CN115337238A

CN115337238A - Preparation method and application of coffee fermented product

Info

Publication number: CN115337238A
Application number: CN202211023767.7A
Authority: CN
Inventors: 潘丹阳; 陈慧芳; 何敬愉
Original assignee: Guangzhou Huanya Cosmetic Science and Technology Co Ltd
Current assignee: Guangzhou Huanya Cosmetic Science and Technology Co Ltd
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2022-11-15
Anticipated expiration: 2042-08-24
Also published as: CN115337238B

Abstract

The invention belongs to the technical field of cosmetics, and discloses a preparation method and application of a coffee ferment. The preparation method comprises the following steps: the coffee bean, milk and honey are taken as fermentation raw materials, and the coffee bean, milk and honey are subjected to high-pressure homogenization, and then are subjected to anaerobic fermentation and aerobic fermentation in sequence, wherein the pressure of the high-pressure homogenization is 10-100MPa. The invention adopts high-pressure homogeneous extraction technology, combines biological two-stage fermentation, takes coffee beans, milk and honey as main raw materials, and carries out anaerobic fermentation and aerobic fermentation to fully release active substances of the coffee beans, the milk and the honey into a fermented product, and the prepared coffee fermented product has good effects of oxidation resistance, inflammation resistance and cell activity improvement, is more beneficial to absorption and utilization of skin, and has excellent anti-aging and relieving effects.

Description

Preparation method and application of coffee fermented product

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of a coffee fermentation product.

Background

Aging (Aging), also known as Aging, is a vital step in the process of any living organism. As the outermost protective layer of the human body, the aging characteristics of the skin mainly comprise skin relaxation and wrinkle appearance, the skin of the exposed part of the body becomes rough, and the wrinkles deepen and thicken; the skin becomes loose after elasticity is stopped; vasodilatation, epidermal keratosis, etc. Since aging has complex psychological and physiological effects on people's life and work, it can cause a series of psychological problems such as anxiety, depression, and self-inferior. Therefore, anti-aging treatment, especially for skin, has been one of the focuses of research. Causes of skin aging include free radical damage, reduced cell renewal, damage caused by ultraviolet radiation, protein glycosylation, elevated matrix metalloproteinase levels, DNA mutations, telomere shortening, mitochondrial damage, and the like.

Coffee is a plant of Rubiaceae (Rubiaceae) coffee (Coffea), is the most common beverage plant, has great economic value, and is a main beverage popular in the world together with cocoa and tea. According to records of Chinese materia medica, coffee is slightly bitter, astringent and flat; has effects of inducing resuscitation, promoting urination and invigorating stomach, and can be used for treating listlessness, anorexia, and is often used as refreshing, promoting urination and invigorating stomach medicine.

Coffee contains alkaloids, phenolic acids, flavonoids, terpenoids, sterol esters, volatile components and the like, and caffeine (1,3,7-trimethyl xanthine, caffeine) is a main alkaloid component in coffee fruits, is a source of coffee bitterness and widely exists in tea leaves, cocoa and coffee. Caffeine is a representative substance of caffeine and is one of the most important active ingredients in coffee beans. Caffeine has stable chemical structure, can be dissolved in water, and has various biological activities. Research shows that caffeine relieves oxidative stress caused by D-galactose by inhibiting increase of COX-2, NOS-2, TNF-alpha and IL-1 beta, thereby relieving neuroinflammation; in addition, caffeine slows oxidative stress-induced aging by activating SIRT 3/MAPK/autophagy. At present, coffee beans are mostly applied to the edible field, and are still not fully and effectively developed and utilized in the aspect of skin care efficacy.

Therefore, the application of coffee beans in the field of skin care needs to be further developed, and the coffee beans are utilized to prepare skin care products with anti-aging and relieving effects.

Disclosure of Invention

The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides a preparation method and application of a coffee fermentation product. The coffee fermented product has good effects of resisting oxidation, resisting inflammation and improving cell activity, and has antiaging and soothing effects.

In a first aspect of the invention, a method for preparing a coffee ferment is provided.

Specifically, the preparation method of the coffee leavening comprises the following steps:

the coffee bean, milk and honey are used as fermentation raw materials, and the coffee bean, milk and honey are subjected to high-pressure homogenization, and then are subjected to anaerobic fermentation and aerobic fermentation in sequence, wherein the pressure of the high-pressure homogenization is 10-100MPa.

Preferably, the pressure of the high-pressure homogenization is 35-100MPa, and the time of the high-pressure homogenization is 20-60min; further preferably, the pressure of the high-pressure homogenization is 50-90MPa, and the time of the high-pressure homogenization is 25-40min.

Preferably, an anaerobic strain is added during the anaerobic fermentation.

Preferably, the concentration of the anaerobic strain is 10 ⁵ -10 ⁹ CFU/mL。

Preferably, the inoculation amount of the anaerobic strain is 0.1-1.0% by volume percentage. The inoculation amount means that the volume of the inoculated anaerobic strain liquid accounts for 0.1-1.0% of the volume of the fermentation raw material. Further preferably, the inoculum size of the anaerobic strain is 0.3% -0.8%.

Preferably, the anaerobic strain is selected from at least one of lactobacillus plantarum, bifidobacterium longum, or streptococcus thermophilus; further preferably, the anaerobic strain comprises lactobacillus plantarum and streptococcus thermophilus; more preferably, in the anaerobic strain, the inoculation amount of lactobacillus plantarum accounts for 30% or more of the total inoculation amount of the anaerobic strain; most preferably, in the anaerobic strain, the amount of lactobacillus plantarum inoculated accounts for 45% or more of the total amount of the anaerobic strain inoculated.

Preferably, an aerobic strain is added during the aerobic fermentation.

Preferably, the aerobic strain is selected from at least one of kluyveromyces lactis or saccharomyces cerevisiae; further preferably, the aerobic strains are Kluyveromyces lactis and Saccharomyces cerevisiae.

Preferably, the concentration of the aerobic strain is 10 ⁵ -10 ⁹ CFU/mL。

Preferably, the inoculation amount of the aerobic strain is 0.1-1.0 percent by volume percentage. Namely the volume of the inoculated aerobic bacterial liquid accounts for 0.1 to 1.0 percent of the volume of the fermentation raw material. Further preferably, the inoculation amount of the aerobic strain is 0.3% -0.8%.

Preferably, the fermentation feedstock further comprises water; the fermentation raw materials comprise, by weight, 5-60 parts of coffee beans, 5-60 parts of milk, 0.5-10 parts of honey and 5-80 parts of water; further preferably, the fermentation raw materials comprise 5-50 parts of coffee beans, 5-50 parts of milk, 1-8 parts of honey and 20-70 parts of water in parts by weight; more preferably, the fermentation raw materials comprise 15-30 parts of coffee beans, 15-40 parts of milk, 1-5 parts of honey and 30-60 parts of water by weight.

Preferably, the fermentation raw material further comprises at least one of potato, glucose and peptone. The total weight of the potatoes, the glucose and the peptone is 2 to 10 parts by weight.

Preferably, the fermentation raw materials comprise the following components in parts by weight: 15-30 parts of coffee beans, 15-30 parts of milk, 1-8 parts of honey, 40-60 parts of water and 3-8 parts of glucose; further preferably, the fermentation raw materials comprise, by weight: 20 parts of coffee beans, 20 parts of milk, 5 parts of honey, 50 parts of water and 5 parts of glucose.

Preferably, the coffee beans are at least one of unroasted, light roasted, medium roasted, deep roasted coffee beans. Further preferably, the coffee beans are deep roasted coffee beans.

Preferably, the temperature of the anaerobic fermentation is 30-40 ℃, and the time of the anaerobic fermentation is 6-24h; preferably, the temperature of the anaerobic fermentation is 35-40 ℃, and the time of the anaerobic fermentation is 8-18h.

Preferably, the temperature of the aerobic fermentation is 26-35 ℃, and the time of the aerobic fermentation is 18-72h; preferably, the temperature of the anaerobic fermentation is 26-32 ℃, and the time of the aerobic fermentation is 24-60h.

Preferably, after the anaerobic fermentation and the aerobic fermentation are both completed, sterilization treatment is performed.

More specifically, a preparation method of coffee leavening comprises the following steps:

(1) Pulverizing coffee bean, mixing with milk, mel and water, and homogenizing under high pressure to obtain coffee homogenate;

(2) Mixing the coffee homogenate with an anaerobic strain, and performing anaerobic fermentation to obtain a primary coffee fermented product;

(3) And mixing the primary coffee fermented product with an aerobic strain, carrying out aerobic fermentation, and filtering to obtain the coffee fermented product.

The invention also provides the application of the preparation method of the coffee leavening in preparing cosmetics.

Specifically, the coffee fermented product prepared by the preparation method is applied to cosmetics.

A cosmetic comprises coffee fermented product prepared by the preparation method.

Preferably, the cosmetic is essence water.

Further preferably, the essence water comprises the coffee fermentation product prepared by the preparation method, 1,2-pentanediol, beta-glucan, glycerol, hydrogenated lecithin, 1,2-hexanediol, disodium EDTA, polyglycerol-10 oleate, glyceryl polyether-26 and xanthan gum.

The milk is rich in protein, vitamins, basic acid and trace elements, and has skin caring effect. The milk protein is also called cow milk protein, is a general name of a mixture of a plurality of proteins in milk, and is Lactoferrin (LF) separated from cow milk, has the effects of resisting lipid peroxidation and removing hydroxyl radicals and DPPH radicals, can be used as a humectant, a nutrient and a conditioner to be added into cosmetics, and can increase the drug effects of other active ingredients. Besides a large amount of saccharides, the honey also contains polyphenol compounds, vitamins, bioactive enzymes, proteins, amino acids, mineral elements and the like, and various components of the honey endow the honey with antibacterial, anti-inflammatory, antioxidant and wound healing promoting activities. The invention destroys coffee fiber by physical high-pressure homogenizing technology, which makes the effective component in coffee bean fully released and fused with milk and honey. The adopted two-stage fermentation is firstly carried out anaerobic fermentation and then aerobic fermentation, thus fully playing the biotransformation capacity of different microorganisms, further improving the content of effective substances in coffee beans, reducing the irritation of raw materials, effectively increasing the types and the content of active substances in fermentation products, and leading the obtained coffee fermentation product to have good effects of oxidation resistance, inflammation resistance and cell activity improvement. By selecting zymophyte in the two-stage fermentation, polysaccharide, amino acid, enzyme and some small molecular components generated by fermentation and transformation can better play a role in nourishing the skin, and have obvious effects of resisting aging and relieving.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts high-pressure homogeneous extraction technology, combines biological two-stage fermentation, takes coffee beans, milk and honey as main raw materials, and carries out anaerobic fermentation and aerobic fermentation to fully release active substances of the coffee beans, the milk and the honey into a fermented product, and the prepared coffee fermented product has good effects of oxidation resistance, inflammation resistance and cell activity improvement, is more beneficial to absorption and utilization of skin, and has excellent anti-aging and relieving effects.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

In the following examples and comparative examples, lactobacillus plantarum was used at a concentration of about 10 ⁷ CFU/mL, bifidobacterium longum concentration of about 10 ⁷ CFU/mL, concentration of Streptococcus thermophilus about 10 ⁷ CFU/mL, kluyveromyces lactis concentration of about 10 ⁷ CFU/mL, saccharomyces cerevisiae concentration of about 10 ⁷ CFU/mL. Other materials, reagents or apparatus may be conventionally commercially available or may be prepared by methods known in the art, unless otherwise specifiedThe method is used for obtaining the compound.

Example 1

The embodiment provides a preparation method of a coffee fermented product, which comprises the following steps:

(1) According to the mass parts, 20 parts of deeply roasted coffee beans are crushed and then mixed with 20 parts of milk, 5 parts of honey, 50 parts of water and 5 parts of glucose, and high-pressure homogenization is carried out to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

(2) Mixing coffee homogenate with 0.5% (the volume of the bacteria solution accounts for 0.5% of the volume of the coffee homogenate) bacteria solution (composed of 0.1% Lactobacillus plantarum, 0.2% Bifidobacterium longum, and 0.2% Streptococcus thermophilus), fermenting at 37 deg.C for 12 hr, after fermentation is completed, sterilizing at 90 deg.C for 20min to obtain coffee primary ferment;

(3) Mixing the primary coffee ferment with 0.5% (the volume of the bacteria solution accounts for 0.5% of the volume of the primary coffee ferment) aerobic strain bacteria solution (composed of 0.25% of Kluyveromyces lactis and 0.25% of Saccharomyces cerevisiae), fermenting at 180r/min at 28 deg.C for 48h, and filtering to obtain the coffee ferment.

Example 2

(1) Pulverizing 20 parts by mass of moderately roasted coffee beans, mixing with 20 parts by mass of milk, 5 parts by mass of honey, 50 parts by mass of water and 5 parts by mass of glucose, and homogenizing under high pressure to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

(2) Mixing the coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) bacterial liquid (consisting of 0.1% of lactobacillus plantarum, 0.2% of bifidobacterium longum and 0.2% of streptococcus thermophilus), fermenting at 37 deg.C for 12h, and after the fermentation is completed, sterilizing at 90 deg.C for 20min to obtain coffee primary fermentation product;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacteria liquid accounts for 0.5% of the volume of the primary coffee fermented product) aerobic bacteria liquid (composed of 0.25% of Kluyveromyces lactis and 0.25% of Saccharomyces cerevisiae), fermenting at the rotation speed of 180r/min at 28 deg.C for 48h, and filtering to obtain the coffee fermented product.

Example 3

(1) According to the mass parts, 20 parts of lightly baked coffee beans are ground, mixed with 20 parts of milk, 5 parts of honey, 50 parts of water and 5 parts of glucose, and subjected to high-pressure homogenization to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

Example 4

(1) Pulverizing 20 parts by mass of unbaked coffee beans, mixing with 20 parts by mass of milk, 5 parts by mass of honey, 50 parts by mass of water and 5 parts by mass of glucose, and homogenizing under high pressure to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

Example 5

(2) Mixing the coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) bacterial liquid (consisting of 0.25% of lactobacillus plantarum and 0.25% of streptococcus thermophilus), fermenting at 37 deg.C for 12h, and after fermentation, sterilizing at 90 deg.C for 20min to obtain primary coffee ferment;

Example 6

(2) Mixing the coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) Lactobacillus plantarum bacterial liquid, fermenting at 37 deg.C for 12h, sterilizing at 90 deg.C for 20min after fermentation is completed, and making into primary coffee ferment;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the primary coffee fermented product) Kluyveromyces lactis bacterial liquid, fermenting at 28 deg.C for 48 hr at 180r/min, and filtering to obtain the coffee fermented product.

Example 7

(1) Grinding 20 parts by mass of deeply roasted coffee beans, mixing with 20 parts by mass of milk, 5 parts by mass of honey, 50 parts by mass of water and 5 parts by mass of glucose, and homogenizing under high pressure to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

(2) Mixing coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) Streptococcus thermophilus bacterial liquid, fermenting at 37 deg.C for 12 hr, sterilizing at 90 deg.C for 20min after fermentation is completed, and making into primary coffee ferment;

Example 8

(2) Mixing coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) Bifidobacterium longum bacterial liquid, fermenting at 37 deg.C for 12h, sterilizing at 90 deg.C for 20min after fermentation is completed, and making into primary coffee ferment;

Example 9

(2) Mixing coffee homogenate with 0.5% (the volume of the bacteria solution accounts for 0.5% of the volume of the coffee homogenate) bacteria solution (composed of 0.25% Lactobacillus plantarum and 0.25% Streptococcus thermophilus), fermenting at 37 deg.C for 12 hr, sterilizing at 90 deg.C for 20min after fermentation is completed, and making into primary coffee ferment;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacterial liquid is 0.5% of the primary coffee fermented product) Kluyveromyces lactis bacterial liquid, fermenting at 28 deg.C for 48 hr at 180r/min, and filtering to obtain the final product.

Example 10

(1) Grinding 5 parts by mass of deeply roasted coffee beans, mixing with 34 parts by mass of milk, 1 part by mass of honey, 50 parts by mass of water and 10 parts by mass of glucose, and carrying out high-pressure homogenization to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

(3) Mixing the primary coffee ferment with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the primary coffee ferment) Kluyveromyces lactis bacterial liquid, fermenting at the rotation speed of 180r/min and the temperature of 28 ℃ for 48h, and filtering to obtain the coffee ferment.

Example 11

(1) According to the mass parts, 50 parts of deeply roasted coffee beans are crushed and then mixed with 5 parts of milk, 5 parts of honey, 30 parts of water and 10 parts of glucose, and high-pressure homogenization is carried out to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

Example 12

(1) Grinding 5 parts by mass of deeply roasted coffee beans, mixing with 50 parts by mass of milk, 5 parts by mass of honey, 30 parts by mass of water and 10 parts by mass of glucose, and carrying out high-pressure homogenization to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the coffee homogenate) of Kluyveromyces lactis bacterial liquid, fermenting at 28 deg.C for 48 hr at 180r/min, and filtering to obtain coffee fermented product.

Comparative example 1

The present comparative example provides a method of preparing a coffee ferment comprising the steps of:

(1) Grinding 20 parts by mass of deeply roasted coffee beans, and mixing with 20 parts by mass of milk, 5 parts by mass of honey, 50 parts by mass of water and 5 parts by mass of glucose to prepare coffee homogenate;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacteria liquid accounts for 0.5% of the volume of the coffee homogenate) aerobic strain bacteria liquid (composed of 0.25% of Kluyveromyces lactis and 0.25% of Saccharomyces cerevisiae), fermenting at 180r/min at 28 deg.C for 48h, and filtering to obtain coffee fermented product.

Comparative example 2

The present comparative example provides a method of preparing a coffee mix, comprising the steps of:

(2) Mixing the coffee homogenate with 0.5% (water volume is 0.5% of coffee homogenate volume) deionized water, standing at 37 deg.C for 12 hr, and sterilizing at 90 deg.C for 20min to obtain coffee primary mixture;

(3) The coffee primary mix was mixed with 0.5% (water volume 0.5% of the coffee primary mix) deionized water, mixed at 28 ℃ for 48h at 180r/min and filtered to produce a coffee mix.

Comparative example 3

(1) Grinding 20 parts by mass of deeply roasted coffee beans, mixing with 75 parts by mass of water and 5 parts by mass of glucose, and carrying out high-pressure homogenization to obtain coffee homogenate; wherein the homogenizing pressure is 70MPa, and homogenizing for 30min;

Comparative example 4

(3) Mixing the primary coffee fermented product with 0.5% (water volume accounts for 0.5% of the primary coffee fermented product volume) deionized water, fermenting at 28 deg.C for 48 hr at 180r/min, and filtering to obtain coffee fermented product.

Comparative example 5

The present comparative example provides a method for preparing a coffee ferment, comprising the steps of:

(2) Mixing the coffee homogenate with 0.5% deionized water, fermenting at 37 deg.C for 12 hr, and sterilizing at 90 deg.C for 20min to obtain primary coffee fermented product;

Comparative example 6

(2) Mixing the coffee homogenate with 0.5% (the volume of the bacterial liquid accounts for 0.5% of the volume of the primary coffee fermented product) aerobic bacterial liquid (composed of 0.25% of Kluyveromyces lactis and 0.25% of Saccharomyces cerevisiae), fermenting at 180r/min at 28 deg.C for 48h, and sterilizing at 90 deg.C for 20min to obtain primary coffee fermented product;

(3) Mixing the primary coffee fermented product with 0.5% (the volume of the bacterial liquid is 0.5% of the volume of the coffee homogenate) bacterial liquid (composed of 0.25% Lactobacillus plantarum and 0.25% Streptococcus thermophilus), fermenting at 37 deg.C for 12 hr, and filtering to obtain coffee fermented product.

Application example 1

The application embodiment provides a skin care essence water added with coffee fermentation, and the formula composition of the skin care essence water is shown in the following table 1 in percentage by mass:

TABLE 1

Composition (I)	Mass fraction (%)
		Deionized water	Balance of
Example 5 coffee fermentation	10.00
		1,2 pentanediol	5.00
Beta-glucan	4.00
		Glycerol	2.00
Hydrogenated lecithin	1.00
		1,2 hexanediol	1.00
EDTA disodium salt	0.02
		Polyglycerol-10 oleate	0.5
Glycerol polyether-26	0.5
		P-hydroxyacetophenone	0.30
Chinese gum	0.20

Application comparative example 1

The application comparative example provides skin care essence water, and the formula composition of the skin care essence water is shown in the following table 2 in percentage by mass:

TABLE 2

Composition (I)	Mass fraction (%)
		Deionized water	Allowance of
1,2 pentanediol	5.00
		Beta-glucan	4.00
Glycerol	2.00
		Hydrogenated lecithin	1.00
1,2 hexanediol	1.00
		EDTA disodium salt	0.02
Polyglycerol-10 oleate	0.5
		Glycerol polyether-26	0.5
P-hydroxyacetophenone	0.30
		Chinese gum	0.20

Comparative application example 2

The application comparative example provides skin care essence water, and the formula composition of the skin care essence water is shown in the following table 3 in percentage by mass:

TABLE 3

Composition (I)	Mass fraction (%)
		Deionized water	Balance of
Comparative example 4 coffee ferment	10.00
		1,2 pentanediol	5.00
Beta-glucan	4.00
		Glycerol	2.00
Hydrogenated lecithin	1.00
		1,2 hexanediol	1.00
EDTA disodium salt	0.02
		Polyglycerol-10 oleate	0.5
Glycerol polyether-26	0.5
		P-hydroxyacetophenone	0.30
Chinese gum	0.20

Comparative application example 3

The application comparative example provides skin care essence water, and the formula composition of the skin care essence water is shown in the following table 4 in percentage by mass:

TABLE 4

Application comparative example 4

The application comparative example provides skin care essence water, and the formula composition of the skin care essence water is shown in the following table 5 in percentage by mass:

TABLE 5

Composition (I)	Mass fraction (%)
		Deionized water	Balance of
Comparative example 6 coffee fermentation product	10.00
		1,2 pentanediol	5.00
Beta-glucans	4.00
		Glycerol	2.00
Hydrogenated lecithin	1.00
		1,2 hexanediol	1.00
EDTA disodium salt	0.02
		Polyglycerol-10 oleate	0.5
Glycerol polyether-26	0.5
		P-hydroxyacetophenone	0.30
Chinese gum	0.20

Product effectiveness testing

1. The caffeine content of the coffee ferments or coffee blends prepared in examples 1 to 12 and comparative examples 1 to 6, as well as DPPH scavenging ability, hydroxyl radical scavenging ability, and total antioxidant activity were measured.

(1) Caffeine content determination

The caffeine content was measured by high performance liquid chromatography using a 4.6 x 150mmC18 column with mobile phases: acetonitrile (A) and 0.2% phosphoric acid water (B), the flow rate is 1mL/min, the wavelength is 280nm, and the specific method comprises the following steps:

0-10 minutes, 10% a;10-30 minutes, 10-8%A;30-31 minutes, 8-80% a;31-40 minutes, 80% a;40-41 min, 80-10% A;41-45 minutes, 10% A.

(2) Evaluation of hydroxyl radical scavenging ability

The following tests were carried out on the samples prepared in examples 1 to 12 and comparative examples 1 to 6.

1.0mL of FeSO is added into the reaction system ₄ Solution (9.0 mmol/L), 1mL salicylic acid-ethanol solution (9.0 mmol/mL), 1.0mL sample solution, and 1.0mL H ₂ O ₂ (8.8 mmol/mL) and reacted in 37 ℃ water bath for 30min, absolute ethyl alcohol is used as a blank control, the absorbance is measured at 510nm, and the removal rate of OH of each extracting solution is calculated according to the following formula (1):

Y(％)＝[1-(A1-A2)/A3]×100％ (1)

in the formula, A1 is the absorbance of an extracting solution reaction system; a2 is H or not ₂ O ₂ Absorbance of the sample (absolute ethanol instead of H) ₂ O ₂ ) (ii) a A3 is the absorbance of the blank control in absolute ethanol.

(3) Evaluation of DPPH radical scavenging ability

The ability to scavenge DPPH free radicals reflects to some extent the antioxidant capacity of the material. The greater the free radical clearance rate, the stronger the antioxidant capacity and the stronger the anti-aging capacity. Therefore, the anti-aging effect of the DPPH can be judged by researching the capability of the sample to remove DPPH free radicals. The following tests were carried out on the samples prepared in examples 1 to 12 and comparative examples 1 to 6.

Adding 2.0mL of DPPH test solution with the mass concentration of 45.8mg/L and a certain amount (such as 0.5 mL) of sample to be tested into a test tube, supplementing the total volume to 3mL by 50% ethanol (v/v), shaking up, reacting in a dark place for 30min, and measuring the absorbance at the wavelength of 517nm by using A1 cm cuvette and marking as A1; adding 2mL of absolute ethyl alcohol and a sample to be detected with a corresponding volume into the test tube, supplementing the total volume to 3mL by 50% ethanol (v/v), and recording the detected absorbance as A2; 2mL of DPPH test solution and 1mL of 50% ethanol (v/v) were added to the tube, and the absorbance measured was designated as A3. Calculating the DPPH free radical clearance rate (Y) of the liquid to be detected according to the following formula (2);

the DPPH free radical clearance is calculated by the formula: y (%) = [1- (A1-A2)/A3 ]. Times 100% (2)

In the formula, A1 is the absorbance value of the system after the blank control is cleared of DPPH, A2 is the absorbance value of the system after the sample is cleared of DPPH, and A3 is the absorbance value of the reaction system before the medicine is not added.

(4) Determination of total antioxidant activity by FRAP method

The following tests were carried out on the samples prepared in examples 1 to 12 and comparative examples 1 to 6, with the following steps:

1) And (3) determination of a sample: adding 100 μ L sample solution (diluted appropriately according to the condition), adding 3mL FRAP working solution, mixing, reacting at 37 deg.C for 15min, measuring absorbance at 593nm, and repeating each experiment three times.

2)FeSO ₄ Determination of the standard curve: according to step 1), at 0.1-1.6mmol/L of FeSO ₄ The standard solution of (2) was used in place of the sample to draw a standard curve.

3) And (3) calculating the result: final total antioxidant activity of the sample as FeSO ₄ Expressed in mmol/L as FeSO ₄ Obtaining corresponding FeSO on a standard curve according to the A value after reaction for a standard solution ₄ Is defined as the FRAP value. The greater the FRAP value, the greater the antioxidant activity.

The test results are shown in Table 6.

TABLE 6 caffeine content and hydroxyl radical clearance, DPPH clearance, total antioxidant Activity results

The results in Table 6 show that examples 1 to 12, compared to comparative examples 1 to 6, both of high pressure homogenization and two-stage fermentation techniques improve the release of caffeine, which is an active substance in coffee beans, and that deeply roasted coffee has a higher caffeine content and thus may be advantageous in terms of caffeine dissolution when roasted at high temperatures for a long period of time during roasting. In example 11, the caffeine content and the antioxidant effect were increased by increasing the ratio of coffee, but the utilization of the effective active substance in coffee was decreased as compared with example 5. Compared with other fermentations, the effect is better when lactobacillus plantarum and streptococcus thermophilus are used together in the anaerobic fermentation stage. When lactobacillus plantarum and streptococcus thermophilus are adopted in the anaerobic fermentation stage, and the inoculation amount ratio of lactobacillus plantarum is more than 40%, the caffeine dissolution rate in the fermented coffee product obtained through fermentation is the highest (the final caffeine content is higher when the same amount of coffee beans are fermented), and the effects of the final fermented product on hydroxyl radical clearance, DPPH clearance and total antioxidant activity are the best.

2. Evaluation of erythrocyte hemolytic Properties

The red blood cell hemolysis (RBC) experiment is one of the alternatives of the rabbit eye irritation experiment (Draizetest), and the basic principle is to evaluate the damage of chemicals to eye tissue cells by measuring the dissolution amount and the denaturation degree of hemoglobin. The RBC test is used internationally for evaluating eye irritation studies on chemicals such as cosmetics and raw materials.

The samples prepared in examples 1 to 12 and comparative examples 1 to 6 were used in the hemolysis test of red blood cells according to the RBC test method and the grading Standard of European Alternatives validation center (ECVAM). Wherein HD50 is the sample concentration at which 50% of erythrocytes are hemolyzed, DI is the protein denaturation index, and L/D is the ratio of HD50 to DI, and the specific test standards are shown in Table 7.

TABLE 7 erythrocyte hemolysis test Standard

L/D	Grading
		L/D>100	Has no irritation
10<L/D≤100	Micro-stimulation property
		1<L/D≤10	Mild irritation
0.1<L/D≤1	Irritation due to poisoning
		L/D≤0.1	Severe irritation

TABLE 8 results of the hemolysis test of erythrocytes

The results of the erythrocyte hemolysis test are shown in Table 8, and example 5 shows that the mixture or fermentation obtained by carrying out no fermentation or only anaerobic fermentation, and changing the order of the two-stage fermentation, increases the cell irritation as compared to comparative examples 2, 4 and 6. The coffee fermentation product obtained by fermenting coffee by adopting high-pressure homogenization and two-stage fermentation (anaerobic fermentation and aerobic fermentation) is safe and non-irritant. Example 5 in combination with comparative example 3 shows that the addition of both milk and honey reduces the irritation of the coffee fermentate.

3. Determination of hyaluronidase inhibition capacity by hyaluronidase in vitro inhibition

taking 0.1mL of CaCl ₂ The solution (0.25 mmol/L) and 0.5mL hyaluronidase solution (100U/mL) are cultured for 20min at 37 ℃ in water bath at constant temperature; adding 0.5mL of sample solution to be tested, and keeping the temperature for 20min; adding 0.5mL sodium hyaluronate solution (0.5 mg/mL), culturing in water bath at 37 deg.C for 30min, taking out, and standing at room temperature for 5min; adding 0.1mL NaOH solution (0.4 mol/L) and 0.5mL acetylacetone solution (3.5 mL acetylacetone dissolved in 50mL 1.0mol/L sodium carbonate solution), heating in boiling water bath for 15min, immediately transferring to ice water bath, and cooling for 5min; 1.0mL of an Ellisib reagent (0.8 g p-dimethylaminobenzaldehyde dissolved in 15mL concentrated hydrochloric acid and 15mL absolute ethanol) was added dropwise, and diluted with 3.0mL absolute ethanol, left at room temperature for 20min for color development, and the absorbance value at a wavelength of 540nm was measured with a spectrophotometer. The calculation formula of the sample for measuring the hyaluronidase inhibition rate is as follows:

hyaluronidase inhibition = [ (A-B) - (C-D) ]/(A-B). Times.100% (3);

in the above formula, A-absorbance value of control solution (replacing sample solution with acetic acid buffer solution),

b-control blank solution absorbance value (the sample solution and the enzyme solution are replaced by acetic acid buffer solution),

c is the absorbance value of the sample solution,

d-absorbance value of sample blank solution (replacing enzyme solution with acetic acid buffer solution).

The test results are shown in table 6.

4. Study of the Effect of coffee fermentates on HaCaT cell survival after UV irradiation

Inoculation of HaCaT cells into 6-well plates, 5% CO at 37% ₂ The incubator of (a) was incubated overnight. When the plating rate of the cells in the 6-well plate reached 50% -60%, the cell culture medium was added to the blank control group and the negative control group, the cell culture medium containing 5% of the test sample (samples prepared in examples 1-12 and comparative examples 1-6) was added to the test sample group, pre-treated for 1 hour, and then subjected to UVB irradiation (30 mJ/cm) ² ) And (4) 40min. Further at 37 deg.C, 5% CO ₂ Is continuously cultured in the incubatorAfter 24h of incubation, the medium was aspirated, washed 2-3 times with PBS, and the cell viability was determined by MTT method, the results of which are shown in Table 9.

TABLE 9 inhibition of hyaluronidase and results of cell relative survival assays

The results in table 9 show that the coffee fermentations prepared in the examples and the comparative examples have certain inhibition rate on hyaluronidase, the coffee fermentations fermented by two-stage fermentation and added with milk and honey can further improve the inhibition rate on hyaluronidase, and the fermented coffee fermentations have better protection effect on HaCat cells after UV irradiation and can promote the growth of the cells.

5. Evaluation test of human body efficacy

Randomly selecting 40 volunteers with healthy skin and no history of dermatosis and allergy, wherein the age is 20-45 years, and 20 volunteers are male and female respectively. Before testing, volunteers were allowed to calm down at room temperature (25 + -2) ° c, relative Humidity (RH) (50 + -2)% and the area tested was cleaned with a cleaning product, wiped with alcohol, and then allowed to air dry for 15min. The skin care essence lotion prepared in application example 1 was used as an experimental group for the left face area of each volunteer, and the skin care essence lotion prepared in application example 1 was used for the right face area of each volunteer for comparison. The skin care essence is applied once every morning, noon and evening, 1g of the product is applied to the face every time, the face is massaged for 2 minutes, the period is 4 weeks, and the visits in the first, second and fourth weeks are performed.

Facial skin test: the change condition of facial wrinkles is analyzed by a VISIA whole-face analyzer through photographing, the change condition of facial melanin and hydration degree is analyzed by an MPA580 skin tester, and the change condition of skin sensitivity is determined by a lactic acid stimulation experiment. The operation flow of the lactic acid stimulation experiment comprises the following steps: before the test, the volunteers uniformly use mild cleanser to clean facial skin, then filter paper containing 10% lactic acid is attached to the nasolabial sulcus, the pricking senses of the volunteers at 2.5min and 5min are recorded by a 4-point method, the pricking senses are 0 point without pricking, 1 point with mild pricking, 2 points with moderate pricking and 3 points with severe pricking, and the volunteers evaluate the relaxing (0-5 points) effect of the samples in the using process, wherein the highest point is 5, the effect is obvious, and the lowest point is 0, the effect is not effective and can be accurately 0.5. The test results are shown in table 10.

Table 10 skin lotion efficacy test results

As shown by the results of table 10, the number of facial wrinkles was significantly improved after using application example 1 containing coffee ferment compared to application comparative examples 1 to 4, indicating that the application of coffee ferment as an effective active ingredient in cosmetics can effectively reduce wrinkles. The hydration degree of the facial skin is increased by 20-35% after the application of the coffee fermented product 1, so that the coffee fermented product is used as an effective active ingredient in cosmetics to improve the problem of dry skin and improve the sense of skin tightness. Compared with application comparative examples 1-4, the facial skin sensitivity is obviously improved after the application example 1 is used, and the fact that the coffee fermentation product is used as an effective active ingredient in cosmetics can effectively reduce the skin sensitivity and enhance the skin barrier function.

Claims

1. A method for preparing a coffee ferment is characterized by comprising the following steps:

2. The method according to claim 1, wherein the pressure of the high-pressure homogenization is 35 to 100MPa, and the time of the high-pressure homogenization is 20 to 60min.

3. The method according to claim 1, wherein an anaerobic strain is added during the anaerobic fermentation; the anaerobic strain comprises at least one of lactobacillus plantarum, bifidobacterium longum or streptococcus thermophilus; preferably, the anaerobic strain comprises lactobacillus plantarum and streptococcus thermophilus.

4. The process according to claim 1, wherein an aerobic strain is added during the aerobic fermentation; the aerobic strain is at least one of Kluyveromyces lactis or Saccharomyces cerevisiae.

5. The method according to claim 3, wherein the amount of the anaerobic strain inoculated is 0.1 to 1.0% by volume; the temperature of the anaerobic fermentation is 30-40 ℃, and the time of the anaerobic fermentation is 6-24h.

6. The preparation method according to claim 4, wherein the inoculation amount of the aerobic strain is 0.1-1.0% by volume percentage; the temperature of the aerobic fermentation is 26-35 ℃, and the time of the aerobic fermentation is 18-72h.

7. A preparation method according to claim 1 or 2, wherein the coffee beans are at least one of unroasted, light roasted, medium roasted, and deep roasted coffee beans.

8. The method of any one of claims 1-4, wherein the fermentation feedstock further comprises water; the fermentation raw materials comprise, by weight, 5-60 parts of coffee beans, 5-60 parts of milk, 0.5-10 parts of honey and 5-80 parts of water.

9. Use of a process for the preparation of a coffee ferment as claimed in any one of claims 1 to 8 in the preparation of a cosmetic product.

10. A cosmetic comprising the coffee fermented product produced by the production method according to any one of claims 1 to 8.