CN111789773A

CN111789773A - Small molecular water cluster composition and preparation method thereof

Info

Publication number: CN111789773A
Application number: CN202010277428.6A
Authority: CN
Inventors: 李茂锐
Original assignee: Guangzhou Hengguang Composite Material Co ltd
Current assignee: Guangzhou Hengguang Composite Material Co ltd
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-10-20

Abstract

The invention relates to the technical field of biological medicines, in particular to a small molecular water cluster composition and a preparation method thereof. The micromolecular water cluster composition is prepared by adopting micromolecular water and biological enzyme, the specific biological enzyme is selected to stabilize the micromolecular water for a long time, and meanwhile, the micromolecular water cluster composition prepared by the invention can play a role in enhancing a skin barrier by promoting the terminal differentiation of keratinocytes, the up-regulation expression of transcription levels of intercellular connexin and keratinocyte envelope protein, can remarkably promote antibacterial humoral response, humoral immune response, antibacterial peptide-mediated antimicrobial humoral immune response, bacterial defense response and genes related to other biological defense response, and has an immunoregulation effect.

Description

Small molecular water cluster composition and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a small molecular water cluster composition and a preparation method thereof.

Background

Water of nature is not a single water molecule (H)₂O) form, water molecule H₂The O cannot vary in size and is composed of two hydrogen H atoms and one oxygen O atom. But the water molecules do not exist independently, and the water molecules are mutually attracted by means of hydrogen bonds (hydrogen bonds) and exist in the form of cohesive masses. The V-shaped structure of the HOH molecule and the polarity of the O-H bond cause asymmetric distribution of charges, and this polarity degree causes attraction between water molecules, so that water molecules are associated together to a considerable extent to form a water molecule cluster, commonly called a water molecule cluster, also called a water cluster, a small molecule cluster running water, or a small water molecule cluster (micro cluster water), or become a structured small molecule cluster water (structured water). The water molecule cluster is a non-continuous oneThe water molecule cluster compound formed by the continuous hydrogen bond structure. In the market, the small molecular group water is also often referred to as "acidic water", "alkaline water", "activated water", "small molecular group water", "functional water", "energy water", "oxygen-enriched water", and the like.

The water is usually a water molecule group consisting of more than 12 water molecules, called macromolecular group water or dead water, such as rainwater, lake water, tap water, bottled water, purified water and distilled water. The natural small molecular group water consists of 5-8 water molecules, is formed under rare natural conditions, and has few producing areas; the water in the longevity village, the natural flowing non-polluted mountain spring water, the original glacier water and the water which are all small molecular groups are also called as the running water. Small cluster water is distinguished from regular water, and water above 100Hz is generally stated to be regular water, and water below 100Hz is defined as small cluster water. The small molecular group water is active water with high penetrability, high diffusibility and high dissolving power, can bring more water and nutrition into cells, and simultaneously better discharges waste and poison. Since the small molecular group water is more easily absorbed and utilized by each cell in the body, the biological utilization rate of the small molecular group water is much higher than that of common water, and the small molecular group water can enhance the efficiency and the function of metabolism, enhance the information transfer between each cell and surrounding organizations, obviously improve the transmission supply of nutrients in the whole body, improve the transportation capacity of oxygen in the whole body and the like. However, the small molecular water has poor stability due to small particle size, and generally will not agglomerate to form large molecular water in more than 4 hours.

Aiming at the problems, the invention adopts micromolecular water and specific biological enzyme to prepare the micromolecular water cluster composition, and the biological enzyme can stabilize the micromolecular water for a long time; meanwhile, the small molecular water can guide biological enzymes with the functions of catalyzing, decomposing and mastering life activities to act on the skin and pores, and the most original and effective mode is carried out for deep cleaning, skin repairing and moistening, so that the elasticity and luster of the skin are kept. The small molecular water cluster composition prepared by the invention can play a role in enhancing the skin barrier by promoting the terminal differentiation of keratinocytes, the up-regulated expression of intercellular connexins and the transcription level of keratin-derived proteins. Meanwhile, the immune response can be obviously improved, and the immune response can also obviously improve the antibacterial humoral response, the humoral immune response, the antimicrobial humoral immune response mediated by antibacterial peptide, the bacterial response, the bacterial defense response and the genes related to the defense response of other organisms, and has the immune regulation effect.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a cell viability curve;

FIG. 2 is a graph showing the results of the cell morphology assay;

FIG. 3 is a differential gene distribution volcano plot;

FIG. 4 is a bar graph of differential gene GO enrichment with GO Term on the abscissa and the significance level of GO Term enrichment on the ordinate;

FIG. 5 is a graph of differential gene GO enrichment bubbles, with the abscissa being the ratio of the differential gene factors annotated to GO Term to the total number of differential genes and the ordinate being GO Term;

FIG. 6 is a KFGG enrichment analysis result graph of up-regulated genes, the abscissa is a KEGG pathway, the ordinate is the significance level of pathway enrichment, and the higher the numerical value is, the more significant the significance is;

FIG. 7 is a plot of the KFGG enrichment analysis results of up-regulated genes, with the abscissa being the ratio of the differential basis factors annotated to the KEGG pathway to the total number of differential genes, the ordinate being the KEGG pathway, the size of the dots representing the basis factors annotated to the KEGG pathway, and the color from gray to black representing the significance of the enrichment;

FIG. 8 is a list of skin barrier-associated genes;

FIG. 9 shows skin barrier-associated genes.

Disclosure of Invention

In order to solve the above problems, a first aspect of the present invention provides a small molecule water cluster composition, comprising at least the following components in parts by weight: 0-200 parts of micromolecular water and 0-2 parts of biological enzyme; the weight parts of the micromolecule water and the biological enzyme are not 0.

As a preferable technical scheme, the paint at least comprises the following components in parts by weight: 50-150 parts of micromolecular water and 0-2 parts of biological enzyme; the weight part of the biological enzyme is not 0.

As a preferable technical scheme, the paint at least comprises the following components in parts by weight: 80-120 parts of micromolecular water and 0.5-1.8 parts of biological enzyme.

As a preferable technical scheme, the paint at least comprises the following components in parts by weight: 90-110 parts of small molecular water and 1-1.5 parts of biological enzyme.

As a preferable technical scheme, the paint at least comprises the following components in parts by weight: 95-105 parts of micromolecular water and 0.8-1.2 parts of biological enzyme.

As a preferable technical scheme, the paint at least comprises the following components in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

As a preferred technical scheme, the biological enzyme comprises at least one of protease, amylase and cellulase.

In a preferred embodiment, the protease is at least one selected from bromelain, papain, ficin, soybean protease, actinidin, and siraitia grosvenorii protease.

In a second aspect, the present invention provides a method for preparing a small molecule water cluster composition, comprising at least the following steps: mixing small molecular water and biological enzyme.

In a third aspect, the invention provides a small molecule water cluster composition for use in cosmetics.

Has the advantages that: the micromolecular water cluster composition is prepared by adopting micromolecular water and biological enzyme, the specific biological enzyme is selected to stabilize the micromolecular water for a long time, and meanwhile, the micromolecular water cluster composition prepared by the invention can play a role in enhancing a skin barrier by promoting the terminal differentiation of keratinocytes, the up-regulation expression of transcription levels of intercellular connexin and keratinocyte envelope protein, can remarkably promote antibacterial humoral response, humoral immune response, antibacterial peptide-mediated antimicrobial humoral immune response, bacterial defense response and genes related to the defense response of other organisms, and has an immunoregulation effect.

Detailed Description

The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, but the scope of protection is not limited thereto.

"preferred", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may, in some instances, provide certain beneficial results. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

The invention provides a small molecular water cluster composition, which at least comprises the following components in parts by weight: 0-200 parts of micromolecular water and 0-2 parts of biological enzyme; the weight parts of the micromolecular water and the biological enzyme are not 0.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 20-180 parts of small molecular water and 0-2 parts of biological enzyme; the weight part of the biological enzyme is not 0.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 50-150 parts of micromolecular water and 0-2 parts of biological enzyme; the weight part of the biological enzyme is not 0.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 80-120 parts of micromolecular water and 0.5-1.8 parts of biological enzyme.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 90-110 parts of small molecular water and 1-1.5 parts of biological enzyme.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 95-105 parts of micromolecular water and 0.8-1.2 parts of biological enzyme.

In a preferred embodiment, at least the following ingredients are included in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

In a preferred embodiment, the weight ratio of the small molecule water to the biological enzyme is (98-100): 1.

Small molecule water

In a preferred embodiment, the preparation method of the small molecule water comprises the following steps: sending the highly mixed hydrogen and water mixed solution with the pressure of 7-9kg into a micromolecular water preparation device, penetrating through a permanent magnetic field of 40-50 kilogausses at the flow speed of 12m/s, and vertically cutting magnetic lines to obtain the hydrogen and water mixed solution.

In a more preferred embodiment, the preparation method of the small molecule water comprises the following steps: sending the highly mixed hydrogen and water mixed solution with the pressure of 8kg into a micromolecular water preparation device, passing through a 45 kilogauss permanent magnetic field at the flow speed of 12m/s, and vertically cutting magnetic lines of force to obtain the hydrogen and water mixed solution.

In a preferred embodiment, the volume ratio of hydrogen to water is 1: 1.

the micromolecule water has the following seven characteristics of orderly arranged molecular structures, high density, high oxygen content, no free charge, high cohesion energy and the like:

(1) structure matching: the human cell wall is provided with aquaporin, the diameter of the channel is only 2 nanometers and is hexagonal, the structure of the small molecular water is also hexagonal, and the diameter of the small molecular water is only 0.5 nanometer, so that the small molecular water can bring nutrient substances into cells through the channel, enhance the activity of the cells, and is a good carrier of nutrient components.

(2) The permeability is strong: the micromolecule water has small particles and strong permeability, can be directly absorbed by skin, can quickly enter human cells, ensures that the cells have sufficient clean water, ensures that intracellular water and extracellular water reach a balanced state, promotes metabolism to be normally carried out, and ensures that the whole human body is in a healthy state.

(3) Strong dissolving power: the water solubility of the micromolecule water is more than 30 percent higher than that of the common water. Firstly, nutrient substances can be decomposed and conveyed to be brought into cells, the nutrient substances taken in by a human body are in a macromolecular group structure, micromolecular water can decompose decomposable substances into micromolecular group structures, and undegradable substances are emulsified and coagulated to be brought into the cells; the residues and toxic and harmful substances remained in cells and on the vessel wall for a long time can be dissolved and taken out of the body; thirdly, the alcohol can be effectively neutralized and decomposed, the alcohol content in the body is reduced, and the drunkenness is prevented; fourthly, the toxin expelling capability of the body is improved, and the cardiovascular and cerebrovascular diseases can be prevented and improved.

(4) Strong emulsifying power: the small molecular water can emulsify redundant cholesterol and triglyceride in blood and is discharged out of the body through metabolic absorption.

(5) Strong metabolic capacity: metabolic force is the ability to be excreted and is of great importance to human health. The micromolecule water can promote the discharge of vivotoxin to a certain extent, so that the body has a clean environment and a healthy state, and has certain defense and treatment effects on diseases.

(6) Strong cleaning ability: the micro-molecular water belongs to alkalescent water, can neutralize and remove acidic waste, maintain alkalescent levels of body fluid, blood and cell sap, promote the circulation of human body capillaries to be accelerated, improve the nourishing supply, toxin expelling and detoxifying abilities of the body and promote metabolism.

(7) Strong activating force: the small molecular group water has high oxygen content, has a large amount of energy and high movement speed, is called as active water, enters each corner of a human body and cells, continuously activates the cells, enables the inside and the outside of the cells to be filled with clean, active and rich-nutrition liquid, and can carry more nutrients, mineral substances and oxygen which are beneficial to the human body, thereby greatly promoting the growth and the development of the cells, enabling the cells of the human body to be more active and enabling the human body to be in a normal running state at any time.

However, small molecular water has poor stability due to its small particle size and will not agglomerate in more than 4 hours.

Biological enzymes

The enzyme exists in all living plants, is a substance with special biological activity and consists of amino acid, and has strong catalytic action on a plurality of organic chemical reactions and reactions which are reviewed in organisms.

In a preferred embodiment, the biological enzyme comprises at least one of a protease, an amylase, and a cellulase.

In a more preferred embodiment, the biological enzyme comprises a protease.

In a preferred embodiment, the protease is selected from at least one of bromelain, papain, ficin, glycanase, actinidin, and luo han guo protease.

In a more preferred embodiment, the protease is bromelain, papain.

In a preferred embodiment, the weight ratio of bromelain to papain is (1-3): 1.

In a more preferred embodiment, the weight ratio of bromelain to papain is 2: 1.

Bromelain

In a preferred embodiment, the method for preparing bromelain comprises the following steps:

(1) cleaning pineapple peel, juicing, filtering to remove large particle impurities, pre-cooling, and adding NaHSO₃Cysteine, ethylene diamine tetraacetic acid and vitamin B1, centrifuging for 10-30min at 2000-;

(2) adding polyacrylic acid into the supernatant obtained in the step (1), placing the mixture at the temperature of 2-6 ℃ for cold coagulation for 8-12h, centrifuging the mixture for 10-30min at the speed of 2000-4000r/min, and collecting precipitates;

(3) adding a calcium acetate aqueous solution into the precipitate obtained in the step (2), centrifuging at 2000-4000r/min, taking the supernatant, adding a buffer solution, dialyzing at 2-6 ℃ for 8-12 hours, and finally freeze-drying to obtain the calcium acetate.

In a more preferred embodiment, the method for preparing bromelain comprises the following steps:

(1) cleaning pineapple peel, squeezing to obtain juice, and filteringFiltering to remove large particle impurities, pre-cooling, adding powder NaHSO₃Centrifuging at 3000r/min for 20min with powdered cysteine, powdered ethylenediamine tetraacetic acid and powdered vitamin B1, and collecting supernatant;

(2) adding polyacrylic acid into the supernatant obtained in the step (1), condensing and polymerizing for 10 hours at 4 ℃, centrifuging for 20 minutes at 3000r/min, and collecting precipitates;

(3) slowly adding a calcium acetate aqueous solution into the precipitate obtained in the step (2) until the precipitate is completely dissolved, centrifuging at 3000r/min, taking the supernatant, adding a buffer solution, dialyzing at 4 ℃ for 10 hours, and finally freeze-drying to obtain the calcium phosphate.

In a preferred embodiment, the temperature of the pre-cooling treatment is 0-5 ℃, and the time of the pre-cooling treatment is 10-20 h.

In a more preferred embodiment, the temperature of the pre-cooling treatment is 3 ℃, and the time of the pre-cooling treatment is 15 h.

In a preferred embodiment, the powder NaHSO is contained in the pineapple juice per unit volume₃The addition amount of (A) is 0.1-0.5 mg/L.

In a more preferred embodiment, the powder NaHSO is contained in the pineapple juice per unit volume₃The amount of (B) added was 0.3 mg/L.

In a preferred embodiment, the powdered NaHSO₃The weight ratio of the powder cysteine to the powder ethylenediamine tetraacetic acid to the powder vitamin B1 is (0.1-0.5): (0.02-0.06): (0.3-0.7): (0.01-0.05).

In a more preferred embodiment, the powdered NaHSO₃The weight ratio of the powder cysteine to the powder ethylene diamine tetraacetic acid to the powder vitamin B1 is 0.3: 0.05: 0.5: 0.03.

in a preferred embodiment, the polyacrylic acid is added in an amount of 0.5 to 1% by weight based on the supernatant obtained in step (1).

In a more preferred embodiment, the polyacrylic acid is added in an amount of 0.8% by weight of the supernatant obtained in step (1).

In a preferred embodiment, the concentration of the aqueous calcium acetate solution is from 35 to 45 mmol/L.

In a more preferred embodiment, the concentration of the aqueous calcium acetate solution is 40 mmol/L.

In a preferred embodiment, the buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

In a preferred embodiment, the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer has a concentration of 0.04 to 0.06mol/L and a pH of 6.5 to 7.

In a preferred embodiment, the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer has a concentration of 0.05mol/L and a pH of 6.8.

In a preferred embodiment, the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is added in an amount of 2 to 6 times the volume of the supernatant in step (3).

In a more preferred embodiment, the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is added in an amount of 4 times the volume of the supernatant in step (3).

In a preferred embodiment, the freeze-drying temperature is-40 ℃.

The CAS number of the cysteine is 4371-52-2.

The vitamin B1 has a CAS number of 59-43-8.

The CAS number of the polyacrylic acid is 9003-01-4.

Papain

In a preferred embodiment, the method for preparing papain comprises the following steps:

(a) cleaning and juicing peeled and deseeded papaya, adding complex enzyme to react at 40-50 ℃ for 1-2h, filtering, removing filter residue, centrifuging at 2000-4000r/min for 10-30min, taking supernatant, and refrigerating at 0-5 ℃ for later use;

(b) adding an ethanol water solution into the supernatant obtained in the step (a), then adding cysteine, ethylenediamine tetraacetic acid and vitamin B1, adjusting the pH value to 6-8 by using a sodium hydroxide water solution, standing for 8-12h, centrifuging at 2000-4000r/min, taking the supernatant, and finally freeze-drying to obtain the compound.

In a more preferred embodiment, the method for preparing papain comprises the following steps:

(a) cleaning papaya after peeling and removing seeds, adding water with the same amount as papaya, juicing, adding complex enzyme, reacting at 45 ℃ for 1.5h, performing suction filtration, removing filter residue, centrifuging at 3000r/min for 20min, collecting supernatant, and refrigerating at 3 ℃ for later use;

(b) adding an equal volume of ethanol aqueous solution into the supernatant obtained in the step (a), then adding powdered cysteine, powdered ethylenediamine tetraacetic acid and powdered vitamin B1, adjusting the pH value to 7 by using a sodium hydroxide aqueous solution, standing for 10h, centrifuging at 3000r/min, taking the supernatant, and finally freeze-drying to obtain the compound vitamin A.

In a preferred embodiment, the addition amount of the compound enzyme is 0.3-0.8% of the weight of the papaya.

In a more preferred embodiment, the complex enzyme is added in an amount of 0.5% by weight of the papaya.

In a preferred embodiment, the complex enzyme is selected from at least one of pectinase, cellulase, amylase and glucanase.

In a more preferred embodiment, the complex enzyme is cellulase or glucanase.

In a preferred embodiment, the weight ratio of the cellulase to the glucanase is (2-4): 1.

in a more preferred embodiment, the cellulase to glucanase weight ratio is 3: 1.

in a preferred embodiment, the concentration of the aqueous ethanol solution is 70 to 80 wt%.

In a more preferred embodiment, the concentration of the aqueous ethanol solution is 75 wt%.

In a preferred embodiment, the amount of powdered cysteine added per unit volume of the supernatant obtained in step (a) is 1 to 3 g/L.

In a more preferred embodiment, the amount of powdered cysteine added per unit volume of the supernatant obtained in step (a) is 2 g/L.

In a preferred embodiment, the weight ratio of the powder cysteine to the powder ethylenediaminetetraacetic acid to the powder vitamin B1 is (1-3): (0.5-1.5): (0.2-0.4).

In a more preferred embodiment, the weight ratio of powdered cysteine, powdered ethylenediaminetetraacetic acid, powdered vitamin B1 is 2: 1: 0.3.

in a preferred embodiment, the concentration of the aqueous sodium hydroxide solution is 0.5 to 2 mol/L.

In a preferred embodiment, the concentration of the aqueous sodium hydroxide solution is 1 mol/L.

In a preferred embodiment, the freeze-drying temperature is-40 ℃.

The CAS number of the cellulase is 9012-54-8.

The CAS number of the glucanase is 9025-70-1.

In a preferred embodiment, the method for preparing the biological enzyme comprises the following steps: mixing bromelain, papain and water, performing aerobic fermentation at 36-38 deg.C for 10-30 days, stirring once every 10-20 hr after fermentation period, stirring for 10-30min, stopping stirring after 5-15 days, standing for 5-15 days, and filtering.

In a more preferred embodiment, the method for preparing the biological enzyme comprises the following steps: mixing bromelain, papain and water, performing aerobic fermentation at 37 deg.C for 20 days, stirring every 15 hr for 20min after fermentation, stopping stirring after 10 days, standing for 10 days, and filtering.

In a preferred embodiment, the weight of the water is 1-2 times the sum of the weight of the bromelain and the weight of the papain.

In a more preferred embodiment, the weight of water is 1.5 times the sum of the weight of bromelain and the weight of papain.

The applicant unexpectedly finds that when the bromelain and the papain prepared by the method are compounded and the biological enzyme is prepared by aerobic fermentation, the weight ratio of the small molecular water to the biological enzyme is limited to (98-100): 1, the weight ratio of the pineapple proteinase to the papain is (1-3): 1, the prepared small molecular water cluster composition has excellent stability, the excellent stability can be still maintained even if the composition is diluted to 10% by deionized water, and the stability of the small molecular water cluster composition is not influenced by the addition of preservatives such as phenoxyethanol and the like. This is probably because under specific conditions, the activity of the biological enzyme is high and can exist stably in small molecule water; meanwhile, ions generated in the system have strong paramagnetism due to the existence of superoxide radicals and free protons in the system, and can stabilize small molecular water for a long time through continuous shearing action.

The micromolecular water cluster composition is prepared from micromolecular water and biological enzyme, and the prepared micromolecular water cluster composition is non-toxic, free of side effect and pure natural. The small molecular water cluster composition is alkalescent, can neutralize harmful acidic metabolites in vivo, makes body fluid in alkalescence, and achieves prevention and health care effects; has a six-ring structure, consists of 7-9 water molecules (8 water molecules on average), has small diameter, fast permeation and fast cell membrane passing, and is beneficial to water exchange and harmful metabolite elimination; the calcium ion-rich water-soluble fertilizer is rich in calcium ions, can be combined with acid radicals to neutralize harmful substances in the body, has strong dissolving power, can take away wastes in the body and helps excretion; has negative potential (-86-105mV) higher than that of fresh fruits and vegetables, is beneficial to normal metabolism of organisms, eliminates excessive free radicals in vivo and improves the immunity of human bodies; the oxygen carrying ratio is high, the oxygen content of the water is 2 times of that of fresh tap water, so that complete oxidation and metabolism of nutrient substances (solute) in cells are facilitated, and the generation of acidic waste is reduced.

The micromolecular water cluster composition can be widely applied to the fields of cosmetics and the like, has a good synergistic effect with biological enzymes, can guide the biological enzymes with the functions of catalyzing, decomposing and mastering the life activities to act on skin and pores, and can carry out deep cleaning and skin repair and moistening in the most original and effective mode, so that the elasticity and luster of the skin are kept; the body fluid of a person can be weakly alkaline, and the body can be adjusted to the optimal state; active oxygen (oxygen free radical) is detoxified, the healthy internal environment is ensured, and the invasion of harmful factors is resisted; activating cells, ensuring normal metabolism and reducing blood fat; purifying blood and eliminating venom; relieving fatigue and maintaining vigorous energy; the regulator regulates the balance of the autonomic nervous system and ensures the normal operation of internal organs; enhancing disease resistance and reducing the occurrence of diseases; has analgesic and tranquilizing effects; improving allergic constitution, and preventing immune allergic diseases.

The micromolecular water cluster composition has multiple effects, improves the biological utilization rate of water, and is easy to be absorbed and utilized by each cell; improving metabolism efficiency and ability, improving nutrient supply, promoting oxygen transport ability, promoting discharge of intracellular waste and toxin, regulating and maintaining immune system function, and slowing down organism aging; normal information exchange among cells is reestablished, and the self-healing function of the human body is improved. Enhancing the information transfer between each cell and the surrounding tissue structure and promoting the normal gene information transfer; the micro-molecular water contained in the product has strong physiological activity (dissolving power, diffusion power, permeability, emulsifying power, metabolism power and cleaning power), and can meet the physiological requirements of human body. It also has effects of activating various enzymes in vivo, and is beneficial for human to prevent cardiovascular and cerebrovascular diseases.

Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that these examples are merely illustrative and not restrictive. In addition, all the raw materials are commercially available if not particularly limited.

Examples

Example 1

The embodiment 1 of the invention provides a small molecular water cluster composition, which comprises the following components in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The preparation method of the micromolecular water comprises the following steps: sending the highly mixed hydrogen and water mixed solution with the pressure of 8kg into a micromolecular water preparation device, penetrating through a 45 kilogauss permanent magnetic field at the flow speed of 12m/s, and vertically cutting magnetic lines to obtain the hydrogen and water mixed solution.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is bromelain or papain.

The weight ratio of the bromelain to the papain is 2: 1.

the preparation method of the bromelain comprises the following steps:

(1) cleaning pineapple peel, juicing, filtering to remove large particle impurities, pre-cooling, and adding powder NaHSO₃Centrifuging at 3000r/min for 20min with powdered cysteine, powdered ethylenediamine tetraacetic acid and powdered vitamin B1, and collecting supernatant;

The temperature of the pre-cooling treatment is 3 ℃, and the time of the pre-cooling treatment is 15 h.

The powder NaHSO in the pineapple juice per unit volume₃The amount of (B) added was 0.3 mg/L.

The powder NaHSO₃The weight ratio of the powder cysteine to the powder ethylene diamine tetraacetic acid to the powder vitamin B1 is 0.3: 0.05: 0.5: 0.03.

the adding amount of the polyacrylic acid is 0.8 percent of the weight of the supernatant obtained in the step (1).

The concentration of the calcium acetate aqueous solution is 40 mmol/L.

The buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.

The concentration of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 0.05mol/L, and the pH value is 6.8.

The disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added in an amount of 4 times the volume of the supernatant in step (3).

The freeze-drying temperature was-40 ℃.

The preparation method of the papain comprises the following steps:

The addition amount of the compound enzyme is 0.5 percent of the weight of the papaya.

The complex enzyme is cellulase and glucanase.

The weight ratio of the cellulase to the glucanase is 3: 1.

the concentration of the ethanol aqueous solution is 75 wt%.

The addition amount of the powdery cysteine in the supernatant obtained in the step (a) per unit volume is 2 g/L.

The weight ratio of the powder cysteine to the powder ethylene diamine tetraacetic acid to the powder vitamin B1 is 2: 1: 0.3.

The concentration of the sodium hydroxide aqueous solution is 1 mol/L.

The freeze-drying temperature was-40 ℃.

The preparation method of the biological enzyme comprises the following steps: mixing bromelain, papain and water, performing aerobic fermentation at 37 deg.C for 20 days, stirring every 15 hr for 20min after fermentation, stopping stirring after 10 days, standing for 10 days, and filtering.

The weight of the water is 1.5 times of the sum of the weight of the bromelain and the weight of the papain.

The preparation method of the small molecular water cluster composition comprises the following steps: mixing small molecular water and biological enzyme.

Example 2

The embodiment 2 of the invention provides a small molecular water cluster composition, which comprises the following components in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is bromelain.

The preparation method of the bromelain comprises the following steps:

The concentration of the calcium acetate aqueous solution is 40 mmol/L.

The freeze-drying temperature was-40 ℃.

The preparation method of the biological enzyme comprises the following steps: mixing bromelain and water, performing aerobic fermentation at 37 deg.C for 20 days, stirring every 15 hr for 20min after fermentation period, stopping stirring after 10 days, standing for 10 days, and filtering.

The weight of the water is 1.5 times of the weight of the bromelain.

Example 3

The embodiment 3 of the invention provides a small molecular water cluster composition, which comprises the following components in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is papain.

The preparation method of the papain comprises the following steps:

The complex enzyme is cellulase and glucanase.

The weight ratio of the cellulase to the glucanase is 3: 1.

the concentration of the ethanol aqueous solution is 75 wt%.

The concentration of the sodium hydroxide aqueous solution is 1 mol/L.

The freeze-drying temperature was-40 ℃.

The preparation method of the biological enzyme comprises the following steps: mixing papain and water, performing aerobic fermentation at 37 deg.C for 20 days, stirring every 15 hr for 20min after the fermentation period, stopping stirring after 10 days, standing for 10 days, and filtering.

The weight of the water is 1.5 times of that of the papain.

Example 4

Embodiment 4 of the present invention provides a small molecule water cluster composition, comprising the following components, by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is bromelain or papain.

The weight ratio of the bromelain to the papain is 2: 1.

the bromelain was purchased from de yang xinuo seikaga pharmaceutical co.

The preparation method of the papain comprises the following steps:

The complex enzyme is cellulase and glucanase.

The weight ratio of the cellulase to the glucanase is 3: 1.

the concentration of the ethanol aqueous solution is 75 wt%.

The concentration of the sodium hydroxide aqueous solution is 1 mol/L.

The freeze-drying temperature was-40 ℃.

Example 5

Embodiment 5 of the present invention provides a small molecule water cluster composition, comprising the following components, by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is bromelain or papain.

The weight ratio of the bromelain to the papain is 2: 1.

the preparation method of the bromelain comprises the following steps:

The concentration of the calcium acetate aqueous solution is 40 mmol/L.

The freeze-drying temperature was-40 ℃.

The papain was purchased from Shanxi Yuning Biotech Ltd.

Example 6

Embodiment 6 of the present invention provides a small molecule water cluster composition, comprising the following components, by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

the biological enzyme comprises a protease.

The protease is bromelain or papain.

The weight ratio of the bromelain to the papain is 5: 1.

the preparation method of the bromelain comprises the following steps:

The concentration of the calcium acetate aqueous solution is 40 mmol/L.

The freeze-drying temperature was-40 ℃.

The preparation method of the papain comprises the following steps:

The complex enzyme is cellulase and glucanase.

The weight ratio of the cellulase to the glucanase is 3: 1.

the concentration of the ethanol aqueous solution is 75 wt%.

the concentration of the sodium hydroxide aqueous solution is 1 mol/L.

The freeze-drying temperature was-40 ℃.

Example 7

Embodiment 7 of the present invention provides a small molecule water cluster composition, comprising the following components, by weight: 99 parts of micromolecular water and 0 part of biological enzyme.

The volume ratio of the hydrogen to the water is 1: 1.

performance evaluation

One and half width test

The small molecule water cluster compositions prepared in examples 1-7 were tested for half width according to JY/T007-1996 after being left at room temperature for 3 months. If the half width is less than 100Hz, the product is recorded as qualified, otherwise, the product is not qualified.

The results are shown in Table 1.

TABLE 1 half Width test results

Wherein A represents the small molecular water cluster composition prepared by the invention; b represents that the micromolecular water cluster composition prepared by the invention is diluted to 50 percent by using deionized water; c represents that the micromolecular water cluster composition prepared by the invention is diluted to 30 percent by using deionized water; d represents that the micromolecular water cluster composition prepared by the invention is diluted to 10 percent by using deionized water; e represents diluting the small molecular water cluster composition prepared by the present invention to 30% with deionized water, and adding 0.5% phenoxyethanol (E essentially means 30 wt% small molecular water cluster composition +0.5 wt% phenoxyethanol +69.5 wt% deionized water).

According to experimental results, the small molecular water cluster composition prepared by the invention has excellent stability, and can maintain excellent stability even if being diluted to 10% by adding deionized water; the addition of preservatives such as phenoxyethanol does not affect the stability of the small molecule water cluster composition.

Second, efficacy test

The small molecule water cluster composition prepared in example 1 was subjected to efficacy test using human keratinocytes, which were obtained by primary culture and passage of Guangdong Boxi Biotech limited.

1. Cytotoxicity assays

(1) Cell inoculation: cells were seeded at a seeding density of 1E 4/well in 96-well plates and incubated overnight in an incubator (37 ℃, 5% CO2, 95% RH).

(2) Grouping tests: the test was set with a solvent control group, a positive control group and a sample group. Each sample was set with 8 concentration gradients, with 3 replicate wells set under each concentration gradient.

(3) Preparing liquid: working solutions of test substances with different concentrations were prepared according to the experimental design (table 2).

TABLE 2 test concentration setting table

(4) Administration: and (3) when the cell plating rate in the 96-well plate reaches 40-60 percent, the administration is carried out. Adding 200 mu L of cell culture solution into each hole of the solvent control group; adding 200 mu L of culture solution containing 10% DMSO into each well of the positive control group; adding 200 mu L of culture solution containing the test object with corresponding concentration into each hole of the sample group; null wells were seeded without cells and only 200 μ L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO)₂95% RH) for 24 h.

(5) And (3) detection: after the incubation, the culture solution was discarded, MTT working solution (0.5mg/mL, now ready for use) was added, incubation was carried out at 37 ℃ for 4h in the dark, after the incubation, MTT was discarded, 150. mu.L of DMSO was added to each well, and the OD was read at 490 nm.

(6) Calculating the relative activity of the cells: according to the formula, the relative cell viability (%) is (sample well-zero hole)/(blank control well-zero hole) × 100%, 8 concentrations selected from the small molecule water cluster composition prepared in example 1 of the present invention are plotted as abscissa and the relative cell viability value is plotted as ordinate.

The samples are set to 8 dosing concentrations from high to low, cytotoxicity detection experiments are carried out on keratinocytes, MTT detection results are shown in table 3, and the cell viability change trend is shown in figure 1.

TABLE 3 MTT assay results

According to the MTT experimental result, the following steps are shown: when the concentration of the small molecular water cluster composition prepared in example 1 was 10%, the relative viability value of the cells was 100.89% compared to the solvent control, and there was no cytotoxicity.

2. Morphological experiments

(1) Cell inoculation: and (3) setting a sample group and a solvent control group, wherein each group is provided with two multiple holes. The cells were inoculated at the corresponding inoculation density into 24-well plates, incubators (37 ℃ C., 5% CO)₂95% RH) for 24 h.

(2) Preparing liquid: according to the MTT detection result, determining the morphological observation concentration of the detection sample, and preparing the working solution of the test object with different concentrations, which is shown in Table 4.

Table 4 morphological test working concentrations

(3) Administration: when the cell plating rate of the 24-well plate reaches 40%, the drug is administered, the samples are added with the test substances with different concentrations, the solvent control group is added with the culture solution, and the incubator (37 ℃, 5% CO)₂95% RH) for 24 h.

(4) And (3) cell observation: after incubation, cell morphology was observed under a microscope and photographed.

The results of the morphological examination are shown in figure 2.

According to the morphological results, it can be seen that: when the concentration of the small molecular water cluster composition prepared in the embodiment 1 of the invention is 10%, the cell edge contour is clear, the morphology is regular, no obvious particulate matters exist in cells, and the cell state is not obviously different from that of a solvent control group. Therefore, based on the MTT results and morphological results, it is believed that the sample of the small molecule water cluster composition prepared in example 1 of the present invention does not exhibit significant cytotoxicity in the 10% concentration range based on human keratinocytes.

3. Transcriptome harvest

(1) Inoculation: inoculating cells into 6-well plate at a certain cell density, and culturing in incubator (37 deg.C, 5% CO)₂95% RH) overnight.

(2) Preparing liquid: the test substances were prepared in different concentrations according to the experimental design (table 5).

TABLE 5 Experimental groups

(3) Administration: when the cell plating rate in the 6-hole plate reaches about 60%, the medicine is administered according to the experimental design, and each group is provided with 3 multiple holes.

(4) Collecting a sample: 37 ℃ and 5% CO₂And (3) an incubator, after incubation and culture for 24h, removing the culture solution, adding 1ml of LTrizol into each hole, blowing and cracking the cells, and collecting the samples.

4. Transcriptome analysis

Based on an Illumina technology sequencing platform, the complete transcriptome analysis of the sample is completed by using a double-ended sequencing (Paired-End) method.

The RNA-seq technical process mainly comprises two parts, namely library construction, sequencing and data analysis. From the RNA sample to the final data acquisition, each link of sample detection, library establishment and sequencing influences the data quality and quantity, and the data quality directly influences the result of subsequent information analysis.

Transcriptome analysis, library building and sequencing procedures were as follows: mRNA enrichment → double-stranded cDNA synthesis → end repair plus A and linker → fragment selection and PCR enrichment → library quality detection → Illumina sequencing.

(1) RNA extraction and detection

The RNA is extracted from tissues or cells by adopting a standard extraction method, and then the strict quality control is carried out on an RNA sample, wherein the quality control standard mainly comprises the following 3 aspects.

Agarose gel electrophoresis: samples were analyzed for RNA integrity and for the presence of DNA contamination.

NanoPhotometer spectrophotometer: RNA purity (OD260/280 and OD260/230 ratio) was determined.

Agilent 2100 bioanalyzer: the integrity of RNA is accurately detected.

(2) Library construction and quality control

After the sample is qualified, the mRNA of the eukaryote is enriched by binding the pIyA tail of the mRNA through the complementary pair of A-T by using magnetic beads with oligo (dT). Fragmentation buffer was then added to break the mRNA into short fragments, single-stranded cDNA was synthesized using a hexabasic random primer (random hexamers) using mRNA as a template, and double-stranded cDNA was synthesized by adding buffer, dNTPs and DNA polymerase I, followed by purification of the double-stranded cDNA using AMPure XP beads. And (3) carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection by using AMPure XP beads, and finally carrying out PCR enrichment to obtain a final cDNA library.

(3) Warehouse inspection

After the library is constructed, firstly using Qubit2.0 to carry out preliminary quantification, diluting the library to 1.5ng/ul, then using Agilent 2100 to detect the length of an insert fragment (insert size) of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library (the effective concentration of the library is more than 2nM) so as to ensure the quality of the library.

(4) Sequencing on machine

And after the library is qualified, carrying out HiSeq sequencing on different libraries according to the effective concentration and the requirement of the target offline data volume. The basic principle of Sequencing is Sequencing by Synthesis (Sequencing by Synthesis). Adding four fluorescent-labeled dNTPs, DNA polymerase and a joint primer into a sequenced flowcell for amplification, releasing corresponding fluorescence every time one fluorescently-labeled dNTP is added when each sequencing cluster extends a complementary chain, and acquiring sequence information of a fragment to be detected by a sequencer by capturing a fluorescence signal and converting an optical signal into a sequencing peak through computer software.

5. Information analysis process

The core of RNA-seq is significance analysis of gene expression difference, a statistical method is used for comparing gene expression difference under two conditions or under a plurality of conditions, specific genes related to the conditions are found out from the gene expression difference, then the biological significance of the specific genes is further analyzed, and the analysis process comprises six links of quality control, comparison, quantification, difference significance analysis and function enrichment.

(1) Quality inspection results of RNA samples

The results of quality inspection of the RNA samples are shown in Table 6.

TABLE 6 RNA sample quality test results

Remarking: the classification requirements of the detection result categories are as follows:

a type: the sample quality meets the requirement of library construction and sequencing, and the total amount meets the requirement of library construction for 2 times or more than 2 times;

b type: the sample quality meets the requirement of library construction and sequencing, and the total amount meets the requirement of 1-time but less than 2-time library construction;

class C: sample quality does not completely meet the requirements of library construction and sequencing, and a library can be constructed in an attempt;

and D type: the sample quality does not meet the requirements of library construction and sequencing at all, and the sample used is not recommended.

The quality test results show that the RNA quality test results of the solvent control group and the sample group in the example 1 are A, so that the sample quality meets the requirement of library construction sequencing, and the following library construction sequencing can be carried out.

(2) Results of differential Gene analysis

Differential expression gene screening principle: for experiments without biological duplication, to eliminate biological variation, evaluation was performed from both fold-of-difference and significance level, and differential genes were screened, with thresholds set as: pvalue <0.05| log2FoldChange | > 0.0. The volcano plot visually demonstrates the differential gene distribution for each comparative combination, as shown below. The abscissa in the figure represents the fold change in expression of the gene in the treated and control groups (log2FoldChange) and the ordinate represents the significance level of the difference in expression of the gene in the treated and control groups (-log10padj or-log 10 pvalue). For up-regulated genes, red dots are used and for down-regulated genes, green dots are used.

The volcano pattern of the differential gene distribution is shown in figure 3, and the statistical analysis result of the differential gene shows that 407 genes are obviously up-regulated and 451 genes are obviously down-regulated after the sample is treated compared with the solvent control group.

(3) GO functional enrichment analysis

Gene Ontology (GO for short, http:// www.geneontology.org /) is an international standard classification system for Gene function. The functional enrichment of GO is divided into three parts, Molecular Function (Molecular Function), biological process (bioprocess), and Cellular composition (Cellular Component). The gene or protein can find the GO number corresponding to the gene or protein through an ID correspondence or sequence annotation method, and the GO number can be used for corresponding to Term, namely, a functional class or a cell location.

After screening the differential genes according to the test purpose, the distribution condition of the differential genes in the Gene Ontology is researched by enrichment analysis so as to clarify the embodiment of the sample difference on the Gene function in the experiment. The principle of common GO enrichment analysis is super-geometric distribution, the super-geometric distribution relation between the differential genes and certain specific branches in GO classification is calculated according to the selected differential genes, a specific p-value is obtained through hypothesis verification, and whether the differential genes are enriched in the GO is judged.

And selecting the most significant 30 Term from GO enrichment analysis results to draw a histogram for display, and if the number of the Term is less than 30, drawing all the terms. In the graph, the abscissa is GO Term, the ordinate is the significance level of GO Term enrichment, the higher the numerical value is, the more significant the value is, and different colors respectively represent three GO subclasses of BP, CC and MF. The results are shown in FIG. 4.

The results of analysis of genetic up-regulation on both Biological processes (Biological processes) and cellular compositions (cellular components) show that genes related to epidermal cell differentiation (cornification), keratinocyte differentiation (keratinocidation), keratinization (kerationalization), epidermal development (epidermal development), epidermal cell differentiation (epidermal cell differentiation), skin development (skinelevation) and keratinocyte envelope protein (cornified envelope) are all significantly up-regulated, which indicates that the small-molecule water cluster composition prepared in example 1 of the present invention can achieve the effect of enhancing the skin barrier by promoting keratinocyte differentiation, skin development and expression of the keratinocyte envelope related genes.

The results of GO bioprocess (Biological Process) analysis of up-regulated genes showed that significant up-regulation occurred in genes related to antibacterial humoral response (antibacterial humoral response), humoral immune response (humoral immune response), antibacterial peptide-mediated antimicrobial humoral immune response (antibacterial humoral immune response), bacterial response (response to bacteria), bacterial defense response (defense to bacteria), and defense response to other organisms (defense) indicating that the small molecular water cluster composition prepared in example 1 of the present invention has immune regulation effect.

(4) KEGG pathway enrichment assay

KEGG (Kyoto Encyclopedia of Genes and genomes) is a comprehensive database that integrates genomic, chemical, and system functional information. The KEGG channel enrichment takes padj less than 0.05 as a threshold value of significance enrichment, the most significant 20 KEGG channels are selected from KEGG enrichment results to draw a bar chart for displaying, if the number of the KEGG channels is less than 20, all the channels are drawn, and the enrichment results are shown in figure 5.

The KEGG analysis result of the up-regulated gene shows that the inflammation related pathway gene (IL-17signaling pathway), the Cytokine-Cytokine interaction and the Mineral absorption (Mineral absorption) are obviously up-regulated.

(5) Skin barrier-associated gene analysis

Based on the analysis result of the differential genes after the treatment of the small molecular water cluster composition prepared in the embodiment 1 of the invention, the skin barrier related genes are subjected to detailed analysis, and the analysis result is shown in the attached figures 6 and 7.

The analysis result shows that the Keratin (KRT) family gene is significantly up-regulated after the treatment of the small molecular water cluster composition prepared in the example 1 of the invention compared with the solvent control group (SC), such as KRT1, KRT13, KRT24 and the like, which indicates that the small molecular water cluster composition prepared in the example 1 of the invention has the effect of promoting the expression of keratin; in addition, differentiation related proteins, intercellular junctions and keratinocyte proteins are also remarkably up-regulated and expressed, such as SPRR2D, S100A7, IVL, SPRR2E, DSG4, SPRR2G, SPRR2A, LCE3C and the like, and the small molecular water cluster composition prepared in the embodiment 1 has the effect of promoting the up-regulation of the expression of the terminal differentiation related proteins, intercellular junctions and keratinocyte proteins at the transcriptome level.

From the above experimental results, it can be seen that the small molecular water cluster composition prepared in example 1 of the present invention can enhance the skin barrier by promoting the terminal differentiation of keratinocytes, the up-regulated expression of the transcription levels of intercellular connexins and keratinocyte block proteins. The micromolecular water cluster composition prepared in the embodiment 1 of the invention can obviously improve antibacterial humoral response, humoral immune response, antibacterial peptide-mediated antimicrobial humoral immune response, bacterial defense response and defense response related genes to other organisms, and has an immunoregulation effect.

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. The use of some numerical ranges in the claims also includes sub-ranges within their range, and variations in these ranges are also to be construed as being covered by the appended claims where possible.

Claims

1. The small molecular water cluster composition is characterized by at least comprising the following components in parts by weight: 0-200 parts of micromolecular water and 0-2 parts of biological enzyme; the weight parts of the micromolecular water and the biological enzyme are not 0.

2. The small molecule water cluster composition of claim 1, comprising at least the following ingredients in parts by weight: 50-150 parts of micromolecular water and 0-2 parts of biological enzyme; the weight part of the biological enzyme is not 0.

3. The small molecule water cluster composition of claim 2, comprising at least the following ingredients in parts by weight: 80-120 parts of micromolecular water and 0.5-1.8 parts of biological enzyme.

4. The small molecule water cluster composition of claim 3, comprising at least the following ingredients in parts by weight: 90-110 parts of small molecular water and 1-1.5 parts of biological enzyme.

5. The small molecule water cluster composition of claim 4, comprising at least the following ingredients in parts by weight: 95-105 parts of micromolecular water and 0.8-1.2 parts of biological enzyme.

6. The small molecule water cluster composition of claim 5, comprising at least the following ingredients in parts by weight: 99 parts of micromolecular water and 1 part of biological enzyme.

7. The small molecule water cluster composition of any one of claims 1-6, wherein the biological enzyme comprises at least one of a protease, an amylase, and a cellulase.

8. The small molecule water cluster composition of claim 7, wherein the biological enzymes include at least one protease selected from the group consisting of bromelain, papain, ficin, glycanain, actinidin, and mogroside.

9. A method of preparing a small molecule water cluster composition as claimed in any one of claims 1 to 8, comprising at least the steps of: mixing small molecular water and biological enzyme.

10. Use of a small molecule water cluster composition as claimed in any one of claims 1 to 8 in cosmetics.