Daidai fruit fermentation product and preparation method and application thereof

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to a fermented product of daidai guo, a preparation method thereof and an application thereof. The fermented product of daidai guo described in the present invention is prepared by fermentation in a fermentation system including daidai guo, lactobacillus, a carbon source and a nitrogen source. The present invention performs lactobacillus fermentation on daidai guo, thereby increasing the content of effective substances in daidai guo and reducing its toxic and side effects. At the same time, the present invention also provides an application of the fermented product of daidai guo in the cosmetics industry, which is applied to cosmetic raw materials and brings important economic value to the cosmetics industry.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a fermented daidai fruit, and a preparation method and application thereof. Background Art

[0002] Daidai (Citrus Aurantium L.var daidai Tanaka), also known as Daidai and Huiqing Orange, belongs to the subgenus Citrus of Rutaceae, is a variant of sour orange, and is a plant with medicinal and edible properties. Daidai fruit is slightly cold in nature, bitter and sour in taste, and has the functions of promoting qi, relieving stomach discomfort, digesting food, and resolving phlegm. Folks use Suzhi Fructus as a medicine for treating chest and abdominal fullness, distension and pain, indigestion, phlegm, gastroptosis, rectal prolapse, uterine prolapse and other diseases. Daidai fruit is also rich in flavonoids, sugars, volatile oils, alkaloids, tannins, organic acids and other substances, which make it have anti-inflammatory, antioxidant, anti-tumor, antibacterial, hypoglycemic, gastrointestinal regulation, lipid metabolism and other effects, and also has anti-depression, anti-anxiety, cardiovascular system improvement and other effects. As a variety of the sour orange plant of the Rutaceae family, the orange contains chemical components and pharmacological effects similar to those of the Citrus aurantium and Citrus aurantium. The alkaloids and flavonoids it contains are higher than those of other species in the same family. It has great medicinal and health value and application prospects.

[0003] Microbial fermentation is a process of fermenting raw materials with microorganisms as substrates under appropriate conditions. It mainly relies on the transformation of microorganisms and the synergistic effect of microorganisms and raw materials to increase the content of active ingredients in raw materials, enhance their efficacy or produce new active ingredients, and reduce toxicity or side effects. Active ingredients are mainly present in the cytoplasm and cell walls of plant cells. Microbial (bacteria, fungi, etc.) enzyme systems are of various types and have strong enzyme activity, mainly including cellulases, hemicellulases, pectinases, etc. These enzymes can decompose and utilize the dense structural components in plant cell walls or convert them into other components, while reducing the resistance to the release of active ingredients and making them easy to precipitate from cells. Microorganisms often produce a variety of biologically active substances during their growth and metabolism, such as various metabolic enzymes, primary metabolites, secondary metabolites, etc. Among them, some primary metabolites and secondary metabolites can use the active ingredients or non-active ingredients in traditional Chinese medicine as precursors, and modify the structures of these substances through various biological transformations such as isomerization, methylation, acetylation, etc. to produce new precursor compounds, or decompose them to produce new precursor compounds. In addition to the above characteristics, the genus Lactobacillus also has diverse fermentation methods, rich metabolites, and common special metabolic pathways. It also has the characteristics of simple structure, environmental sensitivity, and easy improvement.

[0004] The application of microbial fermentation technology in the cosmetics industry has many advantages, such as enriching effective substances, producing new effects, reducing harmful substances, low energy consumption, low pollution, and reducing production costs. Nowadays, the requirements for the safety and efficacy of cosmetics are getting higher and higher, and microbial fermentation can be carried out under milder conditions, which can more comprehensively and highly retain the efficacy of plant active ingredients, and can make skin care effects better. Summary of the invention

[0005] The object of the present invention is to provide a fermented product of daidai guo and a preparation method thereof, and to subject daidai guo to lactobacillus fermentation, thereby increasing the content of effective substances in daidai guo and reducing its toxic and side effects. At the same time, the present invention also provides an application of the fermented product of daidai guo in the cosmetics industry, which is applied to cosmetics raw materials, bringing important economic value to the cosmetics industry.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The daidai guo fermented product of the present invention is prepared by fermenting a fermentation system comprising daidai guo, lactobacillus, a carbon source and a nitrogen source.

[0008] Preferably, the lactobacillus is Lactobacillus casei.

[0009] Preferably, the carbon source is soluble starch.

[0010] Preferably, the nitrogen source is urea.

[0011] The method for preparing the fermented daidai fruit of the present invention comprises mixing daidai fruit, a carbon source and a nitrogen source, adding water, and then inoculating a lactobacillus liquid to form a fermentation system, and fermenting to obtain the product.

[0012] Preferably, the daidai fruit needs to be ground and crushed before fermentation to improve the fermentation efficiency.

[0013] Preferably, the amount of carbon source used per liter of fermentation system is 10-90 g, preferably 70 g.

[0014] Preferably, the amount of nitrogen source used per liter of fermentation system is 1-6 g, preferably 4 g.

[0015] Preferably, water is added to adjust the material-liquid ratio to 1:3-17 (g / mL), preferably 1:15.157 (g / mL).

[0016] Preferably, the OD of the inoculated Lactobacillus culture is 600 The inoculation amount of the lactobacillus liquid is 1-8% of the total volume of the fermentation system, preferably 2%.

[0017] The lactobacillus culture liquid is obtained by activating and expanding lactobacillus. The culture medium formula used for the culture is as follows: 10.0g / L casein digest, 10.0g / L beef extract powder, 4.0g / L yeast extract powder, 2.0g / L 6 H 5 O 7 (NH 4 ) 3 、5.0g / LCH 3 COONa, 0.2 g / L MgSO 4 7H 2 O, 0.05g / L MnSO 4 ·4H 2 O, 2.0g / LK 2 HPO 4 , 20g / L glucose, 1.0g / L Tween-80; the culture conditions were temperature 37°C, rotation speed 200rpm, and time 48h.

[0018] Preferably, the fermentation time is 12-120h, preferably 108h;

[0019] Preferably, the fermentation temperature is 25-46°C, preferably 43.6°C.

[0020] Preferably, after the fermentation is completed, 0.22 μm filter membrane is used for filtration to obtain the fermented daidai fruit.

[0021] The present invention also provides application of the fermented product of the daidai fruit in preparing cosmetics.

[0022] Preferably, the fermented product of the daidai fruit is used to prepare cosmetics with antioxidant and anti-inflammatory effects.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The preparation method of the fermented daidai fruit of the present invention is simple, efficient, low in energy consumption, green and pollution-free; it can promote the full analysis of effective substances in daidai fruit, and convert macromolecular substances into small molecules that are beneficial to human absorption; probiotics are used to ferment medicinal and edible plants, which is harmless to the human body; the fermented product is rich in active substances such as flavonoids, polysaccharides, polyphenols, etc., and can replace the use of chemical raw materials in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , standard curve of naringin;

[0026] Figure 2 , glucose standard curve;

[0027] Figure 3 , standard curve of gallic acid;

[0028] Figure 4 , Experimental statistical chart of flavonoid content in fermented products of daidai fruit of different strain groups;

[0029] Figure 5 , Experimental statistical chart of polysaccharide content of fermented products of daidai fruit from different strain groups;

[0030] Figure 6 , Experimental statistical chart of polyphenol content in fermented products of daidai fruit from different strain groups;

[0031] Figure 7 , Experimental statistical chart of free radical scavenging rate of daidai fruit fermentation products from different strain groups;

[0032] Figure 8 , a is an experimental statistical graph of flavonoids content in fermented daidai fruit with different carbon source types, b is an experimental statistical graph of flavonoids content in fermented daidai fruit with different carbon source addition amounts;

[0033] Fig. 9 , a is an experimental statistical graph of flavonoids content in fermented daidai fruit with different nitrogen sources, b is an experimental statistical graph of flavonoids content in fermented daidai fruit with different nitrogen source addition amounts;

[0034] Fig.10 , Experimental statistical chart of flavonoids content in fermented daidai fruit at different fermentation times;

[0035] Fig.11 , Experimental statistical chart of flavonoid content in fermented products of daidai fruit at different fermentation temperatures;

[0036] Fig.12 , Experimental statistical chart of flavonoids content in fermented daidai fruit with different material-liquid ratios;

[0037] Fig.13 , Experimental statistical chart of flavonoid content in fermented products of daidai fruit with different inoculation amounts of bacterial strains;

[0038] Fig.14 , response surface diagram and contour diagram of the effects of fermentation temperature and fermentation time on the flavonoid content of the fermented product;

[0039] Fig.15 , response surface diagram and contour diagram of the effects of solid-liquid ratio and fermentation time on the flavonoid content of the fermented product;

[0040] Fig.16 , response surface diagram and contour diagram of the effects of fermentation temperature and material-liquid ratio on the flavonoid content of the fermented product;

[0041] Fig.17 , a is a statistical graph showing the effect of different concentrations of daidai fruit fermentation products on the cell viability of RAW 264.7 cells, b is a standard curve of TNF-α content, and c is a statistical graph showing the effect of different concentrations of daidai fruit fermentation products on the production of TNF-α in RAW 264.7 cells;

[0042] Fig.18 a is a statistical graph showing the effect of different concentrations of daidai fruit fermentation products on the cell viability of HaCaT cells, b is a statistical graph showing the effect of different concentrations of H 2 O 2 c is the cell viability statistics of the treated HaCaT cells, c is the cell viability statistics of the cell oxidation model treated with different concentrations of daidai fruit fermentation products;

[0043] Fig.19 , negative control and different concentrations of Dai Dai fruit fermentation products after contacting the chicken embryo chorioallantoic membrane (CAM) for 3 minutes;

[0044] Fig. 20 Experimental diagram of positive control and benchmark control after contact with chicken embryo chorioallantoic membrane (CAM) for 3 minutes. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0046] 1. Experimental Materials

[0047] Experimental materials: Daidai fruit, produced in Guilin, Guangxi;

[0048] Fermentation strains: Bacillus subtilis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, and yeast, provided by the Basic Medical College of Guangdong Pharmaceutical University, activated and passaged in our laboratory;

[0049] Experimental reagents: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), ethanol, phenol, concentrated sulfuric acid, glucose, naringin, methanol, gallic acid, Folin reagent, anhydrous Na 2 CO 3 , were purchased from Guangzhou Boqilin Biotechnology Co., Ltd.; distilled water was made by the Molecular Biology Laboratory of the Basic College of Guangdong Pharmaceutical University;

[0050] Experimental instruments: analytical balance, microplate reader, UV-visible spectrophotometer, desktop high-speed refrigerated centrifuge, full-temperature oscillating incubator, water-proof constant temperature incubator, digital chicken embryo incubator, single-person single-sided clean workbench, portable high-pressure steam sterilizer, digital display blast drying oven, electric constant temperature water bath.

[0051] 2. Experimental steps

[0052] The present invention develops an optimal preparation method for the fermented daidai fruit, comprising the following steps:

[0053] S1: Pick colonies from the storage slopes of the tested strains (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, and yeast) and inoculate them on respective activation culture media for activation to obtain activated strains.

[0054] The activation medium for Bacillus subtilis is a nutrient agar medium, which is prepared as follows: 10.0 g of peptone, 5.0 g of sodium chloride, 3.0 g of beef extract powder, 15.0 g of agar, 1 L of distilled water, autoclaved at 121°C for 15 min, poured into a flat plate, solidified, and stored at room temperature. The activation conditions are 37°C for 48 hours.

[0055] The activation medium for Lactobacillus was MRS broth, which was prepared as follows: 10.0 g casein digest, 10.0 g beef extract powder, 4.0 g yeast extract powder, 2.0 g C 6 H 5 O 7 (NH 4 ) 3 5.0gCH 3 COONa, 0.2 g MgSO 4 7H 2 O, 0.05gMnSO 4 ·4H 2 O, 2.0gK 2 HPO 4 , 20g glucose, 1.0g Tween-80, dissolve with appropriate amount of distilled water, then make up to 1L, and sterilize by high pressure at 121℃ for 15min.

[0056] Yeast activation medium was prepared as follows: 20 g peptone, 10 g yeast extract, 70 mL molasses, 1 g MgSO 4 7H 2 O, 0.5 g KH 2 PO 4 , 2gFeSO 4 7H 2 Dissolve in appropriate amount of distilled water, add 15 mL of 0.2% adenine solution, and then adjust the volume to 1 L, and sterilize by autoclave at 121°C for 15 min. The activation conditions are 30°C, 220 rpm, and 48 h.

[0057] S2: The activated strains obtained in S1 were inoculated into their respective seed culture media for expansion and culture until OD 600 =0.6-0.8, to obtain seed solution;

[0058] The seed culture medium of Bacillus subtilis is LB liquid culture medium, which is prepared as follows: 10.0 g of tryptone, 10.0 g of sodium chloride, 5.0 g of yeast extract powder, 1 L of distilled water, and sterilized by high pressure steam at 121° C. for 15 min. The culture conditions are 37° C., 200 rpm, and 48 h.

[0059] The preparation method of the seed culture medium of Lactobacillus plantarum, Lactobacillus casei and Lactobacillus brevis is the same as the activation culture medium of Lactobacillus in S1. The culture conditions are 37°C, 200 rpm, and 48 hours.

[0060] The preparation method of the yeast seed culture medium is the same as the yeast activation culture medium in S1. The culture conditions are 30°C, 200 rpm, and 48 hours.

[0061] S3: Grind the daidai guo and mix it with water to obtain an aqueous solution containing daidai guo, wherein the water is distilled water. Take out the seed liquid obtained in S2 and inoculate it into the aqueous solution containing daidai guo for fermentation as the experimental group, and fermentation without adding strains is set as the blank control group. After the fermentation is completed, filter with a 0.22 μm filter membrane to obtain daidai guo fermentation. According to the comparison of flavonoid content, polysaccharide content, polyphenol content and free radical scavenging rate of the fermentation, the most suitable fermentation strain is screened.

[0062] S4: Based on the most suitable bacterial strains for daidai fruit fermentation selected in S3, single-factor experiments were conducted on the fermentation conditions to obtain the optimal fermentation values ​​of each single-factor experiment, including six factors: optimal carbon source and addition amount, optimal nitrogen source and addition amount, fermentation time, fermentation temperature, material-liquid ratio, and strain inoculation amount.

[0063] S5: Based on S4, the values ​​on both sides of the optimal values ​​of the six factors are selected as the level range of the Plackett-Burman test (hereinafter referred to as "PB test"). Through the PB-experimental design, three main effect factors are determined among the six factors.

[0064] S6: Based on the three main effect factors screened in S5, a Box-Behnken test (hereinafter referred to as "BB test") was designed. Taking flavonoids content as the response surface value, a three-factor three-level response surface experiment was designed to optimize the optimal fermentation conditions and finally obtain the fermented product of daidai fruit.

[0065] 3. Test methods

[0066] 3.1 Determination of flavonoid content

[0067] (1) Reagents

[0068] Preparation of naringin solution: Weigh 8 mg of naringin, dissolve it in methanol and add the diluted volume to a 50 mL volumetric flask to obtain a 0.160 mg / mL naringin standard solution.

[0069] (2) Experimental procedures

[0070] Standard curve drawing: Pipette 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mL of naringin standard solution into a 10 mL volumetric flask, dilute to the scale with methanol, use methanol as blank, measure the absorbance at a wavelength of 284 nm, and draw a standard curve.

[0071] Determination: Dilute the fermented broth with methanol and measure the absorbance at 284nm. Compare the absorbance value with the standard curve equation to calculate the content of flavonoids in the fermented product.

[0072] 3.2 Determination of polysaccharide content

[0073] (1) Reagents

[0074] Accurately weigh 10 mg of glucose and add it to a 100 mL volumetric flask to prepare a 0.1 mg / mL stock solution. Use distilled water to prepare 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL glucose standard solutions respectively.

[0075] (2) Experimental procedures

[0076] Standard curve drawing: Pipette 1mL of 0, 0.02, 0.04, 0.06, 0.08, 0.1mg / mL glucose standard solution, add 1mL of 5% phenol, and quickly add 5mL of concentrated sulfuric acid, let stand for 10min. Shake well, let stand at 30℃ for 30min, measure OD value at 490nm, draw standard curve with glucose content as horizontal axis and OD value as vertical axis.

[0077] Sample content determination: Pipette 1.0 mL of fermentation broth sample, determine the OD value according to the above steps, and substitute it into the standard curve to calculate the polysaccharide content in the sample.

[0078] 3.3 Determination of polyphenol content

[0079] (1) Reagents

[0080] Preparation of gallic acid solution: Weigh 0.010 g of gallic acid into a 100 mL volumetric flask to obtain a 0.1 mg / mL gallic acid standard solution.

[0081] 20% Na 2 CO 3 Solution: weigh 20.0g Na 2 CO 3 , dissolve in distilled water and transfer to a 100 mL volumetric flask.

[0082] 50% Folin reagent: Pipette 15 mL of Folin reagent, add 15 mL of distilled water, and mix thoroughly.

[0083] (2) Experimental procedures

[0084] Drawing of standard curve: Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of gallic acid standard solution into a test tube, add distilled water to make up to 2 mL; add 1.5 mL of 20% Na 2 CO 3 solution, mix well, and let stand for 2 minutes; add 0.5mL 50% Folin reagent, add distilled water to 5mL, mix well, let stand for 30 minutes in the dark, measure the absorbance at 750nm, and use the gallic acid concentration as the control absorbance to draw a standard curve.

[0085] Determination: Add 1.5 mL of 20% Na 2 CO 3 Solution, mix well, let stand for 2 minutes; add 0.5mL 50% Folin reagent, add distilled water to 5mL, mix well, let stand for 30 minutes in the dark, and measure the absorbance at 750nm. The absorbance value is compared with the standard curve equation to calculate the content of polyphenols in the fermentation.

[0086] 3.4 Determination of free radical scavenging rate of fermentation products by DPPH method

[0087] Preparation of DPPH reagent: Place 4 mg of DPPH accurately weighed in a beaker, first dissolve it with a small amount of anhydrous ethanol by shaking, then transfer it to a 50 mL brown volumetric flask and make up the volume. The storage time should not exceed 4 hours.

[0088] (1) Take 100 μL of fermentation product and mix it with DPPH reagent in equal volumes, incubate it in a gas bath at 37°C in the dark for 30 min, take 200 μL of the mixture and measure the absorbance at 517 nm.

[0089] (2) Take 100 μL of the fermentation product and mix it with an equal volume of anhydrous ethanol, incubate it in a gas bath at 37°C in the dark for 30 min, take 200 μL of the mixture and measure the absorbance at 517 nm.

[0090] (3) Mix 100 μL of DPPH reagent and anhydrous ethanol in equal volumes, incubate in a dark environment at 37°C for 30 min, and measure the absorbance of 200 μL of the mixture at 517 nm.

[0091] The free radical scavenging rate I was calculated according to the formula:

[0092] I=[1-(Ai-Aj) / A0]×100%

[0093] Example 1

[0094] This example is a blank control group (control group). The steps are as follows:

[0095] The aqueous solution containing daidai fruit is fermented according to the following fermentation conditions: fermentation temperature is 37° C., the solid-liquid ratio is 1:20, and the fermentation time is 72 hours.

[0096] Example 2

[0097] This example discusses the dominant strains. The content is as follows:

[0098] S1-S2 are the same as S1-S2 in “2. Experimental procedures” above;

[0099] S3: taking out the strain seed solution obtained in S2 and inoculating it into the aqueous solution containing Dai Dai fruit;

[0100] S4: The fermentation conditions were as follows: the fermentation temperature was 37°C, the fermentation time was 48 h (yeast fermentation group), 60 h (Bacillus subtilis fermentation group), and 72 h (Lactobacillus fermentation group), the solid-liquid ratio was 1:20, and the inoculation amount was 2.0%. After the fermentation, the flavonoid content, polysaccharide content, polyphenol content, and free radical scavenging rate were determined according to "3. Experimental method". The standard curves of naringin, glucose, and gallic acid were shown in Table 1. Figure 1-Figure 3 The experimental results are as follows Figure 4-7 As shown, it can be seen that the most suitable bacteria species for daidai guo fermentation is Lactobacillus casei.

[0101] Example 3

[0102] This example discusses the optimal fermentation carbon source type and its addition amount. The content is as follows:

[0103] S1: Take the colony from the lactobacillus preservation slant and inoculate it on the activation medium for activation culture until OD 600 =0.6, and the activated strain was obtained.

[0104] S2: inoculating the activated strain obtained in S1 into a seed culture medium for expansion culture to obtain a seed solution;

[0105] S3: taking out the seed solution obtained in S2 and inoculating it into the aqueous solution containing daidai fruit;

[0106] S4: The fermentation conditions are: fermentation temperature 37°C, material-liquid ratio 1:20, bacterial inoculation amount 2.0%, fermentation time 72h, carbon source addition amount 10g / L, carbon source types are glucose, lactose, fructose, maltose, sucrose, soluble starch. The flavonoid content of the fermentation product was determined according to "3. Experimental method". The experimental results are as follows Figure 8 As shown in a, it can be seen that the best fermentation carbon source is soluble starch.

[0107] Example 4

[0108] This example discusses the optimal amount of fermentation carbon source added. The content is as follows:

[0109] S1-S3 are the same as in Example 3;

[0110] S4: The fermentation conditions are: fermentation temperature 37°C, material-liquid ratio 1:20, bacterial inoculation amount 2.0%, fermentation time 72h, soluble starch addition amounts 0, 10g / L, 30g / L, 50g / L, 70g / L, 90g / L. The flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Figure 8 As shown in b, it can be seen that the optimal fermentation carbon source addition amount is 70g / L.

[0111] Example 5

[0112] This example discusses the optimal type of nitrogen source for fermentation and its addition amount. The content is as follows:

[0113] S1-S3 are the same as in Example 3;

[0114] S4: The fermentation conditions are: fermentation temperature 37°C, material-liquid ratio 1:20, bacterial inoculation amount 2.0%, fermentation time 72h, soluble starch addition amount 70g / L, nitrogen source addition amount 2g / L, and the types of nitrogen sources are yeast extract, trypsin, urea, beef extract, and ammonium sulfate. The flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Fig. 9 As shown in a, it can be seen that the best nitrogen source for fermentation is urea.

[0115] Example 6

[0116] This example discusses the optimal amount of nitrogen source added for fermentation. The content is as follows:

[0117] S1-S3 are the same as in Example 3;

[0118] S4: The fermentation conditions are: fermentation temperature 37°C, material-liquid ratio 1:20, bacterial inoculation amount 2.0%, fermentation time 72h, soluble starch addition amount 70g / L, urea addition amount 0, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L. The flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Fig. 9 As shown in b, it can be seen that the optimal amount of nitrogen source added for fermentation is 4g / L.

[0119] Example 7

[0120] This example discusses the optimal fermentation time. The content is as follows:

[0121] S1-S3 are the same as in Example 3;

[0122] S4: The fermentation conditions are: fermentation temperature 37°C, material-liquid ratio 1:20, bacterial inoculation amount 2.0%, soluble starch addition amount 70g / L, urea addition amount 4g / L, fermentation time 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h; the flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Fig.10 As shown, it can be seen that the optimal fermentation time is 108h.

[0123] Example 8

[0124] This example discusses the optimal fermentation temperature. The content is as follows:

[0125] S1-S3 are the same as in Example 3;

[0126] S4: The fermentation conditions are: the fermentation temperatures are 25℃, 28℃, 31℃, 34℃, 37℃, 40℃, 43℃, 45℃, respectively; the solid-liquid ratio is 1:20; the inoculation amount of the strain is 2.0%; the amount of soluble starch added is 70g / L; the amount of urea added is 4g / L; the fermentation time is 108h; the flavonoid content of the fermentation product is determined according to "3. Experimental method". The experimental results are as follows Fig.11 As shown, it can be seen that the optimal fermentation temperature is 43°C.

[0127] Example 9

[0128] This example discusses the optimal fermentation feed-liquid ratio. The content is as follows:

[0129] S1-S3 are the same as in Example 3;

[0130] S4: The fermentation conditions are: fermentation temperature 43°C, material-liquid ratio 1:3, 1:5, 1:7, 1:9, 1:11, 1:13, 1:15, 1:17, bacterial inoculation amount 2.0%, soluble starch addition amount 70g / L, urea addition amount 4g / L, fermentation time 108h; the flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Fig.12 As shown, it can be seen that the optimal fermentation feed-liquid ratio is 1:15.

[0131] Example 10

[0132] This example discusses the optimal fermentation strain inoculation amount. The content is as follows:

[0133] S1-S3 are the same as in Example 3;

[0134] S4: The fermentation conditions are: fermentation temperature 43°C, material-liquid ratio 1:15, bacterial inoculation amounts of 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, respectively, fermentation time 108h, soluble starch addition amount 70g / L, urea addition amount 4g / L; the flavonoid content of the fermented product was determined according to "3. Experimental method". The experimental results are as follows Fig.13 As shown, it can be seen that the optimal bacterial inoculation amount is 2%.

[0135] Example 11 PB test

[0136] This example explores the significant effects of the above six single factors on the flavonoid content of the fermented product. The content is as follows:

[0137] S1: Take the colony from the lactobacillus preservation slant and inoculate it on the activation medium for activation culture until OD 600 =0.6, and the activated strain was obtained.

[0138] S2: inoculating the activated strain obtained in S1 into a seed culture medium for expansion culture to obtain a seed solution;

[0139] S3: taking out the seed solution obtained in S2 and inoculating it into the aqueous solution containing daidai fruit;

[0140] S4: The effects of six influencing factors (urea concentration, soluble starch concentration, fermentation time, fermentation temperature, material-liquid ratio and bacterial inoculation amount) in the single factor experiment on the flavonoid content of the fermented product of Dai Dai fruit were analyzed and statistically analyzed. According to the results of the single factor experiment, the low level (-) and high level (+) were taken as the level range of the PB experiment, as shown in Table 1. Then the PB experiment design was carried out using Design-Expert10.0 software, as shown in Table 2. Taking the flavonoid content as an indicator, the flavonoid content under each group of experimental conditions was determined.

[0141] The multivariate linear model equation constructed based on the PB test results is: R = +419.78 + 2.38A + 5.50B + 13.80C + 22.74D - 10.89E + 14.53F, where R represents the model estimated value of the flavonoid content of fermented daidai fruit. The variance analysis of the six factors using Design-Expert 10.0 software shows that the model R 2 =0.9851, indicating that the model fits the actual test well, and the experimental prediction value is highly correlated with the actual value. The model P value is 0.0002, indicating that this model is significant within a 95% confidence level. The influence of the six factors on the fermentation results is as follows: fermentation temperature> liquid ratio> fermentation time> inoculation amount> soluble starch concentration> urea concentration.

[0142] Table 1. Range of levels in the Plackett-Burmen experimental design

[0143] serial number factor unit Low level (-1) High level (1) A Urea concentration ％ 0.3 0.5 B Soluble starch concentration ％ 5 9 C Fermentation time h 96 120 D Fermentation temperature ℃ 40 46 E Inoculation amount ％ 1 3 F Material-liquid ratio g / mL 1:13 1:17

[0144] Table 2. Plackett-Burmen test design table and test results

[0145]

[0146] Table 3. Plackett-Burmen test variance analysis table

[0147]

[0148]

[0149] Example 12BB test

[0150] This example discusses the optimal fermentation strain inoculation amount. The content is as follows:

[0151] S1-S3 are the same as in Example 11;

[0152] S4: According to the results of Example 11, three factors (fermentation temperature, material-liquid ratio, fermentation time) that have a significant impact on the flavonoid content of the fermented product were selected, and the flavonoid content was used as the response surface value to design a 3-factor 3-level response surface experiment, as shown in the BB experiment factor level table (Table 4). Design-Expert 10.0 software was used to perform BB experiment design, and the BB experiment design and results are shown in Table 5. At the same time, the data results of Table 5 were multivariately fitted by Design-Expert 10.0 software to obtain the quadratic polynomial equation model of each factor and the response value: R 1 =+407.80+3.86A+5.55B+5.07C+8.73AB-8.98AC-6.12BC-14.15A 2 -16.46B 2 -14.51C 2 , where R 1 Flavonoid content.

[0153] From the results of variance analysis of the response surface equation (see Table 6), it can be seen that the regression model has extremely significant differences, while the lack of fit term is not significant, indicating that the model has a good degree of fit. The regression model is established and can be used to predict and analyze the flavonoids content in the fermentation process of daidai fruit.

[0154] The response surface diagram is drawn according to the regression equation. It is a three-dimensional spatial surface of the interaction between the response value and each factor. It is mainly used to intuitively analyze the influence of two interactions. When one of the three factors of fermentation temperature, liquid ratio and fermentation time is fixed, the influence of the other two factors and their interaction on the comprehensive score of fermented daidai fruit is analyzed, and the response surface and contour lines of the model are obtained, such as Figure 14-16 shown.

[0155] The equation model was analyzed by Design-Expert12 software. The optimal parameters for fermenting daidai fruit with Lactobacillus casei were fermentation temperature of 43.6℃, ​​material-liquid ratio of 1:15.157, and fermentation time of 108h. Experimental verification was carried out under these conditions. The average value of three parallel experiments was taken. The actual flavonoid content after optimization was measured to be 412.01mg / g, which was close to the predicted maximum flavonoid content of 408.89mg / g, indicating that the model was credible. After experimental comparison of the flavonoid content before and after fermentation optimization, it was found that the flavonoid content after optimization was 36.71% higher than that before optimization.

[0156] Table 4. Box-Behnken test factor level table

[0157]

[0158] Table 5. Box-Behnken test design and results

[0159]

[0160] Table 6. Response surface method variance analysis table

[0161]

[0162] Note: P<0.01 indicates extremely significant difference; P>0.05 indicates no significant difference.

[0163] By examining the effects of various factors on the flavonoids content of the fermented product of daidai fruit, it can be seen that the optimal fermentation conditions are: fermentation temperature 43.6℃, ​​solid-liquid ratio 1:15.157, bacterial inoculation amount 2.0%, soluble starch addition amount 70g / L, urea addition amount 4g / L, and fermentation time 108h.

[0164] 4. Establish an LPS-induced mouse mononuclear macrophage cell line RAW 264.7 cell inflammation model to explore the inhibitory effect of daidai fruit fermentation on inflammation

[0165] The specific steps are as follows:

[0166] (1) Materials and reagents

[0167] Mouse mononuclear macrophage cell line RAW 264.7 cells were preserved by Guangdong Pharmaceutical University and used in this laboratory after passage. Fetal bovine serum, RPMI-1640 medium, penicillin-streptomycin solution, 0.25% trypsin-EDTA solution, dimethyl sulfoxide (DMSO), bacterial lipopolysaccharide (LPS), dexamethasone, MTT cell proliferation and cytotoxicity detection kit, human tumor necrosis factor α (TNF-α) ELISA scientific detection kit.

[0168] (2) Steps

[0169] ① Cell culture

[0170] Mouse mononuclear macrophage cell line RAW 264.7 cells were cultured in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in a cell culture incubator (37°C, 5% CO). 2 ) were routinely cultured, the medium was changed once every 1-2 days, and the cells in the logarithmic growth phase were digested with 0.25% trypsin-EDTA solution and used in the experiment.

[0171] ② Determination of safe concentration of fermented daidai fruit by MTT method

[0172] Take cells in logarithmic growth phase and adjust the cell density to (5-6)×10 5 / mL, inoculated in 96-well plates, 100 μL / well, the edge wells were filled with PBS, and placed at 37°C, 5% CO 2 The cells were cultured overnight in an incubator. A blank control group and an experimental group were set up. According to the volume fraction, each well of the experimental group was added with fermentation products at final concentrations of 0.625%, 1.25%, 2.5%, 5%, 10%, 20%, and 40%, respectively. Each group had 6 replicate wells. After the administration, the 96-well plate was placed in a CO 2 Culture in an incubator for 24 h ± 2 h. Use the MTT method to determine the safe concentration of the fermentation product using cell viability as an indicator.

[0173] Cell viability (%) = (experimental group - blank group) / (control group - blank group) * 100%

[0174] like Fig.17 As shown in a, the experimental concentration of the fermented product of the daidai fruit was screened by the MTT method, and it was found that when the concentration was 0.625%-2.5%, the cell viability of RAW 264.7 cells was above 90%. Therefore, this concentration range was used to determine the TNF-α content.

[0175] ③Effect of fermented daidai fruit on TNF-α content in mouse mononuclear macrophage cell line RAW 264.7 cells stimulated by LPS

[0176] Take cells in logarithmic growth phase and adjust the cell density to (4-5)×10 5 pc / mL, inoculated into 96-well plates, 100 μL / well, incubated at 37°C, 5% CO 2 The cells were cultured overnight in an incubator. A blank control group (without LPS stimulation), a model group stimulated by LPS, a positive group (100 μg / ml dexamethasone) and an experimental group (0.625%, 1.25%, 2.5%) were set up, with 3 replicate wells in each group. After the administration, the 96-well plate was placed in a CO 2 Culture in an incubator for 24 h ± 2 h. After the culture is completed, collect the cell culture supernatant in a sterile centrifuge tube and test it according to the operating instructions of the human tumor necrosis factor α (TNF-α) ELISA scientific research detection kit. In addition, the concentration of TNF-α standard (20pg / mL-200pg / mL) is used as the vertical axis, and OD 450 As the horizontal axis, make a standard curve based on the OD of the sample 450 , find out the corresponding concentration. 450 Calculate the regression equation of the standard curve, such as Fig.17 As shown in b; the OD of each group 450 Substitute into the equation to calculate the intracellular TNF-α content. Finally, take the average value of three replicate wells in each group as the final TNF-α content result.

[0177] (3) Results

[0178] As shown in 17c, with the increase of the concentration of the fermented product of daidai fruit, the content of TNF-α gradually decreased. This proves that the fermented product of daidai fruit can inhibit the production of TNF-α in RAW 264.7 cells and has an anti-inflammatory effect. Note: *** (P<0.01), compared with the blank control group.

[0179] 5. By establishing H 2 O 2 Induced oxidation model of human immortalized epidermal cells (HaCaT) to explore the antioxidant protection ability of daidai fruit fermentation products

[0180] The specific steps are as follows:

[0181] 1) Materials and Reagents

[0182] Human immortalized epidermal cells (HaCaT) were preserved by the Biochemistry Laboratory of Guangdong Pharmaceutical University and used in our laboratory after passage. Fetal bovine serum, DMEM high glucose medium, penicillin-streptomycin solution, 0.25% trypsin-EDTA solution, MTT cell proliferation and cytotoxicity detection kit, dimethyl sulfoxide (DMSO), hydrogen peroxide (H 2 O 2 ), ascorbic acid (Vc).

[0183] 2) Steps

[0184] ① Cell culture

[0185] Human immortalized epidermal cells (HaCaT) were routinely cultured in a cell culture incubator (37°C, 5% CO2) using DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. The medium was changed once every 2-3 days. Cells in the logarithmic growth phase were digested with 0.25% trypsin-EDTA solution and used in the experiment.

[0186] ② Determination of safe concentration of fermented daidai fruit

[0187] Take cells in logarithmic growth phase and adjust the cell density to (5-6)×10 5 / mL, inoculated in 96-well plates, 100 μL / well, the edge wells were filled with PBS, and placed at 37°C, 5% CO 2 The cells were cultured overnight in an incubator. A blank control group and an experimental group were set up. According to the volume fraction, each well of the experimental group was added with fermentation products at final concentrations of 0.625%, 1.25%, 2.5%, 5%, 10%, 20%, and 40%, respectively. Each group had 6 replicate wells. After the administration, the 96-well plate was placed in a CO 2 Culture in an incubator for 24 h ± 2 h. Use the MTT method to determine the safe concentration of the fermentation product using cell viability as an indicator.

[0188] like Fig.18 As shown in a, the experimental concentration of the fermented product of the daidai fruit was screened by the MTT method. It was found that when the concentration was 0.625%-2.5%, the cell viability of HaCaT cells was above 90%, so this concentration range was used to determine the antioxidant damage protection ability. Note: **(P<0.1); ***(P<0.01), compared with the blank control group.

[0189] ③Construction of oxidation model of human immortalized epidermal cells (HaCaT)

[0190] Take cells in logarithmic growth phase and adjust the cell density to (5-6))×10 5 pc / mL, inoculated into 96-well plates, 100 μL / well, incubated at 37°C, 5% CO 2 The cells were cultured in an incubator overnight. A blank control group and different concentrations of H 2 O 2 (0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mmol / L). After administration, the 96-well plate was placed in a CO 2 The cells were cultured in an incubator for 4 h. The MTT method was used to determine the cell viability. 2 O 2The concentration of HaCaT oxidation model was constructed, and the results were as follows Fig.18 b. The concentration of oxidant in the oxidative damage model is selected so that the number of damaged cells is about 50%. Fig.18 b It can be seen that when H 2 O 2 When the concentration was 0.8mmol / L, the cell viability was 47.43%, so 0.8mmol / L H 2 O 2 Note: ***(P<0.01), compared with the blank control group.

[0191] ④ Antioxidant damage protection ability of daidai fruit fermentation products

[0192] Take cells in logarithmic growth phase and adjust the cell density to (4-5)×10 5 pc / mL, inoculated into 96-well plates, 100 μL / well, incubated at 37°C, 5% CO 2 Cultured overnight in an incubator. Set up a blank control group, a model group, a positive group (25 μg / ml ascorbic acid) and an experimental group (0.625%, 1.25%, 2.5%). First, the blank control group and the model group were added with DMEM high-glucose culture medium, the positive group was added with 25 μg / ml ascorbic acid, and the experimental group was added with 0.625%-1.25% fermentation products to act on the cells for 24 hours. After the action, the supernatant was discarded and a final concentration of 0.8 mmol / L H 2 O 2 After stimulating HaCaT cells for 4 h, cell viability was detected by MTT assay.

[0193] 3) Results

[0194] like Fig.18 As shown in Figure c, it was found that the cell viability of the cell oxidation model treated with 0.625%-1.25% of the fermented product of Dai Dai fruit increased, and with the increase of concentration, the cell viability increased, proving that the fermented product of Dai Dai fruit has a protective effect on the oxidative damage of cells. Note: ***(P<0.01), compared with the blank control group.

[0195] 6. Chicken embryo chorioallantoic membrane test on eye irritation / corrosion of daidai fruit fermentation products

[0196] (1) Materials

[0197] White laghorn chicken SPF grade fertilized chicken embryo, fatty alcohol ether sodium sulfate mixture (ASV), sodium chloride (NaCl), sodium hydroxide (NaOH), acetic acid (CH 3 COOH).

[0198] (2) Steps

[0199] ①Chicken embryo preparation

[0200] Place the eggs in a Rcom MARU MA× incubator with the air chamber facing upwards, incubate at a temperature of 37.5℃±0.5℃ and a relative humidity of 50%±1%, and automatically turn the eggs 3 times per hour at a horizontal angle of 45°. Use them for testing on the 9th day of incubation.

[0201] ②Chicken embryo chorioallantoic membrane test for eye irritation / corrosion

[0202] The chicken embryos cultured to 9 days old were examined by egg candling and the air chambers were opened. The negative control group used a 0.9% NaCl solution, the positive control group used 0.3%, 3%, 30% acetic acid solution and 0.2%, 0.3%, 0.5% NaOH solution, and the reference substance positive control group used a 0.5%, 1%, 5% fatty alcohol ether sulfate sodium salt mixture (ASV). The experimental group used a final concentration of 0.625%, 1.25%, 2.5% of the fermented material of the daidai fruit, and the experiment was repeated 6 times.

[0203] Since the fermented product of the daidai fruit is a colored liquid, the endpoint scoring method was used. After each group of solutions contacted the chorioallantoic membrane for 3 minutes, the bleeding, coagulation and vascular lysis were observed and recorded, and the endpoint score (ES) was calculated. Each chicken embryo was scored as the sum of the bleeding, coagulation and vascular lysis observed in each chicken embryo. The ES was the average of the mathematical sum of the scores of the 6 chicken embryos. The eye irritation of the test substances was classified according to the ES values ​​according to Table 7.

[0204] Table 7 Endpoint scoring method results evaluation

[0205] End point scoring Stimulus Classification ES≤12 No / mild irritation 12<ES<16 Moderate irritation ES≥16 Strong irritant / corrosive

[0206] (3) Results

[0207] like Fig.19 As shown in the figure, a is the negative control, (bd) are the experimental groups of 0.625%, 1.25%, and 2.5% of the fermented material of the daidai fruit after contacting the chicken embryo chorioallantoic membrane (CAM) for 3 minutes. Fig. 20 As shown, the positive control and the benchmark control were exposed to the chicken embryo chorioallantoic membrane (CAM) for 3 minutes, which was used to evaluate the safety performance of the fermented product of Dai Dai fruit.

[0208] As shown in Table 8, the irritation of the fermented daidai fruit at a concentration of 0.625%-2.5% is non-irritating, which demonstrates the safety performance of the fermented daidai fruit.

[0209] Table 8 End point scoring results of daidai fruit fermentation

[0210] Sample name ES value Stimulus Classification Negative control 0 Non-irritating 0.625％ 0 Non-irritating 1.25％ 0 Non-irritating 2.5％ 0 Non-irritating

[0211] In summary, the present invention uses a variety of probiotics to ferment daidaiguo, screens out the most suitable strain for daidaiguo fermentation, optimizes its fermentation conditions, and finally obtains a daidaiguo fermentation product. This daidaiguo fermentation product contains more flavonoids, polyphenols and polysaccharides, among which microbial fermentation is the key to increasing the content of effective substances. Then, we used mouse mononuclear macrophage cell line RAW 264.7 cells to conduct anti-inflammatory experiments and found that this daidaiguo fermentation product can effectively inhibit the production of inflammatory factor TNF-α in RAW 264.7 cells. Subsequently, H 2 O 2 Induced oxidation model of human immortalized epidermal cells (HaCaT), it was found that the fermented product of the daisy fruit had a strong ability to protect against oxidative damage. Finally, the chicken embryo chorioallantoic membrane test was used to verify that the fermented product of the daisy fruit was non-irritating or corrosive within the effective concentration range. Therefore, the fermented product of the daisy fruit has good effects in both anti-inflammatory and anti-oxidation, and is safe and effective. The present invention provides a new idea for the production of natural plant functional raw materials and provides new raw materials for the production of cosmetics.

[0212] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing a fermented product of daidai fruit, characterized in that: It is to mix the daidai fruit, carbon source and nitrogen source, add water, Then inoculate the lactobacillus liquid to form a fermentation system, and ferment to obtain the product; The lactobacillus is Lactobacillus casei; The carbon source is soluble starch; The nitrogen source is urea; The amount of carbon source used per liter of fermentation system is 10-90g; The amount of nitrogen source used per liter of fermentation system is 1-6g; Add water to adjust the material-liquid ratio to 1:3-17 (g / mL); The OD600 of the inoculated Lactobacillus liquid is 0.6-0.8, and the inoculation amount of the Lactobacillus liquid is 1-8% of the total volume of the fermentation system.

2. Use of the fermented product of daidai fruit prepared by the preparation method as claimed in claim 1 in the preparation of cosmetics.

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