Antimicrobial composition containing an extract of a coffee composition and an antibiotic, and its use

Abstract

To provide a novel antimicrobial product that enables the management of animal diseases while reducing reliance on conventional antibiotics. [Solution] The present invention relates to an antimicrobial composition comprising an extract of a coffee composition and an antibiotic, and to the use of the antimicrobial composition in the manufacture of antimicrobial drugs. The extract of the coffee composition comprises a solid component and water, the solid component comprising coffee beans and auxiliary materials. The antibiotic is selected from the group consisting of penicillin antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, tetracycline antibiotics, chloramphenicol antibiotics, aminoglycoside antibiotics and combinations thereof, and the weight ratio of the coffee composition to the antibiotic is from 1.5:1 to 25000:1. The antimicrobial composition of the present invention can exhibit a superior antibacterial effect compared to the use of antibiotics alone, and this antibacterial effect can also be exerted against drug-resistant bacteria.

Description

[Technical Field]

[0001] This invention relates to an antimicrobial composition comprising an extract of a coffee composition and an antibiotic. Furthermore, this invention relates to the use of the antimicrobial composition in the manufacture of antimicrobial drugs. [Background technology]

[0002] To maintain the health and promote the growth of economic animals such as poultry, livestock, and aquaculture animals, farmers often use antimicrobial agents to prevent disease transmission between animals. Examples include antibiotic growth promoters (AGPs). Under conditions of high stocking density or inadequate sanitary conditions, AGPs effectively reduce animal morbidity and mortality, contributing to production efficiency and profitability. It has been reported that the elimination of AGPs can lead to decreased productivity; for example, discontinuing AGP use after weaning results in an increase of $0.86 per animal in feed costs, while discontinuing it during the fattening stage results in an increase of $3.11 per animal (Cardinal et al., 2021).

[0003] However, the long-term or excessive use of antimicrobial agents can lead to the emergence of antibiotic-resistant bacteria, reducing the effectiveness of antimicrobial agents. While it is common practice to add low doses of antibiotics to feed and aquaculture water for preventative purposes, this method of use can cause bacteria in the animals to develop resistance to the antimicrobial agent, rendering previously effective drugs ineffective. With a growing understanding of antibiotic overuse, increased awareness of health risks, and a global demand for reduced antimicrobial use, many countries and consumers are seeking more sustainable aquaculture methods as alternatives to traditional antibiotic-dependent farming practices.

[0004] A study targeting low- and middle-income countries (Boeckel et al., 2019) investigated antibiotic resistance in microbial organisms in livestock such as chickens, pigs, and cattle from 2000 to 2018, finding a particularly significant increase in resistant strains in chickens and pigs. The proportion of antimicrobial drugs with resistance rates exceeding 50% was reported to have increased two to three times. The impact of antibiotic resistance is not limited to livestock but also extends to aquaculture (Bogaard et al., 2001). For example, antibiotic use is higher in broiler chicken and turkey farming compared to laying hens, and consequently, antibiotic resistance of E. coli in the feces of turkey and broiler farmers and slaughterhouses has been confirmed to be higher than that of laying hens. This suggests the possibility that E. coli antibiotic resistance genes are being widely transmitted from poultry to humans.

[0005] According to a 2022 report by the U.S. Food and Drug Administration (FDA), antibiotic use in broiler chickens decreased year by year from 2016 to 2022, and antibiotic use in beef cattle and pork was also lower between 2017 and 2022 than in 2016. Furthermore, data from the ESVAC (European Surveillance of Veterinary Antimicrobial Consumption) program shows that sales of polymyxins and fluoroquinolone antibiotics among veterinary antibiotics in 25 European countries decreased significantly between 2011 and 2018, indicating a shift in the European veterinary antibiotic market.

[0006] Therefore, there is a strong need for the development of novel antimicrobial products and alternatives that enable the management of animal diseases while reducing reliance on conventional antibiotics. This is expected to resolve the difficulties in disease management caused by restrictions on the use of antimicrobial agents and ensure the sustainable development of animal health and the aquaculture industry. [Overview of the Initiative]

[0007] Based on the above needs, one object of the present invention is to provide a composition having antibacterial effects. Compared to the use of conventional antibiotics, this composition can more efficiently suppress bacterial growth and reduce the amount of antibiotics used, and furthermore, it exhibits an inhibitory effect even against antibiotic-resistant bacteria.

[0008] Another object of the present invention is to provide the use of the aforementioned antimicrobial composition.

[0009] To achieve the aforementioned objectives, the present invention provides an antimicrobial composition, An extract of a coffee composition, wherein the coffee composition contains 92% (wt%) to 95 wt% solid components and 5 wt% to 8 wt% water, the solid components contain 20 wt% to 70 wt% coffee beans and 30 wt% to 80 wt% auxiliary materials, and the carbon-nitrogen ratio of the coffee composition is 35 to 50; Antibiotics selected from the group consisting of penicillins, cephalosporins, fluoroquinolones, tetracyclines, chloramphenicols, aminoglycosides, and combinations thereof. Includes, Here, the weight ratio of the coffee composition to the antibiotic is 1.5:1 to 25000:1, and the extract of the coffee composition includes an aqueous extract of the coffee composition, an alcohol extract of the coffee composition, or a combination thereof.

[0010] The present invention further provides the use of the antimicrobial composition in the manufacture of antimicrobial drugs. In this use, bacterial infections can be suppressed by administering the antimicrobial composition to animals in need.

[0011] In this invention, by extracting a coffee composition with alcohol or water and incorporating the resulting extract into an antimicrobial composition, a synergistic antimicrobial effect is generated between the coffee composition and the antibiotic, resulting in a superior antibacterial effect compared to the use of the antibiotic alone. Furthermore, this antibacterial effect is also effective against antibiotic-resistant bacteria, overcoming the problem that conventional antimicrobial agents lose effectiveness with increasing frequency of use.

[0012] In the present invention, the carbon-nitrogen ratio of a coffee composition refers to the ratio of the mass of carbon to the mass of nitrogen contained in the coffee composition. In some examples, coffee beans are green coffee beans, roasted coffee beans, or recycled coffee grounds, and green coffee beans and roasted coffee beans include whole coffee beans, crushed coffee beans, or coffee bean powder.

[0013] In some examples, the extract of the coffee composition includes an alcohol extract of the coffee composition.

[0014] In some examples, the particle size of the coffee beans ranged from 500 μm to 8300 μm.

[0015] In some examples, the particle size of the recovered coffee grounds was 500 μm to 2000 μm. In other examples, the particle size of the whole coffee beans was 5000 μm to 8300 μm, the particle size of the crushed coffee beans was 1000 μm to 5000 μm, and the particle size of the coffee bean powder was 500 μm to 1000 μm.

[0016] In some examples, the coffee composition is a ground coffee composition with a particle size of 8300 μm or less. In other examples, the particle size of the ground coffee composition is 500 μm to 8300 μm, 500 μm to 8000 μm, 500 μm to 800 μm, 1000 μm to 2000 μm, 2000 μm to 3000 μm, 3000 μm to 4000 μm, 4000 μm to 5000 μm, 300 μm to 2000 μm, 500 μm to 1000 μm, 200 μm to 1000 μm, 100 μm to 830 μm, or 1000 μm to 7500 μm. In some embodiments, the ratio of the coffee composition to the antibiotic is in the range of 6.25:1 to 2500:1, 12.5:1 to 1250:1, 25:1 to 625:1, or 50:1 to 250:1.

[0017] In some embodiments, the alcohol extract is obtained by extracting the coffee composition with an aqueous ethanol solution having a concentration of 60% to 80%, and the ratio of the coffee composition to the aqueous ethanol solution is 1 g:3 mL to 1 g:7 mL. In other embodiments, the ratio is 1 g:4 mL to 1 g:6 mL, or 1 g:5 mL.

[0018] In some embodiments, the auxiliary materials include corn crushings, beet crushings, rice husks, defatted soybean meal, crushed rice, or combinations thereof.

[0019] In some embodiments, the coffee composition is an unfermented coffee composition or a coffee composition that has been fermented by Aspergillus oryzae.

[0020] According to the present invention, the coffee composition is produced by the following steps: (1) Mixing 40 wt% to 60 wt% of a solid component and 40 wt% to 60 wt% of water to form a mixture; (2) Heating the mixture for 20 to 60 minutes under conditions of a temperature of 115°C to 125°C and a pressure of 1.0 bar to 1.5 bar to obtain a substrate; and (3) Cooling the obtained substrate and drying it until the moisture content becomes 5 wt% to 8 wt% to obtain a coffee composition.

[0021] In some embodiments, step (3) includes the following: Cooling the substrate, inoculating Aspergillus oryzae at a density of 1×10 7 spores / g to 1×10 8 spores / g, and fermenting at 25°C to 35°C for 3 to 7 days; then drying the substrate to make the moisture content be 5 wt% to 8 wt% to obtain a coffee composition.

[0022] In some examples, penicillin antibiotics are selected from the group consisting of penicillin G, penicillin V, ampicillin, amoxicillin, and amoxicillin-clavulanic acid.

[0023] In some examples, the cephalosporin antibiotic is selected from the group consisting of cephalexin, cephadroxil, and ceftiofur.

[0024] In some examples, the fluoroquinolone antibiotic is selected from the group consisting of enrofloxacin, ofloxacin, danofloxacin, ciprofloxacin, and norfloxacin.

[0025] In some examples, the tetracycline antibiotic is selected from the group consisting of oxytetracycline, chlortetracycline, and doxycycline.

[0026] In some examples, the chloramphenicol antibiotics are selected from the group consisting of chloramphenicol, thiamphenicol, and florfenicol.

[0027] In some examples, the aminoglycoside antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, gentamicin, spectinomycin, and apramycin.

[0028] According to the present invention, the antibacterial composition has an inhibitory effect against bacteria selected from the group consisting of Salmonella spp., Escherichia spp., Pasteurella spp., Streptococcus spp., Gallibacterium spp., Staphylococcus spp., Riemerella spp., Pseudomonas spp., and Klebsiella spp.

[0029] In some examples, the bacteria are selected from the group consisting of Salmonella enterica, Salmonella choleraesuis, Escherichia coli, Pasteurella multocida, Glaesserella parasuis, Streptococcus suis, Gallibacterium anatis, Staphylococcus hyicus, Riemerella anatipestifer, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0030] In some examples, the bacteria are drug-resistant.

[0031] According to the present invention, coffee compositions reduce the inflammatory response induced by lipopolysaccharide (LPS). This inflammatory response includes the production of proteins such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by cells, as well as nitric oxide (NO) by macrophages.

[0032] In some embodiments, the animals are livestock, poultry, or aquaculture animals. Livestock include pigs, beef cattle, dairy cows, sheep, or goats; poultry include chickens, ducks, or geese; and aquaculture animals include fish or shrimp. [Brief explanation of the drawing]

[0033] [Figure 1A] These are photographs of E. coli colonies cultured with florfenicol in each group in Test Example 1. [Figure 1B] These are photographs of E. coli colonies cultured with doxycycline in each group in Test Example 1. [Figure 2A] This bar graph shows the number of E. coli bacteria cultured with florfenicol in each group in Test Example 1. [Figure 2B] This bar graph shows the number of E. coli bacteria cultured with doxycycline in each group in Test Example 1. [Figure 3A] These are photographs of E. coli colonies cultured with cephalexin in each group in Test Example 2. [Figure 3B] These are photographs of E. coli colonies cultured with amoxicillin in each group in Test Example 2. [Figure 3C] These are photographs of E. coli colonies cultured with oxytetracycline in each group in Test Example 2. [Figure 3D] These are photographs of E. coli colonies cultured with doxycycline in each group in Test Example 2. [Figure 3E]These are photographs of E. coli colonies cultured with florfenicol in each group in Test Example 2. [Figure 3F] These are photographs of E. coli colonies cultured with gentamicin in each group in Test Example 2. [Figure 3G] These are photographs of Salmonella colonies cultured with cephalexin in each group in Test Example 2. [Figure 3H] These are photographs of Salmonella colonies cultured with amoxicillin in each group in Test Example 2. [Figure 3I] These are photographs of Salmonella colonies cultured with oxytetracycline in each group in Test Example 2. [Figure 3J] These are photographs of Salmonella colonies cultured with doxycycline in each group in Test Example 2. [Figure 3K] These are photographs of Salmonella colonies cultured with florfenicol in each group in Test Example 2. [Figure 3L] These are photographs of Salmonella colonies cultured with gentamicin in each group in Test Example 2. [Figure 4A] This bar graph shows the number of E. coli bacteria cultured with cephalexin in each group in Test Example 2. [Figure 4B] This bar graph shows the number of E. coli bacteria cultured with amoxicillin in each group in Test Example 2. [Figure 4C] This bar graph shows the number of E. coli bacteria cultured with oxytetracycline in each group in Test Example 2. [Figure 4D] This bar graph shows the number of E. coli bacteria cultured with doxycycline in each group in Test Example 2. [Figure 4E] This bar graph shows the number of E. coli bacteria cultured with florfenicol in each group in Test Example 2. [Figure 4F] This bar graph shows the number of E. coli bacteria cultured with gentamicin in each group in Test Example 2. [Figure 5A] This bar graph shows the number of Salmonella bacteria cultured in cephalexin for each group in Test Example 2. [Figure 5B] This bar graph shows the number of Salmonella bacteria cultured with amoxicillin in each group in Test Example 2. [Figure 5C] This bar graph shows the number of Salmonella bacteria in each group cultured with oxytetracycline in Test Example 2. [Figure 5D] This bar graph shows the number of Salmonella bacteria cultured with doxycycline in each group in Test Example 2. [Figure 5E] This bar graph shows the number of Salmonella bacteria in each group cultured with florfenicol in Test Example 2. [Figure 5F] This bar graph shows the number of Salmonella bacteria cultured with gentamicin in each group in Test Example 2. [Figure 6] These are photographs of the E. coli colonies from each group in Test Example 3. [Figure 7] These are photographs of the E. coli colonies from each group in Test Example 4. [Figure 8A] This bar graph shows the number of E. coli bacteria cultured with cephalexin in each group in Test Example 4. [Figure 8B] This bar graph shows the number of E. coli bacteria cultured with florfenicol in each group in Test Example 4. [Figure 8C] This bar graph shows the number of E. coli bacteria cultured with doxycycline in each group in Test Example 4. [Figure 9A] This bar graph shows the MIC90 of porcine-derived Escherichia coli cultured with florfenicol in each group in Test Example 5. [Figure 9B] This bar graph shows the MIC90 of porcine-derived Escherichia coli cultured with doxycycline for each group in Test Example 5. [Figure 10A] This bar graph shows the MIC90 of chicken-derived Escherichia coli cultured with florfenicol in each group in Test Example 5. [Figure 10B] This bar graph shows the MIC90 of chicken-derived Escherichia coli cultured with enrofloxacin for each group in Test Example 5. [Figure 10C]This bar graph shows the MIC90 of chicken-derived Escherichia coli cultured with doxycycline for each group in Test Example 5. [Figure 11A] This bar graph shows the MIC90 of porcine-derived Salmonella bacteria cultured with florfenicol in each group in Test Example 5. [Figure 11B] This bar graph shows the MIC90 of porcine-derived Salmonella bacteria cultured with enrofloxacin in each group in Test Example 5. [Figure 11C] This bar graph shows the MIC90 of porcine-derived Salmonella bacteria cultured with doxycycline in each group in Test Example 5. [Figure 12A] This bar graph shows the MIC90 of chicken-derived Salmonella bacteria cultured with florfenicol in each group in Test Example 5. [Figure 12B] This bar graph shows the MIC90 of chicken-derived Salmonella bacteria cultured with enrofloxacin in each group in Test Example 5. [Figure 12C] This bar graph shows the MIC90 of chicken-derived Salmonella bacteria cultured with doxycycline in each group in Test Example 5. [Figure 13A] This bar graph shows the MIC90 of Pasteurella maltosida cultured with florfenicol in each group in Test Example 5. [Figure 13B] This bar graph shows the MIC90 of Pasteurella maltosida cultured with doxycycline in each group in Test Example 5. [Figure 14A] This bar graph shows the MIC90 of Glaesserella parasuis cultured with florfenicol in each group in Test Example 5. [Figure 14B] This bar graph shows the MIC90 of each group of *Cerella parasuis* bacteria cultured with enrofloxacin in Test Example 5. [Figure 14C] This bar graph shows the MIC90 of each group of *Cerella parasuisine* cultured with doxycycline in Test Example 5. [Figure 15A] This bar graph shows the number of E. coli bacteria in each group in Test Example 6. [Figure 15B] This bar graph shows the number of Salmonella bacteria in each group in Test Example 6. [Figure 15C] This bar graph shows the number of pentose lactic acid bacteria in each group in Test Example 6. [Figure 15D] This bar graph shows the number of Lactobacillus reuteri bacteria in each group in Test Example 6. [Figure 16A] This bar graph shows the absorbance at 625 nm for each group in Test Example 7. [Figure 16B] This bar graph shows the absorbance at 260 nm for each group in Test Example 7. [Figure 16C] This is a bar graph showing the protein concentrations in each group in Test Example 7. [Figure 16D] This bar graph shows the absorbance at 420 nm for each group in Test Example 7. [Figure 17A] These are images of Escherichia coli from each group in Test Example 8, taken with a scanning electron microscope. [Figure 17B] These are images of Salmonella bacteria from each group in Test Example 8, taken with a scanning electron microscope. [Figure 18A] This is a gel image of a DNA damage test using E. coli in Test Example 9. [Figure 18B] This is a gel image from a DNA damage test using Salmonella bacteria in Test Example 9. [Figure 18C] These are gel images of test groups 9-4 to 9-9 in the protein damage test of Test Example 9. [Figure 19A] This bar graph shows the number of E. coli bacteria in each group in Test Example 10. [Figure 19B] This bar graph shows the number of E. coli bacteria in each group in Test Example 10. [Figure 20A] This is a bar graph showing the IL-6 concentrations in each group in Test Example 11. [Figure 20B] This bar graph shows the nitric oxide (NO) production rate for each group in Test Example 11. [Figure 20C] This is a bar graph showing the TNF-α concentrations in each group in Test Example 11. [Figure 20D]These are microscopic images of each group in Test Example 11. [Figure 21] This bar graph shows the nitric oxide (NO) production rate for each group in Test Example 12. [Modes for carrying out the invention]

[0034] The present invention will be described in more detail below with reference to several embodiments, accompanied by drawings. Those skilled in the art will readily understand the advantages and effects of the present invention based on the following embodiments. Those skilled in the art should understand that the embodiments shown herein are illustrative and do not limit the scope of the present invention. Those skilled in the art may, based on their ordinary knowledge, carry out or apply the present invention by making various modifications and changes without departing from the spirit of the invention.

[0035] Manufacturing Example 1: Extract of a fermented coffee composition produced from green or roasted coffee beans

[0036] (a) Raw material mixing A first solid component with a carbon-nitrogen ratio of 35-50 was formed by mixing 20-40 wt% green or roasted coffee beans, 40-60 wt% crushed corn, and 20-40 wt% crushed sugar beets. Water was then added to this first solid component to form a first mixture with a moisture content of 40-60 wt%. The green and roasted coffee beans include whole coffee beans, crushed coffee beans, or coffee bean powder. The term "crushed material" refers to granular material with uneven particle size obtained after crushing the raw materials (coffee beans, corn, or sugar beets, etc.).

[0037] (b) Sterilization and fermentation: 2-3 kg of the first mixture was placed in a fermentation vessel and sterilized at a temperature of 121°C and a pressure of 1.2 bar for 30 minutes to obtain the first substrate. After the first substrate was cooled to room temperature, 1.5 g-3 g of koji mold powder (6.5 × 10⁶ bacteria) was added. 7Spores ( / g, Kyokan Biotechnology FG003 strain, Aspergillus oryzae, identified by the Food Industry Development Institute) were added, and the mixture was fermented at 25°C to 35°C for 3 to 7 days to obtain the first product. Subsequently, it was dried at 40°C to 70°C for 12 to 14 hours to reduce the moisture content to 5 wt% to 8 wt%, and the dried first product was ground to pass through an 830 μm sieve to obtain the fermented coffee composition of Production Example 1.

[0038] (c) Extraction 5 g of fermented coffee composition was placed in a 50 mL centrifuge tube, and 25 mL of 70% ethanol or water was added to make a 200 mg / mL solution. The mixture was mixed using a Vortex Mixer test tube shaker, and the centrifuge tube was placed in a 30°C incubator and shaken at 120 rpm (Revolutions Per Minute) for 2 hours. After processing, the centrifuge tube was removed and centrifuged at 10,000 rpm for 10 minutes, and the obtained first supernatant was collected. Next, this first supernatant was sterilized at 121°C for 20 minutes, and then centrifuged again at 10,000 rpm for 10 minutes, and the obtained second supernatant was collected. This second supernatant was filtered through a 0.22 μm pore size filter to obtain the extract of Production Example 1. Here, the extract is either a water extract or an ethanol extract.

[0039] Manufacturing Example 2: Extract of fermented coffee composition produced from recovered coffee grounds

[0040] The method for producing the extract in Production Example 2 was generally the same as in Production Example 1, but differed in the following respects. Specifically, in step (a) of Production Example 2, 40 wt% to 70 wt% of recovered coffee grounds and 30 wt% to 60 wt% of crushed corn were mixed to obtain a second solid component with a carbon-nitrogen ratio of 35 to 50. Water was added to this to obtain a second mixture with a water content of 40 wt% to 60 wt%. Through these steps, the extract of Production Example 2 (water extract or ethanol extract) was obtained. Here, the extract is either a water extract or an ethanol extract.

[0041] Production Example 3: Extract of an unfermented coffee composition prepared using roasted coffee beans

[0042] The method for producing the extract in Production Example 3 was generally the same as in Production Example 1, but differed in the following respects. Specifically, in step (a) of Production Example 3, 20 wt% to 40 wt% of crushed roasted coffee beans, 40 wt% to 60 wt% of crushed corn, and 20 wt% to 40 wt% of crushed sugar beets were mixed to form a third solid component with a carbon-nitrogen ratio of 35 to 50. Next, water was added to this third solid component to obtain a third mixture with a water content of 40 wt% to 60 wt%. In step (b), the fermentation step was omitted, and the third mixture was sterilized, cooled, and left to stand at 25°C to 35°C for 3 to 7 days. After that, a drying step and a grinding step were carried out. The extract of Production Example 3 was produced by the above steps. The extract is either a water extract or an ethanol extract.

[0043] Manufacturing Example 4: Antibiotic Solution

[0044] 2 mg of antibiotic was weighed into a 1.5 mL microcentrifuge tube, and under sterile conditions, 1 mL of Tryptic Soy Broth (TSB) or Mueller Hinton II Broth (MH II Broth) was added and mixed. The mixture was then filtered through a 0.22 μm filter to obtain the antibiotic solution of Preparation Example 4. Antibiotic solutions were prepared using florfenicol, doxycycline, cephalexin, amoxicillin, amoxicillin clavulanate, oxytetracycline, or gentamicin, each at a concentration of approximately 2 mg / mL.

[0045] Manufacturing Example 5: Extracts from the control group

[0046] The method for producing the extract in Production Example 5 was generally the same as in Example 1, but differed in the following respects. Specifically, in Production Example 5, steps (a) and (b) were omitted, and in step (c), the fermented coffee composition was not added to the centrifuge tube. Instead, only 25 mL of 70% ethanol or water was used, and the mixture was shaken in a test tube shaker before the subsequent processing. The extract in this production example was essentially obtained by performing only the extraction operation without adding the coffee composition, and was used as a sample for the control group. In this specification, "extract concentration" means the amount of coffee composition (unfermented or fermented) in milligrams per 1 mL of extract solution.

[0047] Example 1: Antimicrobial composition containing the extract from Production Example 1 and an antibiotic

[0048] The extract from Production Example 1 and either the antibiotic solution prepared according to Production Example 4 were mixed, and TSB medium was added to adjust the concentration to obtain the antimicrobial composition of Example 1. The weight ratio of coffee composition to antibiotic in the antimicrobial composition was 1.5:1 to 25000:1.

[0049] Example 2: Antimicrobial composition containing the extract from Production Example 2 and an antibiotic

[0050] The method for producing the antimicrobial composition in Example 2 was generally the same as in Example 1, but differed in the following respects. Specifically, in Example 2, the antimicrobial composition was prepared by mixing the extract from Production Example 2 with one of the antibiotic solutions obtained in Production Example 4.

[0051] Example 3: Antimicrobial composition containing the extract from Production Example 3 and an antibiotic

[0052] The method for preparing the antimicrobial composition in Example 3 was generally the same as in Example 1, but differed in the following respects. Specifically, in Example 3, the antimicrobial composition was prepared by mixing the extract from Production Example 3 with one of the antibiotic solutions prepared in Production Example 4.

[0053] The following tests on the antibacterial effect were conducted using the aforementioned examples and manufacturing examples.

[0054] Test Example 1: Cooperative antibacterial effect of the antibacterial composition of Example 3 (ethanol extract of unfermented coffee composition and antibiotic)

[0055] 1.Bacteria drop observation

[0056] 1.5 g of TSB medium was weighed out and placed in a 100 mL Erlenmeyer flask. 50 mL of reverse osmosis (RO) water was added, and the flask was sterilized at 121 °C for 20 minutes, after which it was cooled to room temperature. Next, approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) was added to the Erlenmeyer flask, and the mixture was cultured in a 37 °C incubator at 120 rpm for 24 hours with shaking to obtain an Escherichia coli solution. Based on Table 1, test groups 1-1 to 1-14 were prepared using the ethanol extract from Production Example 3 and an antibiotic (florfenicol (FF) or doxycycline (DC)). 3 mL of the ethanol extract from Production Example 5 was used for the control group (C). Test groups 1-1 and 1-2 used 3 mL of the ethanol extract from Production Example 3, while test groups 1-3, 1-6, 1-9, and 1-12 used 3 mL of the antibiotic solution from Production Example 4 (an antibiotic solution of florfenicol or doxycycline, adjusted in concentration with TSB medium). Furthermore, test groups 1-4, 1-5, 1-7, 1-8, 1-10, 1-11, 1-13, and 1-14 used 3 mL of the antimicrobial composition from Example 3 (the concentration and type of antibiotic are shown in Table 1). Note that "N / A" means "Not Applicable".

[0057] Table 1: Ethanol extract concentration, antibiotic concentration, and type included in test groups 1-1 to 1-14 and the control group in Test Example 1. [Table 1]

[0058] The compositions for the control group and test groups 1-1 to 1-14 were each dispensed into 15 screw-cap tubes. Separately, the E. coli solution was diluted with TSB medium, and 1 mL was added to each of the control group and test groups 1-1 to 1-14 to determine the number of E. coli in each group (1 × 10⁻⁶). 5 The concentration was defined as Colony Forming Units (CFU) / mL. The total volume of each test tube was 4 mL. These test tubes were then placed in a 37°C incubator and incubated with shaking at 120 rpm for 24 hours to prepare E. coli cultures for the control group and test groups 1-1 to 1-14.

[0059] 9 g of TSB medium and 4.5 g of agar were weighed out, placed in a 500 mL serum bottle, 300 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes to obtain Tryptic Soy Agar (TSA) medium. After the temperature of the TSA medium had decreased to 45 °C to 50 °C, 20 mL was poured into a culture dish, and four compartments were marked on the bottom of the dish with an oil-based pen. The E. coli culture solutions from each group were diluted 100-fold with 0.85% saline to obtain dilutions for the control group and test groups 1-1 to 1-14. 10 μL of each dilution was inoculated into different compartments of the culture dish and uniformly spread using an inoculation loop. The inoculated culture dishes were inverted and incubated for 24 hours in a 37 °C incubator, and the distribution of E. coli colonies after incubation was observed. The results are shown in Figures 1A and 1B.

[0060] 2. Bacteria count calculation

[0061] One mL of the E. coli culture solution from each of the aforementioned groups was taken and sequentially diluted tenfold with 0.85% saline solution. One mL of each concentration in the dilution series was added to a culture dish, and then 20 mL of TSA medium was added. After the medium in the culture dish solidified, the culture dish was placed in a 37°C incubator and incubated for 24 hours. After incubation, the number of bacteria settled in each culture dish was counted, and the number of growing E. coli bacteria from each group was calculated by multiplying by the dilution factor. The results are shown in Figures 2A and 2B.

[0062] As is clear from Figures 1A and 2A, antibiotic solutions or antimicrobial compositions containing florfenicol (test groups 1-3 to 1-8) failed to suppress the growth of E. coli, regardless of whether or not an ethanol extract of an unfermented coffee composition was used in combination. Furthermore, no antimicrobial effect was obtained even when the ethanol extract of an unfermented coffee composition and florfenicol were used simultaneously. As is clear from Figures 1B and 2B, when the composition contained only the ethanol extract of Production Example 3 (test groups 1-1 and 1-2), the growth of E. coli was not suppressed. On the other hand, in antimicrobial compositions containing doxycycline (test groups 1-9 to 1-14), as in test groups 1-10, 1-11, 1-13, and 1-14, the growth of E. coli was suppressed when the ethanol extract of an unfermented coffee composition and doxycycline were used in combination. These results indicate that a synergistic antibacterial effect exists between the ethanol extract of the unfermented coffee composition and doxycycline, and that using them together yields a superior antibacterial effect compared to doxycycline alone (test groups 1-9, 1-12).

[0063] Test Example 2: Cooperative antibacterial effect of the antibacterial composition of Example 1 (ethanol extract of fermented coffee composition and antibiotic)

[0064] 1.Bacteria drop observation

[0065] 1.5 g of TSB medium was weighed out, placed in a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Next, approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) or approximately 0.1 g of freeze-dried human-derived Salmonella powder (purchased from the Food Industry Development Research Institute, BCRC15464) was added to the Erlenmeyer flask, and the mixture was cultured in a 37 °C incubator at 120 rpm for 24 hours with shaking to obtain either an Escherichia coli solution or a Salmonella solution. Based on Table 2, test groups 2-0 to 2-47 were prepared using the ethanol extract of Production Example 1 and antibiotics (cephalexin (CEX), amoxicillin (AMX), oxytetracycline (OTC), doxycycline (DC), florfenicol (FF), or gentamicin (GEN)). 3 mL of the ethanol extract of Production Example 5 was used for the control group (control or con). Test groups 2-0 and 2-1 were used with 3 mL of the ethanol extract of Production Example 1 (ME or M). For the other test groups, even-numbered groups were used with 3 mL of the antibiotic solution of Production Example 4 (antibiotic solution of cephalexin, amoxicillin, oxytetracycline, doxycycline, florfenicol, or gentamicin, adjusted in concentration with TSB medium), and odd-numbered groups were used with 3 mL of the antimicrobial composition of Example 1 (concentration and type of antibiotic are shown in Table 2). Furthermore, the fermented coffee composition of Production Example 1 used in Test Groups 2-0, 2-1, and the odd-numbered groups in the other test groups was obtained by fermenting for 5 days in process (b).

[0066] Table 2: Ethanol extract concentration, antibiotic concentration, and type included in test groups 2-0 to 2-47 and the control group in Test Example 2. [Table 2-1] [Table 2-2]

[0067] The compositions for the control group and test groups 2-0 to 2-47 were dispensed into 49 screw-cap tubes. Separately, the E. coli solution or Salmonella solution was diluted using TSB medium (diluted 10-fold in succession with sterile saline). 2 Double it, and then 10 2 When 10 μL of the diluted solution is applied to a flat plate, the dilution ratio at that point is 10 2 (This doubles the original concentration), 1 mL was taken from this dilution series and added to the control group and test groups 2-0 to 2-47, respectively. This resulted in a reduction of the number of E. coli or Salmonella bacteria in each group to 1 × 10⁻⁶. 5 The concentration was set to CFU / mL. The total volume of each test tube was 4 mL. These test tubes were then placed in a 37°C incubator and cultured with shaking at 120 rpm for 24 hours to form E. coli cultures or Salmonella cultures for the control group and test groups 2-0 to 2-47.

[0068] 15 g of TSB medium and 7.5 g of agar were weighed out, placed in a 1000 mL serum bottle, 500 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes to obtain TSA medium. After the temperature of the TSA medium had decreased to 45 °C to 50 °C, 20 mL was poured into a culture dish, and two compartments were marked on the bottom of the culture dish with an oil-based pen. The E. coli culture solution or Salmonella culture solution from each group was diluted 100-fold with 0.85% saline to obtain dilutions for the control group and test groups 2-0 to 2-47, which contained E. coli or Salmonella. For E. coli, 10 μL each of the dilutions for the control group and test groups 2-1, 2-6, 2-7, 2-10, 2-11, 2-20, 2-21, 2-28, 2-29, 2-36, 2-37, 2-42, and 2-43 was collected. For Salmonella, 10 μL each of diluted solutions was collected from the control group and test groups 2-0, 2-1, 2-6~2-9, 2-16, 2-17, 2-30~2-33, 2-42, and 2-43. These diluted solutions were inoculated into different compartments of the culture dish and spread uniformly using an inoculation loop. After inoculation, the culture dish was inverted and incubated for 24 hours in a 37°C incubator. The bacterial settlement distribution of E. coli after incubation is shown in Figures 3A~3F, and the bacterial settlement distribution of Salmonella is shown in Figures 3G~3L.

[0069] 2. Bacteria count calculation

[0070] One mL of the E. coli culture solution or Salmonella culture solution from each of the aforementioned groups was taken and sequentially diluted 10-fold with 0.85% saline solution. One mL of each concentration in the dilution series was added to a culture dish, and then 20 mL of TSA medium was added. After the medium in the culture dish solidified, the culture dish was placed in a 37°C incubator and incubated for 24 hours. After incubation, the number of bacteria settled in each culture dish was counted, and the number of growing E. coli bacteria from each group was calculated by multiplying by the dilution factor. The results are shown in Figures 4A to 4F, and the number of growing Salmonella bacteria from each group are shown in Figures 5A to 5F. The symbols in the figures indicate the following: *: P<0.05, **: P<0.01, ***: P<0.001.

[0071] Furthermore, for each test group, the number of growing E. coli bacteria obtained in the two replicate groups and the log reduction compared to the control group were calculated, and the results are shown in Table 3. Similarly, the number of growing Salmonella bacteria and the log reduction compared to the control group are shown in Table 4. The bacterial counts are expressed as mean ± standard deviation (SD).

[0072] Table 3: Growing number and logarithmic decrease of E. coli [Table 3]

[0073] Table 4: Number of Salmonella bacteria and logarithmic decrease [Table 4]

[0074] As is clear from Figures 3A to 3L, Table 3, and Table 4, when the antimicrobial composition contained an ethanol extract of a fermented coffee composition made from green or roasted coffee beans, a synergistic antibacterial effect occurred with the six antibiotics used in this test. The bacterial distribution of Escherichia coli (test groups 2-7, 2-11, 2-21, 2-29, 2-37, 2-43) and Salmonella (test groups 2-7, 2-9, 2-17, 2-31, 2-33, 2-43) formed after culturing was clearly sparse, and the number of growing Escherichia coli (test groups 2-5, 2-11, 2-21, 2-29, 2-37, 2-45) and Salmonella (test groups 2-7, 2-9, 2-17, 2-31, 2-33, 2-45) decreased. These results demonstrate that the antimicrobial composition containing both the ethanol extract and the antibiotic of the present invention has an inhibitory effect against both Escherichia coli and Salmonella. Furthermore, the synergistic inhibitory effect resulting from the combined use of the ethanol extract and the antibiotic can be confirmed in Figures 4A to 4F and 5A to 5F.

[0075] Test Example 3: Cooperative antibacterial effect of the antibacterial composition of Example 2 (ethanol extract of fermented coffee composition and antibiotic amoxicillin)

[0076] The test method for Test Example 3 was generally the same as that of Test Example 1 in terms of bacterial settlement observation, but in Test Example 3, test groups 3-1 to 3-7 were prepared using ethanol extracts (with different fermentation times) from Production Example 2 and amoxicillin (AMX) based on Table 5, and these were dispensed into seven screw-cap tubes. In addition, 3 g of TSB medium and 1.5 g of agar were weighed out, placed in a 500 mL serum bottle, 100 mL of RO water was added, and the TSA medium was obtained by sterilization. The control group (C) of this test example used 3 mL of ethanol extract from Production Example 5. Test group 3-1 used 3 mL of amoxicillin antibiotic solution from Production Example 4, adjusted to a concentration of 1000 μg / mL in TSB medium. Test groups 3-2, 3-4, and 3-6 used 3 mL of ethanol extract from Production Example 2, and test groups 3-3, 3-5, and 3-7 used 3 mL of the antimicrobial composition from Example 2. The fermented coffee compositions of Production Example 2 used in test groups 3-2 to 3-7 were obtained in step (b) with different fermentation times as shown in Table 5 (fermentation times are shown in Table 5).

[0077] Table 5: Ethanol extract concentration, coffee composition fermentation time, antibiotic concentration, and type of antibiotic for test groups 3-1 to 3-7 and the control group in Test Example 3. [Table 5]

[0078] Figure 6 shows the bacterial distribution of E. coli after culture. From this, it was found that when the antibacterial composition contains an ethanol extract of a fermented coffee composition produced from recovered coffee grounds (test groups 3-3, 3-5, 3-7), the synergistic antibacterial effect formed by the combined use of the ethanol extract and amoxicillin effectively suppresses the growth of E. coli, regardless of whether the fermented coffee composition was obtained by fermentation for 3, 4, or 5 days. Furthermore, a higher antibacterial effect was obtained compared to when the ethanol extract was used alone (test groups 3-2, 3-4, 3-6).

[0079] Test Example 4: Cooperative antibacterial effect of the antibacterial composition of Example 1 (water extract of fermented coffee composition and antibiotic)

[0080] The test method for Test Example 4 was generally the same as that for Test Example 1, except that, based on Table 6, water extracts from Production Example 1 (in different forms, such as whole beans, crushed beans, or powder) and antibiotics (cephalexin (CEX), florfenicol (FF), or doxycycline (DC)) were used to prepare test groups 4-1 to 4-15, which were then dispensed into 15 screw-cap tubes. In addition, 6 g of TSB medium and 3 g of agar were weighed out, placed in a 500 mL serum bottle, 200 mL of RO water was added, and the mixture was sterilized to obtain the TSA medium. The control group (con) in this test example used 3 mL of water extract from Production Example 5. Test groups 4-1 to 4-3 used 3 mL of water extract from Production Example 1. Test groups 4-4, 4-8, and 4-12 used 3 mL of the antibiotic solution from Production Example 4 (an antibiotic solution of cephalexin, florfenicol, or doxycycline, adjusted in concentration with TSB medium). Test groups 4-5 to 4-7, 4-9 to 4-11, and 4-13 to 4-15 used 3 mL of the antimicrobial composition from Example 1 (the concentration and type of antibiotic are shown in Table 6). When preparing the aqueous extract from Production Example 1 and the antimicrobial composition from Example 1, the fermented coffee composition from Production Example 1 used in test groups 4-1 to 4-3, 4-5 to 4-7, 4-9 to 4-11, and 4-13 to 4-15 was obtained by using different forms of green coffee beans in step (a) and fermenting them for 5 days in step (b) (the forms of the beans are shown in Table 6).

[0081] Table 6: Water extract concentrations, coffee bean form of coffee composition, antibiotic concentration, and type of antibiotic used in Test Groups 4-1 to 4-15 and the control group in Test Example 4. [Table 6]

[0082] Figure 7 shows the bacterial distribution of E. coli after culture, and Figures 8A to 8C show the number of growing E. coli in each group obtained by bacterial count measurement. As is clear from Figures 7 and 8A to 8C, when the antibacterial composition contained a water extract of a fermented coffee composition made from green coffee beans (test groups 4-5 to 4-7, 4-9 to 4-11, 4-13 to 4-15), the growth of E. coli was effectively suppressed by the synergistic antibacterial effect formed by the water extract and cephalexin, florfenicol, and doxycycline, regardless of whether the green coffee beans were whole beans (C), crushed beans (D), or powder (E). Furthermore, a superior antibacterial effect was obtained compared to when antibiotics were used alone (test groups 4-4, 4-8, 4-12).

[0083] Test Example 5: Minimum Inhibitory Concentration (MIC) against resistant bacteria in the antibacterial composition of Example 1 (ethanol extract of fermented coffee composition and antibiotic)

[0084] Mueller-Hinton II Broth (MH II Broth), prepared to a concentration of 22 g / L, was sterilized at 121°C for 15 minutes, then cooled to room temperature. The sterilized MH II Broth was dispensed into rows 2-12 of a 96-well plate. Based on Table 7, test groups 5-1 to 5-9 were prepared using the ethanol extract and antibiotic (florfenicol (FF), enrofloxacin (EF), or doxycycline (DC)) from Preparation Example 1. Test groups 5-1, 5-4, and 5-7 used the antibiotic solution from Preparation Example 4 (antibiotic solutions of florfenicol, enrofloxacin, or doxycycline, with concentration adjusted using MH II Broth). Test groups 5-2, 5-3, 5-5, 5-6, 5-8, and 5-9 used the antimicrobial composition from Example 1 (concentration and type of antibiotic shown in Table 7). The fermented coffee composition of Production Example 1 used in test groups 5-2, 5-3, 5-5, 5-6, 5-8, and 5-9 was obtained by fermenting for 5 days in process (b).

[0085] Table 7: Ethanol extract concentration, antibiotic concentration, and antibiotic type of test groups 5-1 to 5-9 in Test Example 5

Table 7

[0086] 200 μL of the compositions of test groups 5-1 to 5-9 were respectively added to the first row of a 96-well plate. Then, starting from n = 1, 100 μL of the solution in each well of the nth column was aspirated and added to the (n + 1)th column, and this operation was performed up to the 12th column of the compositions of test groups 5-1 to 5-9. Here, the concentration of florfenicol contained in the 12th column of the compositions of test groups 5-1 to 5-3 was 0.5 μg / mL, the concentration of enrofloxacin contained in the 12th column of the compositions of test groups 5-4 to 5-6 was 0.25 μg / mL, and the concentration of doxycycline contained in the 12th column of the compositions of test groups 5-7 to 5-9 was 0.25 μg / mL. Based on the McFarland turbidity standard 0.5 (McFarland 0.5), drug-resistant Porcine E. coli (provided by the Department of Veterinary Medicine, National Chiayi University), Chicken E. coli (provided by the Department of Veterinary Medicine, National Chiayi University), Porcine Salmonella enterica (provided by the Department of Veterinary Medicine, National Chiayi University), Chicken Salmonella enterica (provided by the Department of Veterinary Medicine, National Chiayi University), Pasteurella multocida (provided by the Department of Veterinary Medicine, National Chiayi University), and Glässerella parasuis (provided by the Department of Veterinary Medicine, National Chiayi University) were respectively used to prepare bacterial solutions with a concentration of 1.5×10 8 CFU / mL. 5 μL of these bacterial solutions were respectively added to each well of the 96-well plate, and then the 96-well plate was cultured in a 37°C constant temperature incubator for 16 - 24 hours. After that, the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of the compositions of each test group was determined.

[0087] Regarding the compositions of test groups 5-1 to 5-3 and 5-7 to 5-9, the MIC (MIC 90The concentrations are shown in Figures 9A and 9B, respectively. The MICs for compositions in test groups 5-1 to 5-9 against chicken-derived Escherichia coli with antidote activity are as follows. 90 The concentrations are shown in Figures 10A to 10C, respectively. The MICs for compositions in test groups 5-1 to 5-9 against drug-resistant porcine-derived Salmonella were determined. 90 The concentrations are shown in Figures 11A to 11C, respectively. The MICs for compositions 5-1 to 5-9 against pesticide-resistant chicken-derived Salmonella were determined. 90 The concentrations are shown in Figures 12A to 12C, respectively. The MICs for compositions in test groups 5-1 to 5-3 and 5-7 to 5-9 against the drug-resistant Pasteurella maltosida fungus were determined. 90 The concentrations are shown in Figures 13A and 13B, respectively. The MICs for compositions in test groups 5-1 to 5-9 against the antibiotic-resistant *Cerella parasuisine* were determined. 90 The concentrations are shown in Figures 14A to 14C, respectively.

[0088] According to the results in Figures 9A to 14C, when florfenicol, enrofloxacin, or doxycycline were used alone (test groups 5-1, 5-4, and 5-7), the concentration required to suppress bacterial count by 90% was high. On the other hand, when each antibiotic was used in combination with the fermented coffee composition (test groups 5-2, 5-3, 5-5, 5-6, 5-8, and 5-9), the MIC for each antibiotic was low. 90 The concentration reaching the MIC can be significantly reduced, and for drug-resistant E. coli of porcine and chicken origin, and Salmonella of porcine origin, the MIC increases as the concentration of the ethanol extract of the fermented coffee composition increases. 90 The concentration showed a more pronounced tendency to decrease. Therefore, it can be concluded that the antibacterial effect of antibiotics, which is reduced by their antidote properties, can be enhanced by a synergistic effect with the ethanol extract of the fermented coffee composition.

[0089] Test Example 6: Inhibitory effect of ethanol extract and antibiotics from fermented coffee composition on different bacterial species.

[0090] 1.5 g of TSB medium or 2.75 g of lactic acid bacteria medium (Lactobacilli MRS Broth, MRSB) was weighed into a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the flask was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) or approximately 0.1 g of freeze-dried human-derived Salmonella powder (purchased from the Food Industry Development Research Institute, BCRC15464) was added to the Erlenmeyer flask containing the TSB medium. Approximately 0.1 g of freeze-dried Lactobacillus pentosus (a porcine-derived pentose lactic acid bacterium, strain number BL010, identified by Genshi Biotechnology) or approximately 0.1 g of freeze-dried Lactobacillus reuteri (a porcine-derived lactic acid bacterium, strain number BL011, identified by Genshi Biotechnology) was added to Erlenmeyer flasks containing the aforementioned MRSB medium. These Erlenmeyer flasks were placed in a 37°C incubator, and Escherichia coli and Salmonella were cultured with shaking at a rotation speed of 120 rpm for 24 hours, while the pentose lactic acid bacterium and reuteri lactic acid bacterium were cultured statically for 24 hours to obtain solutions of Escherichia coli, Salmonella, pentose lactic acid bacterium, and reuteri lactic acid bacterium. Based on Table 8, test groups 6-1 to 6-6 were prepared using the ethanol extract from Production Example 1 and oxytetracycline (OTC). The control group used 3 mL of the ethanol extract from Production Example 5, test groups 6-1 to 6-3 used 3 mL of the ethanol extract from Production Example 1, and test groups 6-4 to 6-6 used 3 mL of the antibiotic solution from Production Example 4 (an antibiotic solution of oxytetracycline, whose concentration was adjusted using TSB medium or MRSB). The fermented coffee composition from Production Example 1 used in test groups 6-1 to 6-3 was obtained by fermenting for 5 days in step (b).

[0091] Table 8: Ethanol extract concentration, antibiotic concentration, and type included in test groups 6-1 to 6-6 and the control group in Test Example 6. [Table 8]

[0092] Ethanol extracts or antibiotic solutions from the control group and test groups 6-1 to 6-6 were dispensed into seven screw-cap tubes each. Separately, E. coli solution and Salmonella solution were diluted using TSB medium, and pentose lactic acid bacteria solution and reuteri lactic acid bacteria solution were diluted using MRSB medium. 1 mL of each solution was then added to each group, and the number of E. coli, Salmonella, pentose lactic acid bacteria, or reuteri lactic acid bacteria in each group was determined to be 1 × 10⁶. 5 The solution was adjusted to a CFU / mL concentration. The total volume of each screw-cap tube was 4 mL. These screw-cap tubes were placed in a 37°C incubator. Escherichia coli and Salmonella were cultured with shaking at 120 rpm for 24 hours, while Lactobacillus pentose and Lactobacillus reuteri were cultured statically for 24 hours to obtain the Escherichia coli culture, Salmonella culture, Lactobacillus pentose culture, or Lactobacillus reuteri culture for the control group and test groups 6-1 to 6-6.

[0093] 6 g of TSB medium and 3 g of agar were weighed into a 500 mL serum bottle, 200 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes to obtain TSA medium. 11 g of MRSB and 3 g of agar were weighed into a 500 mL serum bottle, 200 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes to obtain MRS-Agar (MRSA) medium. 1 mL of the bacterial culture solution from each group described above was taken and sequentially diluted 10-fold with 0.85% saline. After dilution, 1 mL of each dilution was added to a culture dish, and 20 mL of TSA medium or MRSA medium was added. After the medium solidified, the culture dish was placed in a 37 °C incubator and incubated for 24 hours. After culturing, the number of bacteria settled in each culture dish was counted and multiplied by the dilution factor to calculate the number of E. coli bacteria in each group, which is shown in Figure 15A, the number of Salmonella bacteria in Figure 15B, the number of Pentoses lactic acid bacteria in Figure 15C, and the number of Lactobacillus reuteri bacteria in Figure 15D.

[0094] As shown in Figures 15A to 15D, the ethanol extract of the fermented coffee composition inhibited the growth of Escherichia coli and Salmonella (test groups 6-1 to 6-3). However, this antibacterial effect did not affect beneficial bacteria such as Pentose lactic acid bacteria and Lactobacillus reuteri, while antibiotics act indiscriminately on all bacterial species (test groups 6-4 to 6-6), thus killing beneficial bacteria as well. Therefore, compared to the use of antibiotics, the ethanol extract of the fermented coffee composition does not adversely affect beneficial bacteria and exhibits a selective antibacterial effect.

[0095] Test Example 7: Bacterial Cell Membrane Damage Test

[0096] 1.5 g of TSB medium was weighed into a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) or approximately 0.1 g of freeze-dried human-derived Salmonella powder (purchased from the Food Industry Development Research Institute, BCRC15464) was added to the Erlenmeyer flask, and the mixture was cultured with shaking at a rotation speed of 120 rpm in a constant temperature culture chamber at 37 °C for 24 hours to obtain either an Escherichia coli solution or a Salmonella solution. Test groups 7-1 to 7-3 (without antibiotics) were prepared using the ethanol extract from Production Example 1, with ethanol extract concentrations of 12.5 mg / mL, 25 mg / mL, and 50 mg / mL, respectively. 3 mL of the ethanol extract from Production Example 5 was used for the control group. The fermented coffee composition of Production Example 1 used in test groups 7-1 to 7-3 was obtained by fermenting for 5 days in process (b).

[0097] Ethanol extracts from the control group and test groups 7-1 to 7-3 were each dispensed into four test tubes. Separately, E. coli solution or Salmonella solution was diluted using TSB medium, and 1 mL was added to each group. The number of E. coli or Salmonella bacteria in each group was then determined to be 1 × 10⁶. 8The solutions were prepared to achieve a CFU / mL concentration. The total volume of each test tube was 4 mL. These test tubes were placed in a 37°C incubator and incubated with shaking at a rotation speed of 120 rpm for 24 hours to obtain E. coli or Salmonella cultures for the control group and test groups 7-1 to 7-3. One mL of the obtained E. coli or Salmonella culture was taken and centrifuged at a rotation speed of 8000 rpm for 5 minutes, and the supernatant and bacterial cells were collected, respectively.

[0098] 1 mL of phosphate-buffered saline (PBS) was added to the bacterial cells, and the cells were centrifuged at 8000 rpm for 5 minutes to remove the PBS. This washing process was repeated three times. After washing, 0.5 mL of propidium iodide (PI) was added to the bacterial cells to disperse them, and the cells were allowed to stand at room temperature and protected from light for 15 minutes before being stained fluorescently. The cells were centrifuged at 8000 rpm for 5 minutes to remove the PI, 1 mL of PBS was added, and the cells were centrifuged again at 8000 rpm for 5 minutes to remove the PBS. This washing process was repeated three times. 1 mL of PBS was added to suspend the bacterial cells, and the fluorescence intensity within the cells was measured using a fluorescence analyzer. The excitation wavelength was 495 nm and the fluorescence emission wavelength was 625 nm. The analysis results are shown in Figure 16A.

[0099] The supernatant was used for the following purposes: (i) the nucleic acid concentration was analyzed by directly measuring the OD value using ultraviolet light at a wavelength of 260 nm; (ii) the protein concentration was calculated by reacting it with Coomassie Blue for 5 minutes and then measuring the OD value at 595 nm; (iii) 950 μL of the supernatant was placed in a microtube, 50 μL of ONPG (ortho-nitrophenyl-β-galactoside) at a concentration of 1 mmol / liter (mM) was added, and the mixture was reacted at room temperature for 2 hours. After that, 200 μL was dispensed into a 96-well plate, and the β-galactosidase concentration was analyzed by measuring the OD value at 420 nm. The results of these analyses (i) to (iii) are shown in Figures 16B to 16D, respectively.

[0100] In test groups 7-1 to 7-3, the ethanol extract of the fermented coffee composition disrupted the bacterial cell membrane, creating pores. This allowed propidium iodide to pass through the cell membrane and bind to nucleic acids within the bacterial cells, emitting red fluorescence (Figure 16A). When the cell membrane ruptures, DNA, RNA, and proteins also leak out of the cell, allowing them to be recovered from the supernatant after centrifugation of the bacterial culture (Figures 16B and 16C). Furthermore, β-galactosidase present in the bacterial cytoplasm is also released outside the cell, preventing the breakdown of lactose into glucose and galactose, thus inhibiting energy production. The β-galactosidase released outside the cell is yellow, as shown in Figure 16D. From the results in Figures 16A to 16D, it was confirmed that the higher the concentration of the ethanol extract of the fermented coffee composition, the greater the damage to the bacterial cell membrane and the greater the amount of material leaked from the bacterial cells. This clearly demonstrates that the ethanol extract of the fermented coffee composition effectively inhibits bacteria through cell membrane disruption.

[0101] Test Example 8: Scanning Electron Microscope (SEM) Image Analysis

[0102] 1.5 g of TSB medium was weighed into a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) or approximately 0.1 g of freeze-dried human-derived Salmonella powder (purchased from the Food Industry Development Research Institute, BCRC15464) was added to the Erlenmeyer flask, and the mixture was cultured with shaking at a rotation speed of 120 rpm in a constant temperature culture box at 37 °C for 24 hours to obtain the Escherichia coli solution or the Salmonella solution. Test groups 8-1 to 8-3 were prepared using the ethanol extract and cephalexin from Production Example 1. The control group used 3 mL of the ethanol extract from Production Example 5, while test groups 8-1 and 8-2 used 3 mL of the ethanol extract from Production Example 1 (without added antibiotics), with concentrations of 25 mg / mL and 50 mg / mL, respectively. Test group 8-3 used 3 mL of the antibiotic solution from Production Example 4 (a cephalexin antibiotic solution, adjusted to 30 μg / mL in TSB medium). The fermented coffee composition from Production Example 1 used in test groups 8-1 to 8-3 was obtained by fermenting for 5 days in step (b).

[0103] Ethanol extracts or antibiotic solutions from the control group and test groups 8-1 to 8-3 were dispensed into four test tubes each. Separately, E. coli solution or Salmonella solution was diluted using TSB medium and 1 mL was added to each group to determine the number of E. coli or Salmonella bacteria in each group (1 × 10⁶). 8 The solutions were adjusted to a CFU / mL concentration. The total volume of each test tube was 4 mL. These test tubes were placed in a 37°C incubator and incubated with shaking at a rotation speed of 120 rpm for 24 hours to obtain E. coli or Salmonella cultures for the control group and test groups 8-1 to 8-3. One mL of the obtained E. coli or Salmonella culture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed to collect the bacterial cells.

[0104] The aforementioned bacterial cells were washed with 1 mL of PBS and centrifuged at 8000 rpm for 5 minutes to remove the PBS. This procedure was repeated three times to wash the bacterial cells, and then 0.5 mL of PBS was added to suspend them. The resulting bacterial suspension was fixed, dehydrated, and coated, and the bacterial cells were imaged using a scanning electron microscope (SEM). The morphology of each group of Escherichia coli is shown in Figure 17A, and the morphology of Salmonella is shown in Figure 17B.

[0105] As shown in Figures 17A and 17B, the ethanol extracts or antibiotic solutions of test groups 8-1 to 8-3 caused pores in the cell membranes of Escherichia coli and Salmonella (indicated by red arrows), released intracellular substances adhered to the outside of the cell membrane to form mucus, roughened the bacterial surface (indicated by green arrows), and further caused atrophy and fission of the bacterial cells (indicated by blue arrows). Compared to antibiotics alone (test group 8-3), culture with a 25 mg / mL ethanol extract made the bacteria more prone to external changes such as pore formation and rough surface development (test group 8-1), and culture with a 50 mg / mL ethanol extract further promoted atrophy or fission of the bacterial cells (test group 8-2). This demonstrates that ethanol extracts can achieve superior antibacterial effects compared to antibiotics.

[0106] Test Example 9: Electrophoretic Analysis

[0107] 1. DNA damage test

[0108] 1.5 g of TSB medium was weighed into a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of human-derived freeze-dried Escherichia coli powder (BCRC11509) or human-derived freeze-dried Salmonella powder (BCRC15464), purchased from the Food Industry Development Research Institute, was added to the Erlenmeyer flask and placed in a 37 °C incubator. The mixture was cultured with shaking at a rotation speed of 120 rpm for 24 hours to obtain either an Escherichia coli solution or a Salmonella solution. Test groups 9-1 to 9-3 (without antibiotics) were prepared using the ethanol extract from Production Example 1, with ethanol extract concentrations of 50 mg / mL, 25 mg / mL, and 12.5 mg / mL, respectively. 3 mL of the ethanol extract from Production Example 5 was used for the control group. The fermented coffee composition of Production Example 1 used in test groups 9-1 to 9-3 was obtained after a fermentation period of 5 days in process (b).

[0109] Ethanol extracts from the control group and test groups 9-1 to 9-3 were each dispensed into four test tubes. Furthermore, 1 mL of either the E. coli solution or the Salmonella solution was diluted with TSB medium, and this was added to the control group and test groups 9-1 to 9-3, respectively, to determine the number of E. coli or Salmonella bacteria present in each group (1 × 10⁻⁶). 8 The concentration was set to CFU / mL. Each test tube contained 4 mL of culture solution and was incubated with shaking at 120 rpm in a 37°C incubator for 24 hours to form E. coli or Salmonella culture solutions for the control group and test groups 9-1 to 9-3. 1 mL of either the E. coli or Salmonella culture solution was centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed to collect the bacterial cells.

[0110] 1 mL of PBS was added to the bacterial cells, and the cells were centrifuged at 8000 rpm for 5 minutes. After removing the PBS, this washing procedure was repeated three times. Nucleic acids were isolated from the bacterial cells using a nucleic acid extraction kit (Blood / Cultured Cell Genomic DNA Extraction Mini Kit, ALPHAGEN APBGR200), and samples for the control group and test groups 9-1 to 9-3 were prepared. 0.8 g of agarose was weighed into a serum bottle, 0.5 × TBE buffer was added, and the mixture was heated in a microwave oven to dissolve and form a gel substrate. The gel substrate was cooled to 60°C, 0.005% (v / v) nucleic acid stain (HealthView Nucleic Acid Stain, Genomics) was added, and the mixture was allowed to stand for 25-30 minutes to solidify, obtaining a 0.8% gel. The obtained gel was transferred to an electrophoresis tank, and the sample and 6× sample stain (Loading Dye, Genomics) were uniformly mixed and added to each well of the gel along with a DNA molecular weight marker (DNA Marker, SIGMA). Electrophoresis was performed at a voltage of 100 volts (V) for 25-30 minutes, after which the gel was imaged using an ultraviolet light irradiator. The electrophoresis results using E. coli from each group are shown in Figure 18A, and the electrophoresis results using Salmonella are shown in Figure 18B.

[0111] As shown in Figures 18A and 18B, after being exposed to the ethanol extract of the fermented coffee composition, Escherichia coli and Salmonella showed band-like smears (indicated by red arrows) in electrophoresis, indicating that structural changes occurred in the bacterial DNA. DNA damage inactivates the bacteria through changes in metabolic function, and this tendency becomes more pronounced as the concentration of the ethanol extract of the fermented coffee composition increases.

[0112] 2. Protein damage test

[0113] 1.5 g of TSB medium was weighed into a 100 mL Erlenmeyer flask, 50 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (BCRC11509), purchased from the Food Industry Development Research Institute, was added to the Erlenmeyer flask, and the mixture was placed in a 37 °C incubator and cultured with shaking at a speed of 120 rpm for 24 hours to obtain an Escherichia coli solution. Test groups 9-4 to 9-9 (without antibiotics) were prepared using 3 mL of the ethanol extract from Production Example 1, and the concentration of the ethanol extract in test groups 9-4 to 9-9 was set to 50 mg / mL. The fermented coffee composition from Production Example 1 used in these test groups was obtained after a fermentation period of 5 days in step (b).

[0114] Test groups 9-4 to 9-9 were each dispensed into six test tubes. The E. coli solution was then diluted with TSB medium, and 1 mL was added to each test group 9-4 to 9-9. The reaction time shown in Table 9 was then elapsed, and the number of E. coli in each group was calculated to be 1 × 10⁻⁶. 8 The concentration was set to CFU / mL. The total volume of each test tube was 4 mL. These test tubes were placed in a 37°C incubator and incubated with shaking at a speed of 120 rpm for 24 hours to form the E. coli cultures of test groups 9-4 to 9-9. 1 mL of the E. coli culture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed to collect the bacterial cells.

[0115] Table 9: Duration of action of test groups 9-4 to 9-9 and E. coli in the protein damage test [Table 9]

[0116] 1 mL of PBS was added to the bacterial cells, and the mixture was centrifuged at 8000 rpm for 5 minutes to remove the PBS. This washing procedure was repeated three times. TMTotal protein was extracted from bacterial cells using a protein extraction kit (Taiding Biotechnology), and samples from test groups 9-4 to 9-9 were prepared. SDS-PAGE was prepared using a 4% stacking gel and a 10% resolving gel, and the samples and the protein molecular weight marker (SIGMA) were added to the wells of the gel, respectively. Electrophoresis was performed at a voltage of 100V for 120 minutes, and then images were taken using a camera. The electrophoresis results are shown in Figure 18C.

[0117] As shown in Figure 18C, the longer the ethanol extract of the fermented coffee composition acts on E. coli, the lighter the band color becomes, indicating a decrease in protein expression. Furthermore, the bands in test groups 9-4 to 9-9 show discontinuous breaks between 100 kilodaltons (kDa) and 140 kDa, indicating that the ethanol extract of the fermented coffee composition interferes with and inhibits protein synthesis in E. coli, thereby affecting the activity of the bacterial cells.

[0118] Test Example 10: Reactive Oxygen Species (ROS) Analysis

[0119] 1.5 g of TSB medium was weighed out and placed in a 100 mL Erlenmeyer flask. 50 mL of RO water was added, and the mixture was sterilized at 121 °C for 20 minutes, then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) was added to the Erlenmeyer flask and incubated in a 37 °C incubator with shaking at 120 rpm for 24 hours to obtain an E. coli solution. Test groups 10-1 to 10-6 were prepared using the ethanol extract from Production Example 1, and test groups 10-7 to 10-9 were prepared using the ethanol extract from Production Example 3 (without adding antibiotics to any of the samples). The concentration of the ethanol extract in test groups 10-1 to 10-9 was 50 mg / mL. The control group used the ethanol extract from Production Example 5. Ethanol extracts from test groups 10-1 to 10-9 were prepared as 3 mL reaction solutions after adding catalase or peroxidase according to the conditions shown in Table 10 and undergoing further different treatments. Here, treatment in a 95°C warm water bath for 5 minutes was intended to inactivate the activity of catalase or peroxidase.

[0120] Table 10: Preparation conditions for reaction solutions of test groups 10-1 to 10-9 in Test Example 10 [Table 10]

[0121] After cooling the reaction solutions of each test group to room temperature, the reaction solutions of the control group and test groups 10-1 to 10-9 were separated and placed into 10 test tubes each. Furthermore, the E. coli solution was diluted with TSB medium, and 1 mL of this solution was added to the control group and test groups 10-1 to 10-9, respectively, to determine the number of E. coli bacteria in each group (1 × 10⁻⁶). 7 CFU / mL ~ 1 x 10 8 The concentration was set to CFU / mL, and the total volume of each test tube was set to 4 mL. These test tubes were placed in a 37°C incubator and incubated with shaking at 120 rpm for 18 hours to obtain E. coli cultures of 10-1 to 10-9 for the control group and the test group.

[0122] One mL of the E. coli culture solution from each of the aforementioned groups was taken and sequentially diluted 10 times with 0.85% saline solution. Then, one mL of each concentration level was placed in a culture dish, and 20 mL of TSA medium was added. After waiting for the liquid in the culture dish to solidify, the culture dishes were placed in a 37°C incubator and incubated for 24 hours. Based on the culture results, the number of bacteria that settled in each culture dish was calculated, and the number of E. coli bacteria growing in each group, obtained by multiplying by the dilution factor, is shown in Figures 19A and 19B.

[0123] According to the results in Figures 19A and 19B, the ethanol extract of unfermented coffee compound showed no antibacterial activity (test groups 10-7 to 10-9), and the antibacterial effect of fermented coffee compound disappeared upon the addition of peroxidase or catalase (test groups 10-3, 10-5). However, when these enzymes were inactivated by a hot water bath, the antibacterial effect was still observed (test groups 10-4, 10-6). Peroxidase or catalase prevents the excessive accumulation of ROS within cells and protects cells from oxidative damage. From these findings, it can be seen that the antibacterial effect of the ethanol extract of fermented coffee compound is exerted by disrupting the oxidation-reduction balance within bacteria, creating a state where the pro-oxidant effect exceeds the antioxidant effect, and inactivating bacteria through oxidative stress caused by the accumulation of reactive oxygen species (ROS).

[0124] Test Example 11: Cellular Inflammatory Response Test

[0125] Porcine intestinal epithelial cells IPEC-J2 and mouse macrophages RAW264.7 were seeded in 6-well plates, and 2 mL of complete medium was added to each well. Dulbecco's Modified Eagle Medium (DMEM) (SH30243, HyClone), containing 10 vol% fetal bovine serum (FBS) and 1 vol% triple antibiotic (Pencillin / Streptomycin / Amphotericin B, PSA), was used as the complete medium. After the cells had completely adhered to the bottom of the wells, the complete medium was replaced with 2 mL of serum-free DMEM. The cells were divided into negative control groups, positive control groups 1-3, and test groups 11-1-11-9. Lipopolysaccharide (LPS) and the ethanol extract from Preparation Example 1 were added to the cells of each group according to the conditions in Table 11, and the concentrations of LPS and ethanol extract shown in Table 11 were prepared. The fermented coffee composition of Production Example 1 used in test groups 11-1 to 11-9 was obtained after 5 days of fermentation in process (b).

[0126] Table 11: Concentrations of lipopolysaccharide and ethanol extract in cell culture medium for each group in Test Example 11 [Table 11]

[0127] Cells were cultured in a culture incubator for 1 hour (positive control group 1, test groups 11-1 to 11-3), 15 hours (positive control group 3, test groups 11-7 to 11-9), or 24 hours (positive control group 2, test groups 11-4 to 11-6), after which the supernatant was collected from each group. IL-6 protein concentration was measured using the Pig IL-6 ELISA Kit (MBS2701081, MyBioSource), NO production rate was measured by the Griess assay, and TNF-α protein concentration was measured using the Porcine TNF-α ELISA Kit (MBS2701342, MyBioSource). Since the amount of each of these three inflammatory factors increases when induced by lipopolysaccharide (LPS) differs, the induction times also differ for each. Figure 20A shows the IL-6 concentration measurement results in IPEC-J2 cells, Figure 20B shows the NO production rate measurement results in RAW264.7 cells, and Figure 20C shows the TNF-α concentration measurement results using IPEC-J2 cells.

[0128] Using a microscope, cultured RAW264.7 cells from the negative control group, positive control group 2, and test groups 14-4 and 14-6 were observed at 200x magnification. The observed cell morphology is shown in Figure 20D.

[0129] As shown in Figures 20A to 20C, in the inflammatory response induced by lipopolysaccharide, IL-6 and TNF-α expression increased in IPEC-J2 cells, and NO production was promoted in macrophages. On the other hand, the ethanol extract of the fermented coffee composition decreased IL-6 concentration (test groups 14-1 to 14-3) and TNF-α concentration (test groups 14-7 to 14-9), and further reduced the NO production rate (test groups 14-4 to 14-6). Furthermore, the decrease in IL-6 concentration and NO production rate was more pronounced as the content of the ethanol extract of the fermented coffee composition increased. As shown in Figure 20D, normal RAW264.7 cells are spherical (blue arrow), but when an inflammatory response is induced, they exhibit a pseudopod-like morphology (red arrow) or extend outward (green arrow). However, pseudopod formation was not observed in cells treated with the ethanol extract of the fermented coffee composition. The above results demonstrate that the ethanol extract of the fermented coffee composition has the effect of reducing the inflammatory response induced by lipopolysaccharides.

[0130] Test Example 12: Bacterial Inflammatory Response Test

[0131] Mouse-derived macrophages RAW264.7 were seeded in 96-well plates, with 100 μL of complete medium in each well. The complete medium was DMEM (SH30243, HyClone) containing 10 vol% fetal bovine serum (FBS) and 1 vol% triple antibiotic (Penicillin / Streptomycin / Amphotericin B, PSA). After the cells had completely adhered to the bottom of the 96-well plate, this complete medium was replaced with 100 μL of serum-free DMEM, and the cells were divided into control and test groups 12-1 to 12-6.

[0132] 1.5 g of TSB medium was weighed into a 100 mL Erlenmeyer flask, 50 mL of reverse osmosis (RO) water was added, and the mixture was sterilized at 121 °C for 20 minutes and then cooled to room temperature. Approximately 0.1 g of freeze-dried human-derived Escherichia coli powder (purchased from the Food Industry Development Research Institute, BCRC11509) was added to the Erlenmeyer flask and cultured with shaking at 120 rpm for 24 hours in a 37 °C incubator to obtain an Escherichia coli solution. Bacterial culture solutions for test groups 12-1 to 12-6 were prepared using the ethanol extract from Production Example 1 and antibiotics (amoxicillin (AMX) or amoxicillin-clavulanic acid (AMX-CA)) according to Table 12 below. The control group was given the ethanol extract from Production Example 5, test group 12-2 was given the ethanol extract from Production Example 1, test groups 12-3 and 12-5 were given the antibiotic solution from Production Example 4 (an antibiotic solution of amoxicillin or amoxicillin-clavulanic acid, adjusted in concentration with TSB medium), and test groups 12-4 and 12-6 were given the antimicrobial composition from Example 1 (the concentration and type of antibiotic are shown in Table 12). The fermented coffee composition from Production Example 1 used in test groups 12-2, 12-4, and 12-6 was obtained after a fermentation period of 5 days in step (b).

[0133] Table 12: Ethanol extract concentration, antibiotic concentration, and type of bacterial culture solution for each group in Test Example 12 [Table 12]

[0134] The control group and test groups 12-1 to 12-6 were each placed in seven test tubes. Separately, the E. coli solution was diluted with TSB medium, and 1 mL was added to each of the control group and test groups 12-1 to 12-6. The number of E. coli in each group was then calculated to be 1 × 10⁻⁶. 8 The concentration was set to CFU / mL. Each test tube was 4 mL in volume, and these test tubes were incubated in a 37°C incubator at 120 rpm for 24 hours with shaking to form the E. coli cultures for the control group and test groups 12-1 to 12-6.

[0135] The control group and test groups 12-1 to 12-6 were centrifuged at 8000 rpm for 5 minutes to remove bacterial cells, and the supernatant from each group was added to a 96-well plate containing RAW264.7 cells. After culturing the cells in an incubator for 24 hours, the supernatant was collected, and the NO production rate of the cells in each group was measured.

[0136] Antibiotics inhibit cell wall synthesis in E. coli, causing bacterial lysis and the release of lipid polysaccharides. The resulting inflammatory response promotes NO production in macrophages. Based on the results in Figure 21, test group 12-2, treated with an ethanol extract of fermented coffee compound, had a lower NO production rate than test group 12-1. Furthermore, in test groups 12-4 and 12-6, the simultaneous use of ethanol extract of fermented coffee compound and antibiotics significantly reduced NO production compared to test groups 12-3 and 12-5, which used the corresponding antibiotics alone. From these findings, it can be seen that ethanol extract of fermented coffee compound can mitigate the inflammatory response caused by the release of lipid polysaccharides induced by antibiotics.

[0137] From the above results, the antibacterial composition provided by the present invention comprises a coffee composition extracted with alcohol or water and an antibiotic. The extract of the coffee composition has the effect of mitigating the inflammatory response caused by LPS by reducing the expression of cell-derived proteins such as IL-6 and TNF-α and NO production in macrophages. Furthermore, a synergistic antibacterial effect is obtained by using the extract of the coffee composition and the antibiotic in combination, and the antibacterial composition of the present invention exhibits a superior antibacterial effect compared to the use of the antibiotic alone. Moreover, since this antibacterial effect also acts on antibiotic-resistant bacteria, it is possible to overcome the problem that conventional antibiotics suffer from a decrease in antibacterial activity with increasing frequency of use.

Claims

1. An antibacterial composition, An extract of a coffee composition, wherein the coffee composition comprises 92 wt% to 95 wt% solid components and 5 wt% to 8 wt% water, the solid components comprising 20 wt% to 70 wt% coffee beans and 30 wt% to 80 wt% auxiliary materials, and the carbon-nitrogen ratio of the coffee composition is 35 to 50. Antibiotics selected from the group consisting of penicillin antibiotics, cephalosporin antibiotics, fluoroquinolone antibiotics, tetracycline antibiotics, chloramphenicol antibiotics, aminoglycoside antibiotics, and combinations thereof. Includes, Herein, the weight ratio of the coffee composition to the antibiotic is 1.5:1 to 25000:1, and the extract of the coffee composition comprises an aqueous extract of the coffee composition, an alcohol extract of the coffee composition, or a combination thereof, in an antimicrobial composition.

2. The antibacterial composition according to claim 1, wherein the extract of the coffee composition comprises an alcohol extract of the coffee composition.

3. The antibacterial composition according to claim 1, wherein the coffee beans are green coffee beans, roasted coffee beans, or recycled coffee grounds.

4. The antimicrobial composition according to claim 1, wherein the auxiliary material comprises crushed corn, crushed sugar beet, rice bran, hulled soybean meal, broken rice, or a combination thereof.

5. The coffee composition is (1) A step of mixing 40 wt% to 60 wt% of the solid component with 40 wt% to 60 wt% of water to form a mixture; (2) A step of heating the mixture at 115°C to 125°C and 1.0 bar to 1.5 bar for 20 to 60 minutes to obtain a substrate; and (3) A step of cooling the substrate and drying it until the moisture content is 5 wt% to 8 wt% to obtain the coffee composition. The antimicrobial composition according to claim 1, manufactured by [method].

6. The antibacterial composition according to claim 1, wherein the alcohol extract is obtained by extracting the coffee composition with an aqueous ethanol solution having a concentration of 60% to 80%, and the ratio of the coffee composition to the aqueous ethanol solution is 1 g:3 mL to 1 g:7 mL.

7. The antibacterial composition according to claim 1, wherein the coffee composition is an unfermented coffee composition or a coffee composition that has undergone fermentation with Aspergillus oryzae.

8. Penicillin antibiotics are selected from the group consisting of penicillin G, penicillin V, ampicillin, amoxicillin, and amoxicillin-clavulanate; Cephalosporin antibiotics were selected from the group consisting of cephalexin, cefuroxime, and ceftiofur; Fluoroquinolone antibiotics are selected from the group consisting of enrofloxacin, ofloxacin, danofloxacin, ciprofloxacin, and norfloxacin; Tetracycline antibiotics are selected from the group consisting of oxytetracycline, chlortetracycline, and doxycycline; Chloramphenicol antibiotics are selected from the group consisting of chloramphenicol, thianphenicol, and florfenicol; The antimicrobial composition according to claim 1, wherein the aminoglycoside antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, gentamicin, spectinomycin, and apramycin.

9. The antimicrobial composition according to claim 1, wherein the antimicrobial composition has an inhibitory effect on bacteria, and the bacteria are selected from the group consisting of Salmonella, Escherichia, Pasteurella, Streptococcus, Lysabacterium, Staphylococcus, Reichelcia, Pseudomonas, and Klebsiella.

10. The antimicrobial composition according to claim 9, wherein the bacteria are selected from Salmonella, Salmonella choleraesuis, Escherichia coli, Pasteurella maltosida, Greiserella parasuis, Streptococcus suis, Galibacterium anatis, Staphylococcus hiix, Liemella anatipestifer, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

11. Use of the antimicrobial composition according to any one of claims 1 to 10 for the manufacture of an antimicrobial drug.

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