A coffee fermentation product, and a method of making and using the same

Abstract

This invention belongs to the field of bio-fermentation and skincare technology, specifically relating to a coffee fermentation product, its preparation method, and its application. The preparation method is as follows: green coffee beans are ground into powder, sieved, mixed with water, and then treated with a compound enzyme for enzymatic hydrolysis. Then, fructooligosaccharides and vitamins are added, mixed thoroughly, and sterilized to obtain culture medium 1. Coffee grounds are homogenized with green tea powder, sucrose, and water, and sterilized to obtain culture medium 2. A compound of *Bifidobacterium breve* and *Saccharomyces cerevisiae* is inoculated into culture medium 1 for fermentation. Then, culture medium 2 is added, and *Lactobacillus plantarum* is inoculated for further fermentation. After fermentation, the residue powder is removed by filtration to obtain a fermentation filtrate. Acidic protease is added to the obtained fermentation filtrate for enzymatic hydrolysis, followed by centrifugation, sterilization, and filtration to obtain the coffee fermentation product. This invention, by using green coffee beans and coffee grounds for synergistic fermentation and using green tea powder as an auxiliary fermentation component, can synergistically enhance the soothing and antioxidant effects of the coffee fermentation product.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation and skin care technology, specifically relating to a coffee fermentation product, its preparation method, and its application. Background Technology

[0002] Coffee is an evergreen shrub or small tree belonging to the Rubiaceae family and the Coffea genus. It is widely popular due to its unique flavor and its anti-fatigue and energizing properties. Coffee, along with cocoa and tea, is known as one of the world's three major beverages.

[0003] Current research on coffee mainly focuses on two aspects: one is coffee pre-processing to enhance its flavor, and the other is the application of coffee in daily chemical products.

[0004] Coffee grounds, as the residue after coffee brewing, have great application potential. Even after brewing, coffee grounds still contain abundant active substances such as polysaccharides, lignin, and proteins. Among them, phenolic substances are the main contributors to the antioxidant effect of coffee grounds.

[0005] Patent publication number KR20170097837A describes a cosmetic composition obtained by fermenting a solution of dried coffee powder, coffee bean powder, and sugar with *Lactobacillus acidophilus*, *Lactobacillus casei*, and *Lactobacillus plantarum*. By utilizing these three microorganisms to ferment the dried coffee powder and coffee bean powder, when applied to cosmetics or soaps, it can exert beneficial effects on the skin, such as antioxidant and whitening effects, while also reducing skin irritation and providing a superior user experience.

[0006] Patent publication number KR20140016764A discloses a coffee bean fermentation broth or coffee by-product fermentation broth, obtained by anaerobic fermentation of coffee beans or coffee by-products using lactic acid bacteria. The resulting fermentation broth, after drying, yields an ethanol extract that, when used in cosmetic compositions, exhibits skin cell proliferation, antioxidant, moisturizing, wrinkle-improving, skin-whitening, and anti-inflammatory effects.

[0007] However, the soothing and antioxidant effects of the aforementioned coffee fermentation products still need further improvement. Providing a different preparation process that can significantly enhance the activity and efficacy of coffee fermentation products is the technical problem that this invention needs to solve. Summary of the Invention

[0008] In view of the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a method for preparing coffee fermentation products.

[0009] Another object of the present invention is to provide a coffee fermentation product prepared by the above method.

[0010] Another object of the present invention is to provide the application of the above-mentioned coffee fermentation product in skin care products.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A method for preparing a coffee fermentation product includes the following preparation steps:

[0013] (1) Grind the green coffee beans into powder, sieve them, mix the obtained coffee powder with water, add a compound enzyme for enzymatic hydrolysis, add oligofructose and vitamins to the enzymatic hydrolysis solution, mix well, sterilize, and obtain culture medium 1.

[0014] (2) Mix coffee grounds (a conventional raw material, the solid residue left after grinding, brewing or boiling coffee beans) with green tea powder, sucrose and water, and sterilize to obtain culture medium 2;

[0015] (3) Inoculate a compound of Bifidobacterium breve and Trichosporon sp. into culture medium 1 and carry out fermentation under anaerobic conditions. After fermentation, add culture medium 2 and inoculate Lactiplantibacillus plantarum for micro-anaerobic fermentation with a gas phase oxygen content of 0.5~1 vt.%. After fermentation, filter to remove residual powder and obtain fermentation filtrate.

[0016] (4) Add acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifuge, sterilize, and filter to obtain coffee fermentation product.

[0017] Furthermore, the sieving mentioned in step (1) refers to sieving through a 200-mesh sieve.

[0018] Further, the complex enzyme mentioned in step (1) is a complex enzyme of cellulase, pectinase and glucanase; more preferably, the mass ratio of cellulase: pectinase: glucanase is 1:3:1.

[0019] Furthermore, the enzymatic hydrolysis treatment in step (1) is carried out at a temperature of 35~45℃ for 1~3h.

[0020] The main function of cellulase in the compound enzyme of this invention is to degrade cellulose on the cell wall and increase the release of coffee active ingredients; the main function of pectinase is to help clarify the liquid; and glucanase can degrade the bitterness in coffee and release coffee aroma substances.

[0021] Furthermore, the vitamin mentioned in step (1) is vitamin B1.

[0022] The purpose of adding fructooligosaccharides and vitamins to culture medium 1 is to provide carbon sources and vitamins for the fermentation process of the complex bacteria.

[0023] Furthermore, the mass fraction of each raw material in step (1) is as follows:

[0024] 25-35 parts coffee powder, 85-100 parts water, 0.5-1.2 parts compound enzyme, 8-15 parts fructooligosaccharides, and 0.5-0.9 parts vitamins.

[0025] Furthermore, the sterilization mentioned in steps (1) and (2) refers to sterilization at 115°C for 20 minutes.

[0026] Furthermore, the mass proportions of each raw material in step (2) are as follows:

[0027] 15-20 parts coffee grounds, 9-13 parts green tea powder, 8-14 parts sucrose, and 32-47 parts water.

[0028] This invention uses coffee grounds as one of the main fermentation raw materials, which has the advantage of low cost. Furthermore, coffee grounds have already undergone pre-brewing or brewing treatment, and further fermentation can release and produce active ingredients different from those produced by coffee powder fermentation, thereby synergistically enhancing the soothing effects of coffee fermentation products. By further using green tea powder as an auxiliary fermentation ingredient, the antioxidant effects of coffee fermentation products can be synergistically enhanced. The purpose of adding sucrose is to provide a carbon source for the micro-anaerobic fermentation of *Lactobacillus plantarum*.

[0029] Further, in step (3), the volume ratio of Bifidobacterium breve to Saccharomyces cerevisiae in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 1~5vt%, preferably 3vt.

[0030] The fermentation process using a compound of Bifidobacterium breve and Mycosaccharomyces cerevisiae in culture medium 1 of this invention has the following advantages:

[0031] 1. They complement each other metabolically. As a facultative anaerobe, *Mycosaccharomyces cerevisiae* can consume oxygen in the early stages of fermentation, thus protecting *Bifidobacterium breve* from the toxic effects of oxygen.

[0032] 2. The enzymes produced by the bacteria are different. Mycosacchari yeast decomposes macromolecules, while Bifidobacterium produces some organic acids and substances with unique flavors.

[0033] 3. They produce unique flavors. Bifidobacteria produce acids, and pyrogens can produce proteases or lipases to generate unique esters.

[0034] Further, the mass ratio of culture medium 1 and culture medium 2 in step (3) is 100~150:30~40.

[0035] Further, the fermentation treatment under anaerobic conditions in step (3) is carried out at a temperature of 35~40℃ for a time of 40~60h; more preferably, the fermentation treatment temperature is 37℃ for a time of 48h.

[0036] Furthermore, the inoculation amount of Lactobacillus plantarum in step (3) is 1~5 vt%, more preferably 3 vt.

[0037] The fermentation treatment of culture medium 2 using Lactobacillus plantarum has the following advantages:

[0038] 1. Strong resistance: As a dominant species among lactic acid bacteria, Lactobacillus plantarum has strong vitality and can adapt to various living environments.

[0039] 2. High substrate utilization rate: After the first fermentation, it contains a large number of secondary metabolites and primary fermentation cells, which can be utilized efficiently and quickly.

[0040] Further, the temperature of the micro-anaerobic fermentation in step (3) is 35~40℃ and the time is 40~60h; more preferably, the temperature of the micro-anaerobic fermentation is 37℃ and the time is 48h.

[0041] Further, in step (4), the concentration of the acidic protease added is 0.1~0.75wt%, the temperature of the enzymatic hydrolysis treatment is 35~45℃, and the time is 1~3h.

[0042] The effect of adding acidic protease for enzymatic hydrolysis in this invention is as follows:

[0043] 1. This process clarifies the final liquid and degrades any remaining large protein molecules.

[0044] 2. Lowering viscosity is beneficial for filtration in post-processing.

[0045] Furthermore, the sterilization in step (4) is carried out at 85°C for 30 minutes; the filtration is carried out using a 0.1μm filter membrane.

[0046] A coffee fermentation product is prepared by the above method.

[0047] The application of the above-mentioned coffee fermentation products in skin care products.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] (1) By using green coffee beans and coffee grounds for co-fermentation, not only can the cost be reduced and the aroma of coffee be increased, but the effective substances in coffee can also be released better, thus enhancing its soothing effect.

[0050] (2) By using green tea powder as an auxiliary fermentation ingredient, the antioxidant effect of coffee fermentation products can be synergistically improved. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0052] The Bifidobacterium breve involved in the following examples was purchased from Vikas Biotech Co., Ltd. under the trade name Bifidobacterium breve HC2953.

[0053] The fungus involved is classified as Trichosporon sp., with accession number CGMCC No. 29720, accession date of December 11, 2023, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] The Lactobacillus plantarum involved is classified and named Lactiplantibacillus plantarum, with accession number CGMCC No.29289, accession date of December 11, 2023, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0055] Example 1

[0056] A method for preparing a coffee fermentation product includes the following preparation steps:

[0057] (1) Grind the green coffee beans into powder and pass them through a 200-mesh sieve. Take 30 parts of coffee powder and mix it with 90 parts of water. Add 0.9 parts of a compound enzyme (cellulase: pectinase: glucanase = 1:3:1) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. Add 12 parts of oligofructose and 0.6 parts of vitamin B1 to the enzymatic hydrolysis solution and mix well. Sterilize at 115℃ for 20min to obtain culture medium 1.

[0058] (2) Mix 18 parts coffee grounds (purchased separately) with 10 parts green tea powder, 10 parts sucrose and 40 parts water homogenously, sterilize at 115℃ for 20 minutes to obtain culture medium 2.

[0059] (3) Inoculate culture medium 1 with a complex of 3vt% Bifidobacterium breve and Trichosporon sp. (Bifidobacterium breve: Trichosporon sp. = 2:1) and carry out fermentation under anaerobic conditions. The fermentation temperature is 37℃ and the fermentation time is 48h. After the fermentation is completed, add 36 portions of culture medium 2 and inoculate with 3vt% Lactiplantibacillus plantarum for micro-anaerobic fermentation. The fermentation conditions are: 37℃, gas phase oxygen content 0.75vt.%, micro-stirring, 48h. After the fermentation is completed, filter to remove the residue powder and obtain the fermentation filtrate.

[0060] (4) Add 0.5 parts of acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. After the enzymatic hydrolysis is completed, centrifuge, sterilize at 85℃ for 30min, cool to about 37℃, and filter through a 0.1μm filter membrane to obtain coffee fermentation product.

[0061] Example 2

[0062] A method for preparing a coffee fermentation product includes the following preparation steps:

[0063] (1) Grind the green coffee beans into powder and pass them through a 200-mesh sieve. Take 25 parts of coffee powder and mix it with 85 parts of water. Add 1.1 parts of a compound enzyme (cellulase: pectinase: glucanase = 1:3:1) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. Add 8 parts of oligofructose and 0.5 parts of vitamin B1 to the enzymatic hydrolysis solution and mix well. Sterilize at 115℃ for 20min to obtain culture medium 1.

[0064] (2) Mix 20 parts coffee grounds (purchased separately) with 13 parts green tea powder, 14 parts sucrose and 47 parts water homogenously, sterilize at 115℃ for 20 min to obtain culture medium 2.

[0065] (3) Inoculate culture medium 1 with a complex of 2vt% Bifidobacterium breve and Trichosporon sp. (Bifidobacterium breve: Trichosporon sp. = 2:1) and ferment under anaerobic conditions at 37°C for 48 hours. After fermentation, add 40 portions of culture medium 2 and inoculate with 4vt% Lactiplantibacillus plantarum for micro-anaerobic fermentation. Fermentation conditions: 37°C, gas phase oxygen content 0.5vt.%, micro-stirring, 48 hours. After fermentation, filter to remove residual powder and obtain fermentation filtrate.

[0066] (4) Add 0.2 parts of acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. After the enzymatic hydrolysis is completed, centrifuge, sterilize at 85℃ for 30min, cool to about 37℃, and filter through a 0.1μm filter membrane to obtain coffee fermentation product.

[0067] Example 3

[0068] A method for preparing a coffee fermentation product includes the following preparation steps:

[0069] (1) Grind the green coffee beans into powder and pass them through a 200-mesh sieve. Take 35 parts of coffee powder and mix it with 100 parts of water. Add 0.6 parts of a compound enzyme (cellulase: pectinase: glucanase = 1:3:1) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. Add 15 parts of oligofructose and 0.9 parts of vitamin B1 to the enzymatic hydrolysis solution and mix well. Sterilize at 115℃ for 20min to obtain culture medium 1.

[0070] (2) Mix 15 parts coffee grounds (purchased separately) with 9 parts green tea powder, 8 parts sucrose and 32 parts water homogenously, sterilize at 115℃ for 20 min to obtain culture medium 2.

[0071] (3) Inoculate 4vt% of a compound of Bifidobacterium breve and Trichosporon sp. (Bifidobacterium breve: Trichosporon sp. = 2:1) into culture medium 1 and carry out fermentation under anaerobic conditions at 37℃ for 48h. After fermentation, add 40 portions of culture medium 2 and inoculate 2vt% of Lactiplantibacillus plantarum for micro-anaerobic fermentation. Fermentation conditions: 37℃, gas phase oxygen content 1vt.%, micro-stirring, 48h. After fermentation, filter to remove residual powder and obtain fermentation filtrate.

[0072] (4) Add 1.0 part of acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. After the enzymatic hydrolysis is completed, centrifuge, sterilize at 85℃ for 30min, cool to about 37℃, and filter through a 0.1μm filter membrane to obtain coffee fermentation product.

[0073] Comparative Example 1

[0074] A method for preparing a coffee fermentation product, which, compared with Example 1, does not involve adding culture medium 2 for fermentation treatment, includes the following preparation steps:

[0075] (1) The preparation of culture medium 1 is the same as in Example 1.

[0076] (2) Inoculate culture medium 1 with a complex of 3vt% Bifidobacterium breve and Trichosporon sp. (Bifidobacterium breve: Trichosporon sp. = 2:1) and carry out fermentation under anaerobic conditions. The fermentation temperature is 37℃ and the fermentation time is 48h. After the fermentation is completed, filter to remove the residue powder and obtain the fermentation filtrate.

[0077] (3) Add 0.5 parts of acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 40℃ and the time is 2h. After the enzymatic hydrolysis is completed, centrifuge, sterilize at 85℃ for 30min, cool to about 37℃, and filter through a 0.1μm filter membrane to obtain coffee fermentation product.

[0078] Comparative Example 2

[0079] A method for preparing coffee fermentation products, which is the same as in Example 1 except that green tea powder is not added to culture medium 2.

[0080] Performance evaluation:

[0081] The soothing and antioxidant effects of the coffee fermentation product obtained in this invention (using the product of Example 1 as the test sample) were tested and compared with the control samples of Comparative Example 1 and Comparative Example 2.

[0082] 1. The testing principles and methods for soothing efficacy are as follows:

[0083] principle:

[0084] Hyaluronidase is a specific enzyme that cleaves hyaluronic acid and is involved in allergic reactions, showing a strong correlation with histamine release from mast cells. The soothing effect of a test sample can be determined using the hyaluronidase inhibition rate; a higher inhibition rate indicates a stronger soothing effect, and vice versa.

[0085] method:

[0086] (1) Treatment of control materials and test samples

[0087] Sample group: Dilute with pure water to a sample concentration of 3%;

[0088] Positive control (dipotassium glycyrrhizate, purity ≥98%): diluted with water to a positive control concentration of 3%;

[0089] Negative control: pure water.

[0090] (2) Experimental operation procedures

[0091] Set up a sample group, a sample background group, a solvent group, and a solvent background group. Each group should have 3 replicates. Add different reagent solutions to each of the four groups, shake well, and let stand at room temperature for 30 minutes to develop color. Measure the absorbance value at a wavelength of 528 nm using a UV spectrophotometer.

[0092] Calculation formula:

[0093] Hyaluronidase inhibition rate (%) = (1 - (CD) / (AB)) × 100

[0094] In the formula: A - is the absorbance of the reaction solution without the sample; B - is the absorbance of the reaction solution without the sample and enzyme.

[0095] C - Absorbance of the reaction solution containing the sample and enzyme; D - Absorbance of the reaction solution containing the sample and without the enzyme.

[0096] Evaluation results:

[0097] The hyaluronidase inhibition rate results for different test groups are shown in Table 1 below.

[0098] Table 1. Hyaluronidase inhibition rate results in different test groups

[0099]

[0100] As can be seen from the results in Table 1, both Example 1 and Comparative Example 2 showed a significantly improved hyaluronidase inhibition rate compared to Comparative Example 1, indicating that the co-fermentation treatment of coffee grounds in this invention can synergistically improve the soothing effect of coffee fermentation products.

[0101] 2. The testing principles and methods for antioxidant efficacy are as follows:

[0102] principle:

[0103] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable free radical in organic solvents. Its alcoholic solution is purple and requires low-temperature, light-protected storage. Possessing a single electron, it readily accepts one electron or a hydrogen ion, exhibiting maximum absorption at 517 nm. When the tested solvent has free radical scavenging capabilities, the single atom of DPPH binds, causing the color to lighten and the absorbance at the maximum wavelength to decrease linearly. The degree of absorbance reduction indicates enhanced antioxidant activity, thus evaluating the antioxidant capacity of the test reagent. A higher free radical scavenging rate reflects a stronger antioxidant capacity of the sample.

[0104] method:

[0105] A 96-well plate was used, with three replicates per group and a total volume of 200 μL. For the sample group: 100 μg of the test sample was mixed with 100 μL of pure water, and 100 μL of 0.1 mM DPPH solution was added to the reaction system. Positive control: Vitamin C at a concentration of 10 μg / ml; Negative control: Pure water. After the reaction system was constructed, it was shaken in the dark for 10 min, and the absorbance at 517 nm was measured using a microplate reader.

[0106] The clearance rate is calculated as follows: clearance rate (%) = [(A0-Ax) / A0] × 100%, where A0 is the absorbance of the control group and Ax is the absorbance of the sample group.

[0107] Evaluation results:

[0108] The free radical scavenging rates of different test groups are shown in Table 2 below.

[0109] Table 2. Free radical scavenging rate results of different test groups

[0110]

[0111] As can be seen from the results in Table 2, Example 1 showed a significantly improved free radical scavenging rate compared to Comparative Example 1 and Comparative Example 2, indicating that the use of green tea powder as an auxiliary fermentation ingredient in this invention can synergistically enhance the antioxidant effect of coffee fermentation products.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing coffee fermentation products, characterized in that: The preparation steps include the following: (1) Grind the green coffee beans into powder, sieve them, mix the obtained coffee powder with water, add a compound enzyme for enzymatic hydrolysis, add oligofructose and vitamins to the enzymatic hydrolysis solution, mix well, sterilize, and obtain culture medium 1. (2) Mix coffee grounds with green tea powder, sucrose and water homogenously, sterilize, and obtain culture medium 2; (3) Inoculate a compound of Bifidobacterium breve and Saccharomyces cerevisiae into culture medium 1 and carry out fermentation under anaerobic conditions. After fermentation, add culture medium 2 and inoculate Lactobacillus plantarum to carry out micro-anaerobic fermentation with a gas phase oxygen content of 0.5~1 vt.% and filter to remove residual powder after fermentation to obtain fermentation filtrate. (4) Add acidic protease to the obtained fermentation filtrate for enzymatic hydrolysis. After enzymatic hydrolysis, centrifuge, sterilize, and filter to obtain coffee fermentation product. The complex enzyme mentioned in step (1) is a complex enzyme of cellulase, pectinase and glucanase; The Bifidobacterium breve mentioned in step (3) was purchased from Weikai Hisense Company under the product name Bifidobacterium breve HC2953; the Mycosaccharomyces cerevisiae was Mycosaccharomyces cerevisiae with the preservation number CGMCC No.29720; and the Lactobacillus plantarum was Lactobacillus plantarum with the preservation number CGMCC No.29289.

2. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The enzymatic hydrolysis treatment in step (1) is carried out at a temperature of 35~45℃ for 1~3h.

3. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The mass proportions of each raw material in step (1) are as follows: 25-35 parts coffee powder, 85-100 parts water, 0.5-1.2 parts compound enzyme, 8-15 parts fructooligosaccharides, and 0.5-0.9 parts vitamins.

4. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The mass proportions of each raw material in step (2) are as follows: 15-20 parts coffee grounds, 9-13 parts green tea powder, 8-14 parts sucrose, and 32-47 parts water.

5. The method for preparing a coffee fermentation product according to claim 1, characterized in that: In step (3), the volume ratio of Bifidobacterium breve to Saccharomyces cerevisiae in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 1~5vt; and the inoculation amount of Lactobacillus plantarum is 1~5vt.

6. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The mass ratio of culture medium 1 and culture medium 2 in step (3) is 100~150:30~40.

7. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The temperature for fermentation under anaerobic and micro-anaerobic conditions in step (3) is 35~40℃ and the time is 40~60h.

8. The method for preparing a coffee fermentation product according to claim 1, characterized in that: The concentration of the acidic protease added in step (4) is 0.1~0.75wt%, the temperature of the enzymatic hydrolysis treatment is 35~45℃, and the time is 1~3h.

9. A coffee fermentation product, characterized in that: It is prepared by the method described in any one of claims 1 to 8.

10. The application of the coffee fermentation product according to claim 9 in skin care products.

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