Physical and Chemical Characteristics of Robusta Coffee Beans Fermented by Kefir Using the Controlled Semi-Carbonic Maceration Method

2.1 Calculation of lactic acid bacteria population

The lactic acid bacteria population was calculated using Bacteriological Analytical Methods (BAM). The coffee sample (5 g) was diluted in 45 mL of a physiological solution (ONECARE REAGEN NACL 0.86%, PT Anugerah Santosa Abadi, catalogue number: 1000023). Dilution was performed up to 10⁻⁷. Dilution results were taken from 1 mL of the last three dilution levels and poured into a petri dish, then MRSA (Himedia MRS Agar Granulated, catalogue number: GM641-500G) with 1% CaCO₃ (Nitrakimia, Calsium carbonate for Analysis, Catalogue number: 1.02066.1000) was added as much as 12-15 mL. The petri dish was shaken on a flat surface, forming Figure 8 slowly to mix the sample liquid and agar and let it stand until it solidifies. After solidifying, cells were incubated at 37°C for 48 h.

2.2 Physical characterization

Color analysis was performed using a color reader with the notations Hunter L* (white), a* (red), and b* (yellow).⁵ Density analysis of coffee beans can be performed by inserting coffee beans into a 100 mL measuring cup to the measuring limit and weighing. The analysis was carried out three times for each sample.

2.3 Chemical characterization

2.3.1 Caffeine

Caffeine analysis was based on the Belay method with modifications.⁶ The stages of caffeine analysis were making a standard solution, making a standard curve by taking (0, 50, 150, 300, 450, 600) μL of the standard caffeine solution and putting it into a 25 mL measuring flask, determining the caffeine content by extracting 25 mg of coffee sample in 10 mL of distilled water, and heating at a temperature of 90°C for 10 min. After cooling, the samples were filtered using filter paper. The filtrate obtained was placed in a separating funnel, and 25 mL of dichloromethane (Merck Pro Analysis, Catalogue number: 106050) was added (extracted three times, namely 10 mL, 10 mL, and 5 mL). The bottom layer was calibrated to a volume of 25 ml. The absorbance of the solution was measured at a wavelength of 285 nm using a spectrophotometer.

2.3.2 Reducing sugar

Reducing sugars levels were tested using the Nelson-Somogyi method.⁷ The following are several stages of testing the reducing sugars: 1. Nelson Solution: Nelson reagent was prepared by mixing 25 mL Nelson Reagent A and 1 mL Nelson Reagent B (PT Dwi Lab Mandiri Nessler’s reagent A MSDS, catalogue number: 109012). Mixing was performed every time it was used; 2. Arsenomolybdate solution (acpchem.com A8302-500 ml, Catalogue number: A8302), 3. Preparation of the standard curve; 4. Sample preparation was performed by extracting 2 g of the sample using 80% ethanol. The filtrate was homogenized, and ethanol (Merck Millipore, ethanol MSDS, Catalogue number: 100983) was evaporated until it ran out. Addition of 100 mL of aquadest and 1 g of CaCO₃ (Merck Millipore, CaCO3 99.95%, Catalogue number: 102059), then boiled for 30 minutes. Pb acetate (Merck, Pb acetate 100% CAS 64-19-7, Catalogue number: 100063), and Na oxalate (PT Delta PrimaLab Saintifik, Sodium Oxalate AR 500 g, Catalogue number: 645535-0500) were added until the color became clear. If the color of the solution was clear, filtering was performed, and 100 mL was calibrated using an aquadest 5. The reducing sugars were analyzed by placing 2 mL of the prepared solution into a test tube. Addition of 1 mL Nelson solution and homogenization. The samples were then heated for 20 min and cooled. Arsenomolybdate (1 mL) and distilled water (6 mL) were added and then shaken until homogeneity. Absorbance was read at 540 nm.

2.3.3 pH

The pH test was carried out using a pH meter that had been calibrated with pH buffers (4, 7, and 9). A 5-gram coffee sample was diluted with 50 mL of distilled water (Merck Millipore, CAS 7732-18-5, Catalogue number: 115333) and stirred until homogeneous. The electrode was rinsed with distilled water and dried using a tissue. The pH meter was dipped into the solution until the pH value was stable, and the pH value was read on the screen.

2.3.4 Total titratable acidity

Total titratable acid analysis was carried out by weighing 1 g of the ground sample (60 mesh), placing it in a beaker, glass, and diluting it with 50 mL of distilled water. Filtration using filter paper. The filtered sample filtrate was placed into a 100 mL measuring flask and diluted using distilled water. The diluted sample was taken as much as 5 mL, and 2 drops of 1% phenolphthalein were added, after which the titration was continued. Titration was performed using a 0.01N NaOH solution until the color changed to pink. The total titratable acid concentration was assumed to be the total lactic acid concentration measured from the sample.

2.4 Data analysis

Physical and chemical analyses were performed using Microsoft Excel 2013 and were analysed descriptively. The results are presented in the form of a bar chart.

3.1 Lactic acid bacteria population

The total lactic acid bacteria in robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method are shown in Figure 1.

Figure 1. Total LAB of Robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

Figure 1 shows that the longer the fermentation, the total lactic acid bacteria increased significantly in the 24th hour of fermentation, namely 7.67 log10 cfu/mL at 35°C and 7.72 log10 cfu/mL at 40°C. This indicates that at 24 h, the growth of LAB entered the log or exponential phase. In the exponential phase, cells grow rapidly and divide at a constant rate until they double.⁸ At 48 h, there was a decrease in the number of LAB, which began to enter the stationary phase. In this phase, the growing bacteria reach a point of decline in growth rate. The stationary phase is the phase when the growth rate is the same as the rate of microbial death, so that the overall number of microbes remains the same.⁹

3.2 Physical characterization

3.2.1 Lightness

The lightness value of Robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 30.54 to 41.68, as shown in Figure 2.

Figure 2. Lightness of Robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

The fermentation process causes the color of coffee to change from yellowish to brownish. Longer fermentation and higher temperatures cause the color of coffee to become browner.

3.2.2 Bulk density

The bulk density value of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 0.39 g/mL to 0.50 g/mL, as seen in Figure 3.

Figure 3. Density of Robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

According to Figure 3, it can be seen that the highest density was found in the control sample, which was 0.50 g/mL, decreasing to 0.39 g/mL in 48-hour fermentation at a temperature of 40°C. The longer the fermentation, the lower the density.

3.3 Chemical characterization

3.3.1 Caffeine content

The caffeine contents of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method are shown in Figure 4.

Figure 4. Caffeine content of robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

Based on Figure 4, it can be seen that the temperature and duration of fermentation caused a decrease in caffeine levels from 1.14% in the control sample to 0.46% in 48-hour fermentation at a temperature of 40°C.

3.3.2 Reducing sugar

Reducing sugars consist of all monosaccharides (glucose, fructose, and galactose) and disaccharides (lactose and maltose), except sucrose and starch (polysaccharides) (Irwan et al., 2023). The reducing sugar content of Robusta coffee fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method is shown in Figure 5.

Figure 5. Reducing sugar content of robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

As shown in Figure 5, the longer the fermentation time, the lower the reducing sugar content. In the control sample, the reducing sugar content (2.42%) decreased by 0.29% in 48-hour fermentation at 40°C. The reducing sugar content is related to the microorganisms and carbon sources added during fermentation.

3.3.3 pH

pH is an indicator of the acidity level of coffee. The acidity of a product is influenced by the degradation of sugars into organic acids during fermentation. The pH values of Robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method are shown in Figure 6.

Figure 6. pH of Robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

The pH value of robusta coffee beans fermented with kefir starter decreased from 6.12-4.43. Fermentation reduces the pH value. The longer the fermentation time, the lower the pH, which means that the acidity of coffee increases.

3.3.4 Total titrated acid

The titratable acid value is the percentage of acid in the material determined by titration and expressed as a percentage of lactic acid. The total titratable acid of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 0.40 to 0.99, which can be seen in Figure 7. The lowest total titratable acid was found in control robusta coffee, or without fermentation, which was 0.40, whereas the highest value was found in robusta coffee fermented by kefir at a temperature of 40°C and a fermentation time of 48 h, which was 0.99.

Figure 7. Total titrated acid of Robusta coffee beans fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method.

The total titrated acid is correlated with pH. The longer the fermentation and the higher the fermentation temperature, the higher the total titrated acid in coffee.

4.1 Lactic acid bacteria population

Fermentation of robusta coffee was performed by adding kefir and lactose as the carbon sources. Kefir contains lactic acid bacteria and yeasts. Lactic acid bacteria (LAB) are types of bacteria that can form lactic acid from glucose or carbohydrate metabolism and grow in a low pH environment.¹⁰ Lactic acid bacteria will break down lactose with the enzyme β- galactosidase (lactase) into glucose and galactose.¹¹

Figure 1 shows that the longer the fermentation, the total lactic acid bacteria increased significantly in the 24th hour of fermentation, namely 7.67 log10 cfu/mL at 35°C and 7.72 log10 cfu/mL at 40°C. This indicated that at 24 h, the growth of LAB entered the log or exponential phase. In the exponential phase, cells grow rapidly and divide at a constant rate until they double.⁸ At 48 h, there was a decrease in the number of LAB, which began to enter the stationary phase. In this phase, the growing bacteria reach a point of decline in growth rate. The stationary phase is the phase when the growth rate is the same as the rate of microbial death, so that the overall number of microbes remains the same.⁹

The higher the fermentation temperature, the greater the total lactic acid bacteria. According to previous study, temperature can affect microorganisms in two ways: when the temperature rises, the metabolic rate increases and growth is accelerated, and vice versa; and when the temperature drops, the metabolic rate also decreases and growth is slowed down.¹² However, if fermentation uses the addition of the inoculum, the fermentation temperature follows the optimum growth temperature of the inoculum. The optimum growth of LAB occurs at 37°C and begins at 10°C. There are three groups of microorganism growth temperatures for microorganisms, namely psychrophilic (7-30°C), mesophilic (30-40°C), and thermophilic (45-60°C). LAB can live at temperatures of 10°C and 45°C.⁸

4.2 Physical characterization

4.2.1 Lightness

The lightness value of Robusta coffee fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 30.54 to 41.68 ( Figure 2). The fermentation process causes the color of coffee to change from yellowish to brownish. Longer fermentation and higher temperatures cause the color of coffee to become browner. According to a previous study, the higher the temperature and fermentation time in the fermentor, the higher the number of brown beans.¹³ The browning process occurs because of the oxidation of polyphenols by the polyphenol oxidase enzyme, which produces brown pigments.¹⁴ Chlorogenic acid is present in the phenolic compound group. The browning process can also be caused by the oxidation of chlorogenic acid during the fermentation process acid.¹⁵

4.2.2 Bulk density

The bulk density of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 0.39 g/mL to 0.50 g/mL, as seen in Figure 3. According to Figure 3, it can be seen that the highest bulk density was found in the control sample, which was 0.50 g/mL, decreasing to 0.39 g/mL in 48-hour fermentation at a temperature of 40°C. The longer the fermentation, the lower the bulk density. This causes water to diffuse more easily into the pores of the coffee beans, resulting in the coffee beans expanding and becoming more porous, such that the coffee beans are larger and the density of the coffee will be lower. In addition, the longer the fermentation period, the higher the microbial activity; thus, it is able to produce metabolites, especially organic acids, during fermentation. These organic acids mix with water, diffuse into the pores of the coffee beans, and increase the breakdown of compounds in coffee, such as caffeine, chlorogenic acid, and other compounds, so that the coffee beans are more porous, and when dried, the density decreases.¹⁵

Bulk density is also influenced by differences in fermentation temperature. The higher the temperature, the lower is the density. The bulk density value in this study with a fermentation temperature of 35°C and a fermentation time of 24 hours-48 hours was 0.43 g/mL and 0.41 g/mL, while at a fermentation temperature of 40°C and a fermentation time of 24 hours-48 hours it was 0.41 g/mL and 0.40 g/mL. This is by the previous research research, the density value at fermentation temperatures of 30°C, 35°C, and 40°C, respectively, namely 0.69 g/mL, 0.65 g/mL, and 0.63 g/mL, indicating that the higher the fermentation temperature, the lower the bulk density.¹³ This is because the greater the increase in fermentation temperature, the greater the loss in coffee weight, which can reduce the bulk density of the coffee produced.¹⁶

4.3 Chemical characterization

4.3.1 Caffeine content

The caffeine contents of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method are shown in Figure 4. Based on Figure 4, it can be seen that the temperature and duration of fermentation caused a decrease in caffeine levels from 1.14% in the control sample to 0.46% in 48-hour fermentation at a temperature of 40°C. Fermentation of robusta coffee beans can reduce caffeine levels due to the activity of microorganisms. The reduction in caffeine levels is carried out by microorganisms by degrading caffeine into simpler compounds. Lactobacillus casei and Leuconostoc mesenteroides degrade caffeine to paraxanthine using the N-demethylation method. Paraxanthine is degraded into methylxanthine (1-methylxanthine by Lactobacillus casei, Leuconostoc mesenteroides, and 7-methylxanthine by Rhizopus oryzae and Saccharomyces cerevisiae).¹⁷

The caffeine content decreased with increasing fermentation time. The longer the fermentation time, the more caffeine compounds contained in coffee beans dissolve in the solvent.¹⁹ This is because caffeine compounds are converted into smaller compounds (paraxanthine and methylxanthine) so that they can easily move and diffuse through the cell walls and dissolve in water.²⁰ In addition, caffeine is degraded into uric acid, 7-methylxanthine, and xanthine during fermentation. According to a previous study, stated that caffeine degradation into uric acid begins after 12 h of fermentation.²¹ In other previous study caffeine degradation into uric acid begins at 12-36 hours of fermentation.²²

In addition, the higher the temperature, the lower is the caffeine content. Caffeine is slightly soluble in cold water or at room temperature but it easily dissolves in hot water. Caffeine easily dissolves as the water temperature increases.¹⁸ Caffeine easily dissolves at high temperatures because higher temperatures widen the distance between molecules in coffee solids, resulting in higher solubility of caffeine at high temperatures. In line with the opinion from a previous study, this decrease occurs during the process of dissolving caffeine compounds from coffee beans, which begins with the breakdown of complex caffeine and chlorogenic acid bonds owing to heat treatment.²³ Caffeine compounds are smaller in size. Caffeine moves easily, diffuses easily through cell walls, and then dissolves in the solvent.

4.3.2 Reducing sugar

Reducing sugars consist of all monosaccharides (glucose, fructose, and galactose) and disaccharides (lactose and maltose), except sucrose and starch (polysaccharides). The reducing sugar content of Robusta coffee fermented with kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method is shown in Figure 5.

As shown in Figure 5, the longer the fermentation time, the lower the reducing sugar content. In the control sample, the reducing sugar content (2.42%) decreased by 0.29% in 48-hour fermentation at 40°C. The reducing sugar content is related to the microorganisms and carbon sources added during fermentation. In this study, coffee fermentation was supplemented with kefir and lactose. Lactic acid bacteria found in kefir convert lactose into glucose and galactose; then, glucose is converted into organic acids, resulting in a decrease in reducing sugars and an increase in acidity in coffee beans. Fauzi et al. (2013) stated that, as the fermentation time increased, the reducing sugar content of coffee beans decreased because of the activity of the added microorganisms.⁸

Temperature differences also affect coffee, reducing sugar content; the higher the temperature used, the lower the resulting reducing sugar content. This was due to microbial activity. Microbial activity is influenced by the fermentation temperature, and the optimum temperature increases microbial activity. Lactic acid bacteria can grow at temperatures of 10°C-45°C, but the optimum temperature for lactic acid bacteria is 37°C.²⁴ At a temperature of 40°C, the activity of lactic acid bacteria decreases, but there are still living lactic acid bacteria that the living bacteria utilize the available glucose as a carbon source to decrease the reducing sugar content.

4.3.3 pH

pH is an indicator of the acidity level of coffee. The acidity of a product is influenced by the degradation of sugars into organic acids during fermentation. The pH values of Robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method are shown in Figure 6.

The pH value of robusta coffee beans fermented with kefir starter decreased from 6.12-4.43. Fermentation reduces the pH value. The longer the fermentation time, the lower the pH, which means that the acidity of coffee increases. The decrease in pH during fermentation is influenced by the microbial activity of the added starter. Kefir contains lactic acid bacteria that can help form organic acids, which results in a decrease in pH. Organic acids are metabolite compounds that are formed as a result of the metabolic activity of acid-forming bacteria, especially lactic acid and acetic acid bacteria. According from a previous study, organic acids formed during coffee fermentation are bacterial catabolic products that cause a decrease in pH, including malic acid, acetic acid, chlorogenic acid, and quinic acid.²⁵ Fermented coffee is still suitable for consumption if it has a pH value above 4.²⁶

Based on Figure 6, it can be seen that the higher the fermentation temperature, the lower the coffee pH. The pH value is also related to the optimal temperature for lactic acid bacteria, which is 37°C. Based on previous research, fermentation at 30°C resulted in a pH value that tended to increase as the fermentation time progressed.²⁷ If the temperature is less than 30°C, the growth of acid-producing microorganisms will be slow, thus affecting the quality of the product, and the pH value in coffee is determined by the acid content in coffee.²⁶

4.3.4 Total titrated acid

The titratable acid value is the percentage of acid in the material determined by titration and expressed as a percentage of lactic acid. The total titratable acid of robusta coffee fermented with a kefir starter at different temperatures and fermentation times using the semi-carbonic maceration method ranges from 0.40 to 0.99, which can be seen in Figure 7. The lowest total titratable acid was found in control robusta coffee, or without fermentation, which was 0.40, whereas the highest value was found in robusta coffee fermented by kefir at a temperature of 40°C and a fermentation time of 48 h, which was 0.99.

The total titrated acid is correlated with pH. The longer the fermentation and the higher the fermentation temperature, the higher the total titrated acid in coffee. The increase in the total acid content during fermentation is closely related to the increasing activity of lactic acid bacteria. During growth, bacteria use carbohydrate sources (sugar) and convert them into organic acids. The accumulation of acid produced during fermentation causes a decrease in pH. The LAB group uses sugar as an energy source, mainly lactic acid and acetic acid. A decrease in pH value is followed by an increase in the total titrated acid value, and vice versa. When the pH value increased, the total titrated acid decreased.

The increase in total acidity during fermentation is closely related to the increase in microbial activity and decrease in acidity during fermentation. The formation of organic acids during fermentation occurs because bacteria use sugar during their growth and convert it into organic acids. The pH decreases owing to the accumulation of acid produced during fermentation.²⁷ Longer fermentation times increase the total titrated acid during fermentation because microbes convert lactose into glucose and galactose, which are then broken down into organic acids and alcohol until the sugar in the food runs out, resulting in an acid that increases with the length of fermentation.²⁸