Roman chamomile composite fermentation liquor nano coating and preparation method thereof

Abstract

The invention relates to the technical field of biochemical engineering, and provides a Roman chamomile composite fermentation liquor nano coating and a preparation method thereof, and the preparation method comprises the following steps: respectively extracting Roman chamomile hydrolat, a protein extract, a polysaccharide extract and a fat-soluble purified extract from Roman chamomile by adopting methods of steam distillation, solid state fermentation, water extraction and alcohol extraction; mixing all the extracts, and performing liquid fermentation to obtain Roman chamomile compound fermentation liquor; then treating the Roman chamomile compound fermentation liquor, and coating the outer side of the Roman chamomile compound fermentation liquor with maltodextrin and chitosan to obtain a nano coating; according to the preparation method disclosed by the invention, various effective components in Roman chamomile can be fully extracted, the problem that various extracts are difficult to mix and dissolve is solved through mixed fermentation, and finally, the obtained compound fermentation liquor is subjected to nano-coating physicochemical treatment, so that the stability, the antibacterial property and the moisturizing and soothing effects of the compound fermentation liquor are further improved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a nano-coated product of Roman chamomile composite fermentation liquid and a preparation method thereof. Background Art

[0002] Roman chamomile is a perennial plant that was originally distributed from southern Europe to western Europe. It can now be seen in many countries. The center of the flower is yellow, the petals are white, and the height can grow up to 60 cm. The distilled essential oil is yellow in color and has a sweet herbal flavor. The volatile oil extracted from Roman chamomile can be used in health care and pharmaceutical products. Its main function is to help people sleep and relieve muscle pain, especially pain caused by nerves. At the same time, the antibacterial effect of Roman chamomile volatile oil is also widely used by people and is often used in food and cosmetics.

[0003] At present, the utilization and research of Roman chamomile are mainly concentrated in the food field, and thus the focus is on studying its volatile components, and there are few studies on its macromolecular extracts. Especially in the field of cosmetics, there are few studies on Roman chamomile in the prior art. Therefore, there are few studies involving Roman chamomile macromolecular extracts.

[0004] For example, the Chinese patent with publication number CN113398053A discloses a preparation method and application of a skin moisturizing composition with soothing and moisturizing effects, wherein a method for extracting golden chamomile extract is disclosed: a mixed solution with a volume ratio of ethanol: fermentation liquid = 1:1 to 3:1 is used as an extractant, and the solid-liquid ratio is 1:10 to 30, the extraction temperature is 50 to 70°C for reflux extraction, and the ethanol is concentrated under reduced pressure. The total flavonoids in chamomile are purified with AB-8 resin, and the sample mass concentration is 1.0 to 2.0 mg / mL, and the sample volume flow rate is 0.5 to 2.0 BV / h; the eluent is 50 to 80% ethanol, and the volume flow rate is 0.5 to 2.0 BV / h. The required eluate is collected and concentrated to dryness to obtain the chamomile extract. In addition, the Chinese patent with publication number CN106726923A discloses a cosmetic additive and a preparation method thereof and a cosmetic containing the cosmetic additive, which contains chamomile extract obtained by water extraction;

[0005] However, the various extracts obtained by the above methods have a single chemical composition and a relatively single effect. In addition, there are many impurities in the various extracts, which can easily cause skin allergies. Since Roman chamomile contains many ingredients and the polarity and solubility of various extracts vary greatly, it is difficult to mix the various extracts for use, resulting in incomplete effects of Roman chamomile extracts and easy waste of raw materials. Summary of the invention

[0006] Based on the above problems, the purpose of the present invention is to provide a Roman chamomile composite fermentation liquid nano-coated material and a preparation method thereof, which can fully extract various effective ingredients in Roman chamomile, and finally solve the problem of various extracts being difficult to miscible through mixed fermentation, and finally the obtained composite fermentation liquid is subjected to nano-coating physical and chemical treatment, thereby further improving the stability, antibacterial properties, moisturizing and soothing effects of the composite fermentation liquid.

[0007] The embodiments of the present invention are achieved through the following technical solutions:

[0008] A method for preparing a nano-coated product of Roman chamomile composite fermentation liquid comprises the following steps:

[0009] S1. Roman chamomile hydrosol extract: using Roman chamomile plants as raw materials, cutting them into small pieces with a length of about 1 cm, steaming them for about 2 hours, collecting the distillate, and obtaining Roman chamomile hydrosol;

[0010] S2, solid-state fermentation: collecting the Roman chamomile plant residues after distillation in S1, cooling them to room temperature, adding 0.02% (total weight of the Roman chamomile plant residues) of Bacillus subtilis and 0.02% (total weight of the Roman chamomile plant residues) of Trichoderma viride to carry out solid-state fermentation for 6 days to degrade plant fiber and decompose plant protein;

[0011] S3, water-soluble extract: add purified water 3-5 times the total mass of Roman chamomile plant residue after solid-state fermentation, stir and heat at 50-70°C for 2-4 hours, filter and collect the filtrate and residue, and the obtained filtrate is the water-soluble extract;

[0012] S4, protein extract: add ammonium sulfate to the aqueous extract, and observe the change in turbidity of the extract. When the turbidity of the extract no longer changes, stop adding ammonium sulfate, and then centrifuge the extract at 3000-5000 r / min for 10-20 min to obtain a precipitate and a supernatant, collect the precipitate at the bottom, and obtain Roman chamomile protein extract;

[0013] S5, polysaccharide extract: add barium hydroxide solution to the supernatant obtained after centrifugation in step S4 (the added barium hydroxide will react with ammonium sulfate in the solution to generate barium sulfate precipitate and ammonia water, and the ammonia water will volatilize by heating), and stop adding barium hydroxide until the amount of precipitate in the supernatant no longer increases, and then centrifuge it at 3000-5000r / min for 10-20min to remove the precipitate, heat the supernatant at 40-60°C for 30-50min, until the ammonia water is completely volatilized, and cool it to room temperature;

[0014] Then, 2 times the weight of anhydrous ethanol is added to the treated supernatant, stirred evenly, allowed to stand for 5-7 hours, centrifuged at 3000-5000 r / min for 10-20 minutes, and the precipitate is collected to obtain Roman chamomile polysaccharide extract;

[0015] S6. Purified fat-soluble extract of Roman chamomile: collect the residue filtered out in S3, add ethanol in an amount of 3-5 times the total mass thereof, use ultrasonic-assisted extraction, extract at 40-50°C for 1-3h, filter, collect the filtrate, and concentrate the filtrate under reduced pressure to obtain an ethanol extract concentrate; add 2-4 times the weight of water to the extract, stir until viscous, and then add 5-7 times the weight of the extract (the mixed solvent is ethyl acetate: petroleum ether in a mass ratio of 3:1) for extraction, let stand for 2-4h, collect the mixed solvent extract, and concentrate under reduced pressure to obtain an extract to obtain a purified fat-soluble extract of Roman chamomile.

[0016] S7, liquid fermentation: the extracted Roman chamomile hydrosol, protein extract, polysaccharide extract, fat-soluble purified extract are all mixed, and 5-10% of white sugar, 0.1-0.5% of peptone, 0.1-0.5% of urea, 0.1-0.5% of diammonium phosphate and 0.3-1.0% of yeast are added, and the mixture is fermented at a constant temperature of 35-40° C. for 72 h, and sterilized at a high temperature of 100-120° C. for 30-50 min. After the fermented liquid is cooled to room temperature, it is filtered and centrifuged, and the supernatant is collected to obtain a Roman chamomile composite fermentation liquid;

[0017] S8. Nano-coating: dissolve polyvinyl alcohol in water, then add a certain amount of Roman chamomile composite fermentation liquid dropwise under stirring conditions, treat under high-speed dispersion conditions for a period of time to obtain a crude emulsion, and subject the pre-cooled crude emulsion to high-pressure microfluidization treatment to obtain a nano-emulsion; then evenly mix the nano-emulsion with maltodextrin and chitosan solutions, add an appropriate amount of sodium polyphosphate solution, disperse evenly, and then subject to centrifugal freeze-drying treatment to obtain a nano-nano coating.

[0018] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0019] The present invention performs graded extraction and graded purification on Roman chamomile, and then mixes and ferments various purified extracts to finally obtain a Roman chamomile composite fermentation liquid, and finally performs nano-coating and physical and chemical embedding treatment on the fermentation liquid, so that not only various effective ingredients in Roman chamomile can be fully extracted, but also various impurities can be removed more accurately through graded purification to ensure the safety of the final product, and finally the problem of various extracts being difficult to mix is ​​solved through mixed fermentation, so that the final product can be widely used in various products; more importantly, the Roman chamomile composite fermentation liquid is used as a core material after being processed, and then maltodextrin and chitosan are wrapped on the outside to cooperate as a wall material, which has high thermal stability and water-blocking ability to prevent the internal composite fermentation liquid from escaping to the outside, so as to maintain the stability of the core material quality, antibacterial performance, and moisturizing and soothing effects. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The following is a detailed description of a Roman chamomile composite fermentation liquid nano-coated material and a preparation method thereof provided in an embodiment of the present invention.

[0022] A method for preparing a nano-coated product of Roman chamomile composite fermentation liquid comprises the following steps:

[0023] S1. Roman chamomile hydrosol extract: using Roman chamomile plants as raw materials, cutting them into small pieces with a length of about 1 cm, steaming them for about 2 hours, collecting the distillate, and obtaining Roman chamomile hydrosol;

[0024] The Roman chamomile hydrosol collected by steam distillation is rich in various volatile components of Roman chamomile, such as chamomile blue oil and α-bergamot alcohol, which maintains the original aroma of Roman chamomile and enables the final composite fermentation liquid to have the composite effects of relieving anxiety and soothing.

[0025] S2, solid-state fermentation: collecting the Roman chamomile plant residues after distillation in S1, cooling them to room temperature, adding 0.02% (total weight of the Roman chamomile plant residues) of Bacillus subtilis and 0.02% (total weight of the Roman chamomile plant residues) of Trichoderma viride to carry out solid-state fermentation for 5-10 days to degrade plant fiber and decompose plant protein;

[0026] The plant residues after distillation are solid fermented with a composite strain of Bacillus subtilis and Trichoderma viride. Trichoderma viride can produce highly active cellulase, which can degrade the cellulose in the plant residues during the fermentation process and convert it into sugars, providing sufficient carbon sources for the later fermentation process. Bacillus subtilis can decompose the protein in the plant residues and degrade it into components such as peptides and amino acids. Peptides and amino acids have smaller molecular weights and are easily absorbed by the skin, which can better play their role and enhance the skin care effect of the final product.

[0027] S3, water-soluble extract: add purified water 3-5 times the total mass of Roman chamomile plant residue after solid-state fermentation, stir and heat at 50-70°C for 2-4 hours, filter and collect the filtrate and residue, and the obtained filtrate is the water-soluble extract;

[0028] The plant residues that have undergone solid fermentation can be extracted with purified water to extract soluble proteins, peptides, amino acids, polysaccharides and other components;

[0029] S4, protein extract: add ammonium sulfate to the aqueous extract, and observe the change in turbidity of the extract. When the turbidity of the extract no longer changes, stop adding ammonium sulfate, and then centrifuge the extract at 3000-5000 r / min for 10-20 min to obtain a precipitate and a supernatant, collect the precipitate at the bottom, and obtain Roman chamomile protein extract;

[0030] S5, polysaccharide extract: add barium hydroxide solution to the supernatant obtained after centrifugation in step S4 (the added barium hydroxide will react with ammonium sulfate in the solution to generate barium sulfate precipitate and ammonia water, and the ammonia water will volatilize by heating), and stop adding barium hydroxide until the amount of precipitate in the supernatant no longer increases, and then centrifuge it at 3000-5000r / min for 10-20min to remove the precipitate, heat the supernatant at 40-60°C for 30-50min, until the ammonia water is completely volatilized, and cool it to room temperature;

[0031] Then, 2 times the weight of anhydrous ethanol is added to the obtained supernatant, stirred evenly, allowed to stand for 5-7 hours, centrifuged at 3000-5000 r / min for 10-20 minutes, and the precipitate is collected to obtain a Roman chamomile polysaccharide extract;

[0032] The present invention purifies the protein and polysaccharide in the water extract by graded method, thereby reducing the impurities in the final product and improving the safety of the product.

[0033] S6. Purified fat-soluble extract of Roman chamomile: collect the residue filtered out in S3, add ethanol in an amount of 3-5 times the total mass thereof, use ultrasonic-assisted extraction, extract at 40-50°C for 1-3h, filter, collect the filtrate, and concentrate the filtrate under reduced pressure to obtain an ethanol extract concentrate; add 2-4 times the weight of water to the extract, stir until viscous, and then add 5-7 times the weight of the extract (the mixed solvent is ethyl acetate: petroleum ether in a mass ratio of 3:1) for extraction, let stand for 2-4h, collect the mixed solvent extract, and concentrate under reduced pressure to obtain an extract to obtain a purified fat-soluble extract of Roman chamomile.

[0034] The residue after water extraction is extracted with ethanol to obtain polyphenols, flavonoids and some fat-soluble components in Roman chamomile, so that the effective ingredients in Roman chamomile can be extracted more thoroughly and efficiently;

[0035] S7, liquid fermentation: the extracted Roman chamomile hydrosol, protein extract, polysaccharide extract, fat-soluble purified extract are all mixed, and 5-10% of the total mass of white sugar, 0.1-0.5% of the total mass of peptone, 0.1-0.5% of the total mass of urea, 0.1-0.5% of the total mass of diammonium hydrogen phosphate and 0.3-1.0% of the total mass of yeast are added, and fermented at a constant temperature of 35-40°C for 72h, and sterilized at high temperature at 100-120°C for 30-50min. After the fermented liquid is cooled to room temperature, it is filtered and centrifuged, and the supernatant is collected to obtain Roman chamomile composite fermentation liquid.

[0036] The present invention performs water extraction and ethanol extraction on the plant residues after solid fermentation, respectively, and can extract various active ingredients in Roman chamomile to the greatest extent, improve the utilization rate of Roman chamomile, and enhance the efficacy of back-end products. More importantly, the present invention performs mixed fermentation on Roman chamomile hydrosol, protein extract, polysaccharide extract, and fat-soluble purified extract, which can convert some fat-soluble components into water-soluble components, and can also promote the conversion of some macromolecular proteins into small molecular polypeptides, amino acids and other components, thereby improving the activity of the product. At the same time, the irritating smell of Roman chamomile hydrosol can be reduced, making the overall aroma softer and more pleasant.

[0037] In order to ensure the storage stability of the obtained fermentation broth, the present invention performs nano-coating and chemical embedding treatment on the fermentation broth as follows:

[0038] S7. Nano-coating: dissolve polyvinyl alcohol in water, then add a certain amount of Roman chamomile composite fermentation liquid dropwise under stirring conditions, treat under high-speed dispersion conditions for a period of time to obtain a crude emulsion, and subject the pre-cooled crude emulsion to high-pressure microfluidization treatment to obtain a nano-emulsion; then evenly mix the nano-emulsion with maltodextrin and chitosan solutions, add an appropriate amount of sodium polyphosphate solution, disperse evenly, and then subject to centrifugal freeze-drying treatment to obtain a nano-nano coating.

[0039] The present invention first processes the Roman chamomile composite fermentation liquid as the core material, and then wraps maltodextrin and chitosan on the outside to work together as the wall material, which has high thermal stability and water-blocking ability to prevent the moisture of the internal composite fermentation liquid from escaping to the outside, so as to maintain the quality stability of the core material, antibacterial properties, and moisturizing and soothing effects.

[0040] Example 1

[0041] A method for preparing a nano-coated product of Roman chamomile composite fermentation liquid comprises the following steps:

[0042] [1] Preparation of Roman chamomile hydrosol: Cut the Roman chamomile plant material into small pieces of about 1 cm in length, steam it for 2 hours, collect the distillate, and obtain Roman chamomile hydrosol;

[0043] [2] Solid fermentation: After the Roman chamomile plant residues distilled in step [1] are cooled to room temperature, 0.02% of the total weight of the Roman chamomile plant residues in the amount of Bacillus subtilis and 0.02% of the total weight of the Roman chamomile plant residues in the amount of Trichoderma viride are added to carry out solid fermentation for 6 days;

[0044] 【3】Water extraction: After solid-state fermentation, add purified water 4 times the weight of the plant to the Roman chamomile plant residue, stir and heat at 60°C for 3 hours, filter to obtain the filtrate and residue, and collect the filtrate to obtain the water-soluble extract;

[0045] [4] Protein extraction: Add ammonium sulfate to the aqueous extract and observe the change in turbidity of the extract. When the turbidity of the extract no longer changes, stop adding ammonium sulfate. Centrifuge the extract at 4000 r / min for 10 min to obtain a precipitate and a supernatant. Collect the precipitate at the bottom to obtain Roman chamomile protein extract.

[0046] [5] Removing ammonium sulfate: Add barium hydroxide solution to the supernatant obtained by centrifugation in step [4] until the amount of precipitate in the supernatant no longer increases, then stop adding barium hydroxide, centrifuge at 4000 r / min for 10 min, remove the precipitate, heat the supernatant at 50°C for 40 min until the ammonia water is completely evaporated, and cool to room temperature;

[0047] [6] Extracting polysaccharides: Add 2 times the weight of anhydrous ethanol to the supernatant obtained in step [5], stir evenly, let stand for 6 hours, centrifuge at 4000 r / min for 10 minutes, collect the precipitate, and obtain Roman chamomile polysaccharide extract;

[0048] [7] Ethanol extraction: Add 4 times the weight of ethanol to the residue filtered out in step [3], use ultrasonic-assisted extraction, extract at 45°C for 2 hours, filter, collect the filtrate, and concentrate the filtrate under reduced pressure to obtain an ethanol extract concentrate. Add 3 times the weight of water to the extract and stir until it becomes viscous. Then add 6 times the weight of the extract of a mixed solvent of ethyl acetate and petroleum ether (ethyl acetate: petroleum ether = 3:1) to extract. Let it stand for 3 hours, collect the ethyl acetate and petroleum ether mixed solvent extract, and concentrate it under reduced pressure to obtain an extract to obtain a fat-soluble purified extract of Roman chamomile;

[0049] [8] Liquid fermentation: The obtained Roman chamomile hydrosol, protein extract, polysaccharide extract, and fat-soluble purified extract were mixed, and 8% by weight of white sugar, 0.2% of peptone, 0.2% of urea, 0.2% of diammonium phosphate, and 0.4% of yeast were added, and the mixture was fermented at a constant temperature of 37°C for 72 hours, and sterilized at a high temperature of 100°C for 40 minutes. After the fermented liquid was cooled to room temperature, it was filtered and centrifuged, and the supernatant was collected to obtain a Roman chamomile composite fermentation liquid;

[0050] [9] Preparation of nano-encapsulated materials: 10 g of polyvinyl alcohol was dissolved in 20 g of water, and then 15 g of Roman chamomile composite fermentation broth was added dropwise under stirring conditions. The crude emulsion was obtained after magnetic stirring at 60°C and 700 rpm for 5 h. The pre-cooled crude emulsion was subjected to high-pressure microfluidization treatment to obtain a nano-emulsion. Chitosan was dissolved in 1% glacial acetic acid to prepare a 5 mg / mL chitosan solution. The chitosan solution was filtered with microporous filter paper after stirring at 60°C for 60 min to obtain a chitosan solution. The nano-emulsion was then evenly mixed with maltodextrin and chitosan solution, and an appropriate amount of sodium polyphosphate solution was added. After uniform dispersion, the nano-encapsulated materials were obtained by centrifugal freeze-drying.

[0051] Example 2

[0052] The difference between this embodiment and embodiment 1 is that during the solid fermentation in step [2], the mass ratio of Bacillus subtilis to Trichoderma viride is 1:2, and the amount of Bacillus subtilis added is 0.03% of the total mass of the Roman chamomile plant residue.

[0053] Example 3

[0054] The difference between this embodiment and embodiment 1 is that during the liquid fermentation in step [8], the added mass of white sugar, peptone, urea, diammonium hydrogen phosphate and yeast accounts for 10%, 0.3%, 0.2%, 0.3% and 0.6% of the total mass of the mixed extract, respectively.

[0055] Example 4

[0056] The difference between this embodiment and embodiment 1 is that when preparing the nano-coating in step [9], the mass ratio of polyvinyl alcohol to Roman chamomile composite fermentation liquid is 1:2.

[0057] Experimental Example 1

[0058] The average molecular weights of the Roman chamomile hydrosol, protein extract, polysaccharide extract, fat-soluble purified extract and composite fermentation broth obtained in each step of Example 1 were tested respectively. The test results are shown in Table 1.

[0059] Table 1 - Average molecular weight of solids in each component

[0060] Serial number Serial number Average molecular weight (KDa) 1 Roman Chamomile Hydrosol 0.6 2 Roman Chamomile Protein Extract 32.3 3 Roman Chamomile Polysaccharide Extract 11.6 4 Roman chamomile fat-soluble purified extract 6.9 5 Roman chamomile compound fermentation liquid 4.1

[0061] It can be seen from Table 1 that the average molecular weight of Roman chamomile hydrosol is 0.6KDa. Because most of its components are volatile components and its solid content is very low, the effect on the molecular weight of the mixture after mixing can be ignored. After mixed fermentation, the average molecular weight of solids in the Roman chamomile mixed fermentation liquid is 4.1KDa, which is much lower than the average molecular weight of protein extract, polysaccharide extract and fat-soluble purified extract. Therefore, after mixed fermentation, the molecules in the obtained fermentation liquid are smaller and more conducive to skin absorption.

[0062] Experimental Example 2-Moisturizing Efficacy Test

[0063] The surface of the zebrafish tail fin is covered with a layer of epidermal tissue similar to human skin. When the osmotic pressure of the solution in the environment exceeds the tolerance range of the skin, it can cause dehydration of the skin, which is specifically manifested by the reduction of the tail area. Therefore, the use of hypertonic solution can establish a zebrafish hydration and moisturizing model. The test substance was dissolved in water to treat the model group zebrafish, and the shrinkage area of ​​the zebrafish tail fin was measured to evaluate whether the cosmetics have the effect of hydrating and moisturizing.

[0064] In this experiment, a blank control group, a model group (15‰) and sample groups 1-6 (2.5%) were set as experimental groups. A 24-well plate was used. Three biological replicates were set for each experimental group, and 8 embryos were set for each replicate. 2 mL of the working solution of each concentration group was added to each well.

[0065] Among them, the blank control group is the original zebrafish tail fin surface area; the model group is a zebrafish hydration and moisturizing model established after using a hypertonic solution on the zebrafish tail fin; the working solutions used in sample groups 1-6 are Roman chamomile hydrosol, protein extract, polysaccharide extract, fat-soluble purified extract composite fermentation liquid, and nano-coated material containing composite fermentation liquid prepared in Example 1, respectively;

[0066] Each test group was placed in a constant temperature incubator at 28.5±0.5℃ for 2h. Then, NaCl high salt solution was added to the model group and the sample group at the same time, and gently mixed. After incubation for 1h under the same conditions, the tail fin image of zebrafish embryos was collected using a microscope. The microscope parameters were kept consistent throughout the shooting process. Image J software was used to analyze the area from the cloaca to the end of the tail fin of each 2 ), calculated the inhibition rate of zebrafish embryo tail fin contraction, and performed a significant difference test on the data of each group. The results are shown in Table 2;

[0067] The following formula was used to calculate the inhibition rate of zebrafish embryo tail fin contraction:

[0068] Contraction inhibition rate (%) = (S2-S1) / (S0-S1) × 100%;

[0069] Wherein, S0 is the average tail area of ​​zebrafish embryos in the blank control group; S1 is the average tail area of ​​zebrafish embryos in the model control group; S2 is the average tail area of ​​zebrafish embryos in the sample group.

[0070] Table 2-Inhibition rate of each group of samples on the contraction of zebrafish embryo tail fin

[0071]

[0072] As can be seen from Table 2, each sample group using Roman chamomile hydrosol, protein extract, polysaccharide extract, and fat-soluble purified extract all has a certain soothing effect, but after the components are mixed and fermented, the moisturizing effect of the obtained Roman chamomile composite fermentation liquid is significantly improved; and after the Roman chamomile composite fermentation liquid is subjected to nano-coating and physicochemical embedding treatment, the moisturizing effect of the Roman chamomile composite fermentation liquid is more obvious and efficient. This is mainly due to the fact that when maltodextrin and chitosan are used as wall materials in combination, the storage of the fermentation liquid is more stable, and the internal composite fermentation liquid is kept from escaping to the outside to maintain its quality stability and antibacterial properties, thereby better exerting the moisturizing effect.

[0073] Experimental Example 3- Soothing Effect Test

[0074] The main effector cells of allergic reactions are mast cells. The number of activated mast cells is positively correlated with the degree of allergy. The more mast cells are activated, the more severe the allergy. Tryptase is a neutral protease pre-synthesized in mast cells. It is the most abundant medium and is highly selective in its storage and expression in mast cells. Tryptase can be used as a marker of mast cell activation. When zebrafish have an allergic reaction, mast cells are also induced to secrete Tryptase. Tryptase complexes with the BAPNA substrate and has a maximum absorption peak at 405nm. The activity detection of Tryptase can be used to evaluate the anti-allergic ability of the test substance, thereby indicating whether the sample has soothing and anti-allergic effects.

[0075] In this experiment, a control group, a model group (1 mg / L) and sample groups 1-6 (0.5%) were set as experimental groups. Three biological replicates were set for each experimental group. Fifteen zebrafish were placed in a 96-well plate. 100 μL of the working solution of each concentration group was added to each well.

[0076] Among them, the blank control group was the original zebrafish trypsin activity; the working solution used for the model group on zebrafish was sodium cromoglycate (20 mg / L); the working solutions used in sample groups 1-6 were Roman chamomile hydrosol, protein extract, polysaccharide extract, fat-soluble purified extract composite fermentation broth, and nano-coated material containing composite fermentation broth prepared in Example 1, respectively;

[0077] Place the 96-well plate to be tested in a 28.5±0.5℃ constant temperature incubator and incubate for 1h. Take 80μL of the supernatant and transfer it to the ELISA plate, add an equal volume of BAPNA (500μg / mL), incubate in a 37±0.5℃ constant temperature ELISA reader for 1h, measure the absorbance at 405nm, and calculate the inhibition rate of Tryptase. The results are shown in Table 2;

[0078] The calculation formula of Tryptase inhibition rate is as follows: Tryptase inhibition rate (%) = (MT) / M × 100%; where M- is the mean absorbance value corresponding to the model group; T- is the mean absorbance value corresponding to each sample;

[0079] Table 3-Relative activity and inhibition rate of trypsin in zebrafish of each component

[0080]

[0081] As can be seen from Table 3, each sample group using Roman chamomile hydrosol, protein extract, polysaccharide extract, and fat-soluble purified extract all have a certain soothing effect, but after the components are mixed and fermented, the soothing effect is more significant; and after the Roman chamomile composite fermentation liquid is subjected to nano-coating and physicochemical embedding treatment, the soothing effect of the Roman chamomile composite fermentation liquid is more obvious and efficient. This is mainly due to the fact that when maltodextrin and chitosan are used as wall materials in combination, the storage of the fermentation liquid is more stable, and the internal composite fermentation liquid is kept from escaping to the outside to maintain its quality stability and antibacterial properties, thereby better exerting the soothing effect.

[0082] In summary, the Roman chamomile composite fermentation liquid prepared by the present invention through separate extraction and purification of the components of Roman chamomile and mixed fermentation has a smaller average molecular weight of the components, which is more conducive to skin absorption and utilization. In terms of moisturizing and soothing effects, the composite fermentation liquid after mixed fermentation is more obvious than the effects of each single component.

[0083] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a nano-coated product of Roman chamomile composite fermentation liquid, characterized in that: The following steps are involved: S1. Extracting Roman chamomile hydrosol: cutting the Roman chamomile plant into small pieces, performing steam distillation for a period of time, collecting the distillate, and obtaining Roman chamomile hydrosol; S2, solid-state fermentation: collecting the Roman chamomile plant residue after distillation in S1, cooling it to room temperature, and then adding Bacillus subtilis and Trichoderma viride to carry out solid-state fermentation for a period of time; S3, water-soluble extract: after solid-state fermentation, the Roman chamomile plant residue is added with purified water and heated for a period of time, and the filtrate and residue are collected by filtration. The obtained filtrate is the water-soluble extract; S4, protein extract: adding sulfate ion solution to the aqueous extract, and observing the change in turbidity of the extract. When the turbidity of the extract no longer changes, stop adding ammonium sulfate, and then centrifuge the extract to obtain a precipitate and a supernatant, and collect the precipitate at the bottom to obtain Roman chamomile protein extract; S5, a purified fat-soluble extract of Roman chamomile: collecting the residue filtered out in S3, adding ethanol, and performing ultrasonic-assisted extraction to obtain an ethanol extract concentrate; then extracting the obtained ethanol extract concentrate to obtain a purified fat-soluble extract of Roman chamomile; S6, liquid fermentation: the Roman chamomile hydrosol, protein extract, polysaccharide extract, and fat-soluble purified extract are all mixed to obtain a mixed extract, and white sugar, peptone, urea, diammonium hydrogen phosphate, and yeast are added, and the mixture is fermented at a constant temperature for a period of time and then sterilized at a high temperature. After the fermented liquid is cooled to room temperature, it is filtered and centrifuged, and the supernatant is collected to obtain a Roman chamomile composite fermentation liquid; S7. Nano-coating: dissolve polyvinyl alcohol in water, then add a certain amount of Roman chamomile composite fermentation liquid dropwise under stirring conditions, treat under high-speed dispersion conditions for a period of time to obtain a crude emulsion, and subject the pre-cooled crude emulsion to high-pressure microfluidization treatment to obtain a nano-emulsion; then evenly mix the nano-emulsion with maltodextrin and chitosan solutions, add an appropriate amount of sodium polyphosphate solution, disperse evenly, and then subject to centrifugal freeze-drying treatment to obtain a nano-nano coating.

2. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: The method also includes the extraction of polysaccharides, and the specific steps are as follows: adding a barium ion solution to the supernatant obtained after centrifugation in step S4, and stopping adding the barium ion solution until the amount of precipitation in the supernatant no longer increases, centrifuging it, taking the supernatant, heating it for a period of time until the ammonia water is completely evaporated, and cooling it to room temperature; then adding anhydrous ethanol to the treated supernatant, stirring it evenly and letting it stand for a period of time, centrifuging and precipitating it, to obtain the Roman chamomile polysaccharide extract.

3. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S2, the mass ratio of Bacillus subtilis to Trichoderma viride is 1:1-2; wherein, the added amount of Bacillus subtilis is 0.01-0.05% of the total mass of the Roman chamomile plant residue.

4. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S3, the amount of purified water added is 3-5 times the total mass of the Roman chamomile plant residue; and then heated with stirring at 50-70° C. for 2-4 hours.

5. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S4, the extract is centrifuged at 3000-5000 r / min for 10-20 min.

6. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S5, ethanol is added to the residue, and the mixture is extracted at 40-50° C. for 1-3 hours, filtered, and the filtrate is collected. The filtrate is concentrated under reduced pressure to obtain an ethanol extract. Water 2-4 times its weight is added to the ethanol extract, and the mixture is stirred until it becomes viscous. A mixed solvent 5-7 times the weight of the ethanol extract is added to extract the mixture. The mixture is allowed to stand for a period of time, and the mixed solvent extract is collected. The mixture is concentrated under reduced pressure to obtain a fat-soluble purified extract of Roman chamomile.

7. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S6, the added masses of white sugar, peptone, urea, diammonium phosphate and yeast account for 5-10%, 0.1-0.5%, 0.1-0.5%, 0.1-0.5% and 0.3-1.0% of the total mass of the mixed extract, respectively.

8. The method for preparing the Roman chamomile composite fermentation liquid nano-coated material according to claim 1, characterized in that: In S6, fermentation is carried out at a constant temperature of 35-40°C for 70-75h and high temperature sterilization is carried out at 100-120°C for 30-50min.

9. The method for preparing the nano-coated material of Roman chamomile composite fermentation liquid according to claim 1, characterized in that: In S7, the mass ratio of polyvinyl alcohol to Roman chamomile composite fermentation liquid is 1:1-2.

10. A nano-coated product of Roman chamomile composite fermentation liquid, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 9.

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