Flavor enhancement of decaffeinated robusta beans re-fermentation using a mucilage analog of the blend of watermelon albedo (Citrullus vulagris Schard) and sucrose

Robusta beans have a bitter taste, sour taste, and high caffeine content. Robusta coffee contains 2.2 percent caffeine, while Arabica coffee contains 1.2 percent caffeine. The caffeine level in the coffee beans can be lowered through the decaffeinated process. Optimal caffeine extraction from coffee requires heating via the decaffeinated process. However, high temperatures and long decaffeinated times can reduce flavor due to the degradation of flavor-forming compounds, resulting in decaffeinated coffee with a weak flavor. This research aimed to enhance the flavor of decaffeinated robusta beans by re-fermentation using watermelon (Citrullus vulgaris Schard.) albedo and sucrose. The specific purpose of the study was to analyze the taste of re-fermented decaffeinated robusta beans using cupping methods caffeine levels, reduction sugar levels, and protein levels in advanced re-fermentation decaffeinated coffee using a mucilage analog of the blend of watermelon albedo and sucrose. The study consists of two factors, decaffeinated robusta beans (5% and 10%) and the amount of sucrose (5%, 10%, and 12%). Results indicate improved decaffeinated robusta beans using re-fermentation mucilage analog of the blend of watermelon albedo and sucrose with cupping test 81.75 and with the chemical characteristic of caffeine 1,05%, reduction sugar level, and 2.27% protein level. Flavor decaffeinated robusta beans re-fermentation indicated an increase with the final high score using cupping methods with the flavor produced by brown sugar, sweet potato smell and aftertaste, herbs, and advanced re-fermentation of decaffeinated using mucilage analog showed a change in the chemical characteristic of coffee.


Introduction
Robusta coffee is one of the varieties of coffee with a high yield, producing 51.48 thousand tons annually [1].Compared to Arabica coffee, Robusta coffee has a bitter and sour taste, and contains high caffeine.Based on research [2] that Arabica coffee has a better taste than Robusta coffee and contains 1.2% caffeine.In contrast, the caffeine content in Robusta coffee is 2.2%.For some people who consume it frequently, coffee's high caffeine content can have a negative effect on the human body's health.Excess caffeine consumption can cause insomnia, hypertension, seizures, and nausea [3].
The caffeine content in coffee can be reduced through the decaffeinated process.Based on research [4], decaffeinated coffee beans contain 0.1-0.3%caffeine.Coffee can be decaffeinated using either chemical solvent or water.Using water as a solvent or the water process method is cheap, easy, environmentally friendly, and safe [5].This water extraction method requires heating to extract caffeine from coffee optimally.High temperatures and a long time can reduce flavor due to the degradation of flavor-forming compounds in decaf coffee [6].So that the resulting decaf coffee has a low taste.One of the developments that can be done to increase the flavor of decaf coffee is to carry out re-fermentation using mucilage analogs.Re-fermentation, such as the honey process method, can be carried out, which still contains mucilage in coffee beans with high reduced sugar content [7].The mucilage of coffee beans plays a role in the production of honey, enhancing the flavor of decaf coffee.The mucilage in coffee beans is mucus that coats coffee beans with a high content of protopectin as a carbon and nitrogen source in biotechnology processes to produce organic acids [8].In fermentation, this mucilage layer will form a honey-like flavor and taste in the honey process, which retains the mucilage during fermentation.Re-fermentation of coffee beans can use analog mucilage with high sugar content such as glucose, fructose, and sucrose.The manufacture of analog mucilage from watermelon albedo contains high pectin, the same content as natural mucilage in coffee.The pectin content in watermelon albedo can be an alternative to improving the taste and flavor of decaffeinated robusta.

Research procedure 2.2.1. Coffee decaffeination.
Coffee decaffeination was carried out using the Water Process method.Green beans for robusta coffee were weighed as much as 300 g and soaked in 500 ml of distilled water for 5 hours, then boiled using 900 ml of distilled water at 80 o C for 2 hours to widen the pores of the coffee so that the caffeine can be extracted.Furthermore, the green beans are separated from the cooking water and dried until the green bean water content is 25-30%.Then the boiled water that has been obtained is added with 10% activated carbon and stirred for 1 hour at room temperature, to obtain caffeine-free saturated water.

Decaffeination process.
Green coffee beans that have reached a 25-30% moisture content are put into caffeine-free saturated water, then boiled at 80 o C for 1 hour.After that, the green beans were dried to a moisture content of <12%.

Making analog mucilag.
The watermelon was cut, then peeled the skin and flesh of the watermelon to obtain the albedo.Watermelon albedo was mashed and obtained watermelon pulp or analog mucilage.Furthermore, the resulting pulp is weighed, added sucrose (refined sugar) is added according to the treatment, then stirred until smooth.3 2.2.4.Re-fermentation of decaffeinated coffee.Dried decaffeinated Robusta green bean with a moisture content of <12%, then re-fermented using mucilage analog.Fermentation was carried out openly for 24 hours at room temperature is 27 o C.After that, it dried to a moisture content of <12% to produce re-fermentation decaffeinated Robusta coffee beans.

Observation parameters 2.3.1. Cupping test coffee (Standards Committee of
The Specialty Association of America) [9].Coffee samples are ground for 30 minutes before the cupping process, if this is not possible, the coffee must be stored in an airtight container.Then the grind size must be coarser than the standard coffee grinder, which is 70-75% through the US standard 20 mesh size filter.The optimum ratio is 8.25 g of coffee per 150 ml of water, corresponding to the optimum midpoint of the balanced recipe for the Golden Cup.Then the determination of the volume of water in the cupping glass is adjusted to the weight of the coffee at this ratio +/-0.25 g.The water used for the cupping test is clean, odorless and undistilled.Total dissolved solids should not be less than 100 ppm or more than 250 ppm.When poured into ground coffee, the water is fresh, and the temperature is 93 o C. Hot water is poured directly into the cupping cup, and the coffee grounds should not be disturbed for 3-5 minutes before evaluation.[10].Caffeine was weighed as much as 2 mg and dissolved using 70% ethanol as much as 20 ml so that a standard caffeine solution with a concentration of 0.1 mg/ml was obtained, then determine the wavelength was using a standard solution of caffeine 0.1 mg/ml pipette as much as 0.1 ml, then add 0.9 ml of 70% ethanol, so that a solution concentration of 0.01 mg/ml is obtained.After that, the standard caffeine solution was tested in the 200-400 nm wave range.The maximum wavelength obtained for caffeine is 273 nm.Then, a standard-making solution can be made by taking 0.02; 0.04; 0.06; 0.08; 0.1, and 0.12 ml standard solution of 0.1 mg/ml caffeine, then it was dissolved until a standard solution concentration of 0.002 was obtained; 0.004; 0.006; 0.008; 0.01 and 0.012 mg/ml.After that, the standard caffeine solution was measured using a UV-vis spectrophotometer with a wavelength of 273 nm.After that, a caffeine content test was carried out by weighing 1 g of green bean in 100 ml of 70% ethanol.Then the caffeine was extracted for 24 hours in the dark.After that, it was filtered using filter paper, and the caffeine content was measured using a UV-vis spectrophotometer at a wavelength of 273 nm.Then the caffeine content is calculated using the curve equation y = ax + b. [11].DNS reagent was prepared by dissolving 1 g of 3,5-dinitro salicylic acid, and 30 g of Na-K dissolved in 80 ml of distilled water.After that, 20 ml of 2 N sodium hydroxide, 10 ml of sodium metabisulfite, and 2% phenol were added, measured, then homogenized to obtain a DNS reagent solution.Then, a standard curve solution can be prepared by glucose concentrations of 0.15; 0.2; 0.25; 0.3; 0.35, and 0.4 mg/ml.After that, pipette 1 ml of standard glucose solution from each concentration and add 3 ml of DNS reagent.The solution was heated at 100 o C for 15 minutes, and cooled to room temperature for 5 minutes.After that, the standard solution was measured using a UV-VIS spectrophotometer with a wavelength of 510 nm.Then tested for reduced sugar levels by means of pulverized and weighed samples of as much as 1 g, then dissolved using distilled water as much as 70 ml.The solution was homogenized using a vortex for 15 minutes, and filtered.The filtrate obtained was then measured in a 100 ml volumetric flask.Pipette 1 ml of sample solution and 3 ml of DNS reagent into each tube, heat it using a boiling water bath for 5 minutes and cool it to room temperature.Then the solution was transferred into the cuvette, and the absorbance was measured at a wavelength of 510 nm.Glucose levels will be determined using the regression equation y = ax + b. [12].The preparation of the reagent solution is divided into two stages, i.e Lowry A solution and Lowry B solution.Lowry A solution consists of Folin Ciocalteau solution (Phenol Reagent).Lowry B solution was prepared by means of 2% sodium carbonate in 0.1N NaOH solution in 100 ml and 0.5% copper sulfate in 1% Na-K Tartarate solution.After that, 50 ml of 2% sodium carbonate in NaOH solution was pipetted, and 0.5% copper sulfate was added to 1 ml of Na-K Tartarate solution and homogenized.Then prepare a standard protein solution by means of Bovine Serum Albumin, weigh as much as 0.0025 g, and dissolve it using distilled water as much as 10 ml in a volumetric flask.After that it was homogenized until a standard protein solution of 0.25 mg/l was obtained.A standard curve was then made by pipetting Standard Protein Solution as much as 0.1, 0.2, 0.4, 0.6, 0.8, and 1 ml into a test tube.Then it was dissolved to obtain 25, 50, 75, 100, and 125 mg/ml concentrations.After that, add 4 ml of distilled water and 5.5 ml of Lowry B solution to each test tube.Then homogenize and let stand at room temperature for 10-15 minutes.Then add 0.5 ml of Lowry A solution and mix well.Let stand for 30 minutes until a blue color forms, then measure the absorbance using a wavelength of 650 nm.Grind coffee beans, then weigh as much as 1 g.After that, 9 ml of distilled water was added and homogenized using a vortex.The solution was then filtered and centrifuged at 3000 rpm for 10 minutes until the denatured protein precipitated.The supernatant was decanted for use in the test.Then to test for protein levels, pipette the sample as much as 1 ml into a test tube.After that, add 2 ml of distilled water and 2.75 ml of Lowry B solution to each test tube.Then homogenize and let stand at room temperature for 10-15 minutes.Then add 0.25 ml of Lowry A solution and mix well.Let stand for 30 minutes until a blue color forms, then measure the absorbance using a UV-Vis spectrophotometer at a wavelength of 650 nm.

Research design
The design of this study used control and two factorials.Factor A is the concentration of decaffeinated Robusta Coffee has two levels, i. : 12% (b/b) refined sugar Each treatment was repeated three times.Each sample will be tested for caffeine content, analysis of reduced sugars, protein content, and cupping test.

Data analysis
The data obtained from the research stage will be analyzed with ANOVA (analysis of variance) and continued with Duncan's further tests to see any differences in software used in processing data in Microsoft Excel 2013 and SPSS.

Results and discussion
The flavor and taste test is used to evaluate coffee's taste characteristics.Through a taste test, quality, consistency, damage or defects from processing, selling price determination, and improvement of processing methods can be identified [13].The quality of coffee drinks is based on an international standard cupping test by the Specialty Coffee Association of America (SCAA) to classify coffee drinks [14].The method used in determining the taste of coffee is the SCAA method developed by the Specialty Coffee Association of America, which includes attributes such as a combination of taste characteristics, aroma, consistency of taste in the mouth, consistency of taste in a cup, the balance of coffee taste, level of cleanliness of taste characteristics, sweetness, the overall taste of coffee, coffee texture and acidity assessed by certified testers (Cupper, who holds a Q Grader certificate) to describe the product [15].The results of the cupping test on the treated decaffeinated Robusta coffee refermentation can be seen in Table 1.The taste test results showed that the final scores for decaffeinated Robusta coffee and decaffeinated Robusta coffee for the fermentation treatment were 74.25; 81.75; 81.50; 74.25; 73.25; 72.50, and 73.75.In addition, each score denotes a number ranging from 7-10.This indicates an increase in the quality of the excellent taste of Robusta coffee.The parameters of aroma/aroma, flavor, aftertaste, bitter/sweet, and salt/acid in decaffeinated Robusta coffee decreased with an average value of 6.75.This can occur because decaffeination can dissolve volatile compounds in coffee beans.However, after being given the fermentation treatment, there was an increase.During fermentation, microbial metabolite reactions occur with substances contained in coffee beans so that they can form volatile compounds in coffee related to the taste produced [16].When fermentation occurs, sugar as the main substrate is used by microorganisms as a carbon source, so that the sugar will be adequately reduced to alcohol and acetic acid, giving the coffee a sour taste [17].The formation of flavors in coffee is influenced by the content of protein, caffeine, reduced sugars, chlorogenic acid, and trigonelline which contribute to the resulting taste.Chemical compounds such as chlorogenic acid, caffeine, and trigonelline will experience solubility affecting the sour taste.Some samples experienced an increase in acidity during the fermentation process.The acid content in coffee is a group of carboxylic acids such as folic acid, acetic acid, oxalic acid, citric acid, lactic acid, malic acid, and quinic acid [18].
When fermentation occurs, carbohydrates decompose due to the activity of carbohydrates enzymes and pectinase enzymes into reduced sugars such as glucose and fructose.In addition, protein decomposition occurs in peptide compounds and amino acids [19].Amino acids and glucose play a role in the browning process during the roasting process affecting the taste of the coffee [20].During the roasting process, the Maillard reaction occurs between the carbonyl groups of the reduced sugar with the amino acids, peptides, and proteins [21].This reaction will form melanoidin, which is the main component in the browning process and affects the taste of coffee.In addition, the amino acids that form melanoidin react with some acid-forming compounds, such as tannins and acetic acid which will give the coffee brown color.The roasting process causes pyrolysis, so volatile compounds evaporate and cause aromas in coffee such as volatile furan compounds with caramel flavor, oxazole with sweet hazelnut flavor, phenol with bitter aroma, pyrazine with sweet butter flavor [22].
Fermentation treatment with the addition of 2.5 g and 5 g of sucrose (refined sugar) showed a final score of 81.75 (Specialty) and 81.50 (Specialty).Based on research [23], re-fermented decaffeinated robusta coffee obtained the highest score on the cupping test results of 81.75 (fine).This follows the statement [24] that fermentation greatly determines the quality of brewed coffee and can improve the characteristics of the coffee produced such as body, flavor and aftertaste.There is an increase in the flavor produced during fermentation, due to the reaction of the metabolite of microorganisms with the substances contained in coffee beans capable of forming volatile compounds in coffee [16].Microorganisms suspected to be present during the re-fermentation process are pectolytic microorganisms that synthesize carbohydrates and amino acids.This is in accordance with the statement [25] that pectolytic microbes play a role during the mucilage decomposition process in coffee.In addition to fermentation, the determination of coffee taste and aroma can be influenced during the roasting process, coffee cultivation and serving by baristas [26].Some flavor-forming compounds that appear during the coffee fermentation are palmitic acid, linoleic acid, alcohol, linoleic acid, and hydrocarbon compounds [27].The flavor components in coffee consist of fatty acids such as palmitic acid, lauric acid, linoleic acid, docosahexaenoic acid (DHA), and caffeine, which result from the breakdown of sugar from coffee during fermentation, which can enhance the aroma of coffee [28].
Caffeine (1,3,7-trimethyl xanthin) is a bioactive compound in the form of white powder, usually lumpy, odorless, has a bitter taste, is poorly soluble in water, ethanol, and esters but dissolves readily in chloroform and dilute acid, and has a melting point of 235 o -237 o [29].Caffeine positively impacts the body if consumed at <400 mg, increasing joy, peace, and pleasure [30].However, if consumed in excess, it can have adverse effects such as abnormal heartbeats, feelings of anxiety, tremors, headaches, insomnia, and stomach and digestive disorders [31].So for people with gastric acid, it is not recommended to consume caffeine in excess because it will cause inflammation and accelerate the production of stomach acid in the digestive system.The adverse effects depend on the amount of coffee consumed daily, but according to SNI 01-7152-2006, the maximum limit for consuming caffeine in food or drinks is 150 mg/day or 50 mg/serving.
The caffeine content in Robusta coffee is 1.5-2.7% of the dry weight of coffee beans.Caffeine content can be calculated based on the standard curve, y = 60.863+ 0.0433 (R2 = 0.9926), at a maximum wavelength of 273 nm using UV-Vis spectrophotometer results in Figure 1.Based on Figure 1 shows a decrease in Robusta coffee caffeine levels during the fermentation process.The results of the analysis of variance on the influence of the fermentation factor showed no significant effect on the caffeine content of green bean coffee given the treatment (P>0.05).Green bean without decaffeination treatment has a caffeine content of 1.25% which is higher than the caffeine content that has been re-fermented, an average of 1%, which can be caused by the decaffeination process using the water process method with boiling, which makes the compounds in the green bean diffuse into the water.When it is passed through the activated carbon, the caffeine will be caught on the activated carbon [32].Caffeine is located in the cell wall and cytoplasm of coffee beans.It binds to chlorogenic acid, which is difficult to dissolve in water, so heating is required to remove the bond between caffeine and chlorogenic acid [33].Caffeine compounds will be free with a smaller size and molecular weight, making it easier to diffuse through the cell walls and dissolve in water, reducing caffeine levels in coffee beans [34].The fermentation, requires proteolytic enzymes to break down the proteins in the cell wall so that the caffeine in the cell wall, which is cut off by chlorogenic acid, will be free and soluble in water [4].
Fermentation time also affects the decrease in caffeine content in coffee due to the activity of proteolytic bacteria, which produce pretty high protease enzymes.[35] It was reported that protein breakdown could cause a decrease in coffee caffeine levels and an increase in free amino acids.During fermentation, the caffeine content will break into esters as chlorogenic acid due to the esterification process.The breakdown of caffeine into simpler compounds such as theobromine, paraxanthine, theophylline, uric acid, and 7-methyl xanthine and xanthine [36].
Reduced sugars are a class of carbohydrates that can reduce electron acceptor compounds such as glucose, fructose, galactose, lactose, and maltose.The free aldehyde (-CHO) or ketone group (-CO-) in reduced sugar acts as a reducing agent [37].The reduced property of a sugar molecule is determined by the presence or absence of reactive free hydroxyl (OH) groups [38].The content of reduced sugars in coffee will affect its characteristics, such as flavor formation, color, and taste.During processing, reduced sugars and amino acids from proteins will occur in Maillard reactions [39].The method often used to determine reducing sugars is the Bailey method which uses 3,5-dinitro salicylic acid (DNS) reagent.The principle of the DNS method is that the reducing sugar scan reacts with the DNS reagent to form a brownish-yellow 3-amino-5-nitrosalicylic acid compound [40].Reducing sugar levels can be calculated based on the standard curve y = 2.5006x -0.1846 (R2 = 0.9954) at a maximum wavelength of 510 nm using UV-Vis spectrophotometer results in Figure 2. Based on Figure 2 shows that there is an increase in reduced sugar levels in Robusta coffee during the fermentation.The analysis of variance on the influence of the fermentation factor showed no significant effect on the reduced sugar content of green bean coffee given the treatment (P>0.05).Green bean coffee decaf without addition of sucrose showed a reduced sugar content of 0.20%.This shows a low reducing sugar content, which boiling can cause during decaffeinated process, which causes several flavor-forming compounds to dissolve in water [26].Fermentation with treatment showed an increase in reduced sugar levels.During fermentation, the bacteria will degrade cellulose and hemicellulose in coffee, breaking the sugar content, and affecting coffee beans' organic acid content [41].[42] said sugar would split into lactic acid and other acids such as butyric acid, propionate, and ethanol.Besides that, the addition of sucrose (refined sugar) during fermentation and the presence of mucilage analogs increased by reduced sugars.The increase in reduced sugar in decaffeinated Robusta coffee re-fermented a better taste.Based on the cupping test results, the average sweet/bitter characteristic is at number 7. In addition, from the cupping test results, there are notes which are the effect of the taste and aroma produced after roasting, which shows a sweet taste and aroma in decaffeinated Robusta coffee re-fermented.Adding sucrose can affect a product's reduced sugar content, such as sucrose (refined sugar), which contains 94% sucrose [38].The high pectin content in mucilage analogs, which consists of carbohydrates, is broken down during fermentation.The catalase enzyme in coffee will break protopectin into organic acids and sucrose into butyric acid and propionate [43].During fermentation, reduced sugars and pectin in mucilage are degraded by microorganisms through enzymatic reactions to produce ethanol which is acidic and organic acids.Changes in pectin into organic acids such as pyruvic acid, acetic acid, citric acid, malic acid, and succinic acid due to the activity of pectinolytic enzymes.The activity of microorganisms and enzymes during fermentation adds components to the decomposed coffee beans [44].The reduced sugar content in coffee mucilage consists of glucose and fructose compounds, the high level of reduced sugar indicates that the number of fructose molecules is still in the form of oligofructose, because some of it is hydrolyzed into its monomers [45].
Protein contributes to the bitter flavor of coffee, so the higher the protein content of coffee beans, the more bitter the coffee will taste.According to research [46], coffee beans contain higher protein than roasted coffee based on total amino acids.Several amino acids will react to form coffee's aroma and color components.Therefore, the amino acids in coffee beans determine the qualities of roasted coffee [47].Measuring protein levels in coffee can be done using the Lowry method.The Lowry method is based on the principle of the reaction between Cu 2+ ions and peptide bonds and the reduction of phosphomolybdic acid and phosphotungstic acid by tyrosine and tryptophan, which are included in protein residues which will produce a blue color.The formation of color depends on the levels of tyrosine and tryptophan in the protein [12].Using a UV-Vis spectrophotometer, protein levels can be calculated based on the standard curve y = 0.0043x + 0.0856 (R2 = 0.9809) at a maximum wavelength of 650 nm.The results are in Figure 3. Based on Figure 3 shows that there is an increase in reduced sugar levels in Robusta coffee during fermentation.The analysis of variance on the effect of the fermentation factor showed no significant effect on the protein content of green bean coffee given the treatment (P>0.05).Green beans without treatment showed a protein content of 3.14%.After the fermentation treatment, the protein content increased with an average protein content of 4.67%.The high protein can influence the increase in protein content during fermentation.The protein content in green beans ranges from 8.1-10.2%[46], while the albedo of watermelon contains about 0.53% protein [48].In addition, an increase in the amount of protein can be caused by an increase in the number of microorganisms during the fermentation, which act as single-cell proteins (SCP), which are protein-producing microorganisms [49].During fermentation, there is protease enzyme activity produced by microbes which will break down protein so that it can increase amino acids [35].Based on [50] that during the fermentation, the activity of proteolytic enzymes break down proteins into amino acid, and dissolved nitrogen will increase.During fermentation, the amount of water-soluble nitrogen (N) will increase due to the activity of enzymes that break down proteins into easily soluble fragments and are utilized by microbes for growth, so that the number of microbes increases and contributes to single-cell protein, which will increase the crude protein content in the substrate [51].An increase in substrate protein, can occur due to an increase in yeast biomass (MBP) and an increase in yeast cells that function as single-cell protein agents (PST) [52].

Conclusion
In conclusion, The flavor of decaffeinated Robusta coffee after re-fermentation showed an increase in the 25 g decaffeinated robusta coffee fermentation treatment with the addition of 2.5 g sucrose (refined sugar) with the highest final score of 81.75 (specialty coffee) using the cupping test method with the resulting taste characteristics is Brown Sugar, Sweet Potato Aroma & Aftertaste, Herbal.Refermentation of decaffeinated Robusta coffee using the albedo of watermelon showed a change in the chemical characteristics of the coffee.The fermentation could reduce the levels of caffeine, while the levels of reduced sugars and protein increase as fermentation progresses, affecting the increased taste of the coffee produced.

Figure 1 .
Figure 1.Effect of fermentation on caffeine contents in green bean robusta coffee.

Figure 2 .
Figure 2. Effect of fermentation on reducing sugar contents in green bean robusta coffee.

Figure 3 .
Figure 3.Effect of fermentation on protein levels in green bean robusta coffee.

Table 1 .
Cupping test results for decaffeinated robusta coffee re-fermentation.