INTERNATIONAL JOURNAL OF APPLIED TECHNOLOGY RESEARCH

2024, VOL. 5, NO. 1, pp. 62-70

https://ijatr.polban.ac.id/ POLBAN

The Effect of Pineapple Crude Enzymes and Fermentation Time on

The Decaffeination Process of Robusta Coffee
a
Tri Hariyadi*, aTifa Paramitha, aDwi Irmawati, aSalwa Ainaya Salsabila
a
Department of Chemical Engineering, Politeknik Negeri Bandung, Bandung 40559, Indonesia

Received July 7, 2023; Revised December 7, 2023; Accepted January 4, 2024; Published February 5, 2024

ABSTRACT
KEYWORDS
The decaffeination of robusta coffee can be done through fermentation with
a crude enzyme containing bromelain enzyme from pineapple. The study Robusta coffee
aims to determine the activity of the bromelain enzyme by the Kunitz method, Caffeine
the effect of fermentation time and crude enzyme concentration on the Decaffeination
decaffeination process, and the characteristics of coffee before and after Fermentation
fermentation using FTIR. The fermentation time was varied from 6 to 36
hours and the crude enzyme concentration was varied from 10 to 80%. The
activity of the bromelain enzyme was 36 U/ml. Fermentation time affects the
decaffeination process, wherein the longer the fermentation time from 6
hours to 36 hours obtained caffeine content from 2.39% to 0.07%. Besides
that, the crude enzyme concentration affects the decaffeination process,
which obtained the lowest caffeine content or percent decaffeination at the
crude extract concentration of 80% (v/v). FTIR results showed that the
decaffeination process affected the reduction of caffeine in coffee samples. It
can be shown from the increase in the %T value of the C-N functional group
from 40.731 to 54.85.

INTRODUCTION
Indonesia is one of the largest coffee-producing countries in the world. Based on data released by
the United States Department of Agriculture (USDA) in 2020 [1], Indonesia occupies the third
position as the largest coffee producer in the world. Besides that, coffee production in Indonesia
increases every year. Based on the Central Bureau of Statistics, coffee production in 2021 was
762.380 tons [2] and rose to 786.190 tons in 2021 [3].
One of the ingredients in coffee is caffeine, which is beneficial for the human body. Caffeine
has clinically beneficial pharmacological effects, such as stimulating the central nervous system,
relaxing smooth muscle, especially bronchial smooth muscle, and stimulating cardiac muscle [4].
However, caffeine consumption beyond the maximum limit will have a negative effect on the
human body. Caffeine levels in robusta coffee beans (1.5-2.6%) are greater than in arabica coffee
beans (0.9-1.4%) [5], so the caffeine content in robusta coffee has more potential to cause
negative effects on the human body.
Excessive caffeine consumption can cause nervousness, anxiety, tremors, insomnia,
hypertension, nausea, and seizures [6]. According to the Food Drug Administration (FDA), the

*Corresponding Author: [email protected]

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 INTERNATIONAL JOURNAL OF APLLIED TECHNOLOGY RESEARCH

permissible caffeine level is 100-200 mg/day, while according to SNI 01-7152-2006, caffeine levels
in food are 150 mg/day, and beverages are 50 mg/serving [7]. Many researchers have conducted
studies to improve the quality of coffee by decreasing its caffeine content so that it is safe for
human consumption. One way to decrease caffeine content is by decaffeination [8].
Decaffeination is the process of reducing the caffeine content in coffee beans. A commonly
used decaffeination method is by using water solvents. However, this method has a disadvantage,
namely, volatile compounds are lost during the process, thus affecting the aroma and flavor of
the coffee beans [9]. Another frequently used solvent is chemical solvents, which have the
disadvantage of leaving residues. The fermentation method using enzymes is one of the suitable
options for decaffeination because it has no drawbacks, as mentioned above.
Proteolytic enzymes can be used in the decaffeination process because they can help soften
and break membrane components, thus facilitating caffeine's dissolution. A previous study by
Rohman [10] showed that the use of proteolytic enzyme can decrease caffeine content from 1.89%
to 0.58%. Besides that, a study by Daisa et al. [11] stated that the use of crude papain enzyme
containing proteolytic enzyme can decrease caffeine content from 1.94% to 1.23%. Some types of
proteolytic enzymes are known, such as papain enzyme from papaya, bromelain enzyme from
pineapple, rennin from cattle and pigs, and fisin from ficus tree sap [12]. According to Oktadina
et al. [13], the bromelain enzyme contained in pineapple fruit can break down protein
compounds, accelerating the release of mucus in coffee beans and reducing caffeine content. The
breakdown of protein causes a reduction in bitterness in coffee, makes the smell more fragrant,
and increases free amino acids in coffee [14]. Noviar et al. [15] also explained that besides
reducing caffeine and coffee protein levels, bromelain enzyme can improve the taste of the coffee
produced.
In this study, the fermentation method was used to decrease the caffeine content of robusta
coffee by using a crude enzyme containing bromelain enzyme from pineapple. The operating
conditions of fermentation were varied, namely crude enzyme concentration and fermentation
time. The characteristics of coffee and decaffeinated coffee were analyzed using Fourier
Transform Infra-Red (FTIR).

RESEARCH METHOD
Equipment and materials
The main tools used in this study are shown in Table 1.

Table 1. The main tools used

No. Tools Specifications Quantity Function
1. Split Funnel 250 mL 3 Extraction tools
2. Waterbath Memmerr 1 Evaporating the solution
3. Oven Memmert 1 Drying coffee beans
4. Centrifugation Thermo 1 Separation of solid and solution
Scientific
5. Spectrophotometry UV- Evolution 1 Measurement of caffeine content and
Vis enzyme activity
6. Blender Miyako 1 Mash pineapple
7. Coffee Grinder - 1 Grinding coffee beans
TRI HARIYADI, ET. AL.

The materials used in the study are shown in Table 2.

Table 2. The main materials used

No Material Function
1. Green bean robusta coffee Raw material
2. Pineapple Raw material for crude enzyme
3. Chloroform Separating caffeine from coffee solution
4. Pharmaceutical caffeine Standard solution of caffeine
5. Buffer Tris HCl Enzyme purification

The preparation of crude extract

The preparation of crude extract referred to Koh et al. [16] with modification. Pineapple flesh was
homogenized with cold phosphate buffer at pH 7 for 30 minutes. Then, the pellet and supernatant
were separated by centrifugation for 15 minutes at a speed of 5000 rpm. The supernatant was
used as a crude enzyme for the fermentation process.

The activity test of the bromelain enzyme

To purify the bromelain enzyme from the crude enzyme, the crude enzyme was added with 80%
(b/v) ammonium sulfate, and the mixture was incubated for 24 hours at 4 °C. Then, the mixture
was centrifuged to separate the supernatant and pellet. Centrifuged pellet (deposit) was on
dialysis using cellophane bags and soaked in tris HCl buffer. The Kunitz Method was used to
determine the activity of the bromelain enzyme [17]. The sample from the last steps was added
with 1% (b/v) casein and then incubated at 37℃ for 30 minutes. After that, the mixture was
added with 0,1% (v/v) TCA, homogenized, and then centrifuged. The supernatant was measured
its absorbance at a wavelength of 280 nm.
The fermentation process

Robusta coffee beans were sorted and washed thoroughly, weighed as much as 400 grams, and
put into a jar. Then, the crude enzyme solution was added to the jar. The concentration of crude
enzyme to the solvent (water) (v/v) was varied, namely 10%, 20%, 30%, 40%, 50%, 60%, 70%,
and 80%. Then, it was incubated at room temperature for 36 hours with a sampling every 6
hours. The fermented sample was dried for 3 hours in the oven at 105 ℃. The dried coffee beans
were then roasted with home cooking utensils for ±10 minutes. The roasted coffee beans were
ground and sifted to a particle size of about 60 mesh. The caffeine content of samples was
analyzed using UV-Vis spectrophotometry, and the functional groups of the sample were
characterized by FTIR.

RESULT & DISCUSSION

Total activity of the bromelain enzyme
The activity of the bromelain enzyme was determined by the Kunitz Method. Based on the
result of this study, the total activity of the bromelain enzyme was 36 U/mL. The total activity of
the bromelain enzyme in this sample was slightly higher than the results in the study of Ilyas et
al. [17], namely 34 U/mL. It can happen because this study used Bogor pineapple fruit that was
 INTERNATIONAL JOURNAL OF APLLIED TECHNOLOGY RESEARCH

young and had a green color. Silaban et al. explained that the bromelain activity of young
pineapple fruit was higher than that of ripe pineapple fruit [18].
The effect of fermentation time on caffeine content in robusta coffee beans

The caffeine content of decaffeinated coffees was measured to determine the decreased caffeine
content resulting from the fermentation process. The caffeine content was measured using UV-
Vis Evolution Spectrophotometry with a maximum wavelength of 272.9 nm. The results of the
caffeine content of decaffeinated coffees against the fermentation time are represented in
Figure 1.

Figure 1. Graph of the effect of fermentation time on caffeine content at variation of crude enzyme concentration

Before the fermentation process, the caffeine content of robusta coffee beans was 2.39%.
Figure 1 shows that caffeine content decreases with increasing fermentation time, with the highest
decrease occurring in samples of crude enzyme concentration of 80% (v/v) and fermentation
time of 30 hours. The decrease in caffeine content has been relatively constant at fermentation
time from 30 hours to 36 hours, starting from a crude enzyme concentration of 60% to 80%
(v/v). However, the highest decrease of all samples occurred at the fermentation time of 36 hours,
with decreasing caffeine content from 2.39% to 0.07% as the lowest caffeine content.
The results of this study are in accordance with the study of Oktadina et al. [13]. Based on
this study, the best fermentation time to decrease caffeine content was 36 hours, with variations
in fermentation time for 24 hours, 36 hours, and 48 hours. From this study, it can be concluded
that the longer the enzymatic process between the bromelain enzyme and coffee, the more
substrate will break down because the contact time between the enzyme and the substrate is
longer. However, after exceeding the limit of enzyme effectiveness, the caffeine content becomes
relatively constant [19]. Caffeine content can decrease because the crude enzyme containing
bromelain enzyme will break down proteins in the cell wall so that caffeine in the cell wall will
dissolve in water [20].
According to SNI 01-3542-2004, the maximum caffeine content of ground coffee is in the
range of 0.45-2 % b/w. Based on this study, decaffeinated coffee powder contains caffeine in the
range of 0.07%-2.26%. Decaffeinated coffee powder that can meet SNI 01-3542-2004 produced
from the fermentation process, namely at fermentation time of 12 hours to 36 hours with a crude
enzyme concentration of 10-80% (v/v) and at fermentation time of 6 hours with crude enzyme
concentration of 20-40% and 70%-80%. Based on the research presented by Smith et al. [8],
TRI HARIYADI, ET. AL.

coffee with a relatively low dose of caffeine does not cause side effects on health but can increase
alertness and body performance. However, it is seen less than coffee with high caffeine doses.
The effect of crude enzyme concentration on the percent decaffeination of robusta coffee
beans
Figure 2 shows the effect of crude enzyme concentration on the percent decaffeination of robusta
coffee resulting from the fermentation process. Based on Figure 2, there is a tendency that the
higher the crude enzyme concentration added to the fermentation process, the higher the percent
decaffeination.
The percent decaffeination increased from 5.55% to 96.6% as the crude enzyme
concentration was added from 10% to 80% (v/v). The highest percent decaffeination was
obtained in samples with a crude enzyme concentration of 80% (v/v) and a fermentation time of
30 hours, while the highest average percent decaffeination from all samples occurred in the crude
enzyme concentration of 80% (v/v). It shows that the higher the crude enzyme concentration,
the more the bromelain enzyme is involved in the fermentation process, so the percent
decaffeination becomes higher. This result was in accordance with the research conducted by
Daisa et al. [11], in which more papain enzymes as proteolytic enzymes result in a lower caffeine
content after the fermentation process. During the fermentation process, the papain enzyme will
hydrolyze the proteins in vacuoles into amino acids, and then compounds in vacuoles, such as
caffeine, will come out.

Figure 2. Graph of the effect of crude enzyme concentration on percent decaffeination

The characteristics of robusta coffee before and after the fermentation process
FTIR characterization was performed on coffee samples before and after the fermentation process
with the lowest caffeine content to identify functional groups. Coffee has a very complex content.
Caffeine belongs to the tertiary amine group characterized by a C-N group with a wavenumber
of 1030-1230 cm-1. In coffee samples before fermentation (Figure 3), the C-N functional group
was shown at a wavenumber of 1161.15 cm−1.
 INTERNATIONAL JOURNAL OF APLLIED TECHNOLOGY RESEARCH

Figure 3. IR spectrum of the coffee sample before the fermentation process

Figure 4 shows the FTIR result of the coffee sample after the fermentation process with the
lowest caffeine content. The C-N/tertiary amine functional group was shown at a wavelength of
1161.15 cm-1. Figures 3 and 4 show a change in the %T value of the C-N functional group, which
is 40.731 for the sample before the fermentation process and 54.855 for the sample after the
fermentation process. A higher %T value indicates a lower absorption intensity of the functional
group and vice versa [21]. It suggests that the presence of caffeine in coffee after fermentation
decreased.

Figure 4. IR spectrum of the coffee sample after the fermentation process

CONCLUSION
The crude enzyme of pineapple fruit used in the fermentation process contains a bromelain
enzyme. The activity of the bromelain enzyme in this study was 36 U/ml. The highest decrease of
all samples occurred at the fermentation time of 36 hours, with decreasing caffeine content from
2.39% to 0.07% as the lowest caffeine content. Percent decaffeination increased from 5.55% to
TRI HARIYADI, ET. AL.

96.6% as the crude enzyme concentration increased from 10% to 80% (v/v). There was a change
in the %T value of the tertiary C-N/amine functional group, as indicated by caffeine samples
before and after fermentation, from 40.731 to 54.855. It suggests that the presence of caffeine in
coffee after fermentation decreased.

LIMITATION AND FUTURE RESEARCH

Continuation of research needs to be carried out, including organoleptic tests to compare the taste
and aroma of decaffeinated coffee resulting from the fermentation process using bromelain
enzyme and without the fermentation process.

ACKNOWLEDGEMENTS

The authors would like to thank Politeknik Negeri Bandung, who funded this research.

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