KR20240023373A - Manufacturing method of fermented coffee bean enhancing of biologically active substance and fermented coffee bean manufactured same - Google Patents

Abstract

The present invention relates to a method for producing fermented coffee beans with increased content of bioactive substances and to fermented coffee beans produced by the method. Specifically, fermented coffee beans produced by the production method according to the present invention significantly increase the content of total phenol and flavonoid, as well as the content of hydroxycitric acid or coffee components trigonelline, caffeine, chlorogenic acid, and caffeic acid, As antioxidant activity and α-glucosidase inhibitory activity are enhanced, it can be usefully used as a functional material.

Description

Method for producing fermented coffee beans with increased content of bioactive substances and fermented coffee beans produced by the method

The present invention relates to a method for producing fermented coffee beans with increased content of bioactive substances and to fermented coffee beans produced by the method.

Coffee trees belong to the Coffee genus of the Rubiaceae family, and commercially grown varieties are largely divided into three varieties: Arabica, Robusta, and Liberica. Coffee, which is made from coffee beans collected from these coffee trees, is a favorite beverage made with a variety of flavors such as bitterness, sourness, sweetness, and astringency. Since it began being cultivated in Ethiopia around the 9th century, in 2018, 2.64 million tons were produced in Europe; With 1.57 million tons consumed in the United States and 150,000 tons consumed in Korea, it is a high-value-added product with the second largest volume of trade after oil.

As it has been reported that coffee contains substances that are significantly active in the treatment or improvement of Alzheimer's disease, Parkinson's disease, type 2 diabetes, cholesterol, heart disease, and liver cirrhosis, pharmacological research beyond it as a favorite food is continuing. However, it is known that excessive consumption of coffee can cause emotional anxiety, nervousness, sleep disorders, and gastrointestinal disorders.

Representative ingredients of coffee include caffeine, chlorogenic acid, niacin, potassium, trigonel, and amino acids. Among these, caffeine, known as the main ingredient, is one of the alkaloid compounds, has no odor, has a bitter taste, and is highly soluble in water.

Meanwhile, fermentation includes lactic acid fermentation using lactic acid bacteria and acetic acid fermentation using acetic acid bacteria, and is a widely used process mainly to generate unique flavors in foods or to obtain beneficial effects through the end products of microorganisms. Accordingly, research is being conducted to create unique aromas or enhance beneficial active ingredients in coffee through fermentation. In this regard, Republic of Korea Patent Publication No. 10-2014-0011235 relates to fermented coffee and its manufacturing method. Fermented coffee made from green coffee beans or green coffee beans fermented from endocarp has improved quality due to improved taste and flavor. It is disclosed that it can be used in the production of excellent coffee.

Republic of Korea Patent Publication No. 10-2014-0011235

The purpose of the present invention is to provide a method for producing fermented coffee with increased content of bioactive substances and fermented coffee produced by the method.

In order to achieve the above object, the present invention provides a method for producing fermented coffee beans with increased content of bioactive substances, which includes the step of inoculating coffee beans with fungi and fermenting them.

In addition, the present invention provides fermented coffee beans with an increased content of bioactive substances prepared by the above production method.

Furthermore, the present invention provides a method for increasing the content of bioactive substances in coffee beans, including the step of inoculating coffee beans with fungi and fermenting them.

Fermented coffee beans produced by the production method according to the present invention significantly increase the content of total phenol and flavonoid, as well as the content of hydroxycitric acid or coffee components trigonelline, caffeine, chlorogenic acid, and caffeic acid, and have antioxidant activity and As the α-glucosidase inhibitory activity is enhanced, it can be usefully used as a functional material.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing fermented coffee beans with increased content of bioactive substances, which includes the step of inoculating coffee beans with fungi and fermenting them.

As used herein, the term "coffee bean" refers to the seeds of the coffee tree, a plant belonging to the genus Coffee of the Rubiaceae family. Coffee is obtained by roasting the seeds and extracting them using hot water. Coffee trees bloom two years after being planted, and bear red or yellow fruit about three years later. Coffee beans are the seeds from which the outer skin, pulp, endocarp, and silver skin have been removed from the fruit. The coffee beans may include all types of coffee beans known in the art. For example, the coffee beans may include Arabica, Robusta, etc. Additionally, the coffee beans may include both before and after processing, and specifically, the processing may be roasting.

The fungus may include all types of fungi known in the art. Specifically, the fungus refers to a filamentous fungus whose main body is made of long, thin hyphae like threads, and includes protomycetes, ascomycetes, and imperfect fungi. etc. may be included. Specifically, the fungus may be any one or more selected from the group consisting of Aspergillus genus, Rhizopus genus, and Monascus genus.

The fungus can be inoculated in an appropriate amount by a person skilled in the art. Specifically, the number of spores per 1 ml of the fungal culture medium is 1.0×10 ³ to 1.0×10 ¹⁰ , 1.0×10 ⁴ to 1.0×10 ¹⁰ spores, 1.0×10 ⁵ to 1.0×10 ¹⁰ spores, 1.0×10 ⁶ to 1.0×10 ¹⁰ Number of spores, 1.0×10 ³ to 1.0×10 ⁹ Number of spores, 1.0×10 ⁴ to 1.0×10 ⁹ Number of spores, 1.0×10 ⁵ to 1.0×10 ⁹ Number of spores, 1.0×10 ⁶ to 1.0×10 ⁹ Number of spores, 1.0×10 It can be inoculated with ³ to 1.0×10 ⁸ spores, 1.0×10 ⁴ to 1.0×10 ⁸ spores, 1.0×10 ⁵ to 1.0×10 ⁸ spores, or 1.0×10 ⁶ to 1.0×10 ⁸ spores.

The fermentation can be performed according to methods known in the art. For example, the fermentation may be solid fermentation, liquid fermentation, or a combination thereof. At this time, the fermentation temperature and period can be appropriately selected and performed by a person skilled in the art. Specifically, fermentation is carried out at 23 to 40°C, 23 to 37°C, 23 to 33°C, 25 to 40°C, 25 to 37°C, 25 to 33°C, 28 to 40°C, 28 to 37°C, or 28 to 33°C. 1 to 25 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 7 days, 3 to 25 days, 3 to 20 days, 3 to 15 days, 3 to 10 days or 3 to 7 days. It can be done in one day.

On the other hand, the Rhizopus genus strains include Rhizopus oligosporus , Rhizopus delemar , Rhizopus japonicus, Rhizopus oryzae , and Rhizopus arijus. ( Rhizopus arrhizus ), Rhizopus niveus , Rhizopus azygosporus, Rhizopus microsporus , Rhizopus schipperae and Rhizopus stolonifer. It may be any one or more strains selected from the group consisting of ( Rhizopus stolonifer ). In one embodiment of the present invention, the Rhizopus genus strain may be a Rhizopus oligosporus strain, and more specifically, it may be a Rhizopus oligosporus strain deposited under the accession number KCCM11948P.

In addition, the Monascus genus strains include Monascus kaoliang , Monascus pilosus, Monascus aurantiacus , Monascus floridanus , and Monascus eremophil. It may be any one or more strains selected from the group consisting of Monascus eremophilus , Monascus ruber , Monascus purpureus , and Monascus argentinensis . In one embodiment of the present invention, the Monascus genus strain may be a Monascus purpureus strain, and more specifically, it may be a Monascus purpureus strain deposited under the accession number KCCM13045P.

The method according to the present invention can increase the content of bioactive substances in coffee beans. The bioactive substance refers to a substance that participates in regulating functions in the living body by enhancing or inhibiting the functions of the living body. The bioactive substances may include all substances known in the art. Specifically, the bioactive substances include not only substances known to exist in coffee beans, but also substances that can be newly created through a fermentation process due to fungal inoculation. All may also be included. For example, the bioactive substance may be any one or more selected from the group consisting of hydroxycitric acid, trigonelline, caffeine, chlorogenic acid, and caffeic acid.

In addition, as the bioactive substances of coffee beans produced by the method according to the present invention are enhanced, the effects exhibited by the enhanced bioactive substances can also be enhanced. For example, the hydroxycitric acid is a substance derived from citric acid, and the ACL (ATP-citrate lyases) enzyme involved in the pathway for synthesizing fat from sugar synthesizes acetyl-CoA from citric acid. It is known to inhibit fat synthesis by competitively binding to the enzyme and inhibiting the synthesis of acetyl-CoA. In addition, trigonelline is an alkaloid that is involved in the synthesis of vitamin B3 and is known to have activities such as inhibiting the development of cancer, anti-diabetic activity, and inhibiting cavity-causing bacteria. In addition, caffeine is a central nervous system stimulant of the methylxanthine series, and is known to not only prevent drowsiness caused by adenosine by reversibly blocking the action of adenosine on receptors, but also to increase concentration and reduce body fat. In addition, chlorogenic acid is a compound composed of an ester bond of caffeic acid and quinic acid, which serves as an important intermediate in lignin biosynthesis, and is known to exhibit anti-diabetic activity by delaying the release of glucose into the blood after a meal. Furthermore, caffeic acid is known to exhibit antibacterial, antiviral, anticancer, detoxification, and blood coagulation activities. In other words, coffee beans produced by the method according to the present invention can be used as a functional material to achieve the effects described above.

The present invention provides fermented coffee beans with increased content of bioactive substances prepared by the above production method.

Fermented coffee beans according to the present invention can be produced by a production method having the characteristics described above. For example, the fermented coffee beans can be produced by inoculating fungi and fermenting them. Meanwhile, the bioactive substance may be any one or more selected from the group consisting of hydroxycitric acid, trigonelline, caffeine, chlorogenic acid, and caffeic acid. In other words, the fermented coffee beans can be used as a functional material to achieve the effects produced by bioactive substances.

Furthermore, the present invention provides a method for increasing the content of bioactive substances in coffee beans, including the step of inoculating coffee beans with fungi and fermenting them.

The method for increasing the content of bioactive substances in coffee beans according to the present invention may have the characteristics described above. For example, the enhancement method can be prepared by inoculating and fermenting fungi. Meanwhile, the bioactive substance may be any one or more selected from the group consisting of hydroxycitric acid, trigonelline, caffeine, chlorogenic acid, and caffeic acid.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Anything that has substantially the same structure as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.

Example 1. Production of fermented coffee beans-(1)

Fermented coffee beans were prepared using fungi in the following manner.

First, 1 kg of green coffee beans were immersed in water for 6 hours and then sterilized at 121°C for 15 minutes. Monascus purpureus (KCCM13045P) strain was inoculated into sterilized green coffee beans to produce 2×10 ⁷ spores and fermented for 5 days. After fermentation was completed, the green beans were washed and dried at 60°C for 2 hours to adjust the moisture content to 10 to 11%. Afterwards, the green coffee beans were roasted at medium roast for 17 minutes at 240°C and stored until use.

Example 2. Production of fermented coffee beans-(2)

Fermented coffee beans were prepared under the same conditions and methods as in Example 1, except that 40 g of cinnamon concentrate (65 brix) was added when inoculating and fermenting the Monascus purpureus strain.

Example 3. Production of fermented coffee beans-(3)

Fermented coffee beans were prepared under the same conditions and methods as in Example 1, except that Rhizopus oligosporus (KCCM11948P) strain was used instead of Monascus purpureus strain.

Example 4. Preparation of fermented coffee-(4)

Fermented coffee beans were prepared under the same conditions and methods as in Example 2, except that Rhizopus oligosporus strain was used instead of Monascus purpureus strain.

Comparative Example 1. Preparation of unfermented coffee beans

As described above, green coffee beans were sterilized and dried at 60°C for 2 hours to adjust the moisture content to 10 to 11%. Afterwards, unfermented coffee beans were prepared by roasting the green beans at medium roasting for 17 minutes at 240°C.

Experimental Example 1. Confirmation of hydroxycitric acid (HCA) content

The hydroxycitric acid content present in fermented coffee beans was confirmed using HPLC analysis as follows.

First, 10 g of fermented coffee beans prepared above were placed in 100 ml of water to extract coffee, and the obtained extract was freeze-dried to obtain powder. 30 mg of the powder was suspended in 1 M HCl and centrifuged at 15,000 × g for 10 minutes to obtain a supernatant. The obtained supernatant was filtered through a GHP membrane syringe filter (Pall, USA) with pores of 0.2 μm in size, and then 20 μl was injected into the HPLC column. At this time, the HPLC conditions are shown in Table 1 below, and the HCA quantitative analysis results are shown in Table 2.

condition system Waters 2545 Binary Gradient Module detector 2998 PDA detector column YMC Triart C18 column (5 ㎜, 4.6×150 ㎜) (Milford, USA) flow rate 0.5 ml/min menstruum A: Distilled water (15 mM sulfuric acid) concentration gradient 0-30 minutes: A solvent 100%

HCA (mg/g coffee) Comparative Example 1 5.42±0.16 Example 1 8.11±0.34 Example 2 9.58±0.26 Example 3 9.69±0.07 Example 4 7.45±0.54

As shown in Table 2, the content of HCA significantly increased in fermented coffee beans compared to unfermented coffee beans.

Experimental Example 2. Coffee component analysis

The contents of trigonelline, caffeine, chlorogenic acid, and caffeeic acid, which are coffee components present in fermented coffee beans, were confirmed using HPLC analysis as follows.

First, samples were obtained from coffee beans as described in Experimental Example 1, and HPLC was performed according to the conditions shown in Tables 3 and 4 below. As a result, the results of quantitative analysis of coffee components are shown in Figure 5.

condition system Waters 2545 Binary Gradient Module detector 2998 PDA detector column A normal-phase Phenomenex column (4.6×100 ㎜, 5 ㎛) (Milford, USA) flow rate 0.8 ㎖/min menstruum A: Distilled phosphoric acid (50 mM phosphoric acid)
B: 100% acetonitrile

Time (minutes) Solvent A (%) Solvent B (%) 0 100 0 10 65 35 15 10 90 20 10 90 21 60 40 22 100 0 25 100 0

trigonelline
(mg/g coffee) Caffeine
(mg/g coffee) chlorogenic acid
(mg/g coffee) caffeic acid
(㎍/g coffee) Comparative Example 1 4.71±0.01 4.20±0.09 3.03±0.01 61.96±0.04 Example 1 7.04±0.43 8.39±0.11 6.68±0.22 69.07±3.36 Example 2 6.83±0.04 7.86±0.15 6.94±0.09 70.44±0.63 Example 3 5.77±0.14 6.18±0.26 4.38±0.14 64.94±1.53 Example 4 6.27±0.25 6.95±0.35 4.66±0.19 72.61±1.26

As shown in Table 5, the content of coffee components trigonelline, caffeine, chlorogenic acid, and caffeic acid significantly increased in fermented coffee beans compared to unfermented coffee beans.

Experimental Example 3. Total phenol and flavonoid content

The total phenol and flavonoid contents present in fermented coffee beans were confirmed as follows.

First, to measure the total phenol content, 1 mg of freeze-dried coffee beans was diluted in 1 ml of water, centrifuged at 15,000 × g for 10 minutes, and the supernatant was recovered. 150 μg of the recovered supernatant was placed in a 96-well plate, and 15 μg of folin reagent and 10% Na ₂ CO ₃ were added. After reacting for 30 minutes at 28°C and dark conditions, the absorbance was measured at a wavelength of 765 nm using a spectrophotometer (Molecular Device, Syunnyvale, USA). At this time, a standard graph was prepared using gallic acid (GAE) as a standard material, and the total phenol content was calculated from this.

Meanwhile, to measure the total flavonoid content, 2 mg of freeze-dried coffee beans were diluted in 1 ml of methanol, centrifuged at 15,000 × g for 10 minutes, and the supernatant was recovered. 20 μg of the recovered supernatant was placed in a 96-well plate, and 60 μg of ethanol, 10 μg of 10% AlCl ₃ , 10 μg of 1M CH ₃ COOK, and 100 μg of water were added. After reacting for 30 minutes at 28°C and dark conditions, the absorbance was measured at a wavelength of 415 nm using a spectrophotometer. At this time, a standard graph was prepared using quercetin (QE) as a standard material, and the total phenol content was calculated from this. As a result, the calculated total phenol and flavonoid contents are shown in Table 6.

Total phenol content
(mg GAE/g coffee) Total flavonoid content
(mg QE/g coffee) Comparative Example 1 5.39±0.05 4.11±0.05 Example 1 7.30±0.08 4.63±0.05 Example 2 7.39±0.07 4.55±0.03 Example 3 7.11±0.09 4.68±0.11 Example 4 7.49±0.04 4.32±0.08

As shown in Table 6, the content of total phenols and flavonoids significantly increased in fermented coffee beans compared to unfermented coffee beans.

Experimental Example 4. Antioxidant activity

The antioxidant activity of fermented coffee beans was confirmed as follows.

4-1. DPPH radical scavenging ability

1 mg of freeze-dried coffee beans was diluted in 1 ml of methanol, centrifuged at 15,000 × g for 10 minutes, and the supernatant was recovered. 40 μg of the recovered supernatant was placed in a 96-well plate, and 40 μg of ethanol and 20 μg of DPPH were added. After reacting for 30 minutes at 30°C and dark conditions, the absorbance was measured at a wavelength of 517 nm using a spectrophotometer. The sample concentration (SC ₅₀ ) required to inhibit radical formation by 50% was calculated from the measured absorbance value.

4-2. FRAP analysis

1 mg of freeze-dried coffee beans was diluted in 1 ml of water, centrifuged at 15,000 × g for 10 minutes, and the supernatant was recovered. 6 μg of the recovered supernatant was placed in a 96-well plate, and 18 μg of water, 180 μg of reaction solution (0.3 M CH ₃ COONa buffer (pH 3.6), 0.04 M HCl solution containing 0.01 M TPTZ, and 0.02 M FeCl A mixture of ₃ ·6H ₂ O at a volume ratio of 10:1:1 was added. After reacting for 30 minutes at 37°C and dark conditions, the absorbance was measured at a wavelength of 593 nm using a spectrophotometer. At this time, a standard graph was prepared using FeSO ₄ ·7H ₂ as a standard material, and the concentration of reduced iron was calculated from this. As a result, the results of confirming the antioxidant activity of fermented coffee beans through DPPH radical scavenging ability and FRAP analysis are shown in Table 7.

DPPH
(㎍/㎖, SC ₅₀ ) FRAP
(mM Fe(II)/g coffee) Comparative Example 1 219.94±2.70 171.19±4.49 Example 1 218.11±5.52 216.06±0.58 Example 2 178.11±1.93 229.55±2.93 Example 3 186.67±1.50 216.65±2.06 Example 4 188.50±1.12 238.75±2.89

As shown in Table 7, antioxidant activity was significantly increased in fermented coffee beans compared to unfermented coffee beans.

Experimental Example 5. α-Glucosidase inhibitory activity

The α-glucosidase inhibitory activity of fermented coffee beans was confirmed as follows.

Specifically, 1 mg of freeze-dried coffee beans was diluted in 1 ml of water, centrifuged at 15,000 × g for 10 minutes, and the supernatant was recovered. To 40 μl of the recovered supernatant, 40 μl of 0.5 M phosphate buffer (pH 6.8) and 0.1 U/ml of α-glucosidase were added and reacted at 37°C for 5 minutes. After adding 40 ㎕ of 5 M pNPG substrate and reacting for 8 minutes, the reaction was terminated by adding 300 ㎕ of 250 mM Na ₂ CO ₃ and analyzed at a wavelength of 405 ㎚ using SpectraMax M3. Absorbance was measured. The results of calculating the α-glucosidase inhibitory activity from the measured absorbance values are shown in Table 8.

α-Glucosidase inhibition (㎍/㎖, IC ₅₀ ) Comparative Example 1 359.44±0.48 Example 1 272.19±0.25 Example 2 212.03±1.03 Example 3 215.35±0.26 Example 4 162.32±1.95

As shown in Table 8, the α-glucosidase inhibitory activity was significantly increased in fermented coffee beans compared to unfermented coffee beans. α-Glucosidase inhibits the decomposition of disaccharides into monosaccharides in the body and suppresses the rise in blood sugar, thereby exhibiting anti-diabetic activity. Fermented coffee beans according to the present invention exhibit excellent α-glucosidase inhibitory activity, thereby suppressing anti-diabetic activity. It can be usefully used as an active functional material.

Korea Center for Microbial Conservation (KCCM) KCCM11948P 20161128 Korea Center for Microbial Conservation (KCCM) KCCM13045P 20210831

Claims (9)

A method for producing fermented coffee beans with increased content of bioactive substances, comprising the step of inoculating coffee beans with fungi and fermenting them.
The method of claim 1, wherein the physiologically active substance is at least one selected from the group consisting of hydroxycitric acid, trigonelline, caffeine, chlorogenic acid, and caffeic acid.
The method of producing fermented coffee beans according to claim 1, wherein the fungus is at least one selected from the group consisting of Aspergillus genus, Rhizopus genus, and Monascus genus.
The method of claim 3, wherein the Rhizopus genus is a Rhizopus oligosporus strain.
The method of producing fermented coffee beans according to claim 4, wherein the Rhizopus oligosporus strain is a strain deposited under the accession number KCCM11948P.
The method for producing fermented coffee beans according to claim 3, wherein the Monascus genus is a Monascus purpureus strain.
The method of claim 6, wherein the Monascus purpureus strain is a strain deposited under the accession number KCCM13045P.
Fermented coffee beans with increased content of biologically active substances manufactured by the manufacturing method according to claim 1.
A method of increasing the content of bioactive substances in coffee beans, including the step of inoculating coffee beans with fungi and fermenting them.