice fermented by different strains.

[0037] (4) Preparation of fermentation supernatant

[0038] The fermentation product obtained in step (3) was centrifuged at 6000 rpm for 15 minutes to obtain a centrifugal supernatant, and the strain residue in the centrifugal supernatant was removed by filtration using a 0.22 μm filter membrane to obtain the fermentation supernatant of Daphne dahurica juice.

[0039] Example 2: Detection of substances in the fermentation supernatant of Daphne dahurica juice

[0040] (1) Determination of polyphenol content

[0041] The total polyphenol content in the fermented genus dahurica juice prepared in Example 1 was determined using a plant total phenol (TP) assay kit (microplate method) (purchased from Shanghai Yaji Biotechnology Co., Ltd.; product model TP1121 100T) according to the kit instructions. Unfermented genus dahurica juice served as a control group. The results are shown in Table 1.

[0042] (2) Determination of flavonoid content

[0043] The total flavonoid content in the fermented genus daphne juice prepared in Example 1 was determined using a plant flavonoid colorimetric assay kit (purchased from Shanghai Fusheng Industrial Co., Ltd.; product number AS632168) according to the kit instructions. Unfermented genus daphne juice served as a control group. The results are shown in Table 1.

[0044] (3) Determination of saponin content

[0045] The total saponin content in the fermented genus dahurica juice prepared in Example 1 was determined using a total saponin content microassay kit (purchased from Shanghai Bohu Biotechnology Co., Ltd.; product number BH-9611368) according to the kit instructions. Unfermented genus dahurica juice served as a control group. The results are shown in Table 1.

[0046] (4) Determination of total acid content

[0047] Total acid was determined in the fermented liquid of genista dahurica juice according to GB 12456-2021, "National Food Safety Standard - Determination of Total Acidity in Foods." The results are shown in Table 1.

[0048] (5) Determination of polysaccharide content

[0049] Preparation of phenol test solution: Weigh 10 g of phenol, dissolve it in ultrapure water, shake well and transfer it completely to a 200 mL volumetric flask, add ultrapure water until the mark, then transfer it to a brown bottle, mix and shake well, store in a dark place and use it later (prepare it for immediate use).

[0050] Accurately weigh 0.05g of glucose standard, dissolve in ultrapure water, shake well, and transfer completely to a 50mL volumetric flask. Add ultrapure water to the mark to prepare a 1mg / mL standard stock solution. Then, dilute 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the stock solution to 10mL in five 10mL volumetric flasks to prepare a series of five gradient concentrations: 0.02mg / mL, 0.04mg / mL, 0.06mg / mL, 0.08mg / mL, and 0.10mg / mL. Refrigerate at 4°C until ready to use.

[0051] Accurately measure 1 mL of each of the above-mentioned gradient glucose standard reference solutions and place them in stoppered test tubes. Then, add 1 mL of 5% phenol solution to each solution, mix thoroughly, quickly add 5 mL of concentrated sulfuric acid, shake well, and keep warm in an 80°C water bath for 20 minutes. Take out the solution, cool it in a cold water bath, and measure the absorbance value A. Take 1 mL of ultrapure water as a blank control, add 1 mL of 5% phenol solution, quickly add 5 mL of concentrated sulfuric acid, mix well, and keep warm in an 80°C water bath for 20 minutes. Cool it to room temperature and measure the absorbance value at a wavelength of 490 nm by UV-visible spectrophotometry. Use the absorbance value as the ordinate (y) and the concentration as the abscissa (x) to draw a standard curve. The regression equation y=8.073x+0.0561(R 2 =0.9999).

[0052] Take 1 mL of fermented liquid from different strains of Daphne daphne root juice, add 1 mL of 5% phenol solution, and quickly add 5 mL of concentrated sulfuric acid. After mixing thoroughly, incubate in an 80°C water bath for 20 minutes. Cool to room temperature and measure the absorbance at a wavelength of 490 nm. Substitute the absorbance value into the standard curve to obtain the polysaccharide content.

[0053] Table 1 Determination of polyphenols, flavonoids, saponins, total acid and polysaccharide contents

[0054] As shown in Table 1, the contents of polyphenols, flavonoids, saponins, total acid and polysaccharides were increased after fermentation by Bacillus coagulans CGMCC 1.3220. The contents of flavonoids, polyphenols and saponins reached 887 mg / L, 594 mg / L and 765 mg / L, respectively, which were 19.3%, 14% and 17.1% higher than those of unfermented dahliae juice, respectively. The total acid content was 2.5 times higher than that of unfermented dahliae juice, and the polysaccharide content was 28.1% higher than that of unfermented dahliae juice.

[0055] Example 3: Determination of the whitening ability of the fermented liquid of Daphne dahurica juice

[0056] The whitening ability is usually evaluated based on the tyrosinase activity inhibition rate. The tyrosinase activity inhibition rate of the fermented liquid of Daphne daphne juice prepared in Example 1 is tested respectively. The specific steps are as follows:

[0057] (1) Principle of evaluation of tyrosinase activity inhibition rate:

[0058] Tyrosinase is a key enzyme in the synthesis of melanin in organisms. It catalyzes the hydroxylation of L-tyrosine to form L-dopa, which is then oxidized to form dopaquinone. Dopaquinone undergoes a series of enzymatic and non-enzymatic reactions to form melanin. Therefore, by inhibiting tyrosinase activity, the amount of melanin produced in an organism can be regulated.

[0059] (2) Detection reagents

[0060] PBS buffer (0.2 M, pH 6.8): Measure 51 mL of 0.2 M sodium dihydrogen phosphate solution and 49 mL of 0.2 M sodium dihydrogen phosphate solution and mix well. Tyrosinase (250 U): Dissolve tyrosinase (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) in the above PBS buffer, transfer to a 100 mL volumetric flask, and dilute to the mark with PBS buffer to prepare a 250 U / mL working solution.

[0061] (3) Detection method

[0062] To four centrifuge tubes, A, B, C, and D, add 0.25 mL of L-tyrosine and 0.25 mL of PBS, respectively. Add 0.25 mL of sample solution to tubes B and D. Add 0.25 mL of PBS solution to tubes A and C. Vortex all four tubes to mix thoroughly. After incubating at 37°C for 10 minutes, add 0.25 mL of enzyme solution to tubes C and D. Add the same volume of PBS buffer to tubes A and B to make up the test sample volume. Incubate all four tubes at 37°C for another 20 minutes. Add 200 μL of the above solutions to a 96-well plate and measure the absorbance at 475 nm using a microplate reader.

[0063] (4) Calculation of tyrosine activity inhibition rate:

[0064] Tyrosinase activity inhibition rate % = ((CA)-(DB)) / (CA)*100%

[0065] The results of tyrosinase inhibition rates are shown in Table 2.

[0066] Table 2 Tyrosinase inhibition rate

[0067] Tyrosinase activity inhibition rate Unfermented (control group) 15.9％ Bacillus coagulans CGMCC 1.3220 98.7％ Lactobacillus plantarum CGMCC 1.1856 87.3％ Pediococcus acidilactici CGMCC 1.4 44.8％ Lactococcus lactis CGMCC 1.2030 30.2％ Lactobacillus fermentum CGMCC 1.15608 74.9％

[0068] As shown in Table 2, Bacillus coagulans CGMCC 1.3220 had the highest inhibition rate on tyrosinase activity, reaching 98.7%, which was 82.8% higher than that of unfermented genus dahliae juice, and its effect was better than that of other strains.

[0069] Example 4: Determination of the antioxidant capacity of the fermented liquid of Daphne dahurica juice

[0070] 1. Hydroxyl free radical scavenging rate

[0071] (1) Measurement principle

[0072] It is known that a mixture of FeCl3-EDTA, hydrogen peroxide, and ascorbic acid react at pH 7.4 to produce hydroxyl radicals, which can degrade deoxyribose to form malondialdehyde. At low pH, the products malondialdehyde (MDA) and thiobarbituric acid (TBA) are heated to produce a pink chromophore, and the content of the pink chromophore can be used to determine the hydroxyl radical content.

[0073] (2) Measurement method

[0074] Prepare three groups of reaction reagents, including

[0075] (A) 75 μL 0.8 mM FeCl3 + 75 μL 4 mM EDTA + 600 μL 4 mM KH2PO4-KOH buffer + 2.0565 mL distilled water + 43.5 μL 3% H2O2 + 75 μL 112 mM deoxyribose + 75 μL 4 mM ascorbic acid;

[0076] (B) 75 μL 0.8 mM FeCl3 + 75 μL 4 mM EDTA + 600 μL 4 mM KH2PO4-KOH buffer + 2.0565 mL sample solution + 43.5 μL 3% H2O2 + 75 μL 112 mM deoxyribose + 75 μL 4 mM ascorbic acid;

[0077] (C) 75 μL 0.8 mM FeCl 3 + 75 μL 4 mM EDTA + 600 μL 4 mM KH 2 PO 4 -KOH buffer + 2.0565 mL sample solution + 43.5 μL 3% H 2 O 2 + 75 μL 4 mM ascorbic acid.

[0078] The reaction mixtures of each group were incubated at 37°C in a constant temperature water bath for 1 hour, and then 2 mL of 0.6% TBA was added to the mixture, and the mixture was incubated in a boiling water bath for 15 minutes. After rapid cooling, the mixture was centrifuged and the absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm.

[0079] (3) Calculation method

[0080] Hydroxyl radical scavenging rate (%) = [1-(BC) / A]*100%

[0081] The results of hydroxyl radical scavenging rates are shown in Table 3.

[0082] 2. DPPH free radical scavenging rate

[0083] (1) Principle of determination: DPPH free radical is a single electron free radical with characteristic absorption at 517 nm. Free radical scavengers can pair with the single electron of DPPH free radical to make its absorption at 517 nm disappear. The degree of fading is quantitatively related to the number of electrons it accepts, and the scavenging ability of the free radical scavenger can be inferred based on this.

[0084] (2) Assay method: Prepare and mix three groups of reaction reagents, including:

[0085] (A) 1 mL distilled water + 3 mL 0.1 mM DPPH reaction solution;

[0086] (B) 1 mL sample solution + 3 mL 0.1 mM DPPH reaction solution;

[0087] (C) 1 mL sample solution + 3 mL ethanol.

[0088] After each group was fully shaken, the mixture was reacted at room temperature in the dark for 30 minutes, and then the absorbance at 517 nm was measured.

[0089] (3) Calculation method

[0090] DPPH free radical scavenging ability (%) = [1-(BC) / A] * 100%

[0091] The results of DPPH free radical scavenging ability are shown in Table 3.

[0092] Table 3 Antioxidant capacity test

[0093] As shown in Table 3, Bacillus coagulans CGMCC 1.3220 has the strongest antioxidant capacity, with hydroxyl radical scavenging rates and DPPH radical scavenging rates reaching 64.4% and 83.6%, respectively, which are 51.7% and 66.3% higher than those of unfermented genus daphne juice, and the effect is better than that of other lactic acid strains.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fermented liquid of genus daphne juice, characterized in that: The invention is obtained by fermenting daphne juice with Bacillus coagulans CGMCC 1.3220; the fermentation comprises inoculating the daphne juice with Bacillus coagulans CGMCC 1.3220 and then fermenting the juice at a temperature of 35-40°C for 5-9 days.

2. A method for producing the fermented liquid of genus daphne juice according to claim 1, characterized in that: include: The radix dahuricae is squeezed to obtain radix dahuricae juice, the radix dahuricae juice is fermented with Bacillus coagulans CGMCC 1.3220 for 5 to 9 days, and after fermentation, the residue is removed by filtering to obtain the radix dahuricae juice fermentation clear liquid.

3. The method according to claim 2, characterized in that Bacillus coagulans CGMCC 1.3220 was inoculated into the genus daphne juice at an inoculum rate of 1-5% v / v.

4. The method according to claim 2, characterized in that The fermentation temperature is 35~40℃.

5. Use of the fermented clear solution of Daphne dahurica juice according to claim 1 in the preparation of whitening and antioxidant products.

6. The use according to claim 5, characterized in that The products are external skin products and health care products.

7. A composition, characterized in that The composition contains the fermented clear liquid of genus daphne juice according to claim 1.

8. Use of the composition according to claim 7 in the preparation of whitening and antioxidant products.

9. The use according to claim 8, characterized in that The products are external skin products and health care products.

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