CN115337238B

CN115337238B - Preparation method and application of coffee ferment

Info

Publication number: CN115337238B
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: 2023-09-05
Anticipated expiration: 2042-08-24
Also published as: CN115337238A

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 beans, milk and honey are used as fermentation raw materials, and are prepared by anaerobic fermentation and aerobic fermentation after high-pressure homogenization, wherein the pressure of the high-pressure homogenization is 10-100MPa. The invention adopts the high-pressure homogenizing extraction technology and combines biological double-stage fermentation, takes coffee beans, milk and honey as main raw materials, carries out anaerobic fermentation and then carries out aerobic fermentation, so that active substances of the coffee beans, the milk and the honey are fully released into a fermented product, and the prepared coffee fermented product has good functions of resisting oxidation and inflammation and improving cell activity, is more beneficial to absorption and utilization of skin, and has excellent anti-aging and relieving effects.

Description

Preparation method and application of coffee ferment

Technical Field

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

Background

Aging (Aging), also known as Aging, is an integral stage in the course of any biological vital activity. As the outermost protective layer of the human body, the aging characterization of the skin mainly comprises skin relaxation and wrinkle appearance, and the skin of the exposed part of the body becomes rough, and the wrinkles deepen and thicken; the skin is not elastic and becomes relaxed; vasodilation, dysplastic epidermis, etc. Because aging has complex psychological and physiological effects on life and work of people, the aging can cause a series of psychological problems such as anxiety, depression, spelt and the like. Thus, anti-aging treatments, particularly on the skin, have been one of the focus of research. Causes of skin aging include free radical damage, reduced cell turnover rate, damage caused by ultraviolet irradiation, protein glycosylation, elevated levels of matrix metalloproteinases, DNA mutation, shortening of telomeres, mitochondrial damage, etc.

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

Coffee contains alkaloids, phenolic acids, flavonoids, terpenes, sterols, lipids and volatile components, etc., and caffeine (1, 3, 7-trimethylxanthine, caffeine) is the main alkaloid component in coffee fruits, which is the source of bitter taste of coffee, and is widely found in tea, cocoa and coffee. Caffeine is a representative substance in caffeine, and is one of the most important active ingredients in coffee beans. Caffeine has a stable chemical structure, can be dissolved in water, and has various biological activities. Studies have shown that caffeine reduces oxidative stress caused by D-galactose by inhibiting increases in COX-2, NOS-2, TNF- α and IL-1β, thereby alleviating neuroinflammation; in addition, caffeine slows down oxidative stress induced senescence by activating SIRT 3/MAPK/autophagy. At present, coffee beans are mostly applied to the field of eating, and the skin care effect is not fully and effectively developed and utilized.

Therefore, further development of the application of coffee beans in the field of skin care is needed, and the application of the coffee beans in the field of skin care is utilized to prepare skin care products with anti-aging and soothing effects.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a preparation method and application of a coffee ferment. The coffee ferment has good antioxidant, antiinflammatory and cell activity improving effects, and can be used for resisting aging and relieving.

In a first aspect, the invention provides a method for preparing a coffee ferment.

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

the coffee bean, milk and honey are used as fermentation raw materials, and are subjected to high-pressure homogenization, 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, anaerobic strains are 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. The inoculation amount refers to the volume of the inoculated anaerobic strain bacterial liquid accounting for 0.1-1.0% of the volume of the fermentation raw material. Further preferably, the anaerobic strain is inoculated in an amount of 0.3% to 0.8%.

Preferably, the anaerobic bacterial 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 inoculation amount of lactobacillus plantarum accounts for 45% or more of the total inoculation amount of the anaerobic strain.

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 strain is kluyveromyces lactis or 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% by volume percent. The volume of the inoculated aerobic strain bacterial liquid accounts for 0.1-1.0% of the volume of the fermentation raw material. Further preferably, the inoculum size 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, by weight, 5-50 parts of coffee beans, 5-50 parts of milk, 1-8 parts of honey and 20-70 parts of water; more preferably, the fermentation feedstock comprises, in parts by weight, 15-30 parts of coffee beans, 15-40 parts of milk, 1-5 parts of honey and 30-60 parts of water.

Preferably, the fermentation feedstock further comprises at least one of potato, glucose, peptone. The total weight of the potato, the glucose and the peptone is 2-10 parts based on the important parts.

Preferably, the fermentation raw materials are as follows 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 are as follows in parts 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 unbaked, light roasted, medium roasted, deep roasted coffee beans. Further preferably, the coffee beans are deep-roasted coffee beans.

Preferably, the anaerobic fermentation temperature is 30-40 ℃, and the anaerobic fermentation time is 6-24 hours; preferably, the anaerobic fermentation is carried out at a temperature of 35-40 ℃ for 8-18 hours.

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

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

More specifically, a method for preparing a coffee ferment, comprising the steps of:

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

(2) Mixing the coffee homogenate with anaerobic strains, and carrying out anaerobic fermentation to obtain a primary coffee ferment;

(3) Mixing the primary coffee ferment with an aerobic strain, performing aerobic fermentation, and filtering to obtain a coffee ferment.

In a second aspect, the invention provides the use of a method for preparing a coffee ferment as described above in the preparation of a cosmetic.

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

A cosmetic comprising a coffee ferment prepared by the method.

Preferably, the cosmetic is essence water.

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

Milk contains abundant proteins, vitamins, basic acids and microelements, and has skin caring effect. Milk protein is also called cow milk protein, is a generic name of a plurality of protein mixtures in cow milk, is separated from cow milk, has the functions of resisting lipid peroxidation and scavenging hydroxyl radicals and DPPH radicals, can be used as a humectant, a nutritional agent and a conditioning agent to be added into cosmetics, and can increase the efficacy of other active ingredients. The honey contains a large amount of saccharides, polyphenols, 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 activities. The invention damages the coffee fiber by a physical high-pressure homogenization technology, so that the effective components in the coffee beans are fully released and are fused with milk and honey. The adopted double-stage fermentation is carried out firstly, anaerobic fermentation is carried out again, aerobic fermentation is carried out, the bioconversion capability of different microorganisms is fully exerted, the content of effective substances in the coffee beans is further improved, the irritation of raw materials is reduced, the types and the content of active substances in a fermentation product are effectively increased, and the obtained coffee fermentation product has good effects of resisting oxidation and inflammation and improving the cell activity. By selecting zymophyte in the double-stage fermentation, polysaccharide, amino acid, enzyme and some small molecular components generated by fermentation and conversion can better play a role in nourishing skin, and have remarkable anti-aging and relieving effects.

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

the invention adopts the high-pressure homogenizing extraction technology and combines biological double-stage fermentation, takes coffee beans, milk and honey as main raw materials, carries out anaerobic fermentation and then carries out aerobic fermentation, so that active substances of the coffee beans, the milk and the honey are fully released into a fermented product, and the prepared coffee fermented product has good functions of resisting oxidation and inflammation and improving cell activity, 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 will be presented. It should be noted that the following examples do not limit the scope of the 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 of about 10 ⁷ CFU/mL, kluyveromyces lactis concentration of about 10 ⁷ CFU/mL, saccharomyces cerevisiae at a concentration of about 10 ⁷ CFU/mL. Other materials, reagents or apparatus are available from conventional commercial sources, unless otherwise specified, or may be obtained by methods known in the art.

Example 1

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

(1) According to the mass parts, after 20 parts of deeply roasted coffee beans are crushed, the crushed coffee beans are 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, homogenizing for 30min;

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

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

Example 2

(1) According to the mass parts, after 20 parts of moderately roasted coffee beans are crushed, the crushed coffee beans are 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, homogenizing for 30min;

Example 3

(1) According to the mass parts, after 20 parts of lightly baked coffee beans are crushed, the crushed coffee beans are 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, homogenizing for 30min;

Example 4

(1) According to the mass parts, 20 parts of unroasted coffee beans are crushed, mixed with 20 parts of milk, 5 parts of honey, 50 parts of water and 5 parts of glucose, and homogenized under high pressure to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, homogenizing for 30min;

Example 5

(2) Mixing coffee homogenate and 0.5% (0.5% of the volume of the bacterial liquid is the volume of the coffee homogenate) bacterial liquid (composed of 0.25% lactobacillus plantarum and 0.25% streptococcus thermophilus), fermenting at 37deg.C for 12 hr, and sterilizing at 90deg.C for 20min to obtain primary fermented product of coffee;

Example 6

(2) Mixing coffee homogenate and 0.5% (volume of bacteria liquid is 0.5% of volume of coffee homogenate) lactobacillus plantarum bacteria liquid, fermenting at 37deg.C for 12 hr, sterilizing at 90deg.C for 20min after fermentation, and making into primary fermented product of coffee;

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

Example 7

(2) Mixing coffee homogenate and 0.5% (volume of bacteria liquid is 0.5% of volume of coffee homogenate) Streptococcus thermophilus bacteria liquid, fermenting at 37deg.C for 12 hr, sterilizing at 90deg.C for 20min after fermentation, and making into primary fermented product of coffee;

Example 8

(2) Mixing coffee homogenate and 0.5% (volume of bacteria liquid is 0.5% of volume of coffee homogenate) of Bifidobacterium longum bacteria liquid, fermenting at 37deg.C for 12 hr, sterilizing at 90deg.C for 20min after fermentation, and making into primary fermented product of coffee;

Example 9

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

Example 10

(1) According to the mass parts, 5 parts of deeply roasted coffee beans are crushed, mixed with 34 parts of milk, 1 part of honey, 50 parts of water and 10 parts of glucose, and homogenized under high pressure to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, homogenizing for 30min;

Example 11

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

Example 12

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

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

Comparative example 1

This comparative example provides a method for preparing a coffee ferment comprising the steps of:

(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 to prepare coffee homogenate;

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

Comparative example 2

This 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 37deg.C for 12 hr, and sterilizing at 90deg.C for 20min to obtain primary coffee mixture;

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

Comparative example 3

(1) According to the mass parts, smashing 20 parts of deeply roasted coffee beans, mixing with 75 parts of water and 5 parts of glucose, and homogenizing under high pressure to prepare coffee homogenate; wherein the homogenizing pressure is 70MPa, homogenizing for 30min;

Comparative example 4

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

Comparative example 5

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

Comparative example 6

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

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

Application example 1

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

TABLE 1

Composition of the components	Mass fraction (%)
		Deionized water	Allowance of
Coffee fermentate prepared in example 5	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
		Para hydroxy acetophenone	0.30
Hansheng rubber	0.20

Comparative example 1 was used

The application comparative example provides a skin care essence, which comprises the following components in percentage by mass:

TABLE 2

Composition of the components	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
Para hydroxy acetophenone	0.30
		Hansheng rubber	0.20

Comparative example 2 was used

TABLE 3 Table 3

Composition of the components	Mass fraction (%)
		Deionized water	Allowance of
Comparative example 4 coffee fermentate	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
		Para hydroxy acetophenone	0.30
Hansheng rubber	0.20

Comparative example 3 was used

TABLE 4 Table 4

Comparative example 4 was used

TABLE 5

Composition of the components	Mass fraction (%)
		Deionized water	Allowance of
Comparative example 6 coffee fermentate	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
		Para hydroxy acetophenone	0.30
Hansheng rubber	0.20

Product effect test

1. The caffeine content of the coffee fermented product or coffee mixture obtained in examples 1 to 12 and comparative examples 1 to 6 was examined for DPPH scavenging ability, hydroxyl radical scavenging ability, and total antioxidant activity.

(1) Caffeine content determination

The caffeine content was determined by high performance liquid chromatography using a 4.6 x 150mm c18 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 is as follows:

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

(2) Evaluation of the ability to scavenge hydroxyl radical

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

1.0mL 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 testThe solution was then added with 1.0mL of H ₂ O ₂ (8.8 mmol/mL) was reacted in a water bath at 37℃for 30min, the absorbance was measured at 510nm with absolute ethanol as a blank, and the clearance of OH from each extract was calculated according to the following formula (1):

Y(％)＝[1-(A1-A2)/A3]×100％ (1)

wherein A1 is absorbance of an extracting solution reaction system; a2 is H-free ₂ O ₂ Absorbance of the sample (substitution of absolute ethanol for H) ₂ O ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the A3 is the absorbance of the control absolute ethanol.

(3) DPPH radical scavenging ability evaluation

The DPPH free radical scavenging ability is to some extent the antioxidant capacity of the reactive species. The higher the free radical scavenging rate, the stronger the antioxidant capacity and the stronger the anti-aging capacity. Therefore, the anti-aging effect of the DPPH free radical scavenging ability can be judged by the research of the sample. 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 uniformly, carrying out light-proof reaction for 30min, and measuring absorbance at 517nm wavelength 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 a test tube, supplementing the total volume to 3mL by using 50% ethanol (v/v), and recording the measured absorbance as A2; to the test tube, 2mL of DPPH test solution and 1mL of 50% ethanol (v/v) were added, and the absorbance was measured and designated as A3. Calculating the clearance rate (Y) of the solution to be tested to DPPH free radicals according to the following formula (2);

the calculation formula of the DPPH free radical clearance is as follows: y (%) = [1- (A1-A2)/A3 ] ×100% (2)

Wherein A1 is the absorbance value of the system after the blank 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 added.

(4) FRAP method for determining total antioxidant activity

The samples prepared in examples 1-12 and comparative examples 1-6 were tested as follows:

1) Determination of the samples: 100. Mu.L of sample solution (appropriately diluted according to the situation) was added to the reaction tube, 3mL of FRAP working solution was added thereto, and the mixture was uniformly mixed, reacted at 37℃for 15 minutes, and the absorbance was measured at 593nm, and each experiment was repeated three times.

2)FeSO ₄ Determination of a standard curve: according to step 1), feSO is added in an amount of 0.1 to 1.6mmol/L ₄ Instead of the sample, a standard curve was drawn.

3) Calculation results: sample final total antioxidant Activity as FeSO ₄ Expressed in terms of equivalent concentration in mmol/L, feSO ₄ The corresponding FeSO is obtained on a standard curve according to the A value after the reaction ₄ The concentration (mmol/L) of (C) is defined as the FRAP value. The greater the FRAP value, the greater the antioxidant activity.

The results of the above test are shown in Table 6.

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

The results in Table 6 show that both the high pressure homogenization and the dual stage fermentation techniques used in examples 1-12 and comparative examples 1-6 enhance the release of caffeine as an active ingredient from the beans and that the deeply roasted coffee has a higher caffeine content, which may be beneficial to the dissolution of caffeine during the roasting process at high temperatures and for long periods of time. In example 11, the amount of caffeine and the antioxidant effect were increased by increasing the ratio of coffee added, but the utilization of the active substances in coffee was decreased as compared with example 5. The effect is better when Lactobacillus plantarum and Streptococcus thermophilus are used together in the anaerobic fermentation stage than in other fermentations. In the anaerobic fermentation stage, lactobacillus plantarum and streptococcus thermophilus are adopted, and when the inoculation amount of lactobacillus plantarum is more than 40%, the caffeine dissolution rate in the fermented coffee product obtained by fermentation is highest (the content of caffeine is higher after the same amount of coffee beans are fermented), and the effect of the final fermented product on the hydroxy free radical clearance, DPPH clearance and total antioxidant activity is optimal.

2. Erythrocyte hemolysis evaluation

The red blood cell hemolysis (RBC) test is one of alternative methods of rabbit eye irritation test (draizer), and the basic principle is to evaluate the damage of chemicals to ocular tissue cells by measuring the amount of hemoglobin elution and the degree of denaturation. RBC experiments are mainly used internationally for eye irritation studies for evaluating chemicals such as cosmetics and raw materials.

The samples prepared in examples 1-12 and comparative examples 1-6 were subjected to a erythrocyte hemolysis test in accordance with the RBC test method and fractionation criteria of the european replacement method verification center (ECVAM). Where HD50 is the concentration of the sample at which 50% of the erythrocytes were hemolyzed, DI is the protein denaturation index, and L/D is the ratio of HD50 to DI, and the specific test criteria are shown in Table 7.

TABLE 7 erythrocyte hemolysis test criteria

L/D	Grading
		L/D>100	No irritation
10<L/D≤100	Microstimulation
		1<L/D≤10	Mild irritation
0.1<L/D≤1	Toxicity irritation
		L/D≤0.1	Severe irritation

TABLE 8 erythrocyte hemolysis test results

The results of the erythrocyte hemolysis test are shown in Table 8, and comparison of example 5 with comparative examples 2, 4 and 6 shows that the mixture or fermented product obtained by fermenting without fermentation or by performing only anaerobic fermentation, and by changing the sequence of two-stage fermentation, increases the cell irritation. And the coffee ferment obtained by fermenting the coffee by adopting high-pressure homogenization and double-stage fermentation (anaerobic fermentation and then aerobic fermentation) is safe and non-irritating. Example 5 in combination with comparative example 3 shows that the addition of both milk and honey slows the stimulation of coffee ferments.

3. Measurement of hyaluronidase inhibition ability Using hyaluronidase in vitro inhibition method

0.1mL CaCl was taken ₂ The solution (0.25 mmol/L) and 0.5mL hyaluronidase solution (100U/mL) were incubated at 37℃in a water bath for 20min; adding 0.5mL of sample liquid to be tested, and continuously preserving heat for 20min; then adding 0.5mL sodium hyaluronate (0.5 mg/mL), culturing at 37 ℃ in water bath for 30min, taking out, and standing at normal temperature for 5min; 0.1mL of NaOH solution (0.4 mol/L) and 0.5mL of acetylacetone solution (3.5 mL of acetylacetone was dissolved in 50mL of 1.0mol/L sodium carbonate solution) were added, and immediately after heating in a boiling water bath for 15min, the mixture was transferred to an ice water bath for cooling for 5min; 1.0mL of an Escherichia reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15mL of concentrated hydrochloric acid and 15mL of absolute ethanol) was added dropwise, and the mixture was diluted with 3.0mL of absolute ethanol, left at room temperature for 20min to develop a color, and absorbed at a wavelength of 540nm as determined by a spectrophotometerLuminosity values. The calculation formula for measuring the inhibition rate of the sample to the hyaluronidase is as follows:

hyaluronidase inhibition ratio = [ (a-B) - (C-D) ]/(a-B) ×100% (3);

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

b-control absorbance value of blank solution (acetate buffer solution instead of sample solution and enzyme solution),

c-absorbance value of the sample solution,

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

The test results are shown in Table 6.

4. Study of the Effect of coffee fermentate on HaCaT cell viability after UV irradiation

HaCaT cells were seeded into 6-well plates at 37℃with 5% CO ₂ Is incubated overnight. When the cell plating rate in the 6-well plate reaches 50% -60%, adding cell culture medium into blank control group and negative control group, adding cell culture medium containing 5% of tested sample (samples prepared in examples 1-12 and comparative examples 1-6) into tested sample group, pretreating for 1 hr, and then performing UVB irradiation (30 mJ/cm) ² ) And 40min. Then at 37 ℃ and 5% CO ₂ After the culture was continued for 24 hours, the medium was aspirated, washed 2 to 3 times with PBS, and the cell viability was measured by MTT method, and the measurement results are shown in Table 9.

TABLE 9 results of hyaluronidase inhibition and cell relative viability tests

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

5. Human efficacy evaluation test

40 volunteers with healthy skin, no history of skin diseases and allergies were selected at random, and aged 20-45 years, 20 for men and women. The volunteers were allowed to quietly leave room temperature (25.+ -. 2) and Relative Humidity (RH) (50.+ -. 2)% in the environment before testing, and the test areas were cleaned with a cleaning product and then naturally dried for 15 minutes after being wiped with alcohol. The skin care essence water prepared in application example 1 was used as a test group in the left face area of each volunteer, and the skin care essence water prepared in application comparative example 1 was used as a control in the right face area. Skin care essence is applied once every day in the morning, noon and evening, 1g of the product is smeared on the face each time, the skin care essence is massaged for 2 minutes, the period is 4 weeks, and the return visit is carried out in the first week, the second week and the fourth week.

Facial skin test: and (3) photographing and analyzing the change condition of facial wrinkles by using a VISIA full-face analyzer, analyzing the change condition of facial melanin and hydration degree by using an MPA580 skin tester, and measuring the change condition of skin sensitivity by using a lactic acid stimulation experiment. The lactic acid stimulation experiment operation flow is as follows: the volunteers uniformly use mild cleaning agents to clean facial skin before the test, then a filter paper sheet containing 10% of lactic acid is attached to the nasal labial sulcus, the stinging feeling of the volunteers at 2.5min and 5min is recorded by a 4-point method, no stinging is 0 point, light stinging is 1 point, moderate stinging is 2 points and heavy stinging is 3 points, the volunteers evaluate the soothing (0-5 point) effect of the sample in the use process, wherein the maximum point is 5, the representing effect is obvious, the minimum point is 0, the no effect is represented, and the sample can be precisely reached to 0.5. The test results are shown in Table 10.

Table 10 skin care essence efficacy test results

The results in Table 10 show that the number of facial wrinkles is significantly improved after application example 1 using a coffee ferment containing the coffee ferment as an effective active ingredient in cosmetics, compared with application comparative examples 1 to 4, demonstrating that the wrinkles can be effectively reduced. The facial skin hydration degree after application example 1 was increased by 20% -35%, which indicates that the coffee ferment was applied as an effective active ingredient to cosmetics, improving the skin dryness problem and improving the skin firmness. The facial skin sensitivity after application example 1 was significantly improved compared to application comparative examples 1 to 4, demonstrating that the application of coffee ferment as an effective active ingredient in cosmetics can effectively reduce skin sensitivity and enhance skin barrier function.

Claims

1. A method for preparing a coffee ferment, comprising the steps of:

the method comprises the steps of taking coffee beans, milk and honey as fermentation raw materials, homogenizing under high pressure, and sequentially carrying out anaerobic fermentation and aerobic fermentation, wherein the pressure of the high pressure homogenization is 10-100MPa;

anaerobic strains are added in the anaerobic fermentation process; the anaerobic strain comprises at least one of lactobacillus plantarum, bifidobacterium longum or streptococcus thermophilus;

adding an aerobic strain in the aerobic fermentation process; the aerobic strain is selected from the group consisting of Kluyveromyces lactis and Saccharomyces cerevisiae, or the aerobic strain is selected from the group consisting of Kluyveromyces lactis.

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

3. The method of claim 1, wherein the anaerobic strain comprises lactobacillus plantarum and streptococcus thermophilus.

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

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

6. The method of preparing according to claim 1 or 2, wherein the coffee beans are at least one of unbaked, light roasted, medium roasted, deep roasted.

7. 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.

8. Use of the method for preparing a coffee ferment according to any one of claims 1 to 7 for the preparation of cosmetics.

9. Cosmetic comprising a coffee ferment prepared by the preparation method according to any one of claims 1 to 7.