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Article

Improved Functions of Fermented Coffee by Lactic Acid Bacteria

by
Seon-Gyu Kim
1,†,
Aoun Abbas
1,† and
Gi-Seong Moon
1,2,3,*
1
Department of Biotechnology, Korea National University of Transportation, Jeungpyeong 27909, Republic of Korea
2
4D Convergence Technology Institute, Korea National University of Transportation, Jeungpyeong 27909, Republic of Korea
3
Major in IT·Biohealth Convergence, Department of IT·Energy Convergence, Graduate School, Korea National University of Transportation, Chungju 27469, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2024, 14(17), 7596; https://doi.org/10.3390/app14177596 (registering DOI)
Submission received: 18 July 2024 / Revised: 8 August 2024 / Accepted: 21 August 2024 / Published: 28 August 2024
(This article belongs to the Special Issue Current Progress in Fermentation Technology and Microbial Biotechnology: A Comprehensive Overview of Recent Developments and Innovations)
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Abstract

:
Coffee is one of the most popular beverages in the world, and at present, specialty coffees are developing for better tastes, flavors, or functions. Fermented coffees also reflect this trend and some brands are commercialized. Unlike general fermented coffees, we tried to ferment coffee extract with several lactic acid bacteria. Finally, we selected three strains that persisted in the coffee extract, and show strong antimicrobial activity, for the fermentation starters. The strains were identified as Pediococcus pentosaceus (KNUT 0384), Lacticaseibacillus paracasei (CJNU 1840), and Lactiplantibacillus plantarum (CJNU 0441) based on 16S rRNA gene sequences. During the fermentation process, the total acidities (p < 0.05 vs. the control, non-fermented coffee extract) and cell masses increased, which indicates that the bacteria metabolized properly in the extract. Furthermore, the fermented coffee extracts showed increased antimicrobial activities against Listeria monocytogenes (p < 0.05 vs. the control) and Streptococcus mutans (p < 0.05 vs. the control), increased anti-oxidative activities (p < 0.05 vs. the control, except for the KNUT 0384 sample), decreased caffeine content (p < 0.05 vs. the control, except for the KNUT 0384 sample), and increased chlorogenic acid content (p < 0.05 vs. the control). Taken together, the fermented coffee extracts with the selected lactic acid bacteria could be specialty coffees where several functions are improved when compared with a control coffee extract.

1. Introduction

The functional beverage market has recently been dominated by the emerging trend of producing natural drinks using plant-derived substances such as roots, flowers, fruits, leaves, and seeds. The concept of harnessing nature’s influence to create beverages that provide health benefits to the human body is encompassed by terms such as “functional beverages” or “bionic drinks”, and coffee is among them [1]. Coffee has gained significant global popularity and is extensively consumed due to its delightful taste, aromatic qualities, and the stimulatory effects it offers [2]. The consumption of coffee, believed to have originated in northeast Africa, gradually disseminated to the Middle East during the 15th century, eventually reaching Europe. Subsequent to petroleum, coffee has emerged as the second most valuable commodity on a global scale. At present, coffee ranks among the most widely consumed pharmacologically active beverages, firmly establishing its role as a habitual element of daily life worldwide [3]. According to statistical reports published in 2023 by Statista, in the fiscal year 2020/2021, global coffee consumption amounted to approximately 166.63 million 60 kg bags, exhibiting a modest rise from the previous year’s figure of 164 million bags [4]. Another study conducted in South Korea also revealed that daily coffee consumption increased by 48% among Korean adults from 2001 to 2011 [5]. Developments in out-of-home consumption habits, opportunities provided by online commerce platforms, and the development of creative brewed coffee variants are some of the key reasons propelling the coffee market’s expansion. In addition to their social, environmental, and economic sustainability, consumers are also interested in the quality and provenance of coffee goods [6]. Following these trends, roasteries have brought out special coffee products with special effects on health. In general, high-quality coffee cannot be produced using all types of beans; however, specialty coffee production is distinguished by its approach to controlled conditions of production, from plantation to the selection of raw materials and the cultivation of the products through to different processes like fermentation [7].
The constantly increasing demand for specialty coffee has forced researchers to investigate the impact of coffee on human health along with the quality and taste of the specialty coffee, and many researches have proven the beneficial effects of coffee usage, such as it preventing several chronic diseases like type 2 diabetes mellitus, Parkinson’s disease, Alzheimer’s disease, and liver disease (cirrhosis and hepatocellular carcinoma) [8,9,10]. Antimicrobial, antioxidant, and anti-inflammatory responses are among the other beneficial attributes of coffee; these characteristics of coffee are the result of the availability of the bioactive compounds in coffee that make it a functional beverage [2,11,12]. There are many bioactive extracts present in coffee brews, and their composition differs depending on the brewing techniques utilized [13]. Over the past decade, almost 1000 coffee phytochemicals have been described with different functional and bioactive properties [14].
Some of the key bioactive compounds found in coffee are phenolic compounds, including chlorogenic acids, polyphenols and derivatives, methylxanthines (such as caffeine, theophylline, and theobromine), diterpenes (like cafestol and kahweol), nicotinic acid (vitamin B3) and its precursor trigonelline, as well as magnesium and potassium [13,15]. The concentration of these extracts depends upon the species of coffee plant and the method of extraction used (e.g., Coffea arabica, Arabica, Coffea canephora, and Robusta); nevertheless, the bioactive properties, including antibacterial and antioxidative potential, of these coffee compounds are impacted by various factors such as coffee bean processing like fermentation, brewing techniques, and specific roasting conditions [16]. Chlorogenic acid, which is a recognized antioxidant, has been shown to possess anti-inflammatory, antibacterial, and antiviral properties [17], exhibit anticarcinogenic activity, and provide protective effects on neuronal cells is an important polyphenol present in the human diet, and green coffee beans contain the largest amount of chlorogenic acid found in plants, ranging from 6% to 12% [18]. To harness the vast medicinal, unique qualities, and economic benefits of different types of coffee, blended varieties are commercially available as specialty coffee. For instance, Arabica (Coffea arabica L.) and Robusta (Coffea canephora) are two economically significant coffee species. Arabica is renowned for its aromatic and sweet characteristics, as well as its notable variations in acidity and flavor. In contrast, Robusta is known for its bitterness due to its higher caffeine content, and it has lower sweetness and acidity compared to Arabica [19]. Blends that combine both Arabica and Robusta have traditionally aimed to achieve a harmonious balance between aroma and flavor, integrating the desirable qualities of Arabica with the distinctive bitterness of Robusta [20]. Different concentrations of these two types of coffee can be mixed together to obtain different taste aromas and biological effects; however, 50% Arabica and 50% Robusta was favored in terms of brewing taste in a previous study [21].
One of the most important stages in the post-harvest processing of coffee is fermentation. It has been shown to increase the phenolic and flavonoid content as well as the antioxidant activity of fermented coffee [22]. Thus, fermentation improves the functionality of food [23]. Various studies have been performed on the fermentation of coffee beans, the use of different starter cultures in fermentation, and their impact on coffee quality and functionality [24,25]. Sánchez-Riaño et al. [26] studied the effects of the coffee cherry sanitization method and fermentation times on coffee quality. In their study, the sanitization method influenced fermentation time because microbial loads were different according to the methods. Sanitization with a chemical disinfectant and prolonged fermentation times were favorable for coffee safety and quality. Zhao et al. [27] studied refermentation with yeast and lactic acid bacteria to improve the flavor of coffee beans. Co-inoculation of the two microorganisms after spontaneous coffee bean fermentation with dried coffee pulp as the substrate increased chlorogenic acid content significantly, and certain volatile compounds were found with higher concentrations in the inoculated samples. Fermentation depends on various factors including the type of starter culture [28]. The role of lactic acid bacteria (LAB) in fermentation in coffee is becoming more important as the improved quality of fermented products along with a reduction in fermentation time has been shown [29]. Lactic acid produced in coffee during fermentation can deliver a smoother, more rounded flavor profile, often featuring milky or yogurt-like notes that may add a creamy quality to the coffee [30].
In this study, however, we directly applied LAB to a coffee extract (50% Arabica + 50% Robusta) rather than coffee beans and finally selected three strains of LAB, including Pediococcus pentosaceus KNUT 0384, Lacticaseibacillus paracasei CJNU 1840, and Lactiplantibacillus plantarum CJNU 0441, which are resistant to the extract, for further functional evaluation.

2. Materials and Methods

2.1. Selection of Coffee Extract-Resistant LAB Strains

To isolate coffee extract-resistant LAB, approximately 450 strains were tested. The coffee extract (50% Arabica and 50% Robusta) was supplied by Nextbio Co. (Hoengseong, Korea). The extract was adjusted to be 5, 10, and 20 brix on agar plates, and the LAB strains were spotted on the plates. The strains that showed growth on the plates at all concentrations were finally selected and identified via 16S rRNA gene sequencing. They were also inoculated in 20 brix coffee extract broth and the viable cell counts were checked.

2.2. Acidity Change of the Fermented Coffee Extract with Selected LAB Strains Applied

One milliliter of an overnight grown culture of the LAB strains KNUT 0384, CJNU 1840, or CJNU 0441 was centrifuged (8000 rpm, 10 min, 4 °C), and the cell pellet was washed twice with distilled and demineralized water (ddH2O).
The washed cell pellets were suspended in ddH2O and inoculated in 20 brix coffee extract broth. The culture samples (1 mL) were taken at 0 and 12 h, diluted with 4 mL ddH2O, and titrated against 0.1 N NaOH to reach a pH of 8.3. The lactic acid content was then calculated using the percentage conversion method based on the assumption that lactic acid was the major acid component and by considering dilution and titration with NaOH.
The percentage acidity was measured using the following formula.
Acidity (%) = mL of 0.1 N NaOH × F × 0.009/Weight of sample (g) × 100
where F is the factor of 0.1 N NaOH solution and 0.009 is the lactic acid (g) equivalent to 1 mL of 0.1 N NaOH.

2.3. Change in the Cell Mass of the Fermented Coffee Extract with Selected LAB Strains

KNUT 0384, CJNU 1840, and CJNU 0441 probiotic strains were grown in 2.5 mL of MRS broth at 37 °C for 12 h and transferred to individual conical tubes. The conical tubes holding the cultures were then centrifuged (8000 rpm, 10 min, 4 °C), and the cell pellet was resuspended in 250 µL ddH2O. The bacterial suspensions were then inoculated into 25 mL of 20 brix coffee extract at a 1% concentration, incubated at 37 °C, and 10 mL of cultures was sampled at 0 and 12 h. Each sample was then centrifuged (5000 rpm, 15 min, 4 °C), and the cell pellet was diluted with 1 mL of ddH2O and transferred to an individual conical tube. The conical tube was dried at 80 °C in a dry oven (VS-1202D3-S, Vision Co., Bucheon, Korea), and cell mass was measured by subtracting the weight of the empty conical tube from the measured weight of the dried bacterial cell-containing tube.

2.4. Sample Preparation for Bioactivity Assays

The sample was taken from the fermented 20 brix coffee extract with our selected probiotic strains after 12 h incubation and centrifuged (8000 rpm, 10 min, 4 °C). The supernatants were filtered with a 0.22 µm syringe filter (Agela Technologies, Tianjin, China), and the filtrates were used as samples to check antimicrobial activity and antioxidation activity and for the determination of caffeine and chlorogenic acid content.

2.5. Measurement of the Antimicrobial Activity of the Fermented Coffee Extract

The antimicrobial activity of the coffee extract fermented with different probiotic strains was checked against Listeria monocytogenes KCTC 3569 and Streptococcus mutans KCTC 5244.
One milliliter of the overnight-grown culture of L. monocytogenes KCTC 3569 in MRS broth was taken and centrifuged (8000 rpm, 10 min, 4 °C), and the cell pellet was washed and resuspended with 1 mL of 0.1% peptone water. A 500 µL sample of the previously prepared filtered supernatants from the fermented coffee extract was added into 5 mL of 0.1% peptone water where 1% L. monocytogenes KCTC 3569 suspension had been inoculated. The mixture was incubated at 37 °C, and samples were taken at 0, 6, and 12 h of incubation for measurement of viable cell count on MRS agar plates. S. mutans KCTC 5244 in BHI broth was also prepared as above, and viable cell counts were measured on BHI agar plates.

2.6. Measurement of the Antioxidant Activity of the Fermented Coffee Extract

To evaluate antioxidant activity utilizing the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) (Roche, Mannheim, Germany) radical scavenging test, a modified version of the ABTS cation decolorization assay [31] was used. The procedure involves preparing an ABTS stock solution by mixing 7 mM ABTS dissolved in 0.45 µm filtered ddH2O with 2.45 mM potassium persulfate (Daejung chemicals, Siheung, Korea) dissolved in 0.45 µm filtered ddH2O in a 1:1 ratio and then incubating the solution at room temperature for 24 h in a dark location to allow the reaction to complete. The stock solution was then diluted with 0.45 µm filtered ddH2O to reach an absorbance of 0.70 ± 0.02 at 734 nm, yielding the ABTS working solution.
For the assay, 4 µL of the previously prepared filtered supernatant of 20 brix coffee extract sample was mixed with 196 µL of the ABTS working solution and incubated for 10 min in a dark location. The absorbance was subsequently determined at 734 nm. A standard curve was created by dissolving ascorbate in ddH2O and diluting it to various concentrations, and the absorbance values of the samples were calculated by comparing them with the standard curve.

2.7. Measurement of the Caffeine Content of the Fermented Coffee Extract

For the measurement of caffeine content, a chloroform extraction method was used by adding 25 mL of chloroform along with 5 mL of the sample of the fermented coffee extract to a separatory funnel. The mixture was then gently shaken and left to stand with the cap open for 10 min to separate the lower layer. This separation process was repeated 4 times, where 100 mL of chloroform was used for every 5 mL of the sample to separate only the lower layer. The separated lower layer was then subjected to vacuum concentration at 40 °C to remove the solvent completely. Subsequently, the sample, dissolved in 10 mL of methanol filtered through a 0.45 µm syringe filter (Agela Technologies), was analyzed using HPLC. The peak area values obtained from the analysis were used to calculate the caffeine concentration in the sample by comparing them to the standard curve for caffeine at different concentrations. The HPLC analysis conditions were as follows; temperature: RT; detection: UV detector (278 nm); flow rate: 1 mL/min; sample injection volume: 20 µL; column: C18 (4.6 × 250 mm, 5 µm particle size; Waters, Milford, MA, USA); mobile phase: isocratic, ddH2O:MeOH (50:50).

2.8. Measurement of the Chlorogenic Acid Content of the Fermented Coffee Extract

The already prepared filtered supernatant of 20 brix coffee extract fermented with different probiotic strains was used as a sample. HPLC was used to evaluate the samples after they had been diluted to adequate concentrations with methanol. To calculate the chlorogenic acid concentration in the samples, the peak area values from the analysis were fitted to the standard curve for chlorogenic acid at various concentrations. The HPLC analysis conditions were as follows; temperature: 30 °C; detection: 324 nm; flow rate: 1 mL/min; sample injection volume: 20 µL; column: C18 (4.6 × 250 mm, 5 µm particle size; Waters; mobile phase: gradient composition as shown in Table 1, solvent A (0.002 M H3PO4) and solvent B (acetonitrile).

2.9. Statistic Analysis

The experiments in this study were performed in triplicate and expressed as the mean ± standard deviation (SD), and statistical analyses were performed by using SPSS ver. 25 (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA). Significance was verified via one-way ANOVA, and Duncan’s multiple range test (p < 0.05) was used for post hoc analysis.

3. Results

3.1. Selection of Coffee Extract-Resistant LAB Strains

From the screening of LAB, finally, three strains of KNUT 0384, CJNU 1840, and CJNU 0441 showed that they could form colonies on 5, 10, and 20 brix coffee extract containing agar plates, and the viable cell counts persisted in 20 brix coffee extract broth. Based on 16S rRNA gene sequencing, the KNUT 0384, CJNU 1840, and CJNU 0441 strains were identified as Pediococcus pentosaceus, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum, respectively.

3.2. Acidity Change of the Fermented Coffee Extract

The acidity changes in the 20 brix coffee extract fermented with three LAB strains, P. pentosaceus KNUT 0384, L. paracasei CJNU 1840, and L. plantarum CJNU 0441 were measured. The acidity of 12 h incubation was 1.20, 1.21, and 1.21% for KNUT 0384, CJNU 1940, and CJNU 0441 strain, respectively, whereas that of the control (without fermentation) was 0.9% (Figure 1). The acidity and pH of a substance are inversely proportional to each other, and this pH is affected by a variety of reasons in the fermentation process. Fermentation produces organic acids and absorbs basic amino acids, lowering the pH significantly and increasing the acidity of the fermented product [32]. As the LAB strains used in this experiment produced lactic acid during fermentation, the acidities of the samples increased.

3.3. Increased Cell Mass of the Fermented Coffee Extract

The cell mass of lactic acid bacteria involved in the fermentation of 20 brix coffee extract was measured at the start of fermentation (0 h) and after 12 h of incubation. The cell masses in all of the samples increased after 12 h incubation; in particular, the cell mass of L. paracasei CJNU 1840 showed the highest increase of 2.53 g/L (Table 2). The increase in cell mass indicates that our strains involved in fermentation grew well by utilizing the coffee extract as an energy source.

3.4. Increased Antimicrobial Activity of the Fermented Coffee Extract

The antimicrobial activity of the fermented coffee extract against L. monocytogenes KCTC 3569 and S. mutans KCTC 5244 was investigated. For L. monocytogenes KCTC 3569, all of the fermented coffee extracts showed increased antimicrobial activity, and in particular, the fermented coffee extract with L. plantarum CJNU 0441 showed the strongest activity (Figure 2A). For S. mutans KCTC 5244, all of the fermented coffee extracts also showed increased antimicrobial activity but there was no significant difference among the fermented coffee extracts (Figure 2B).

3.5. Increased Anti-Oxidative Activity of the Fermented Coffee Extract

From the ABTS assay, the anti-oxidative activity of the fermented coffee extract was measured. All of the fermented coffee extract samples showed increased anti-oxidative activity but that of the fermented coffee extract with P. pentosaceus KNUT 0384 was not statistically significant. The fermented coffee extract with L. paracasei CJNU 1840 showed the highest anti-oxidative activity of 4002.06 (mg eq. AA/100 mg), whereas the control (without fermentation) showed 2695.47, indicating that the highest one increased by 1.48 times compared with the control (Figure 3).

3.6. Decreased Caffeine Content of the Fermented Coffee Extract

The caffeine content of the fermented coffee extract decreased when compared with the control. The caffeine content of the control showed 6763.14 ppm, whereas that of the fermented coffee extracts with P. pentosaceus KNUT 0384, L. paracasei CJNU 1840, and L. plantarum CJNU 0441 showed 6226.52, 5925.72, and 6132.26 ppm, respectively. The decrease in the caffeine content of the fermented coffee extract with L. paracasei CJNU 1840 was the highest, followed by the extracts with L. planatrum CJNU 0441 and P. pentosaceus KNUT 0384, as shown in Figure 4.

3.7. Increased Chlorogenic Acid Content in the Fermented Coffee Extract

The chlorogenic acid content increased in the fermented coffee extracts when compared with the control. The content in the control showed 1871.13 ppm, whereas that of the fermented coffee extracts with P. pentosaceus KNUT 0384, L. paracasei CJNU 1840, and L. plantarum CJNU 0441 showed 2439.04, 2805.09, and 2698.69 ppm, respectively. The increase in the chlorogenic acid content of the fermented coffee extract with L. paracasei CJNU 1840 was the highest, followed by the extracts with L. planatrum CJNU 0441 and P. pentosaceus KNUT 0384, as shown in Figure 5.

4. Discussion

Fermentation improves the functionality of foods, and the fermentation process involves several parameters, like the type of microorganisms involved and their cell concentration enzymatic activities, and conditions like temperature, pH, and fermented media. In general, microbial fermentation is divided into four categories, viz., (i) the production of viable cellular material or the generation of biomass; (ii) the generation of metabolites; (iii) the synthesis of enzymes, proteins, and vitamins; and (iv) the production of value-added products from a substrate [25]. In our experiment, we observed the increased acidity and increased biomass of the fermented samples as compared to the non-fermented sample (control). As we used lactic acid bacteria strains CJNU 0441, CJNU 1844, and KNUT 0384, these strains were involved in the production of lactic acid, thus increasing the acidity of the fermented BV 20 brix coffee extract. Lactic acid bacteria are those that produce lactic acid as a major organic acid. Besides that, coffee naturally contains caffeic acids, quinic acids, and chlorogenic acids. The increased acidity could be due to the production of lactic acid by the starter culture and the breakdown of coffee metabolites into acid precursors during the fermentation process. Increased acidity in coffee can significantly alter its sensory properties, influencing both its flavor profile and the overall drinking experience. The increased acidity or low pH can enhance citrus and acidic flavors, which are typically desirable in coffee. However, it is essential to carefully monitor this parameter; excessively low pH levels can result in overly acidic or even disagreeable flavors. Thus, pH, acidity, and type of acid produced serve as a critical control point in the production of specialty coffees [33]. We also observed an increase in biomass in the fermented coffee extract. During fermentation, cells utilize a portion of the substrate and vital nutrients from the fermentation medium. Initially, the cells undergo multiplication and growth. The growth pattern can vary depending on the cell type, whether they are unicellular or molds. For instance, unicellular organisms, which divide when they grow, will increase the number of cells or increase the biomass. During fermentation, the cell mass of bacteria increases due to the production of new cells. In the process of fermentation, bacteria need energy for their metabolism and the production of organic acids such as lactic acids. The production of these compounds requires energy and building blocks that are obtained from carbohydrates. Therefore, bacteria need to produce new cells to maintain their population size and continue the fermentation process [34].
We observed the enhanced oxidative potential and antimicrobial activity of the fermented coffee extract against the Gram-positive bacteria Listeria monocytogenes KCTC 3569 and Streptococcuss mutans KCTC 5244. The mechanism of the antibacterial activity of coffee extract is not well understood. However, coffee contains a wide range of chemicals that may be responsible for its antibacterial effect, such as complex combinations of phenolic compounds [9]. Depending on their composition and concentration, these phenolic chemicals may activate or inhibit microbial development [35]. It has also been observed that fermentation increases the content and bioactivity of phenolic compounds responsible for the anti-oxidative and antibacterial activities of coffee extract [36]. In our experiment, we also observed an increase in the content of phenolic acids, specifically chlorogenic acid. Our results match with the results of a study conducted by Silveira et al. [37] in which the CGA content was increased by 400% via solid-state fermentation of the coffee pulp with different yeast strains. There is a lack of data on the mechanism of increased CGA content during fermentation. However, one little clue on why CGA is increased in fermented coffee is that CGA is formed by the ester linkage of quinic acid and caffeic acid via possible esterification of the esterase enzyme in our selected strains [38]. It has been reported in previous studies that fermentation increases the total polyphenol content of fermented coffee beans because phenolic compounds are naturally coupled with sugar, which decreases their availability to the organism. During fermentation, the starter organism’s proteolytic enzyme hydrolyzes phenolic complexes into simple, soluble-free phenols and biologically more active forms that are easily absorbed [21]. Chlorogenic acid (CGA) is a polyphenol that coffee contains, and it has proven to be an antimicrobial and antioxidative agent [39]. Therefore, the increased CGA content in fermented coffee extract might influence the consumer’s health due to its antimicrobial, anti-inflammatory, and antioxidative potential. From several in vitro, animal, and human studies, it is somehow evident that CGA consumption can improve consumers’ health due to its anti-inflammatory and anti-oxidative activity against various degenerative and non-degenerative diseases [38]. The phenolic compounds’ antibacterial mechanism of action is complicated. They can exert antibacterial activity by altering the cytoplasmatic membrane structure, affecting the proton motive force, electron movement, and active transport. Furthermore, the mechanism of phenolic toxicity may include inhibition by oxidized chemicals, either by reactivity with sulfhydryl groups or more non-specific interactions with proteins [40]. A recent study revealed that CGA may exhibit its antibacterial effects through multiple mechanisms, including the downregulation of ribosomal subunits, modulation of lipid metabolism, and scavenging of intracellular ROS [41]. The increased antioxidation activity is also linked with the increased production of chlorogenic acid; however, during the fermentation process, lactic acid bacteria also secrete secondary metabolites, which are also oxidative agents, such as vitamin C and Vitamin B.
The caffeine content of the fermented coffee extract decreased with the increase in fermentation time in our experiment. It was noted that the caffeine content of the fermented coffee extract showed an inverse relation with the fermentation time. After 12 h of fermentation, the caffeine content decreased while at the start of fermentation, its concentration was high. The decreased content of caffeine in the fermented coffee extract is related to the growth of bacteria involved in fermentation because they can use caffeine for their metabolism [42]. Previous studies suggest that LAB degrade caffeine into dimethylxanthine and then methylxanthine with the help of the caffeine demethylase enzyme by removing the methyl group at N-x terminals. There are three types of dimethylxanthine, i.e., paraxanthine, theobromine, and theophylline. Food microorganisms mostly transform caffeine into paraxanthine via demethylation of a caffeine methyl group at N-3 and the paraxanthine is further demethylated at N-7 and produces 1-methyxanthine [43]. The LAB-degrading potential of caffeine into dimethylxanthine could be due to the toxicity of caffeine. One study suggests that paraxanthine toxicity is lower in animal caffeine models [44]. It could be assumed that low caffeine content is due to the degradation of caffeine into low toxic compounds as caffeine possesses some antimicrobial effects and these antimicrobial effects are reduced through the demethylation of caffeine by bacteria metabolism.

5. Conclusions

In the following study, we examined fermented coffee extracts using three lactic acid bacteria strains, P. pentosaceus KNUT 0384, L. paracasei CJNU 1840, and L. plantarum CJNU 0441. We discovered improved characteristics in the fermented coffee extract. First, there was an increase in antibacterial activity against Listeria monocytogenes and Streptococcus mutans, which are known pathogens. Furthermore, the fermented coffee extract’s antioxidative activity increased, implying the production of phytochemicals with free radical-scavenging characteristics. In addition, (CGA) chlorogenic acid, a bioactive molecule linked to a variety of health advantages, such as antimicrobial, anti-inflammatory, and anti-oxidative effects, was found to be high in the fermented coffee extract. Thus, coffee with high CGA content may be used to make ‘specialty coffee’ to reduce inflammation and alleviate the damage caused by free radicals in consumers’ bodies. Finally, the caffeine concentration was found to be decreased in the extract, which may be of interest to some people who prefer to consume less caffeine or people sensitive to high caffeine concentrations with certain medical conditions such as IBS (irritable bowel syndrome). Overall, these results highlight the positive impact of the fermentation process with lactic acid bacteria on the chemical composition and functional properties of coffee extract, providing valuable insights for potential applications in the food industry, specifically in the specialty coffee industry to produce a specialty coffee with a strong sensory profile along with medicinal features.
As fermentation affects all phenolic compounds in coffee, the profile and bioactivity of compounds other than CGA, like caffeic acid, p-coumaric acid, ferulic acid, catechins, and flavonoids, could be analyzed. However, future research could focus on the mechanism of action of fermented coffee to better understand its functionality in consumers’ health as well as sensory evaluation.

Author Contributions

Conceptualization, G.-S.M.; methodology, S.-G.K. and G.-S.M.; software, S.-G.K. and A.A.; validation, S.-G.K. and G.-S.M.; formal analysis, S.-G.K. and A.A.; investigation, S.-G.K.; resources, G.-S.M.; data curation, S.-G.K., A.A. and G.-S.M.; writing original draft preparation, S.-G.K., A.A. and G.-S.M.; writing review and editing, S.-G.K., A.A. and G.-S.M.; visualization, S.-G.K. and A.A.; supervision, G.-S.M.; project administration, G.-S.M.; funding acquisition, G.-S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2021R1A6A1A03046418).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support the conclusion are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 07596 g001
Figure 1. Increased total acidities of the fermented coffee extracts with lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. (Hoengseong, Korea). Control, coffee extract (50% Arabica + 50% Robusta); KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 10% inocula. *, p < 0.05 vs. the control.
Figure 1. Increased total acidities of the fermented coffee extracts with lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. (Hoengseong, Korea). Control, coffee extract (50% Arabica + 50% Robusta); KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 10% inocula. *, p < 0.05 vs. the control.
Applsci 14 07596 g001
Applsci 14 07596 g002
Figure 2. Increased antimicrobial activities of the fermented coffee extracts with lactic acid bacteria. (A) the activity against Listeria monocytogenes KCTC 3569; (B) the activity against Streptococcus mutans KCTC 5244. The coffee extract was supplied by NEXTBIO Co. Listeria, Listeria monocytogenes KCTC 3569 only; Mutans, Streptococcus mutans KCTC 5244 only; BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. Different letters indicate a significant difference (p < 0.05).
Figure 2. Increased antimicrobial activities of the fermented coffee extracts with lactic acid bacteria. (A) the activity against Listeria monocytogenes KCTC 3569; (B) the activity against Streptococcus mutans KCTC 5244. The coffee extract was supplied by NEXTBIO Co. Listeria, Listeria monocytogenes KCTC 3569 only; Mutans, Streptococcus mutans KCTC 5244 only; BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. Different letters indicate a significant difference (p < 0.05).
Applsci 14 07596 g002
Applsci 14 07596 g003
Figure 3. Increased anti-oxidative activities of fermented coffee extracts with lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Figure 3. Increased anti-oxidative activities of fermented coffee extracts with lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Applsci 14 07596 g003
Applsci 14 07596 g004
Figure 4. Decreased caffeine content of fermented coffee extracts induced by lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Figure 4. Decreased caffeine content of fermented coffee extracts induced by lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Applsci 14 07596 g004
Applsci 14 07596 g005
Figure 5. Increased chlorogenic acid content of fermented coffee extracts induced by lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Figure 5. Increased chlorogenic acid content of fermented coffee extracts induced by lactic acid bacteria. The coffee extract was supplied by NEXTBIO Co. BV, coffee extract (50% Arabica + 50% Robusta) as a control; KNUT 0384, fermented coffee extract with Pediococcus pentosaceus; CJNU 1840, fermented coffee extract with Lacticaseibacillus paracasei; CJNU 0441, fermented coffee extract with Lactiplantibacillus plantarum. The lactic acid bacteria were inoculated in the coffee extract (20 brix) with 1% inocula to make the fermented coffee extract. *, p < 0.05 vs. the control.
Applsci 14 07596 g005
Table 1. The composition of the gradient mobile phase.
Table 1. The composition of the gradient mobile phase.
Time (min)083035405060
Solvent A100%100%0%0%100%100%100%
Solvent B0%0%100%100%0%0%0%
Table 2. Changes of cell mass during the fermentation of the coffee extract with lactic acid bacteria.
Table 2. Changes of cell mass during the fermentation of the coffee extract with lactic acid bacteria.
FermentationCell Mass (g/L)
Pediococcus pentosaceus KNUT 03840 h7.88
12 h8.78
Lacticaseibacillus paracasei CJNU 18400 h8.33
12 h10.86
Lactiplantibacillus plantarum CJNU 04410 h8.23
12 h10.34
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Kim, S.-G.; Abbas, A.; Moon, G.-S. Improved Functions of Fermented Coffee by Lactic Acid Bacteria. Appl. Sci. 2024, 14, 7596. https://doi.org/10.3390/app14177596

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Kim S-G, Abbas A, Moon G-S. Improved Functions of Fermented Coffee by Lactic Acid Bacteria. Applied Sciences. 2024; 14(17):7596. https://doi.org/10.3390/app14177596

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Kim, Seon-Gyu, Aoun Abbas, and Gi-Seong Moon. 2024. "Improved Functions of Fermented Coffee by Lactic Acid Bacteria" Applied Sciences 14, no. 17: 7596. https://doi.org/10.3390/app14177596

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