Effect of Yeast Inoculation on Physicochemical Properties

of Fresh Cacao Beans

Dalia Sukmawati1, Shabrina Nida1, Nuranisa Putri Ariani2, Nadya Kurnia Maharani2,Syifa
Aulia Gunadi2.

1
First author affiliation, Country
2
Second author affiliation, Country

*Corresponding author: daliasukmawati@gmail.com

Abstract. Cocoa is a very important commodity for Indonesia as one of the third major expo
rting countries in international trade. However, in its production, the quality of cocoa b
eans in Indonesia is known to be very low, this is because one of the factors is that coco
a beans are not fermented. Fermentation is a chemical change in organic substances caused
by microbial enzymes. This study aimed to determine the effect of the administration of Sa
ccharomyces cerevisiae, Pichia kudriavzevii, Pichia scutulata and Pichia manshurica isolat
es on the physicochemical properties of wet cocoa bean fermentation. Fermentation was carr
ied out for 48 hours in a styrofoam box by soaking wet cocoa beans into the media of cocoa
husk extract and yeast extract with the addition of 5 inoculum combinations of the four ye
asts. Parameters observed during fermentation included temperature, pH, polyphenols, antio
xidants, reducing sugars and fermentation index. Based on the results of the research at 4
8 hours of fermentation time, the average pH and temperature produced ranged from 4.667 -
5. And 26 - 34°C, then the average yield of reducing sugar was 1.834 - 4.145 mg/mL, polyph
enol content ranged from 2.106667 - 3.79 mg/mL and antioxidant levels ranged from 0.790 -
0.843 and the fermentation index was 0.830 - 1.840.

Keywords: Saccharomyces cerevisiae, fresh cacao beans; fermentation; Pichia kudriavzevii;

Pichia scutulata; Pichia manshurica.
INTRODUCTION
Indonesia is one of the world's maj
or cocoa supplier countries with a percenta
ge of 5% after Côte d'Ivoire (42.3%) and G
hana (19.5%) (ICCO 2018). Abundant coc
oa production and high interest in domestic
chocolate consumption provide opportuniti
es for Indonesian cocoa farmers to improv
e the quality of cocoa beans through
fermentation process. Non-optimal post-ha
rvest handling can cause defects in seed qu
ality. Poor fermentation techniques will ca
use irreparable harm to the taste through su
bsequent processing modifications. Cocoa
beans without fermentation do not produce
the characteristic aroma of chocolate and h
ave an excessively astringent and bitter tast
e (Wahyudi et al. 2008).
Fermentation is the process of over
hauling sugars and citric acids in the pulp i
nto organic acids carried out by microbes o
f fermentation actors (Camu, et al., 2008.,
Meersman et al, 2013). These organic acid
s will induce enzymatic reactions in the se
eds so that biochemical changes occur that
will form compounds that give cocoa arom
a, taste, and color (Apriyanto et al., 2016b;
Afoakwa et al., 2014).
Fermentation occurs in two stages,
namely the anaerobic and aerobic stages. T
he anerobic stage occurs in a state of no ox
ygen, while the aerobic stage occurs in a st
ate of needing a lot of oxygen. The level of
anaerobic and aerobic microbial activity is
highly dependent on the availability of nutr
ients and built-in carbon sources as well as
the results of metabolites (alcohol and lacti
c acid) of fermented cocoa beans (Sukenda
r et al., 2019).
The success of a fermentation proc
ess of fresh cocoa beans is determined by t
he degree of fruit maturity and the length o
f fermentation time. In addition, the minim
um number of fermented beans affects cha
nges in the physico-chemical properties pr
ofile of fermented cocoa beans based on th
e results of the cut test. The precise proces
sing obtained is then applied at the farmer l
evel, especially on a scale of 20 kg / box
(Sukendar et al., 2019).
RESEARCH METHODOLOGY
Location and Time of Study
The study was conducted from
January to April 2022. The research was
conducted at the Microbiology Laboratory,
Faculty of Mathematics and Natural
Sciences, Jakarta State University.
Materials
Sample used in this study were
cocoa fruit peels and cocoa beans (TSH
and Sulawesi) obtained from Bogor.
Medium used was Yeast Mannitol Broth
(YMB). Other materials used in this study
were alcohol 96%, alcohol 70%, aquades,
NaOH, HCl.
Cocoa Beans Preparations
Ripe cocoa beans were split to half.
Cocoa beans and pulp then taken as
samples for the fermentation process.
Yeast Cultivation
Pichia manshurica and
Saccharomyces cerevisiae were grown in
100 mL YMB medium, then incubated for
±22 hours at room temperature (27- 28⸰C)
on a rotary shaker.
Starter Medium Preparation
Kultur dari Pichia manshurica dan
Saccharomyces cerevisiae., masing-
masing pada media YMB, sebanyak 1 ose
diinokulasikan ke dalam 100 mL media
YMB, diinkubasi pada rotary shaker dalam
suhu ruang (27- 28⸰C) selama ±22 jam.
Cocoa Beans Fermentation
Fermentation of the cocoa beans ta
kes place in Styrofoam boxes (40 cm x 40
cm) with a maximum wet cocoa bean coun
t of 2.5 kg/box. Fermentation is done by pl
acing the moist cocoa beans separated fro
m the cocoa pods in a fermentation box (4
0 cm in diameter and 40 cm in height) cov
ered with banana leaves. Wash leaves thor
oughly with antiseptic soap to wash off 96
% and 70% alcohol.
The seeds weigh up to 2.5 kg and
were gradually added with liquid medium
containing pure cultures of yeast isolates u
ntil they are completely absorbed by the co
coa beans. The number of pure cultures of
yeast added (108 cfu/g) was based on the n
umber of pure cultures added to normal co
coa fermentation (Ardhana and Fleet, 200
3) and the amount of water added was wei
ght based on the difference between of fres
h cocoa beans and cocoa beans.
The cocoa beans were placed in fer
mentation boxes. Additionally, each cocoa
bean prepared in the fermentation box is in
oculated with a yeast culture grown in the
starter solution. Fermentation took place at
room temperature with three processing ti
mes of 0 hours, 24 hours and 48 hours. Aft
er fermentation, the cocoa beans were rem
oved and dried in an oven at 50°C.
Characterization of Fermentation
Chart Title
40
35
30
25
0 jam 24 jam 48 jam
ABC ABD ACD
BCD ABCD K(-)
Characterization of fermented beve
rages includes pH and temperature measur
ements (Moore et al. 2011), reducing sugar
content (Miller 1959), polyphenol content,
antioxidant content, and fermentation inde
x. The Total Plate Count (TPC) method is acetic acid bacteria (BAA) predominate (S
used to calculate microbial growth. ukendar et al., 2019).

RESULTS AND DISCUSSION Reducing Sugar Content

pH and Temperature Reducing sugars are the result of m
The quality of fermented cocoa icroorganisms overtaking pectin, starch an
beans is determined mainly by the acidity d sucrose found in pulp and molasses durin
(pH) and temperature of the beans during g fermentation. Reducing sugars not only s
fermentation. The pH value is a parameter erve as raw materials for ethanol formation
for determining the acidity level of but also as potential flavor compounds in
fermented cocoa beans. Acidity comes cocoa beans. Reducing sugar content in co
from organic acids formed due to coa bean fermentation increases at the begi
metabolic reactions during the nning of fermentation, decreases during fer
fermentation process (Sabhannur, 2017). mentation, and remains stable until the end
The results of the pH analysis are of the fermentation stage (Afoakwa et al.,
2013). Figure 3 shows the results of the re
Chart Title
5 ducing sugar analysis.
3 Figure 3. Graph of Reducing Sugar
1
0 jam 24 jam 48 jam

ABC ABD ACD

BCD ABCD K(-)
presented in Figure 1.

Temperature is a parameter that ind

icates the occurrence of fermentation proce
Content of Fermented Cocoa Beans.
sses by microorganisms. These microorgan
isms convert the sugars and citric acid in th
Reducing sugars are primarily aro
e pulp into simpler organic acids. Ferment
matic precursor carbonyl compounds in fer
ation processes are carried out anaerobic a
mented cocoa beans that arise from the hy
nd aerobic using various types of active mi
drolysis of sucrose by the enzyme invertas
croorganisms. In anaerobic conditions, yea
e (Neilsen et al., 2013; Afoakwa, 2010). R
sts that thrive with lactic acid bacteria (BA
educing sugars in cocoa beans at the start o
L) predominate, and in aerobic conditions,
f fermentation increased by 4.5 ± 0.1 to 11.
45 ± 0.1 at 24 h fermentation and further d
ecreased to 10.6 ± 0.1 at 120 h fermentatio
n. Reducing sugars in dry cocoa bean pulp
during fermentation decreased from 4.7 ±
0.1 at the beginning of fermentation to 2.8
± 0.1 after 24 hours of fermentation, and d
ecreased to 1.5 ± 0.1 after 24 hours of ferm
entation and further 120 hours of fermentat
ion. Reducing sugars in fresh cocoa beans
at the start of fermentation increased by 7
± 0.1 to 12.5 ± 0.1 at 24 hours of fermentat
ion and further increased to 15.5 ± 0.1 at 1
20 hours of fermentation. Reducing sugars
in raw cacao bean pulp decreased slightly f
rom 3±0.1 at the start of fermentation to 2.
8±0.1 after 24 hours of fermentation, and f
urther decreased to 1.5±0.1 after 120 hours
of fermentation. Fermentation initiation in
creased sharply up to 24 hours fermentatio
n, decreased slightly up to 48 hours fermen
tation, and increased slightly up to 72 hour
s fermentation.
Fermentation Index
Cocoa beans from good fermentati
on produce cocoa beans with a color index
of ≥ 1, while cocoa beans that do not go th
rough suboptimal fermentation have a colo
r index of < 1 (Schwan and Wheals, 2004).
Mishnavi et al. (2002) found that anthocya
nins are the main components of polyphen
olic compounds in cocoa beans and exhibit
a reddish purple color with a maximum ab
sorbance of 500-550 nm under acidic cond
itions. Color can therefore be used to estim
ate the anthocyanin content of a cocoa bea
n, so the higher the anthocyanin content of
a cocoa bean, the less fermented the cocoa
bean means.
Polyphenol Content
In the cotyledon of the cocoa bean, there
are storage cells containing fats and
proteins as well as pigment cells
containing polyphenolic compounds.
Cocoa bean polyphenols are monomers,
namely epicatechin which is the main
flavanol with content of 34.65 to 43.27
mg/g (about 35% of total phenols). Other
flavanols are catechin, gallocatechin and
epigallocatechin (Kim and Keeney, 1984;
Wollgast and Anklam, 2000).
Based on the results of the total pol
yphenol test data that was conducted, the r
esults showed a decrease in polyphenol co
mpounds per hour. This is possible becaus
e fermentation of fresh cocoa beans for 5 d
ays can reduce polyphenolic compounds.
Camu et al. (2008) also showed that the re
duction of polyphenolic compounds reache
d 10-50%. The same was confirmed by (B
onvehi and Coll, 1997) that the content of
polyphenolic compounds decreased ±63 % Oxidation can be inhibited by a number of
from 150 mg/g to 55 mg/g. This may be methods, including preventing food from
because the polyphenol compounds in being exposed to oxygen and light, adding
cocoa beans are oxidized by an enzymatic antioxidants, storing at low temperatures,
reaction and aided by the presence of inactivating enzymes that react Oxidative
oxygen and the moisture content of the reactions and use of packaging (Pokorný et
beans which is always around 10-40%.. al., 2001; Choe and Min 2009).
Antioxidant Content Antioxidant supplementation is a widely
used method because it is relatively simple
Antioxidants are compounds that
and inexpensive. The uses of antioxidants
can inhibit, delay or prevent oxidative
can be distinguished in biological and food
reactions in biological systems and food
systems. The antioxidants in the biological
systems. Oxidative reactions are caused by
system have a role in fighting free radicals
the presence of free radicals that activate
in the body to prevent the occurrence of
the chain of oxidation reactions. Free
diseases such as cancer, atherosclerosis,
radicals are compounds in the form of
inflammation, cardiovascular disease and
atoms or molecules that have unpaired
tooth decay. Ito et al., 2003; Prior and Gu,
electrons in the outermost orbital so they
2005). Based on the calculated data and
have a mate-seeking reaction. Oxidation in
antioxidant graph, it can be concluded that
foods is caused by a reaction between
there is a but not significant reduction in
oxygen and unsaturated fatty acids,
antioxidant activity. The DPPH radical
proteins, carbohydrates or vitamins (Lee et
inhibitory activity decreased with
al., 2004).
fermentation time, meaning that the
decrease in antioxidant activity was due to
the decrease in polyphenols. Polyphenols
are the major components of cocoa beans
that play a role in antioxidant activity
(Steinberg, 2002). Based on this
explanation, it can be concluded that
antioxidants and polyphenols are closely
linked during the fermentation of cocoa
beans.

CONCLUSION

Based on the data, it can be concl

uded that the main polyphenols of
cocoa beans are flavanols in the f
orm of epicatechin, catechin and p
rocyanidin. The processing steps o
f cocoa beans that affect the tran
sformation of antioxidant compound
s are fermentation and roasting. F
ermentation reduces polyphenol con
tent through diffusion, oxidation,
and condensation. High-temperature
roasting also reduces the polyphen
ol content caused by phenolic degr
adation.

REFERENCE