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Utilizing Coffee Pulp and Mucilage for Producing Alcohol-Based Beverage

Article in Fermentation · April 2021

DOI: 10.3390/fermentation7020053

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 fermentation

Article
Utilizing Coffee Pulp and Mucilage for Producing
Alcohol-Based Beverage
Yadav KC 1 , Raju Subba 1 , Lila Devi Shiwakoti 2 , Pramesh Kumar Dhungana 3 , Rishikesh Bajagain 4 ,
Dhiraj Kumar Chaudhary 5 , Bhoj Raj Pant 6 , Tirtha Raj Bajgai 7 , Janardan Lamichhane 8 , Sampada Timilsina 8 ,
Jitendra Upadhyaya 9, * and Ram Hari Dahal 10, *

1 Central Campus of Technology, Tribhuvan University, Dharan 56700, Nepal; ykcdng504@gmail.com (Y.K.);
ft.subba@gmail.com (R.S.)
2 National Tea and Coffee Development Board, Hile, Dhankuta 56806, Nepal; leela504s@gmail.com
3 School of Agriculture and Food Sciences, University of Queensland, Brisbane 4072, Australia;
prameshdhungana@gmail.com
4 Department of Environmental Engineering, Kunsan National University, Kunsan 54150, Korea;
rbajagain@gmail.com
5 Department of Environmental Engineering, Sejong Campus, Korea University, 2511 Sejong-ro,
Sejong City 30019, Korea; dhirajchaudhary2042@gmail.com
6 Nepal Academy of Science and Technology, Khumaltar, Lalitpur 44700, Nepal; environmentnast@gmail.com
7 Minhas Microbrewery, Distillery and Winery, 1314 44 Ave NE, Calgary, AB T2E 6L6, Canada;
tirraj@yahoo.com
8 Department of Biotechnology, Kathmandu University, Dhulikhel 45200, Nepal; ljanardan@ku.edu.np (J.L.);
sampadatimilsina118@gmail.com (S.T.)
9



Department of Bioresource Engineering, McGill University, Macdonald Campus, Lakeshore Road,

 Ste-Anne-de-Bellevue, Montreal, QC 21111, Canada
10 Department of Microbiology, School of Medicine, Kyungpook National University, Daegu 41944, Korea
Citation: KC, Y.; Subba, R.;
* Correspondence: jitu.upadhyaya@gmail.com (J.U.); ramhari.dahal@gmail.com (R.H.D.)
Shiwakoti, L.D.; Dhungana, P.K.;
Bajagain, R.; Chaudhary, D.K.; Pant,
B.R.; Bajgai, T.R.; Lamichhane, J.; Abstract: Coffee pulp, mucilage, and beans with mucilage were used to develop alcoholic beverages.
Timilsina, S.; et al. Utilizing Coffee The pulp of 45.3% pulp, 54.7% mucilage with seed, and 9.4% mucilage only were obtained during
Pulp and Mucilage for Producing the wet processing of coffee. Musts were prepared for all to TSS (Total soluble solid) 18 ◦ Bx and
Alcohol-Based Beverage. Fermentation fermentation was carried out for 12–16 days until TSS decreased to 5 ◦ Bx at 30 ◦ C. Phenolic charac-
2021, 7, 53. https://doi.org/10.3390/ teristics, chromatic structures, chemical parameters, and sensory characteristics were analyzed for
fermentation7020053 the prepared alcoholic beverages. Methanol content, ester content, aldehyde, alcohol, total acidity,
caffeine, polyphenols, flavonoids, chromatic structure, and hue of the alcoholic beverage from the
Academic Editor: Ogueri Nwaiwu
pulp was 335 mg/L, 70.58 ppm, 9.15 ppm, 8.86 ABV%, 0.41%, 30.94 ppm, 845.7 mg GAE/g dry
extract, 440.7 mg QE/g dry extract, 0.41, and 1.71, respectively. An alcoholic beverage from the
Received: 17 March 2021
pulp was found superior to an alcoholic beverage from mucilage with beans and a beverage from
Accepted: 2 April 2021
Published: 5 April 2021
mucilage in sensory analysis. There is the possibility of developing fermented alcoholic beverages
from coffee pulp and mucilage. However, further research is necessary for quality of the beans that
Publisher’s Note: MDPI stays neutral
were obtained from the fermentation with the mucilage.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: coffee pulp and mucilage; mucilage with beans; alcoholic beverage; sensory analysis
iations.

1. Introduction
Copyright: © 2021 by the authors. Recently, the global demand for ethanol has been steadily increasing. The global
Licensee MDPI, Basel, Switzerland. ethanol production was about 90 billion liters in 2013 and increased to 115 billion liters
This article is an open access article in 2019 [1]. The global output of ethanol in 2020 was strongly impacted by the Covid-19
distributed under the terms and crisis and dropped to 98 billion liters; however, the production is anticipated to a gradual
conditions of the Creative Commons increase by 2021 [2]. Sugarcane is the readily used plant for ethanol production. However,
Attribution (CC BY) license (https:// the demand for obtaining ethanol could not be achieved from sugarcane alone due to its
creativecommons.org/licenses/by/ cost and because raw materials are restricted to areas with special soil for it [3]. In order to
4.0/).

Fermentation 2021, 7, 53. https://doi.org/10.3390/fermentation7020053 https://www.mdpi.com/journal/fermentation

Fermentation 2021, 7, 53 2 of 13

meet the ethanol production demand, alternative materials should be explored to reduce
the burden in sugarcane.
Coffee drink, obtained from the coffee plant (Coffea arabica L.), is one of the most
commonly consumed beverages in the world. It is the second most traded commodity
after oil, and due to the demand for this product, large amounts of waste are generated [3].
The coffee bean is a naturally fruiting cherry mainly composed of hard dicotyledon seed
covered by silver-skin, parchment, mucilage, and pulp. Coffee pulp constitutes 29–50% of
the dry weight of the cherry, which is obtained during wet processing of coffee [4–7]. The
covering materials are removed during processing [8]. Pulp and mucilage, being relatively
rich in sugars, are used for microbial growth. For example, Aspergillus niger was used
for solid-state fermentation of coffee pulp [9]. Bacillus cereus, Bacillus megaterium, Bacillus
subtilis, Candida parapsilosis, Pichia caribbica, Pichia guilliermondii and Saccharomyces cerevisiae
were used as a potential starter culture for enhancing the coffee fermentation process [10].
Coffee pulp has been used for the extraction of caffeine, protein, pectic enzymes,
fertilizers, biogas, and coffee pulp molasses [5,11]. Mucilage is rich in both simple and
complex sugars [5], which can be used in fermentation. Few studies have been conducted
for producing ethanol from a mixture of coffee pulp and mucilage. For example, the ethanol
yield was reported to be equivalent to 77.29% of the theoretical yield (an ethanol yield
of 25.44 kg/m3 , resulting from the 64.40 kg/m3 of total sugars) from a mixture of coffee
pulp and mucilage, commercial baker’s yeast, and panela [12], which showed that the
production of ethanol is viable in small coffee farms using readily available raw materials.
Orrego et al. achieved bioethanol yield of more than 90% of theoretical yield from coffee
mucilage [13]. However, alcoholic fermentation of byproducts of coffee, such as coffee pulp
and mucilage, has rarely been studied. This study utilizes coffee waste during processing,
i.e., coffee pulp and mucilage, for the production of alcohol.
Having high sugar content (2.6–31.26 gL−1 ), the Ethiopian coffee pulp has found to
produce 7.4 gL−1 ethanol [14]. It has been reported that the bioethanol yield was found to
be 0.46 g/g of sugar in wet coffee pulp [15]. In addition, coffee pulp is a good source of
natural antioxidant and it contains hydroxycinnamic acids (chlorogenic, caffeic, and ferulic
acid) [16]. Coffee pulp is one of the food wastes which cause environmental problems.
In order to reduce its environmental impacts, several types of studies are focused on
the extraction of active ingredient and its utilization as animal feed or compost. A non-
fermented drink known as Kisher is produced in Yemen and Somalia from ripe fruit
berries. Coffee pulp is also used for the production of a beverage called cascara (also called
coffee cherry tea) due to its bioactive components [17]. Mucilage and pulp from processed
coffee were used in ethanol production in Rwanda [18]. It has been reported that dry
white wine has been produced from coffee pulp in Central America [19]. Even though it
contains several beneficial compounds such as minerals, amino acids, polyphenols, and
caffeine [16], there are limited works on utilizing coffee pulps and mucilage for human
consumption. The alcohol produced from a biological way by fermentation of sugars
can be a strong candidate for replacing fossil fuels, and are advantageous for their purity,
renewability, have a more complete combustion and less waste [20]. In addition, coffee
pulp and mucilage can be the new valuable, cost-efficient, and eco-friendly raw material
for the beverage industry. Therefore, this study aims to utilize the pulp and mucilage from
wet processing of coffee in preparing a fermented alcoholic beverage instead of leaving
them as waste.

2. Materials and Methods

2.1. Raw Material Collection
Ripe coffee cherries (Coffea arabica L.) were collected from the Bhirgaun, Dhankuta,
Nepal (27◦ 010 12.800 N 87◦ 210 48.300 E, elevation of 1269.0 masl (meters above sea level)).
Coffee cherries were harvested from 7:00 AM to 8:00 AM, packaged in porous polyethylene
bags, and transported to lab of the Central Campus of Technology, Dharan, Nepal. Five
hours after the harvest, pulping and de-mucilizing was carried out. Sugar and wine yeast
Fermentation 2021, 7, 53 3 of 13

Saccharomyces cereviseae (ex bayanus), Lalvin EC-1118, Canada) was obtained from the lab
of the Central Campus of Technology.

2.2. Preparation for Fermentation

Ripe coffee cherries were processed for preparing the must for fermentation (Figure 1).
After rejecting the off standards and degraded coffee by flotation technique, coffee pulping
was done by using a coffee pulper (Total Machinery, Teku, 10 kg hand pulper) and byprod-
ucts of coffee (pulp and mucilage) were treated for fermentation, coded AP for pulp, AM
for mucilage, and AMS for mucilage with beans. In brief, 45.3% of the pulp and beans,
along with mucilage 54.7% and 9.4% of the mucilage only was obtained during processing
which was similar to experiments of Costa Rica [5].
For fermentation, three types of preparations were done (Figure 1): (i) AP: The pulp
with TSS (total soluble solids) 18 ◦ Bx was kept in a fermenting glass. Only 3–4% juice was
obtained. So, distilled water was added to cover all the pulp where TSS decreased to 4 ◦ Bx.
(ii) AMS: After pulping, the beans with the mucilage (TSS 15 ◦ Bx) were collected in the
clean and hygienic bucket. Distilled water at 20 ◦ C was added to beans that were kept in
fermenting glass jar. (iii) AM: Rapid rubbing by hand for 15–20 min was done to extract
the mucilage (TSS 15 ◦ Bx) from beans. For all three preparations, sterilization was done in
a water bath at 65 ◦ C for 15–20 min before maintaining the TSS. Distilled water was added
to cover mucilage where TSS decreased to 9 ◦ Bx. The TSS was raised to 18 ◦ Bx for all musts
before fermentation. The must (100 mL) was taken and heated to lukewarm temperature
(35–40 ◦ C). Wine yeast (Saccharomyces cerevisiae) at the rate of 0.25 g dry yeast/L of must
was added. It was stirred until the effervescence of CO2 appeared. Then pitching was
carried out. The musts were fermented in separate bottles fitted with air-locked corks
until the TSS reached 5 ◦ Bx. After the completion of fermentation, fermented juices for
each sample were separated from the residues and table sugar was added to 10 ◦ Bx for
flavoring purposes. The beverages were allowed to settle for 2 days, siphoned through
sterilized polyethylene pipes in clean sterilized bottles and pasteurized in a water bath at
65 ◦ C for 15–20 min [21]. AP, AM, and AMS were aged at 4 ◦ C for 15 days before analyses
of chemical and sensory parameters.

2.3. Extraction of Phytochemicals

Phytochemicals from pulp and mucilage were extracted by using methanol with slight
modification as described by Gerumu et. al. [22]. Ten grams of samples was steeped in
100 mL of 80% methanol at 65 ◦ C for 10 min. Then it was cooled to room temperature and
homogenized for 3 min using a grinder. Subsequently, it was filtered using Whatman no.
41 filter paper and the residue was re-extracted following the above procedure. The extract
was stored in a screw-capped bottle at 4 ± 2 ◦ C until analysis. Ten milliliters of extract
were evaporated, dried at 80 ◦ C, and the residue was weighed to know its concentration.

2.4. Analytical Methods

Proximate analysis of raw materials was carried out in triplicate. Standard AOAC
methods: (AOAC 935.29) for moisture content, (AOAC 922.06) for crude fat, (AOAC 992.23)
for crude protein, (AOAC 923.03) for total ash, and (AOAC 962.09) for crude fiber were
used. The TSS was analyzed by a hand refractometer (model WYT-32, Zhongyou, Fujian,
China). It was calibrated to give the concentration of total soluble solids in ◦ Brix at a
standard temperature of 20 ◦ C. A digital pH meter (Japsin Industrial Instrumentation, New
Delhi, India) was used to analyze pH.

2.4.1. Alcohol Content and Total Dry Extract

The distillate of the beverage was taken in the specific gravity bottle and its tempera-
ture was measured, then the distillate was completely filled in the bottle and the weight
was measured for further calculations as per FSSAI manual for methods of analysis of
alcoholic beverages [23]. Briefly, 100 mL of the alcoholic drink was taken in the dried tared
Fermentation 2021, 7, 53 4 of 13

beaker and evaporated in a water bath. After wiping the external sides of the beaker, it
was kept in a hot-air oven at 100 ± 5 ◦ C for 1–2 h. The weight of the beaker was taken
after cooling in the desiccator. The experiment was continued until the constant weight
was obtained, and calculation was done as per FSSAI manual for methods of analysis of
alcoholic beverages [23].

2.4.2. Methanol Content

Methanol in each sample was determined by the chromotropic acid spectrophoto-
metric method [23]. Stock solution of methanol was prepared by diluting 1.0 g methanol
(99.99% pure) to 100 mL with 40% (v/v) ethanol (methanol free). Again, 10 mL of this
solution was diluted to 100 mL with 40% ethanol. From the stock solution, methanol
concentrations of 20, 40, 60, 80, and 100 ppm were obtained by diluting with 40% ethanol.
A distilled sample (1 mL) was diluted to 5 mL with distilled water and shaken well. One
mL of this solution, 1 mL of distilled water (for blank), and 1 mL of each of the methanol
standards were taken into 50 mL stoppered test tubes and kept in an ice-cold water bath.
To each test tube, 2 mL of KMnO4 reagent was added and kept aside for 30 min. A little
amount of sodium bisulphite and 1 mL of chromotropic acid solution were added to decol-
orize the solution. After mixing uniformly, 15 mL of sulphuric acid was added slowly with
swirling and placed in hot water bath maintaining 80 ◦ C for 20 min. The color development
from violet to red was observed. After cooling the mixture, the absorbance at 575 nm was
noted and methanol content was calculated as per FSSAI manual for methods of analysis
of alcoholic beverages [23].

Figure 1. Cont.
Fermentation 2021, 7, 53 5 of 13

Figure 1. Flowchart for (a) mucilage extract alcoholic beverage (AM), (b) mucilage with seed alcoholic
beverage (AMS), and (c) pulp alcoholic beverage.

2.4.3. Ester Content

Briefly, to the 50 mL of distillate, 10 mL of 0.1 N NaOH was added and refluxed on
a steam bath for 1 h. After cooling, back titration for the unspent alkali against standard
sulphuric acid was carried out. For blank, 50 mL of distilled water instead of distillate was
taken and experiments were conducted in the same way. The difference in titer value in
milliliters of standard sulphuric acid gives the equivalent ester and was calculated as per
FSSAI manual for methods of analysis of alcoholic beverages [23].

2.4.4. Aldehyde Content

In brief, 50 mL of distillate of liquor was taken in a 250 mL iodine flask and 10 mL of
0.05 N sodium bisulphite solution was added. The flask was kept in a dark place for 30 min
with occasional shaking. Then, 25 mL of standard iodine solution was added and back
titration of excess iodine against 0.05 N standard sodium thiosulphate solution using starch
indicator (1% solution) was conducted to light green end point. The same experiment was
carried out for blank, except distilled water was used instead of distillate. The difference
in titer value in milliliters, of sodium thiosulphate solution gives the equivalent aldehyde
content and was calculated as per FSSAI manual for methods of analysis of alcoholic
beverages [23].

2.4.5. Total Acidity and Volatile Acidity

The pH meter was calibrated using the buffer solutions of pH 4.0, 7.0, and 9.2. Ap-
proximately 100 mL of distilled water was taken in a beaker and turn magnetic stirrer after
placing magnetic bead in it. The electrode of the pH meter was immersed into the water
and titration against standard NaOH solution to pH 8.2 was carried out. Again, titration
against standard NaOH was carried out to pH 8.2 by adding 50 mL of liquor sample to the
pH-adjusted water. Volume of the NaOH was noted for total acidity. To the 50 mL of the
distillate, titration against standard NaOH using phenolphthalein indicator was carried
Fermentation 2021, 7, 53 6 of 13

out for volatile acidity. Total and volatile acidity were calculated as per FSSAI manual for
methods of analysis of alcoholic beverages [23].

2.4.6. Caffeine Content

Caffeine content in the samples was determined by UV-Vis spectrophotometric
method [24,25] with slight modifications. Samples (2.5 g) were poured to 200 mL of boiling
water and stirred for 10 min. After filtering through a cotton wool, the extract was cooled
at a room temperature and volume made to 250 mL with distilled water. This solution was
mixed with dichloromethane in ratio 1:1 (25:25 mL). It was stirred for 10 min and caffeine
was extracted by dichloromethane from the solution with the help of a separating funnel.
Caffeine was extracted 4 times with 25 mL dichloromethane at each round and was stored
in volumetric flasks. The absorbance of the extracted solution was measured at 270 nm on
UV/visible spectrophotometer. The test results were correlated with standard calibration
curve of caffeine (y = 0.035x + 0.1, r2 = 0.996) and it was expressed in percentage (%).

2.4.7. Total Phenolic Content

Total phenolic contents (TPC) were determined using spectrophotometric method
with some modifications [26]. The reaction mixture was prepared by mixing 0.5 mL of plant
extract, 2.5 mL of 10% Folin-Ciocalteu reagent, and 2.5 mL of 7.5% of Na2 CO3 aqueous
solution. The mixture was incubated at 45 ◦ C for 45 min in an oven. The absorbance was
determined at 765 nm using a UV-visible spectrophotometer. The same procedure was
repeated for the standard solutions of gallic acid. A calibration curve was constructed
using the standard data. Based on the measured absorbance of test samples, the total
phenolic content was determined from calibration curve and expressed as mg of gallic acid
equivalent (GAE) per g of dry matter in extract (mg GAE/g).

2.4.8. Total Flavonoid Content

Total flavonoid content (TFC) was determined using a modified aluminum chloride
(AlCl3 ) assay method [27]. Briefly, 2 mL of extract solution was taken in a test tube. 110
Then, 0.2 mL of 5% NaNO3 was added and allowed to stand for 5 min. Later, 0.2 mL of 10%
AlCl3 was added and mixed properly and allowed to stand for 5 min. After this, 2 mL of
1 N sodium hydroxide (NaOH) was added in the tube and the final volume was adjusted
to 5 mL by adding distilled water. The absorbance was measured after 15 min at 510 nm.
The test result was correlated with standard curve of quercetin (20, 40, 60, 80, 100 µg/mL)
and the total flavonoid content was expressed as mg of the quercetin equivalent per gram
(mg QE/g) of dry matter in extract.

2.4.9. Color Measurement

Spectrophotometry was used to analyze color intensity and hue [28]. The triplicate
readings for each sample in three different wavelengths, i.e., 420, 520, and 620 nm were
recorded for calculating color intensity and hue (Equations (1) and (2)).

Colour intensity = A420 + A520 + A620 (1)

A420 (2)
Hue = A520

2.5. Sensory Analysis

Sensory analysis was evaluated with reference to wine [29] by 25 panelists and con-
verting scores of quality parameters in percentage for total quality score of 100. Sensory
parameters were analyzed with a quality score of 15%, 30%, 30%, 15%, and 10% for appear-
ance, aroma, taste, aftertaste, and overall acceptability, respectively. A 2 h training session
was conducted for 14 days to familiarize panel members with sensory attributes.
Fermentation 2021, 7, 53 7 of 13

2.6. Statistical Analysis

The data of each experimental analysis, performed in triplicate, were analyzed by
one-way analysis of variance (ANOVA), and this was carried out by using GenStat Re-
lease 12.1 software (Copyright 2009, VSN International Ltd., Hemel Hempstead, UK).
Means were separated using Tukey’s HSD post-hoc test (p < 0.05). Values are means of
triplicate ± standard deviations.

3. Results
3.1. Chemical Composition of Coffee Pulp and Mucilage
Chemical composition of the coffee pulp was analyzed (Table 1). Protein content for
coffee pulp was similar to findings of Braham and Bressani (1979), but slightly different
in caffeine content and reducing sugar [5]. The caffeine content was found to be lower,
which might be due to the variation of caffeine-extracting solvent. Similarly, reducing
sugar was slightly different, which might be due to the difference in harvesting time and
geography. In contrast, ash content was slightly lower, which might be due to the difference
in geography, harvesting time, and variation of processing technology [30]. The crude fiber
content was found to be higher than industrial waste pulp in Kenya [31], but was lower
than pulp obtained by the semi-washed process in Brazil [32]. The fat content was similar
to the findings of [33] and similar pH value was obtained in the study in Mexico [34].
Crude fat, crude fiber, caffeine content, and TSS of the mucilage were found to be 0.7%,
1.5%, 1.05%, and 15 ◦ Bx, respectively. Belitz et al. (2008) reported 84.2% moisture, 8.9%
protein, 4.1% sugar, and 0.7% ash [35], which are similar to our findings (Table 1). Total
polyphenols, flavonoids, and tannins differed from [36], which might be due to variation
of agronomic practices, climate, geography, and soil conditions.

Table 1. Analysis of coffee pulp and mucilage.

Particulars Coffee Pulp Mucilage

Moisture (%) 75.7 ± 0.2 85.3 ± 0.6
Dry matter (%) 24.3 ± 0.2 14.7 ± 0.6
Crude Protein (%) 8.1 ± 0.36 7.2 ± 0.3
Fat (%) 1.53 ± 0.05 0.7 ± 0.00
Ash (%) 6.4 ± 0.05 1.1 ± 0.1
Crude fiber (%) 6.3 ± 0.2 1.5 ± 0.22
Total sugar (%) 12.06 ± 0.41 4.3 ± 0.4
Reducing sugar (%) 10.9 ± 0.36 -
Caffeine (%) 1.11 ± 0.11 1.05 ± 0.05
TSS (◦ Bx) 18 ± 0.5 15 ± 0.5
pH 4.3 ± 0.15 3.7± 0.1
Polyphenols (mg GAE/g dry extract) 1862.62 ± 4.42 1618.32 ± 3.2
Tannins (mg GAE/g dry extract) 412 ± 4.7 370 ± 3.6
Flavonoid (mg QE/g dry extract) 697.3 ± 2.1 531.54 ± 2.7
Values are means of triplicate ± standard deviations.

3.2. Fermentation Kinetics with Respect to pH and TSS

There was significant variation (p < 0.05) in the pH content of all the samples with
respect to fermentation time. The pH rapidly dropped up to the sixth day, then gradually
increased up to the tenth day, and finally decreased slowly and stabilized. The fall of pH
up to the fourth day was seen maximum in samples of pulp preparation than the other two
samples (Figure 2a). This shows faster conversion of sugar into acids in pulp sample. In
terms of pH, fermentation can be categorized into two phases: it decreases during the first
and then increases [37]. The drop in pH might be due to the consumption of glucose and
production of ethanol and organic acids by yeast. The production of these acids drives the
pH down to acidic conditions [38]. After the sixth day of fermentation, there was significant
increase in the pH. This might have happened due to the lack of the nutrient, and yeast
begins to consume organic acids as the nutrient source. Similarly, nitrogen sources are
Fermentation 2021, 7, 53 8 of 13

cleaved off to ammonia by yeast, which attracts protons to form ammonium in the aqueous
solution, causing increase in pH [37]. The simultaneous consumption of organic acids [37]
and increase in ethanol production (pH of ethanol, 7.33) resulted in alteration of the pH,
as both these factors affect pH value during the sugar fermentation process. There was a
significant difference between the TSS of each product with respect to fermentation days.
The TSS was decreasing in each day. There was rapid decrease of TSS of the pulp sample
than the other two samples (Figure 2b). TSS, i.e., 5 ◦ Bx [39] was achieved in the twelfth day
by pulp samples, which is earlier than the other two samples. This concludes that the rate
of fermentation of pulp is more than the other two samples. The rapid drop in TSS was
due to utilization of the supplied glucose by the yeast [40], which is quite obvious.

Figure 2. Changes during fermentation period. (a) pH. (b) TSS.

3.3. Chemical Analysis

Total dry extract in the AP, AM, and AMS was found to be 215 g/L, 190 g/L, and
183 g/L, respectively. Methanol content was significantly different (p < 0.05) in all the
samples. Among three fermented beverages (AP, AM, and AMS), AP contained the highest
level of methanol content, i.e., 335 mg/L, while AM contained the least, i.e., 298.6 mg/L
(Table 2). Coffee pulp is found to be richer (~1.9 times) in pectin than coffee mucilage [41].
The highest level of the methanol content in AP might be due to the methylated pectin
that gets transferred in the beverage during fermentation. The methanol level in red
wine that can be accepted by the human body is 400 mg/L [42], which concludes that AP,
AM, and AMS are within the range with respect to methanol content. The International
Organization of Vine and Wine has reported the safety limit of methanol for avoiding the
risk to consumers’ health as 150 mg/L for white and rosé and 300 mg/L for red wine.
Regarding this, some modifications, like adding phenolic acids, can be done to reduce the
methanol content [43].
Among the three samples, alcohol content in AP was found higher (8.86%) and was
not significantly different, but AMS differed significantly (p < 0.05). Higher alcohol content
in AP was due to maximum utilization of sugar. The difference in ethanol content may
be due to the difference in the must formation, and also may be due to the difference in
the chemical constituents between pulp and mucilage. Ester content in all samples was
found in the range as stated by [44] but was higher compared the findings of [45]. Esters
are expressed as ethyl acetate whose concentration ranges from about 30–60 mg/L in
“normal” wines to about 150–200 mg/L in defective wines [46]. And our beverages contain
esters in the range where AP contained a little higher. Ester content in AM and AMS was
not significantly different but AP differed significantly (p < 0.05). Higher alcohol content
might be responsible for higher ester content in AP because esters are formed due to the
reaction between the fatty acids and alcohol. The difference in the ester content might be
contributed by a difference in the carbon and nitrogen content between the samples [47].
Fermentation 2021, 7, 53 9 of 13

Aldehyde content was significantly different (p < 0.05) and AP contained quiet less than
AM and AMS. Aldehyde content was less when compared to [45].

Table 2. Chemical and chromatic analysis of alcoholic beverages of coffee.

Particulars AP AM AMS
c a
Methanol content (mg/L) 335 ± 1.21 298.9 ± 0.28 313.2 ± 1.81 b
Esters content (ppm) 70.58 ± 1.45 b 38.21 ± 6.09 a 33.86 ± 3.29 a
Aldehydes (ppm) 9.15 ± 0.877 a 22 ± 0.4 b 42.94 ± 1.5 c
Alcohol (ABV%) 8.867 ± 0.067 b 8.707 ± 0.092 b 8.25 ± 0.026 a
Total acidity (%) 0.411 ± 0.02 ab 0.393 ± 0.005 a 0.443 ± 0.011 b
Volatile acidity (%) 0.013 ± 0.00 c 0.007 ± 0.00 a 0.0094 ± 0.00 b
Caffeine content (ppm) 30.94 ± 0.674 b 21.29 ± 0.643 a 42.44 ± 0.737 c
Polyphenols (mg GAE/g dry extract) 845.7 ± 14.36 c 554 ± 7.93 a 709.7± 4.5 b
Tannin (mg GAE/g dry extract) 305 ± 4 c 235 ± 4 a 268.3 ± 3.5 b
Flavonoid (mg QE/g dry extract) 440.7 ± 5.03 c 349.3 ± 4.5 a 395 ± 3 b
Chromatic structure 0.41 ± 0.00 c 0.27 ± 0.00 a 0.28 ± 0.00 b
Hue 1.71 ± 0.00 c 1.64 ± 0.00 b 1.51 ± 0.00 a
Alcoholic beverages made from pulp (AP), mucilage only (AM) and mucilage with beans (AMS). Values are means of triplicate ± standard
deviations. Values in the rows bearing the different superscripts (a, b, c, and ab) are significantly different (p < 0.05).

Aldehyde content in the fermented beverages is expressed in terms of actaldehyde

and immediately after fermentation; table wines generally have acetaldehyde levels below
75 mg/L [46]. Our beverages contain aldehydes less than 50 ppm. Color of the red
wine is enhanced by polymerization of anthocyanins and phenolics with the assistance
of aldehyde [48]. This might be the reason of minimization of aldehyde content in AP.
Higher content of total acidity in AMS might be due to unconsumed fatty acids for the
production of esters, while lower values in other samples can be related to the utilization
of carboxylic acids in the production of esters. Volatile acidity was found to be relatively
lower. This might be due to the difference in the chemical composition between the raw
materials. Caffeine content was significantly different (p < 0.05) in all the samples. The
minimum value of caffeine content was found in AM and the maximum in AMS, which
might be due to fermentation along with beans. Asfew & Dekebo reported that caffeine
content of coffee beans ranged from 1.21 to 1.43 % in beans and 0.78 to 0.97 % in coffee
pulp [49]. The value of caffeine content in the alcoholic product is much lower as compared
to that in raw pulp and beans. The low values can be attributed to the fact that caffeine
molecules in coffee beans are complex with chlorogenic acids [50], and the hydrogen bonds
between caffeine and chlorogenic acid molecules have to be broken during fermentation.
Raw pulp contained 1762.6 mg GAE/g dry extract, which was between the range of 1809.9
to 489.5 mg GAE/g dry extract [51] and flavonoid content of 1418.2 mg QE/g dry extract
(Table 1). Among the three samples, AP contained the maximum value of polyphenols,
i.e., 845.7 mg GAE/g dry extract. This maximum value was due to the fact that the pulp
contained the highest value of polyphenols than other parts of the pericarp [51]. AM
contained minimum value of polyphenols, i.e., 554 mg GAE/g dry extract, which might
be due to the minimum value of polyphenols in mucilage. The content of flavonoid was
also similar to that of phenolic content, i.e., maximum in AP. The polymeric anthocyanin
color (%) of the coffee pulp was higher [52], which might be the reason for more flavonoids
in AP. Similarly, tannin content was also higher in AP. The effect of pH of the medium
during fermentation might have influenced the metabolism of the yeast for the growth and
degradation of caffeine and tannins [36].

3.4. Colour of Fermented Alcoholic Beverages

The value of chromatic structure and color density was significantly higher in AP
than AM and AMS, causing a redder appearance like red wine. Flavonoid content was
significantly higher in AP (Table 2), which might have contributed to anthocyanins [52].
Fermentation 2021, 7, 53 10 of 13

Being directly in contact with the red-colored exocarp is another reason for the red color
in AP. AM and AMS were whitish in appearances. Hue is the indication of the aging and
oxidation of wine. All wines were aged for a constant time, so difference in hue was due to
the difference in red pigments in each sample. Oxidation and polymerization might also
be the reason for the decrease in hue [53].

3.5. Sensory Analysis

Haziness and dull appearance in all prepared samples was seen which was due to
improper clarification. AP was more reddish than other samples which might be due to
leaching of pigments from skin during fermentation. AMS got more reddish and brownish
pigment from seed contributed redder than AM. Fruity aroma giving esters, chlorogenic
acid, acetic and propionic acids increased throughout the fermentation process [53] caus-
ing higher aroma in AMS, AP and AM. Similarly, the production of ethanol and lactic,
butyric, acetic, and other higher carboxylic acids during the fermentation of pectinaceous
sugars by microorganisms in coffee fermentation [30] and modification of compounds
such as proteins, carbohydrates, chlorogenic acids in green coffee beans [53] caused the
development of unique aroma and taste at the end of fermentation. According to Sera et al.,
coffee fragrance and flavor is related to cholorgenic acids [36]. Higher methanol content
in AP (Table 2) could extract chlorogenic acid in the fermented beverage causing more
score of aroma in AP [54]. Based on taste, the highest mean score of AP, AMS and AM
was due to chlorogenic acid, epicatechin and isochlorogenic acid that got extracted from
mucilage. Though there is no significance difference in the taste in all samples, AP had
highest score which might be influenced by higher amount of tannins and polyphenols
(Table 2) which relates to a positive trait, especially mouth feel of the wines [55]. The
astringent aftertaste was given by phenolic compound that was isolated from coffee mu-
cilage and pulp [56]. This sensation is felt in the mouth after consumption of some wines,
strong tea or un-ripened fruit [56]. AP had significantly higher score of aftertastes (Table 3)
which might be due to more tannins and TPC than AM and AMS that has given natural
aftertaste (Table 2). The scores of AMS were superior to AM as seed was involved in
fermentation contributing more taste, aftertaste, aroma and OA (overall acceptability). The
value of overall acceptability was also higher for AP in compared to AM and AMS. Total
quality score in this study found that the desirable characteristics, color, pleasant flavor,
taste, aftertaste, and overall acceptability were higher in AP compared to AM and AMS
(Figure 3).

Table 3. Sensory analysis of alcoholic beverages of coffee.

Overall
Particulars Appearance Aroma Taste Aftertaste
Acceptability
AM 9.03 ± 2.53 a 18.89 ± 4.9 a 21.21 ± 3.73 a 10.32 ± 2.32 a 6.35 ± 1.25 a
AMS 9.07 ± 2 a 20.11 ± 5.07 ab 21.96 ± 4.17 a 11.11 ± 3.17 ab 6.32 ± 1.56 a
AP 11.07 ± 2.19 b 21.86 ± 3.71 b 23 ± 2.34 a 12.21 ± 1.67 b 8.25 ± 0.7 b
Alcoholic beverages were made from pulp (AP), mucilage only (AM), and mucilage with beans (AMS). Values are means of triplicate ± stan-
dard deviations. Values in the rows bearing the different superscripts (a, b, ab) are significantly different (p < 0.05).
Fermentation 2021, 7, 53 11 of 13

Figure 3. Total quality score for coffee-based alcoholic beverages. Error bars show standard deviation
and error bars bearing different superscript differs (p < 0.05) with one-way ANOVA.

4. Conclusions
Byproducts of coffee (Coffea arabica L.) pulp and mucilage can be used for the prepa-
ration of fermented alcoholic beverages as well as ethanol for energy. This study showed
that the coffee pulp and mucilage could be a novel valuable and eco-friendly raw material
for the beverage industry and could help to reduce the environmental threat caused by
coffee processing. However, further research is necessary for quality assurance of alcoholic
beverages produced from coffee waste.

Author Contributions: Conceptualization, Y.K., L.D.S., D.K.C., B.R.P., T.R.B., J.L., S.T., J.U., and
R.H.D.; data curation, R.S., L.D.S., R.B., B.R.P., T.R.B., and J.L.; formal analysis, P.K.D.; investigation,
D.K.C.; methodology, P.K.D. and R.B.; resources, S.T.; supervision, J.U. and R.H.D.; validation, Y.K.
and R.H.D.; writing—original draft, Y.K.; writing—review and editing, J.L. and R.H.D. All authors
have read and agreed to the published version of the manuscript.
Funding: This study did not receive any funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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