CN111494273A - Rice fermentation extracting solution and preparation method and application thereof - Google Patents
Rice fermentation extracting solution and preparation method and application thereof Download PDFInfo
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- CN111494273A CN111494273A CN202010254419.5A CN202010254419A CN111494273A CN 111494273 A CN111494273 A CN 111494273A CN 202010254419 A CN202010254419 A CN 202010254419A CN 111494273 A CN111494273 A CN 111494273A
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Abstract
A rice fermentation extract and a preparation method and application thereof belong to the technical field of special foods (health food and special medical formula food) and cosmetics. The preparation method of the rice extract comprises fermenting rice with Isaria cicadae, and extracting fermented filtrate. The invention can ensure that the metabolites in the rice are converted more completely, avoids the nutrient loss, reduces the production cost, can improve the immunity of the human body, obtains a new sleep-aiding effect, and can be widely used for special medical food and health care products. Meanwhile, the invention can obviously improve the content of active ingredients of the rice extract, so that the rice fermentation extract has obvious antioxidation and tyrosinase activity inhibition, thereby achieving the effects of resisting aging and whitening; the preparation process of the invention does not need to add chemical substances such as enzyme and alkali, saves the production cost, has high safety, simplifies the production steps, is easy for industrial production, and can fully ensure the stability of the product quality.
Description
Technical Field
The invention belongs to the technical field of special foods and cosmetics, and particularly relates to a rice fermentation extracting solution as well as a preparation method and application thereof.
Background
The rice, which is also called rice, is mild in nature and sweet in taste, is one of main food crops with long planting history in China, can provide rich nutrient components such as vitamins, oryzanol, protein, anthocyanin and the like, and has the effects of tonifying middle-jiao and qi, strengthening spleen and stomach, replenishing vital essence and strengthening mind, regulating five internal organs, promoting blood circulation, improving hearing and eyesight, relieving restlessness, quenching thirst and stopping diarrhea.
At present, the development and research on rice reprocessing products are few, and the rice raw materials are mainly treated by processes such as baking, frying, enzyme degradation and the like, so that heat-labile nutrient component loss can be caused, and the utilization rate of the rice is not high; or directly extracting active substances in the rice by methods such as enzymatic extraction, alkaline extraction, ultrasonic extraction and the like. The most commonly adopted method is an alkaline method, wherein most active substances can be extracted by high-concentration alkaline liquor, but the chemical property change can be caused by the treatment of the alkaline liquor, the Maillard reaction is promoted, the color of the product is dark, and a toxic substance of cyanamide alanine is generated, so that the kidney function is damaged to a certain extent.
Rice is generally used mainly in daily food, and the nutrition and health care function still need to be strengthened. The fermented matter extracting process is to utilize different kinds of microbe to ferment culture medium with rice as main component to convert the active matter in rice biologically and to extract the fermented filtrate to obtain high effect. The different strains cause different effects of the fermentation product. The major fermentation bacteria are yeasts and bifidobacteria. And the existing fermentation methods all adopt liquid fermentation methods, so that the fermentation time is short, the biotransformation is insufficient, and the accumulation of active ingredients is less.
Young and fair skin is sought after. Therefore, the demand for anti-aging and whitening cosmetics has been leading to the demand for various cosmetics.
Aging is a natural process, and organisms are unstable chemical systems and belong to dissipative structures. The various biomolecules in the system have a large number of active groups which must interact with each other to generate chemical reaction, so that the biomolecules are slowly crosslinked to tend to be stable in chemical activity. With the lapse of time, the crosslinking degree is increased continuously, the active groups of the biomolecules are consumed and reduced continuously, the original molecular structure is changed gradually, and the aging phenomenon of the biological tissues can be generated gradually due to the accumulation of the changes.
Antioxidation is a short name for antioxidant free radicals, Anti-Oxidant. The human body continuously generates free radicals in the human body due to continuous contact with the outside, including respiration (oxidation reaction), external pollution, radiation irradiation and other factors. Scientific studies have shown that cancer, aging or other diseases are mostly associated with the production of excess free radicals. Research on antioxidation can effectively overcome the harm caused by the antioxidation, so the antioxidation is listed as one of the main research and development directions by health-care products and cosmetic enterprises, and is also one of the most important functional requirements of the market.
The problem of skin lightening is mainly dependent on the ability of melanocytes to synthesize melanin. Melanocytes are distributed among basal layer cells of human epidermis, and tyrosinase contained in the melanocytes can oxidize tyrosine into polysaccharide, wherein the tyrosinase is polyphenol transferase and belongs to the class of redox enzymes, and finally melanin can be generated through a series of metabolic processes. The more melanin production, the darker the skin; otherwise, the whiter the skin. Tyrosinase has unique dual catalytic functions, is a key enzyme for melanin synthesis in organisms, and has close relation with human aging. Abnormal overexpression of this gene can lead to pigmentation disorders in humans. The tyrosinase inhibitor can be used for treating pigmentation dermatoses such as freckle, chloasma, and senile plaque. Therefore, bearberry extract, vitamin c derivatives, kojic acid and derivatives thereof, green tea extract, licorice extract and other traditional Chinese medicine extracts in the currently popular whitening cosmetics in the market are tyrosinase inhibitors, and the tyrosinase inhibitors mainly inhibit the synthesis of melanin by inhibiting the activity of tyrosinase, thereby playing a role in whitening.
The existing anti-aging and whitening cosmetics mainly have the following problems and defects:
(1) because the cosmetics usually contain chemical components such as acid, alkali, salt, surfactant and the like, part of the cosmetics have irritation to the skin, and after the chemical components act on the skin, the strong membranes of organs and the like, the chemical components often cause irritant skin lesions, also called irritant contact dermatitis, which is acute inflammation which rapidly appears on the local part of the skin. And some components in the product can be deposited in the body and can not be excreted, which can cause the phenomena of unsmooth blood, aging of the skin, acne, color spots, sallow skin, wrinkle increase, relaxation, slow skin metabolism and the like. Even enter the fetus through the placenta, thus harming the development of the nervous system of the fetus and causing adverse effects.
(2) Melanin is produced through a complex series of chemical reactions, and there are several ways to inhibit its production. Most of the whitening products in the current cosmetic market only mainly comprise tyrosinase inhibitors, and new compounds are discovered at a higher speed every year. However, each of the whitening additives can only inhibit one pathway, but cannot fully inhibit melanin.
(3) In order to reflect the effect of the existing whitening cosmetic products, various chemical components and using hormones are intentionally added, although the whitening cosmetic products have whitening effect on skin and can reduce wrinkles, some cosmetic enterprises can add forbidden substances (such as antibiotics) into the products. After long-term use of these hormone-containing cosmetics, the skin is affected by hormone dependence, even systemic damage, and hormone-dependent inflammatory skin reactions such as pigmentation, including erythema, pimple, exudation, telangiectasia, etc., may occur when the cosmetics are not used.
The active ingredients of the rice are mild and safe in nature, have whitening and moisturizing effects, and enable the skin to be smooth and fine. But its efficacy is relatively weak and single. The fermented matter extracting process is to utilize different kinds of microbe to ferment culture medium with rice as main component to convert the active matter in rice biologically and to extract the fermented filtrate to obtain high effect. The different strains cause different effects of the fermentation product. The major fermentation bacteria are yeasts and bifidobacteria. And the existing fermentation methods all adopt liquid fermentation methods, so that the fermentation time is short, the biotransformation is insufficient, and the accumulation of active ingredients is less.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems, the invention provides the rice fermentation extract, the preparation method and the application thereof, which can ensure that the metabolites in the rice are converted more completely, avoid the nutrient loss, reduce the production cost, improve the immunity of the human body, obtain the new effects of helping sleep, resisting oxidation and inhibiting the activity of tyrosinase, and can be used for special foods and cosmetics.
The technical scheme is as follows: a preparation method of rice fermentation extract is characterized in that isaria cicadae is used as a strain to ferment rice, and fermentation filtrate is extracted.
Preferably, the method comprises the steps of:
(1) preparation: the solid culture medium for the zymophyte comprises the following components in parts by weight:
preparing a culture medium according to a proportion, and filling the culture medium into a culture box;
(2) and (3) sterilization: placing the culture box into a sterilization pot, wherein the sterilization temperature is 110-;
(3) inoculating, namely cooling the culture medium to below 20 ℃, inoculating under aseptic condition, and inoculating 15 culture boxes per 300m of L Isaria cicadae seed liquid;
(4) solid fermentation culture: naturally fermenting for 20d to 50d at the temperature of 20 ℃ to 37 ℃;
(5) harvesting: taking out the substances in the culture box, removing the sporophore bundles, drying and cooling the culture medium, vacuum packaging with a film bag, and storing at-20 ℃;
(6) extraction: crushing the culture medium, and extracting for 1-4 hours by using distilled water under reflux;
(7) and (3) filtering: filtering the extracting solution by a 100-sand 500-mesh nylon filter bag, and then taking a filtrate;
(8) alcohol precipitation and concentration: concentrating under vacuum with industrial ethanol 1-5 times the volume of the filtrate; the obtained concentrated solution is fermentation extract.
Preferably, the reflux extraction time in the step (6) is 2 hours.
Preferably, the size of the crushed particles of the culture medium in the step (6) is 10-40 meshes.
Preferably, the ratio of the extracted materials to the extracted liquids in the step (6) is 1: 12.
Preferably, in the step (1), 10 to 25 parts by weight of the rice is replaced by 10 to 25 parts by weight of the rice and 1 to 5 parts by weight of the rice bran.
The rice fermentation extract prepared by the above preparation method.
The extract is applied to the preparation of sleep-aiding products.
The extract can be used for preparing cosmetics.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) enriches the existing rice reprocessing products, and invents a rice fermentation extract.
(2) By the fermentation effect of isaria cicadae, metabolites in rice are converted more completely, active ingredients are accumulated more, and the rice is rich in polysaccharide, cordycepin, amino acid, fatty acid and nucleoside, so that the immunity of a human body can be improved, a new sleep-aiding effect is obtained, and the rice can be used for special medical food and health-care products.
(3) In the preparation process, no foreign matters such as enzyme and alkali are needed to be added, so that the production cost is saved, the production steps are simplified, the industrial production is easy, and the stability of the product quality can be fully ensured.
(4) Avoids the nutrient loss caused by processing techniques such as fine grinding, homogenization and the like, and ensures the nutritive value of the product to the maximum extent.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the specific contents of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
The method for preparing the rice fermentation extracting solution comprises the following steps:
① preparation of PDA solid inclined plane comprises mixing PDA culture medium with culture medium of L per 1000m including potato 200g, glucose 20g and agar 20g, cutting peeled potato, placing in a container, adding 1000m L water, boiling for 20min, filtering, adding glucose and agar (liquid culture medium without agar), adding water to 1000m L, heating to dissolve, packaging into test tubes of 1/4, plugging with cotton plug, sterilizing in a sterilizer under 0.1MPa for 30min, and cooling while hot.
② slant strain is prepared by collecting Isaria cicadae, aseptically activating in PDA liquid culture medium, transferring tube, culturing at 22 deg.C for 3 days, and selecting the strain with good growth vigor;
③ preparation method of PDA conical flask seed liquid comprises culturing potato 200g and glucose 20g in L per 1000m, cutting peeled potato, boiling in 1000m L water for 30min, filtering, adding glucose, adding water to 1000m L, heating to dissolve, packaging in conical flask with a volume of 60% of conical flask volume, plugging cotton plug, wrapping with kraft paper, sterilizing in 0.15MPa for 30min, and cooling to 20 deg.C or below.
Inoculating the PDA liquid culture medium into a conical flask under aseptic condition, culturing at 22 deg.C and oscillation frequency of 100r/min for 5 days until a large amount of mycelium pellets appear in the conical flask;
④ solid fermentation culture:
(1) preparing a rice solid culture medium, namely filling the prepared culture medium into a culture box (the specification of the culture box is that the circular diameter is 9-10 cm, the height is × and the height is 6-10 cm), sealing the culture box by using a breathable sealing film, wherein the mixture ratio is 10 parts by weight of rice matrix, 2 parts by weight of rice bran, 3 parts by weight of silkworm chrysalis meal, 0.05 part by weight of potassium dihydrogen phosphate, 0.3 part by weight of peptone, 0 part by weight of yeast powder and 12 parts by weight of water, and uniformly mixing the raw materials according to the ratio.
(2) And (3) sterilization: placing the culture box into a sterilizing pot, and sterilizing at 0.15MPa for 30 min;
(3) inoculating, namely cooling the solid culture medium to below 20 ℃, inoculating under aseptic conditions, and inoculating 15 culture boxes per 300m of L seed liquid;
(4) solid fermentation culture: transferring the inoculated culture box to a culture room, culturing at 20-25 deg.C and relative humidity of 60-80% for 30d-50d, and maintaining fresh air in the culture room, wherein the environmental temperature is 2-3 deg.C lower than that in the early mycelium growth stage, and ventilation is required at proper time every day;
⑤ harvesting when the height of the culture reaches above 3cm, there is a great amount of conidium on the surface, and the bundle is bent, removing the sealing film, cutting off the bundle with a tool, taking out the solid culture medium, oven drying at 60 deg.C overnight, cooling, vacuum packaging with film bag, and storing at-20 deg.C.
⑥ the rice fermentation extract is obtained by pulverizing dried solid fermentation culture medium, sieving with 20 mesh sieve, adding distilled water at a ratio of 1: 3, extracting under slight boiling for 4 hr by reflux extraction, filtering with 100-mesh 500-mesh nylon filter bag, collecting filtrate, adding 3 times volume of industrial ethanol, precipitating, collecting supernatant, vacuum concentrating at 60 deg.C to original volume, and concentrating to obtain rice fermentation extract.
Example 1 optimization of preparation method of Rice fermentation extract
And (4) optimizing the extraction conditions by adopting orthogonal experimental design.
A. Factor level meter
Protein and total sugar content are taken as investigation indexes, and factors such as reflux extraction time (A), feed-liquid ratio (B), addition amount (C) of 95% ethanol in an extraction solvent, size (D) of crushed particles of a culture medium and the like are obtained through a plurality of preliminary experiments. In order to further study the extraction process of the cordyceps sobolifera culture medium, a three-level four-factor table is designed with A, B, C, D four factors.
TABLE 1 factor level table
B. Analysis of results of orthogonal experiments
TABLE 2 analysis of results of orthogonal experiments
Note that K is mean and R is very poor, composite score ═ protein content/protein content maximum × 50+ total sugar content/total sugar content maximum × 50.
TABLE 3 ANOVA TABLE (composite score)
F0.05(2, 2) ═ 19, denotes 0.05> P >0.01, denotes P <0.01
As can be seen from the table, B has a significant effect on the protein and total sugar content.
And (4) analyzing results: comprehensively considering, comparing with visual analysis and variance analysis, the cicada fungus culture medium has yield of less than 40 mesh when passing through 10 mesh sieve, and A, C, D has no significant influence on protein and total sugar content, so B is selected3C1A1D3Is an optimal extraction process.
Example 2 safety of Rice fermentation extract
L D could not be detected in the rice fermentation extract50The maximum tolerated dose now reflects the toxicity of the test agent.
7 mice (without water prohibition) which had been fasted for 12 hours were subjected to continuous observation for one week at a time (maximum allowable concentration of the gavage needle) corresponding to 100g of crude drug per kg of mouse (injection: unit: mass of drug g/kg of mouse weight). Mice did not die within one week. The appearance, behavior, diet and excreta of the mice have no obvious change. The MTD of the rice fermentation extract is 12 g/kg.
Example 3 direct sleep experiment
① Experimental procedures
The mice are randomly divided into three dose groups of 4 groups, namely a control group and an artificially cultured rice fermentation extract solution, wherein the three dose groups are 1.25 g, 2.5 g and 5g of crude drug/kg of mice, and each group contains 10 mice and half of males and females. Gavage was performed 3 pm daily for 5 days continuously, and the number of mice falling asleep and the duration of sleep of the mice were observed within 1h of the last administration. The index of falling asleep is the disappearance of righting reflex for more than 1 minute.
② results
TABLE 4 Rice fermentation extract solution vs. mouseInfluence of sleep
The experimental result shows that the rice fermentation extract has no direct sleep effect, which indicates that the sleep-aiding effect of the rice fermentation extract is not directly inhibiting the occurrence of central nerves and is not addicted in actual use.
Example 4 subthreshold dose hypnosis experiment of sodium pentobarbital in mice
① Experimental procedure:
the mice are randomly divided into three dose groups of 4 groups, namely a control group, 1.25 artificial culture rice fermentation extract solution, 2.5 artificial culture rice fermentation extract solution and 5g crude drug/kg mouse, and each group contains 10 mice and half of males and females. Gavage administration is carried out every morning for 5 days continuously, after 1 hour of the last administration, the mice are injected with 30mg/kg of sodium pentobarbital in a subthreshold dose into the abdominal cavity (pre-test shows that 10-20% of normal mice can disappear by turning positive reflex), and the number of the animals falling asleep within 30min is recorded by taking the mice with the disappearance of the positive reflex for more than 1min as the standard of falling asleep. And (5) observing whether the tested object can improve the incidence rate of the animals falling asleep at the subthreshold dose of the sodium pentobarbital (the record standard is that the righting reflex disappears for more than 1 min).
② results
Table 5 effect of rice fermentation extract solution on incidence of subliminal dose sleep of sodium pentobarbital (n ═ 10)
Note: comparison with blank group P <0.05
The experimental result shows that the incidence rate of animals falling asleep in a subthreshold dose of the sodium pentobarbital can be obviously improved by the rice fermentation extracting solution under a high dose.
Example 5 experiment for prolonging sleep time of sodium pentobarbital in mice
① Experimental procedures
Grouping and administration of mice are the same as above, after 1h of last administration, pentobarbital sodium (50mg/kg, pre-test shows that normal mice can disappear by 100 percent of righting reflex) is carried out on the abdominal cavity of the mice, and the mice are observed whether the sleep time induced by the pentobarbital sodium is prolonged or not.
② results
TABLE 6 influence of rice fermentation extract solution on sleep time of pentobarbital sodium-induced mice
Note: comparison with blank group P <0.05
The experimental result shows that the rice fermentation extract can obviously prolong the sleep time induced by the pentobarbital sodium under medium and high doses.
Example 6 Properties of fermented extract of rice
The fermented extract prepared in example 1 is dark brown liquid, pH value is 5.2-6.3, soluble solid content is 1-20mg/m L, total bacterial count is less than 10CFU/ml, no pathogenic bacteria are detected, and according to the cosmetic hygiene standard GB7916-1987, total bacterial count of the cosmetic is not more than 1000CFU/ml, so the fermented extract meets the cosmetic quality requirement.
Performing component analysis on the rice fermentation extract, wherein the protein polypeptide detection method refers to GB 5009.5-2010; the crude polysaccharide detection method refers to GB/T5009.8-2008, and the obtained results are as follows:
the rice fermentation extract liquid prepared by the invention contains 14.282% of total protein (peptide) and 15.399% of total sugar content on average.
Example 7 antioxidant Activity of fermented extract of Rice
① determination of hydroxyl radical scavenging rate
9 mmol/L FeSO was added to the tube in the order shown in the following table40.5m L, 9 mmol/L ethanol-salicylic acid 0.5m L, then adding a proper amount of deionized water, and finally adding H2O2Shaking up 0.5m L, heating in water bath at 37 ℃ for 15min, taking out and measuring absorbance A0, wherein the reference solution is a system without adding hydrogen peroxide.
The sample addition system A was determined as described aboveX，AX0。
Table 7 sample reagent addition table
The clearance was calculated as follows:
hydroxyl radical clearance (%) ═ a0-(Ax-Ax0)/A0*100％
Results of the experiment
TABLE 8 scavenging effect of rice fermentation extract with different concentrations on hydroxyl radical
From the above table, it can be seen that the removal rate of hydroxyl radical is improved with the increase of the sample concentration, and when the sample mass concentration is 1.000mg/m L, the removal rate of hydroxyl radical is 91.71%2+1.9662x+0.0205，R20.9901. Calculating the half hydroxyl radical scavenging concentration EC of the rice fermentation extract50＝0.329mg/mL。
② measurement of DPPH radical scavenging Rate
DPPH is an early synthetic organic radical commonly used to evaluate the hydrogen donating ability of antioxidants, is very stable in organic solvents, is purple in color, and has a characteristic absorption peak at 517nm, when encountering a radical scavenger, the lone pair of electrons of DPPH is paired to discolor it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.
270 μ L DPPH-B were added sequentially to 96-well plates according to the following tableAlcohol solution, 30 μ L sample solution to be tested, shaking, standing at room temperature in shade for 30min, and measuring its absorbance A at 517nm1。
The same method is used for measuring the absorbance A at 517nm after mixing 95% ethanol and DPPH-ethanol0。
TABLE 9 sample reagent addition Table
The DPPH radical clearance is calculated according to the following formula.
DPPH free radical clearance (%) - (A0-A1)/A0 × 100%
Results of the experiment
TABLE 10 Effect of rice fermentation extracts of different concentrations on DPPH removal
EXAMPLE 1 EC of rice fermentation extract on DPPH scavenging action501180 microgram/m L, which indicates that the rice fermentation extract has strong antioxidant capacity, can remove free radicals, promote cell metabolism, enhance cell activity, improve the structure and function of skin, and improve the vitality of organisms, thereby delaying cell aging and playing the role of resisting aging.
Example 8 in vitro tyrosinase inhibitory Activity of Rice fermentation extract
Tyrosinase is a key enzyme in melanogenesis, which controls the process of melanogenesis, and its degree of activity plays a major role in pigment deposition. Many whitening and freckle-removing products sold in the market at present achieve the whitening effect by inhibiting tyrosinase, so the strength of the tyrosinase inhibition effect is a main index for evaluating whitening cosmetics.
Test solution group:
adding 0.6m L PBS solution, 0.8m L concentration sample solution and 0.4m L tyrosinase solution into a test tube with a plug as shown in the following table, fully mixing, quickly putting into a constant-temperature water bath kettle at 32 ℃ for heat preservation for 10min, then quickly adding 0.8m L levodopa solution, mixing, measuring an absorbance value A4 at 475nm of wavelength by using a microplate reader, and calculating the average value of 3 groups of parallel data to ensure accuracy.
The following groups were also set as controls.
Control group A1 was prepared by adding 1.4m L PBS solution, 0.8m L deionized water and 0.4m L tyrosinase solution to a stoppered test tube, mixing well, quickly placing in a constant temperature water bath at 32 deg.C, keeping the temperature for 10min, and measuring absorbance A1 at a wavelength of 475nm using a microplate reader.
And in the control group A2, 0.6m L PBS solution, 0.8m L deionized water and 0.4m L tyrosinase solution are added into a test tube with a plug, fully and uniformly mixed, quickly placed into a constant-temperature water bath kettle at 32 ℃ for heat preservation for 10min, then quickly added with 0.8m L levodopa solution, uniformly mixed, and an enzyme labeling instrument is used for measuring the absorbance value A2 at the wavelength of 475 nm.
And in the control group A3, 1.4m L PBS solution, 0.8m L sample solution and 0.4m L tyrosinase solution are added into a test tube with a plug, the mixture is fully and uniformly mixed, the mixture is quickly placed into a constant-temperature water bath kettle at 32 ℃ for heat preservation for 10min, and an absorbance value A3 at 475nm of wavelength is measured by using an enzyme labeling instrument.
TABLE 11 sample reagent addition Table
Tyrosinase inhibition was calculated according to the following formula.
Tyrosinase inhibition (%) - (1- (a)4-A3)/(A2-A1))*100％
Results of the experiment
TABLE 12 influence of Rice fermentation extracts of different concentrations on tyrosinase Activity in vitro
From the above results, when the sample mass concentration is 2.85mg/m L, the inhibition rate of tyrosinase is 89.07%, and the inhibition rate is the highest, according to the test results, the regression equation y is determined to be-13.255 x3+73.367x2-95.526x +70.296, and R2 is determined to be 0.9793, and the half inhibition concentration IC of the rice fermentation extract is obtained501.524mg/m L, and the median tyrosinase inhibitory concentration of nicotinamide determined simultaneously is IC5021.96mg/m L, namely the rice fermentation extract provided by the invention has the whitening effect equivalent to 14 times of that of nicotinamide, and has strong whitening effect.
The above examples are only for illustrating the technical idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (9)
1. A preparation method of rice fermentation extract is characterized in that isaria cicadae is used as a strain to ferment rice, and fermentation filtrate is extracted.
2. The method for preparing the rice fermentation extract according to claim 1, wherein the method comprises the following steps:
(1) preparation: the solid culture medium for the zymophyte comprises the following components in parts by weight:
10-25 parts by weight of rice,
1-6 parts of silkworm chrysalis powder,
0 to 0.2 weight portion of monopotassium phosphate,
0-1 part by weight of peptone,
0 to 3 weight parts of yeast powder,
10-30 parts by weight of water,
preparing a culture medium according to a proportion, and filling the culture medium into a culture box;
(2) and (3) sterilization: placing the culture box into a sterilization pot, wherein the sterilization temperature is 110-;
(3) inoculating, namely cooling the culture medium to below 20 ℃, inoculating under aseptic condition, and inoculating 15 culture boxes per 300m of L Isaria cicadae seed liquid;
(4) solid fermentation culture: naturally fermenting for 20d to 50d at the temperature of 20 ℃ to 37 ℃;
(5) harvesting: taking out the substances in the culture box, removing the sporophore bundles, drying and cooling the culture medium, vacuum packaging with a film bag, and storing at-20 ℃;
(6) extraction: crushing the culture medium, and extracting for 1-4 hours by using distilled water under reflux;
(7) and (3) filtering: filtering the extracting solution by a 100-sand 500-mesh nylon filter bag, and then taking a filtrate;
(8) alcohol precipitation and concentration: concentrating under vacuum with industrial ethanol 1-5 times the volume of the filtrate; the obtained concentrated solution is fermentation extract.
3. The method for preparing rice fermentation extract according to claim 2, wherein the reflux extraction time in the step (6) is 2 hours.
4. The method for preparing rice fermentation extract according to claim 2, wherein the size of the crushed particles of the culture medium in the step (6) is 10-40 mesh.
5. The method for preparing rice fermentation extract according to claim 2, wherein the ratio of the materials to the liquids extracted in the step (6) is 1: 12.
6. The method for preparing rice fermentation extract according to claim 2, wherein 10-25 parts by weight of rice in the step (1) is replaced by 10-25 parts by weight of rice and 1-5 parts by weight of rice bran.
7. A rice fermentation extract prepared by the method according to any one of claims 1 to 6.
8. Use of the rice fermentation extract of claim 7 in the preparation of a sleep-aid product.
9. Use of the rice fermentation extract of claim 7 for the preparation of cosmetics.
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| CN102242070A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Method for artificially culturing paecilomyces cicadae and application of culturing product thereof |
| CN102242069A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Paecilomycescicadae (Miq.)Samson and application thereof |
| CN103655215A (en) * | 2013-11-19 | 2014-03-26 | 安徽农业大学 | Paecilomyces varioti extract with tyrosinase activity and scavenging free radical activity and application thereof |
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2020
- 2020-04-02 CN CN202010254419.5A patent/CN111494273A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242070A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Method for artificially culturing paecilomyces cicadae and application of culturing product thereof |
| CN102242069A (en) * | 2010-05-14 | 2011-11-16 | 浙江泛亚生物医药股份有限公司 | Paecilomycescicadae (Miq.)Samson and application thereof |
| CN103655215A (en) * | 2013-11-19 | 2014-03-26 | 安徽农业大学 | Paecilomyces varioti extract with tyrosinase activity and scavenging free radical activity and application thereof |
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