Edible plant enzyme composition for synergistically improving skin moisture and inflammation

Abstract

The present application provides an edible plant enzyme composition that synergistically improves skin moisture and inflammation. The composition comprises edible plant enzymes and Sophora japonica, and the weight ratio of edible plant enzymes to Sophora japonica is 7:2-29:2. The composition is used in the field of food or health food, and the main dosage forms used are oral liquid, beverage, and soft candy, mainly through direct oral administration. The composition of the present invention can synergistically improve skin moisture and inflammation.

Description

Technical Field

[0001] The present invention belongs to the field of functional foods and medicines, and particularly relates to the preparation and application of an edible plant enzyme composition, which can be used in the production of functional foods such as beverages, drinks, jellies, and gel candies. Background Art

[0002] Plant enzymes are made from fresh vegetables, fruits, Chinese medicinal and edible plants, etc. After a series of processes such as juicing or extraction, yeast, lactic acid bacteria and other fermentation strains are added to produce a mixed fermentation liquid rich in sugars, organic acids, minerals, vitamins, phenols, terpenes and other nutrients as well as some important enzyme bioactive substances. Plant enzymes usually help the human body improve gastrointestinal function, promote cell nutrient absorption, activate cell vitality, neutralize excessive free radicals in the body, and improve human immunity.

[0003] Plant enzymes are widely used to improve digestive health and chronic diseases, but there are few reports on their oral use to improve skin health.

[0004] Chinese patent CN105211863A relates to a composition for making ginger, jujube and yam enzyme and the ginger, jujube and yam enzyme. The patent claims to provide a composition for making ginger, jujube and yam enzyme with the functions of clearing intestines and relieving constipation, beautifying and nourishing the skin, enhancing immunity and losing weight and the ginger, jujube and yam enzyme.

[0005] Chinese patent CN105726383A relates to a method for preparing a sterile enzyme anti-acne lotion, and claims that the method obtains nutritious enzymes by fermenting fruit peels, cloves, cinnamon and mint leaves, which can not only protect the skin but also have antibacterial effects and no side effects.

[0006] Chinese patent CN111956536A relates to the use of soursop fermented product in the preparation of a composition, and claims that the soursop fermented product promotes collagen firming, inhibits protein glycosylation, promotes the expression of specific genes, and / or inhibits the expression of MITF, a gene related to melanin production, thereby helping to delay skin aging.

[0007] Chinese patent CN1931269B relates to a pharmaceutical composition containing rutin, and claims that the composition can be used to treat and / or prevent metabolic syndrome, diabetes and its complications, cardiovascular and cerebrovascular diseases, Alzheimer's disease, hyperlipidemia, and to prevent the recurrence of cardiovascular and cerebrovascular diseases and prevent restenosis after angioplasty. It can also be used to prepare functional health products with auxiliary lipid-lowering function, auxiliary blood sugar-lowering function, auxiliary blood pressure-lowering function, anti-oxidation function, and auxiliary memory improvement function, and used in cosmetics with anti-oxidation effect.

[0008] There is a need in the art for compositions that extend the functionality of plant enzymes. Summary of the invention

[0009] The inventors are based on the following discovery: a composition of edible plant enzymes such as litchi enzymes and Sophora japonica can synergistically improve skin inflammation or synergistically increase skin moisturizing. No research has been reported on combining litchi fermented liquid with Sophora japonica to synergistically increase skin anti-inflammatory and moisturizing. The advantage of this synergistic formula is that the composition can achieve the effect of a high-dose single material at a low dose, which can reflect its application value from multiple angles such as expanding the application range of the dosage form of the material, reducing costs, and saving resources. The principle of the synergistic effect of the composition lies in the complementarity of the targets. Edible plant enzymes such as litchi fermented liquid and Sophora japonica can achieve the anti-inflammatory and / or moisturizing functions of the skin from different targets. The targets of the two materials are complementary; thereby achieving the effect of fully covering the targets and synergistically enhancing the efficacy. In this article, the composition can be used for beauty or skin care, or can be used as an auxiliary for beauty or skin care, or can be used as a food or health product.

[0010] On the one hand, the present invention provides an edible plant enzyme composition, which comprises edible plant enzymes and Sophora japonica flowers, and the weight ratio of edible plant enzymes to Sophora japonica flowers is 7:2-29:2.

[0011] In one embodiment, the edible plant enzyme is litchi enzyme.

[0012] In one embodiment, the edible plant enzyme is in liquid form.

[0013] In one embodiment, the Sophora japonica flower comprises not less than 6 wt % rutin.

[0014] In one embodiment, the composition is an oral composition. In one embodiment, the oral composition is an oral liquid, a beverage or a soft candy.

[0015] In one embodiment, the litchi enzyme is produced by secondary fermentation, which includes sealed static fermentation of a mixed bacterial agent of yeast and lactic acid bacteria, and then static or aerated culture of acetic acid bacteria to obtain it.

[0016] In one aspect, the present invention provides a skin moisturizing composition comprising the edible plant enzyme composition described herein.

[0017] In one embodiment, the skin moisturizing composition further comprises one or more of sodium hyaluronate, resistant dextrin, and fructooligosaccharides.

[0018] In one aspect, the present invention provides a pharmaceutical composition or a health care composition, which comprises the edible plant enzyme composition described herein and a pharmaceutically or health care acceptable excipient.

[0019] In one aspect, the present invention provides a method for moisturizing the skin in a subject, the method comprising administering the edible plant enzyme composition or the skin moisturizing composition described herein to the subject.

[0020] In one aspect, the present invention provides a method for maintaining and / or improving skin health in a subject, the method comprising administering to the subject the edible plant enzyme composition or skin moisturizing composition described herein.

[0021] In one aspect, the present invention provides a method for improving skin inflammation in a subject for non-therapeutic purposes, the method comprising administering to the subject the edible plant enzyme composition or skin moisturizing composition described herein.

[0022] In various methods herein, administration can be oral administration.

[0023] In one aspect, the present invention provides the use of the edible plant enzyme composition or pharmaceutical composition described herein in the preparation of a drug for improving skin inflammation. In one embodiment, the drug is an oral drug.

[0024] Advantages of the present invention include:

[0025] (1) Expand the application of edible plant enzyme products in the functional direction of skin inflammation or moisturizing health care;

[0026] (2) The compositions described herein can synergistically improve skin inflammation;

[0027] (3) The compositions described herein can synergistically increase skin moisture;

[0028] (4) There are few public documents on the specific functions of existing litchi enzymes / litchi fermented liquids, such as hydration and anti-inflammatory. In addition, the hydration function of Sophora japonica is also missing. There are literature reports (CN109820752B) that rutin in Sophora japonica even has a reverse effect when directly acting on the skin. Rutin can discharge the water absorbed by the moisturizer again, thereby causing problems such as skin dehydration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The dilution process of the HA standard stock solution is shown.

[0030] Figure 2 A sodium hyaluronate standard curve is shown.

[0031] Figure 3 A nitric oxide standard curve is shown.

[0032] Figure 4A bar graph showing the experimental data of hyaluronic acid production of the blank group, Sophora japonica group, litchi enzyme liquid group, composition 1 (Sophora japonica: litchi fermentation liquid = 2:7), composition 2 (Sophora japonica: litchi fermentation liquid = 1:7), composition 3 (Sophora japonica: litchi fermentation liquid = 2:29), composition 4 (Sophora japonica: litchi fermentation liquid = 1:3), and composition 5 (Sophora japonica: litchi fermentation liquid = 1:15).

[0033] Figure 5 A bar graph showing the experimental data of hyaluronic acid production of the blank group, osmanthus group, litchi enzyme solution group, and osmanthus and litchi fermentation solution combination (osmanthus: litchi fermentation solution = 1:5).

[0034] Figure 6 A bar graph showing the NO inhibition rate data of the Sophora japonica flower group, litchi enzyme liquid group, composition 1 (Sophora japonica flower: litchi fermentation liquid = 2:7), composition 2 (Sophora japonica flower: litchi fermentation liquid = 1:7), composition 3 (Sophora japonica flower: litchi fermentation liquid = 2:29), composition 4 (Sophora japonica flower: litchi fermentation liquid = 1:3), and composition 5 (Sophora japonica flower: litchi fermentation liquid = 1:15).

[0035] Figure 7 A bar graph showing the NO inhibition rate data of the osmanthus group, litchi enzyme solution group, and osmanthus and litchi fermentation solution combination (osmanthus: litchi fermentation solution = 1:7). DETAILED DESCRIPTION

[0036] In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Although it is believed that those of ordinary skill in the art fully understand the following terms, the following definitions are still stated to help illustrate the subject matter disclosed by the present invention.

[0037] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "Comprising" is a technical term used in claim language, meaning that the elements are present, but other elements may also be added and still form a structure or method within the scope of the claim.

[0038] As used herein, "edible plant enzymes" refer to enzyme products that are edible to humans and contain specific biologically active ingredients, which are made from plants that can be used for food processing as the main raw materials, with or without auxiliary materials, and are obtained through microbial fermentation. The Light Industry Standard of the People's Republic of China QB / T 5323-2018 provides characteristic indicators of edible plant enzymes. Edible plant enzymes can be liquid, semi-solid or solid. For liquid edible plant enzymes, the total acid is calculated as lactic acid (g / 100g) ≥ 0.8; free amino acids (mg / 100g) ≥ 33; organic acids are calculated as lactic acid (g / 100g) ≥ 660; lactic acid (mg / kg) ≥ 550; crude polysaccharides (g / 100g) ≥ 0.1; γ-aminobutyric acid (mg / kg) ≥ 0.03; polyphenols (mg / g) ≥ 0.5; SOD enzyme activity ≥ 15U / L. For semi-solid state, total acid as lactic acid (g / 100g) ≥1.1; free amino acid (mg / 100g) ≥35; organic acid as lactic acid (g / 100g) ≥900; lactic acid (mg / kg) ≥800; crude polysaccharides (g / 100g) ≥0.15; γ-aminobutyric acid (mg / kg) ≥0.039; polyphenols (mg / g) ≥0.6; SOD enzyme activity ≥20U / L. For solid state, total acid as lactic acid (g / 100g) ≥2.4; free amino acid (mg / 100g) ≥97; organic acid as lactic acid (g / 100g) ≥6400; lactic acid (mg / kg) ≥1150; crude polysaccharides (g / 100g) ≥2.8; γ-aminobutyric acid (mg / kg) ≥0.06; polyphenols (mg / g) ≥1.4; SOD enzyme activity ≥30U / L.

[0039] The edible plant enzyme may contain a fermentation strain, such as lactic acid bacteria and / or yeast. Alternatively, the edible plant enzyme may be sterilized without containing a fermentation strain. The edible plant enzyme may be litchi enzyme.

[0040] As used herein, "litchi enzyme" is an enzyme containing specific biologically active ingredients obtained by microbial fermentation using litchi as raw material. The litchi enzyme herein can meet the relevant standards of the above-mentioned edible plant enzymes. Litchi enzyme can be produced from litchi juice and / or litchi pomace through microbial fermentation. The microorganism can include 1, 2 or 3 of yeast, lactic acid bacteria and acetic acid bacteria. The yeast can be one or more of abnormal Hansen yeast, sake yeast, rouxii yeast, baker's yeast, Will's yeast, Pichia pastoris or red yeast. The lactic acid bacteria can be one or more of plant lactobacillus, reuteri lactobacillus, sake lactobacillus, casei lactobacillus, gasseri lactobacillus or paracasei lactobacillus. The acetic acid bacteria can be one or more of Acetobacter pasteurianus, Acetobacter aceticus, Acetobacter ghana, Acetobacter malodorus or Acetobacter aceticus. Litchi enzyme can be produced by secondary fermentation. Secondary fermentation can include the first stage fermentation of a mixed bacterial agent of yeast and lactic acid bacteria (e.g., sealed and left to stand). Secondary fermentation can include the second stage fermentation by acetic acid bacteria (e.g., left to stand or aerated culture). The lychee enzyme may be, for example, the lychee enzyme mentioned in the Chinese invention patent application number 202210565739.1, the invention name of which is “lychee enzyme, preparation method and use thereof”. Since the lychee enzyme in the patent has a better anti-inflammatory effect than other plant enzymes, the composition containing lychee enzyme of the present invention may have a better synergistic effect on skin anti-inflammatory or moisturizing than the composition containing other plant enzymes.

[0041] For example, litchi enzyme can be prepared by the following method, which includes (1) primary fermentation, which includes adding a first carbon source to litchi juice and litchi pomace, inoculating a first bacterial agent, and sealing and standing at 20-45°C for 24-96 hours, wherein the first bacterial agent is a mixed bacterial agent of yeast and lactic acid bacteria; (2) squeezing and filtering the product after the primary fermentation to obtain a primary fermentation filtrate; and (3) secondary fermentation, which includes adding a second carbon source to the primary fermentation filtrate, inoculating a second bacterial agent, standing at 15-40°C or aeration culture for 3-15 days to obtain a secondary fermentation liquid, wherein the second bacterial agent is acetic acid bacteria; (4) filtering and sterilizing the secondary fermentation liquid to obtain litchi enzyme. In one embodiment, the method includes a pre-treatment step of washing and squeezing litchi fruit. In one embodiment, the first carbon source is one or more of fructose, sucrose, brown sugar, maltose, and arabinose. In one embodiment, the addition amount of the first carbon source is 0-5wt%. In one embodiment, the yeast is one or more of Hansen's yeast, sake yeast, Saccharomyces cerevisiae, baker's yeast, Will's yeast, Pichia pastoris or red yeast. Preferably, the yeast is one or more of Hansen's yeast, sake yeast and baker's yeast. In one embodiment, the lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus reuteri, sake lactobacillus, casei, Lactobacillus gasseri or Lactobacillus paracasei. In one embodiment, the weight ratio of yeast: lactic acid bacteria is 50:1-500:1. Preferably, the weight ratio of yeast: lactic acid bacteria is 300:1-500:1. In one embodiment, the addition amount of the first bacterial agent is 0.1‰-10‰ of the total mass of litchi. In one embodiment, the second carbon source is one or more of lactose, brown sugar, glucose, molasses or honey. In one embodiment, the addition amount of the second carbon source is 1%-20% of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is one or more of Acetobacter pasteurianus, Acetobacter aceticus, Acetobacter ghanaiensis, Acetobacter malodorus, or Acetobacter aceticus. In one embodiment, the amount of the second bacterial agent added is 0.1‰-10‰ of the total mass of the primary fermentation filtrate. In one embodiment, the static culture is a static culture sealed with 8 layers of gauze. In one embodiment, the ventilation volume of the aeration culture is 0.1-20mL / min / mL of fermentation liquid.

[0042] In one embodiment, the first bacterial agent is yeast: lactic acid bacteria is 400:1; the yeast is sake yeast; the lactic acid bacteria is Lactobacillus paracasei: Lactobacillus reuteri: Lactobacillus gasseri is 1:1:1. In one embodiment, the addition amount of the first bacterial agent is 0.9‰ of the total mass of litchi. In one embodiment, the first bacterial agent is yeast: lactic acid bacteria is 100:1; the yeast is baker's yeast: Pichia pastoris: Saccharomyces rouxii is 1:1:1; the lactic acid bacteria is Lactobacillus plantarum: Lactobacillus casei: Lactobacillus reuteri is 1:1:1. In one embodiment, the addition amount of the first bacterial agent is 5‰ of the total mass of litchi. In one embodiment, the first bacterial agent is yeast: lactic acid bacteria is 50:1; the yeast is Weill yeast: Red yeast: Saccharomyces rouxii is 1:1:1; the lactic acid bacteria is Lactobacillus sake: Lactobacillus casei is 1:1. In one embodiment, the addition amount of the first bacterial agent is 0.1‰ of the total mass of litchi. In one embodiment, the first bacterial agent is a yeast: lactic acid bacteria ratio of 300:1; the yeast is Hansen yeast; the lactic acid bacteria is Lactobacillus gasseri. In one embodiment, the addition amount of the first bacterial agent is 10‰ of the total mass of litchi. In one embodiment, the first bacterial agent is a yeast: lactic acid bacteria ratio of 500:1; the yeast is baker's yeast: sake yeast ratio of 1:1; the lactic acid bacteria are Lactobacillus plantarum: Lactobacillus gasseri: Lactobacillus sake ratio of 1:1:1. In one embodiment, the addition amount of the first bacterial agent is 8‰ of the total mass of litchi. In one embodiment, the first bacterial agent is a yeast: lactic acid bacteria ratio of 200:1; the yeast is red yeast; the lactic acid bacteria are Lactobacillus plantarum: Lactobacillus sake ratio of 1:1. In one embodiment, the addition amount of the first bacterial agent is 2‰ of the total mass of litchi. In one embodiment, the first bacterial agent is a yeast: lactic acid bacteria ratio of 150:1; the yeast is baker's yeast; the lactic acid bacteria is Lactobacillus casei. In one embodiment, the amount of the first bacterial agent added is 4‰ of the total mass of litchi.

[0043] The secondary fermentation process may include adding a second carbon source to the primary fermentation filtrate, inoculating a second bacterial agent, and standing or aerating and culturing at a second temperature for a second period of time to obtain litchi enzyme, wherein the second bacterial agent is acetic acid bacteria. The second bacterial agent may be one or more of Acetobacter pasteurianus, Acetobacter aceticus, Acetobacter ghanaiensis, Acetobacter malodorus, or Acetobacter aceticus. The second carbon source may be one or more of lactose, brown sugar, glucose, molasses, or honey. The second carbon source may contain two or more sugars, such as two or more sugars with the same weight percentage. The addition amount of the second carbon source is 1%-20% of the total mass of the primary fermentation filtrate, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, or 19%. In one embodiment, the second carbon source is lactose. In one embodiment, the second carbon source is brown sugar. In one embodiment, the second carbon source is molasses: lactose is 1:1. In one embodiment, the second carbon source is glucose. In one embodiment, the second carbon source is honey. In one embodiment, the second carbon source is glucose: honey is 1:1. The second temperature can range from 15-40°C. The second time period can range from 3-15 days. In one embodiment, the second bacterial agent is Acetobacter aceticus: Acetobacter aceticus. In one embodiment, the second bacterial agent is added in an amount of 1‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is Acetobacter pasteurianus. In one embodiment, the addition amount is 5‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is Acetobacter aceticus. In one embodiment, the addition amount is 9‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is Acetobacter Ghanaianus: Acetobacter aceticus: Acetobacter pasteurianus in 1:1:1. In one embodiment, the addition amount is 0.1‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is Acetobacter malodorus: Acetobacter aceticus: Acetobacter pasteurianus in 1:1:1. In one embodiment, the addition amount is 0.9‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is acetic acid bacteria. In one embodiment, the addition amount is 3‰ of the total mass of the primary fermentation filtrate. In one embodiment, the second bacterial agent is Acetobacter ghanaensis. In one embodiment, the addition amount is 10‰ of the total mass of the primary fermentation filtrate.

[0044] As described herein, "Sophora japonica flower" refers to the dried flowers and buds of the leguminous plant Sophora japonica. The 2015 edition of the Chinese Pharmacopoeia stipulates that the total flavonoids contained in Sophora japonica flower, calculated as rutin, shall not be less than 8.0%; the rutin content in Sophora japonica flower shall not be less than 6.0%. In this article, Sophora japonica flower can be in the form of Sophora japonica flower powder. Sophora japonica floret can comprise not less than 6wt% rutin, for example 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, 36wt%, 37wt%, 38wt% or 39wt%.

[0045] The composition described herein may include edible plant enzymes and Sophora japonica. The composition may be an edible composition. The composition may include edible plant enzymes and Sophora japonica, and the weight ratio of edible plant enzymes to Sophora japonica is 7:2-29:2. The weight ratio of edible plant enzymes to Sophora japonica may be 8:2, 9:2, 10:2, 11:2, 12:2, 13:2, 14:2, 15:2, 16:2, 17:2, 18:2, 19:2, 20:2, 21:2, 22:2, 23:2, 24:2, 25:2, 26:2, 27:2 or 28:2.

[0046] The edible plant enzyme composition herein can be prepared as an oral composition. The oral composition can be an oral liquid, a beverage or a soft candy. The edible plant enzyme composition herein can be prepared into a skin moisturizing composition or a pharmaceutical composition after adding applicable ingredients. The skin moisturizing composition can include the edible plant enzyme composition herein. The skin moisturizing composition can further include ingredients for skin moisturizing, such as sodium hyaluronate. The skin moisturizing composition can also include fillers, such as resistant dextrin or oligofructose, etc. The pharmaceutical composition can include the composition described herein and a pharmaceutically acceptable excipient.

[0047] The edible plant enzyme or skin moisturizing composition described herein can be used in a cosmetic method. The method can include a method for moisturizing the skin in a subject, a method for maintaining and / or improving skin health in a subject, or a method for the non-therapeutic purpose of improving skin inflammation in a subject. The method can include applying the edible plant enzyme composition described herein or the skin moisturizing composition described herein to the subject. The mode of application can include oral administration.

[0048] The edible plant enzyme or skin moisturizing composition described herein can be used in an adjunctive therapy method. The adjunctive therapy method includes administering the edible plant enzyme composition or pharmaceutical composition described herein to a subject. The mode of administration can include oral administration.

[0049] Herein, a "subject" may be an object suitable for taking the composition described herein, such as a mammal. Herein, the subject may be a human.

[0050] The present invention will be further explained in detail with reference to the following examples. However, it should be understood by those skilled in the art that these examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0051] Example

[0052] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conditions recommended by normal conditions or manufacturers. The reagents used or the instruments not specified by the manufacturer are all conventional products that can be obtained commercially. Unless otherwise specified, all amounts listed are described in weight percentage based on gross weight. The application should not be construed as being limited to the specific embodiments described.

[0053] Preparation of litchi enzyme:

[0054] (1) Pretreatment: 5 tons of litchi fruits were washed and squeezed;

[0055] (2) Primary fermentation: add 1% sugar to the mixture of fruit juice and pomace, inoculate 1% bacterial agent, and ferment at 25°C in a sealed container for 48 hours;

[0056] (3) Filtration: using a filter press to obtain a primary fermentation filtrate, wherein the mass of the primary fermentation filtrate is greater than 60% of the total mass of the litchi fruit;

[0057] (4) Secondary fermentation: add sugar 2 to the primary fermentation filtrate, inoculate bacterial agent 2, and culture at 40°C for 9 days with aeration;

[0058] (5) Filtration and sterilization.

[0059] In the above preparation method, sugar 1 is fructose: sucrose: brown sugar in a ratio of 1:1:1; the addition amount of sugar 1 is 3% (w / w); bacterial agent 1 is yeast: lactic acid bacteria in a ratio of 500:1; yeast is baker's yeast: sake yeast in a ratio of 1:1; lactic acid bacteria are plant lactobacillus: gasseri lactobacillus: sake lactobacillus in a ratio of 1:1:1; the addition amount of bacterial agent 1 is 8‰ of the total mass of litchi. Sugar 2 is honey; the addition amount of sugar 2 is 20% of the total mass of the primary fermentation filtrate; bacterial agent 2 is malodorous acetic acid bacteria: acetobacter: pasteurian acetobacter in a ratio of 1:1:1; the addition amount of bacterial agent 2 is 0.9‰ of the total mass of the primary fermentation filtrate; the culture method is aeration culture with a ventilation volume of 5mL / min / mL of fermentation liquid. It has been determined that the above litchi enzyme meets the liquid edible plant enzyme standard of the light industry standard QB / T 5323-2018 of the People's Republic of China.

[0060] Preparation of composition

[0061] Sophora japonica flower (Jiangyin Tianjiang Pharmaceutical Co., Ltd.; rutin content not less than 10%); Osmanthus fragrans (Ningxia Vanilla Biotechnology Co., Ltd.); Sophora japonica flower and litchi fermentation liquid composition is prepared by mixing Sophora japonica flower powder and litchi fermentation liquid in proportion.

[0062] Composition 1 (Sophora japonica flower: litchi fermentation liquid = 2:7); ​​Composition 2 (Sophora japonica flower: litchi fermentation liquid = 1:7); Composition 3 (Sophora japonica flower: litchi fermentation liquid = 2:29); Composition 4 (Sophora japonica flower: litchi fermentation liquid = 1:3); Composition 5 (Sophora japonica flower: litchi fermentation liquid = 1:15).

[0063] Example 1: The fermented liquor composition of Sophora japonica and Lychee promotes the production of sodium hyaluronate

[0064] Moisturizing test in human immortalized keratinocytes HaCaT: HA content

[0065] Reagents and instruments:

[0066] Reagents: DMEM culture medium, fetal bovine serum, penicillin-streptomycin double antibiotics, 0.25% trypsin-EDTA digestion solution, PBS solution, HA-ELISA kit.

[0067] Instruments: centrifuge, microscope, multifunctional microplate reader, 12-well plate, CO2 constant temperature incubator, cell culture consumables.

[0068] Experimental methods:

[0069] ELISA detection of HA content in cell supernatant

[0070] 1. Modeling method: Select human immortalized keratinocytes HaCaT, and when HaCaT fills 70% of the 12-well plate, place it in a clean bench and expose it for 20 minutes. The control conditions are windless and the temperature is 25°C, and the cell viability is reduced to 50%-60%;

[0071] 2. Add 0.5 mL of experimental sample material (the sample material is Sophora japonica, litchi enzyme solution or composition 1-5) (according to the ratio of the above composition 1-5, litchi enzyme and Sophora japonica are prepared into a solution with a total concentration of 300 μg / mL. The specific preparation is shown in Table 1. Dissolve the weighed sample and make a fixed volume into 1 mL of solution) and incubate for 24 hours;

[0072] Table 1: Configuration of fermented liquor with different ratios of Sophora japonica flower and litchi

[0073]

[0074] 3. Aspirate the cell supernatant and centrifuge at 3000rpm for 20min to remove cell debris. Since the HA content in the cell supernatant is high, it needs to be diluted 10 times for later use.

[0075] 4. According to the instructions of the ELISA kit (HA-ELISA; Product No.: CSB-E04805h; Wuhan Huamei), draw a standard curve and calculate the corresponding HA content according to the OD value. The specific operation process is as follows:

[0076] Standard curve sample preparation

[0077] 1. Take out a standard from the HA-ELISA kit and centrifuge at 6000-10000rpm for 30 seconds. Dissolve it with 1ml sample diluent and repeatedly pipette 5 times at the bottom of the cryotube to help dissolve it. Mix thoroughly to obtain standard S7 and set aside for later use.

[0078] 2. Arrange 7 1.5ml centrifuge tubes (S0-S6) in sequence and add 250ul of sample diluent to each. Pipette 250ul of standard S7 into the first centrifuge tube (S6) and gently pipette to mix. Pipette 250ul from S6 into the second EP tube (S5) and gently pipette to mix. Repeat this process to dilute the standard sample in multiples.

[0079] Testing process

[0080] All reagents and samples were brought to room temperature before use. Samples were thawed and centrifuged again. All samples and standards were analyzed in duplicate.

[0081] ③ Add 100 μl of standard and sample to each well, cover with the provided gel strip and incubate at 37°C for 2 hours.

[0082] ④Take out the liquid from each hole without cleaning.

[0083] ⑤ Add 100 μl of biotin antibody (1x) to each well, cover with a new gel strip, and incubate at 37°C for 1 hour.

[0084] ⑥Use a multichannel pipette to aspirate and wash each well three times. Then fill each well with washing solution (200 μl) and let it stand for 2 minutes. After the last wash, remove any remaining washing buffer by aspirating. Then turn the 96-well plate upside down and dry it with a clean paper towel.

[0085] ⑦ Add 100 μl (1x) of enzyme-labeled avidin to each well, cover the microtiter plate with a new gel strip, and incubate at 37°C for 1 hour.

[0086] ⑧As in step 6, repeat the aspiration / washing process 5 times.

[0087] ⑨Add 90 μl TMB substrate to each well and incubate at 37°C for 15-30 minutes, away from light.

[0088] ⑩ Add 50 μl of stop solution to each well and tap the plate to ensure thorough mixing.

[0089] Determine the OD value of each well within 5 minutes and read it using a microplate reader set at 450 nm.

[0090] Standard curve results:

[0091] Table 2. Concentration and OD value of sodium hyaluronate standard

[0092]

[0093] Figure 1 The dilution process of the HA standard stock solution is shown. Figure 2 A sodium hyaluronate standard curve is shown.

[0094] 5. Experimental groups: blank group (the sample material added was DMEM culture medium); Sophora japonica group; Litchi enzyme group; Composition 1-5 groups; 3 parallels in each group. The ELISA kit was used to detect the content of sodium hyaluronate (HA) in the supernatant after cell centrifugation with reference to the standard curve.

[0095] result

[0096] Figure 4 Table 3 shows the experimental data of hyaluronic acid production of the blank group, Sophora japonica group, litchi enzyme liquid group, composition 1 (Sophora japonica: litchi fermentation liquid = 2:7), composition 2 (Sophora japonica: litchi fermentation liquid = 1:7), composition 3 (Sophora japonica: litchi fermentation liquid = 2:29), composition 4 (Sophora japonica: litchi fermentation liquid = 1:3), and composition 5 (Sophora japonica: litchi fermentation liquid = 1:15).

[0097] Under the same addition amount, the production amount of sodium hyaluronate (HA) in composition 1 (sophora japonica: litchi fermentation liquid = 2:7), composition 2 (sophora japonica: litchi fermentation liquid = 1:7), and composition 3 (sophora japonica: litchi fermentation liquid = 2:29) is significantly higher than the production amount of sodium hyaluronate (HA) in the case of single materials of sophora japonica and litchi fermentation liquid, while the compositions of sophora japonica: litchi fermentation liquid at other ratios of 1:3 and 1:15 do not have this result. This result proves that litchi fermentation liquid: sophora japonica has a synergistic effect on the production amount of sodium hyaluronate (HA) when the ratio is 7:2 to 15:1.

[0098] Figure 5 Table 4 shows the experimental data of hyaluronic acid production of the blank group, osmanthus group, litchi enzyme liquid group, and osmanthus litchi fermentation liquid composition (osmanthus: litchi fermentation liquid = 1:5). Under the same addition amount, the composition (osmanthus: litchi fermentation liquid = 1:5) produced significantly less sodium hyaluronate (HA) than the litchi fermentation liquid of the same concentration. No synergistic effect of increasing the production of sodium hyaluronate was found in the ratio of osmanthus: litchi fermentation liquid at 1:5.

[0099] Table 3: Hyaluronic acid production experimental data

[0100]

[0101] Table 4: Hyaluronic acid production experimental data

[0102]

[0103] Example 2: Anti-inflammatory effect of the Sophora japonica and litchi fermented liquid composition.

[0104] Anti-inflammatory assay in immortalized human keratinocytes HaCaT: NO release

[0105] Reagents and instruments:

[0106] Reagents: DMEM culture medium, fetal bovine serum, penicillin-streptomycin double antibody, 0.25% trypsin-EDTA digestion solution, dexamethasone, LPS.

[0107] Instruments: centrifuge, microplate reader, 24-well plate, CO2 constant temperature incubator, cell culture consumables.

[0108] Experimental methods:

[0109] 1. When HaCaT cells have grown 30% of the 24-well plate, add 250uL of LPS (concentration 10μg / mL) and 250uL of the test material (sophora flower, osmanthus flower, litchi enzyme solution or combination 1-5), and set up a normal group with 500uL of DMEM and a model group with 500uL of LPS;

[0110] 2. Process for three days;

[0111] 3. Take 50uL of supernatant from each well and add the reagent in the NO release test kit;

[0112] 4. Measure the absorbance at A540nm, make a standard curve based on the standard, and calculate the value of NO release.

[0113] Standard curve preparation process:

[0114] 1. Take out Griess Reagent I and II and return them to room temperature.

[0115] 2. Dilute the standard (1-100 μM) with the solution used for the sample to be tested.

[0116] 3. Add standards and samples to a 96-well plate at 50 μl / well.

[0117] 4. Add 50 μl / well of room temperature Griess Reagent I to each well.

[0118] 5. Add 50 μl / well of room temperature Griess Reagent II to each well.

[0119] 6.540nm absorbance was measured. Standard curve results:

[0120] Table 5: Nitric oxide standard concentration and OD value ( Figure 3 )

[0121]

[0122] Figure 3 A nitric oxide standard curve is shown.

[0123] NO inhibition rate % = (NO release in model group - NO release in sample group) / NO release in model group * 100%

[0124] Experimental groups: LPS-induced inflammatory cell model group (the material to be tested was DMEM culture medium); Sophora japonica group, litchi enzyme liquid group, composition 1 (Sophora japonica: litchi fermentation liquid = 2:7), composition 2 (Sophora japonica: litchi fermentation liquid = 1:7), composition 3 (Sophora japonica: litchi fermentation liquid = 2:29), composition 4 (Sophora japonica: litchi fermentation liquid = 1:3), composition 5 (Sophora japonica: litchi fermentation liquid = 1:15); Osmanthus group; Osmanthus litchi enzyme group (the materials to be tested were Sophora japonica, litchi enzyme liquid, compositions 1-5, Osmanthus and Osmanthus litchi enzyme), with 3 parallels in each group.

[0125] result

[0126] Figure 6The NO inhibition rate data of the Sophora japonica flower group, litchi enzyme liquid group, composition 1 (Sophora japonica flower: litchi fermentation liquid = 2:7), composition 2 (Sophora japonica flower: litchi fermentation liquid = 1:7), composition 3 (Sophora japonica flower: litchi fermentation liquid = 2:29), composition 4 (Sophora japonica flower: litchi fermentation liquid = 1:3), and composition 5 (Sophora japonica flower: litchi fermentation liquid = 1:15) are shown.

[0127] Under the same addition amount, the inhibition rate of NO in inflammatory cells of composition 1 (sophora japonica: litchi fermented liquor = 2:7), composition 2 (sophora japonica: litchi fermented liquor = 1:7), and composition 3 (sophora japonica: litchi fermented liquor = 2:29) was significantly higher than the inhibition rate of NO in inflammatory cells of single materials of sophora japonica and litchi fermented liquor, while the compositions of sophora japonica: litchi fermented liquor at other ratios of 1:3 and 1:15 did not have this result. The composition containing sophora japonica and litchi fermented liquor has a synergistic effect on the inhibition rate of NO in inflammatory cells when the ratio is 7:2-29:2.

[0128] Figure 7 The data of NO inhibition rate of the osmanthus group, litchi enzyme liquid group, and osmanthus litchi fermentation liquid combination (osmanthus: litchi fermentation liquid = 1:7) are shown. Under the same concentration conditions, the combination (sophora flower: litchi fermentation liquid = 1:7) has a significantly higher inhibition rate on inflammatory cell NO than the litchi fermentation liquid at the same concentration. No synergistic effect was found at the ratio of osmanthus: litchi fermentation liquid of 1:7.

[0129] Table 6: Inhibition rate of NO in inflammatory cells

[0130]

[0131] Table 7: Inhibition rate of NO in inflammatory cells

[0132]

[0133] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An edible plant enzyme composition, comprising edible plant enzyme and Sophora japonica flower, wherein the weight ratio of edible plant enzyme to Sophora japonica flower is 7:2-29:2, the edible plant enzyme is litchi enzyme, and the litchi enzyme is litchi fermentation liquid, wherein the litchi fermentation liquid is prepared by the following method: The method comprises (1) primary fermentation, which comprises adding a first carbon source to litchi juice and litchi pomace, inoculating a first bacterial agent, and sealing and standing for fermentation at 20-45° C. for 24-96 hours, wherein the first bacterial agent is a mixed bacterial agent of yeast and lactic acid bacteria, the yeast is baker's yeast and sake yeast, the lactic acid bacteria are plant lactobacillus, gasseri lactobacillus and sake lactobacillus, and the first carbon source is one or more of fructose, sucrose, brown sugar, maltose and arabinose; (2) squeezing and filtering the product after the primary fermentation to obtain a primary fermentation filtrate; and (3) secondary fermentation, which comprises adding a second carbon source to the primary fermentation filtrate, inoculating a second bacterial agent, standing or aerating and culturing at 15-40° C. for 3-15 days to obtain a secondary fermentation liquid, the second bacterial agent is malodorous acetic acid bacteria, acetobacter aceticus and pasteurian acetobacter, and the second carbon source is one or more of lactose, brown sugar, glucose, molasses or honey; The Sophora japonica flower contains no less than 10wt% of rutin.

2. The edible plant enzyme composition according to claim 1, wherein the edible plant enzyme composition is an oral composition.

3. The edible plant enzyme composition according to claim 2, wherein the oral composition is an oral liquid, a beverage or a soft candy.

4. A skin moisturizing composition comprising the edible plant enzyme composition according to any one of claims 1 to 3, wherein the skin moisturizing composition is orally administered.

5. The skin moisturizing composition according to claim 4, further comprising one or more of sodium hyaluronate, resistant dextrin and oligofructose.

6. A pharmaceutical composition comprising the edible plant enzyme composition according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

7. A health-care composition comprising the edible plant enzyme composition according to any one of claims 1 to 3 and an excipient acceptable to health-care foods.

8. Use of the edible plant enzyme composition according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 6 in the preparation of an oral medicament for improving skin inflammation.

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