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J.Exp. Life Sci. Vol. 6 No. 1, 2016 ISSN. 2087-2852
E-ISSN. 2338-1655
52
The Influence of Time Duration in Soybean Husk Fermentation
(Ihtifazhuddin et al.)
The Influence of Fermentation Time in the Physical and Chemical
Composition of Fermented Soybean Husk by Using Aspergillus niger on the
Quality of Raw Feed Materials
Muhammad Ikhwan Ihtifazhuddin1*, Happy Nursyam2, Arning Wilujeng Ekawati3
1Master Program of Aquaculture, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia
2Department of Aquaculture, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia
3Laboratory of Fishery Safety, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia
Abstract
Soybean husk (Glycine max L. Merrill) a soybean processing waste as raw material for tempe obtained after the process
of boiling and soaking soybeans. The main problem in the use of soybean husk (Glycine max L. Merrill) as feed material
is its crude fiber content which is fairly high. This study aimed to observe the fermented soybean husk using Aspergillus
niger to improve the quality of the raw feed materials. This was conducted by using completely randomized design
(CRD) analysis and repeated three times; the time optimization of Aspergillus niger in 2, 4, and 6 days based on
chemical analyses (moisture, protein, fat, ash, crude fiber and feed containing carbohydrates (NFE) and physical
assessment fermentation (smell, texture, moisture and hyphae) were analyzed descriptive qualitatively. The results
showed that 4 days fermentation of soybean husk using A. niger is successful gives the highest score based on physical
characteristics texture, aroma, moisture, and the formed hyphae and the most effective treatment for decrease in
crude fiber is 13% and increase in NFE contained in the largest on 4 days fermented soybean husk by Aspergillus niger
with a long time 4 days.
Keywords: Aspergillus niger, fermentation, soybean husk.
INTRODUCTION
Soybean husk is a waste that is produced
from the process of boiling and soaking soybeans
which were used as the materials to make
tempe. After going through the process, the husk
will be separated and will normally be thrown
away by the tempe producer.
Based on the analysis in the Laboratory of Bi-
ochemistry and Nutrition Fish, Faculty of Fishe-
ries and Marine Sciences, Brawijaya University
that soybean husk (Glycine max L. Merrill) has a
water content of 12.45%, 14.32% protein,
38.35% crude fiber, 2.32% fat, 4.14% ash and
2.42 kkal.g-1 energy. Therefore, soybean husk still
has the potential to be used as a feed for animals
considering that it has a high protein and ener-
gy [1].
The main problem in the use of the soybean
husk (Glycine max L. Merrill) as a raw material is
fairly high cellulose content of around 33.49%
[2]. Further explained that the soybean husk
(Glycine max L. Merrill) contains 10-20% hemicel-
lulose, 29-51% cellulose, 1-4% lignin and 6 -15%
pectin [3].
Correspondence author:
Muhammad Ikhwan Ihtifazhuddin
Email : ichwannfeng@gmail.com
Address : Master Program of Aquaculture, University of
Brawijaya, Veteran Malang, 65145
Technology to improve the quality of mate-
rials the feed is fermented [4]. Generally all fer-
mentation end products usually contain com-
pounds that are simpler and easier to digest than
the original material thus increase the nutritional
value [5]. The use of agricultural waste products
as fermentation substrate is due to the mass-
produced, the cost of which used lower and rich
in nutrients [6].
Cellulase enzyme complex is composed of cel-
lobiohidrolase, endoglucanase and β - glucosi-
dases which all act synergistically to convert
complex carbohydrates lignocellulosic biomass
into glucose efficiently [7]. Cellulase can be pro-
duced by fungi, bacteria, and ruminants. Produc-
tion of commercial enzyme normally uses fungi
or bacteria. Fungi can produce cellulases include
genus Trichoderma, Aspergillus, and Penicillium
[8]. Aspergillus niger has been widely used be-
cause it produces the three fundamental en-
zymes required for cellulolysis [9].
Previous research the use of A. niger can de-
crease crude fiber is already done. Declared by
the proximate analysis note that the content of
crude fiber grout tofu before it is fermented in
the amount of 24.03% and crude fiber content of
the grout tofu out after fermentation between
0.04 to 0.16% [10]. This is supported by other
research results [11], that the content of crude
J.Exp. Life Sci. Vol. 6 No. 1, 2016 ISSN. 2087-2852
E-ISSN. 2338-1655
53
The Influence of Time Duration in Soybean Husk Fermentation
(Ikhwan et al.)
fiber grout tofu 21.29% and after fermented de-
creased to 17.29%. A study mentioned that the
fermentation 2 to 4 days can decrease the crude
fiber [12].
The high fiber content and the lack of other
nutritional content of the constraints of local
feed use this as a source of alternative feed
prospective. Thus, this study aims to observe
different time duration in the physical and chem-
ical composition fermentation soybean husk to
improve the quality of fish feed as the feed mate-
rials.
MATERIALS AND METHODS
Soybean Husk Fermentation
Briefly, dried and ground soybean husk 100 g
each were placed in a 600ml beaker glass and
autoclaved at 121°C for 20 min [13]. After that,
the soybean husk was densified by using dilution
of 106 [14]. The soybean husk (Glycine max
L. Merrill) was then added molasses in the ratio
of 1:1 with the given mold dose [15] and the sub-
strate was then stirred until it became homoge-
neous and mixed with sterile water until the wa-
ter level reaches 70% [11]. Then, the tray was
covered with plastic wrap and laid in the incuba-
tor at a temperature of 30°C with pH 5 [16].
Physical Assessment Fermentation
Physical Assessment Fermentation is used in
order to determine differences in physical quality
which appear, on the soybean husk that fe r-
mented and non-fermented. The scoring media
of fermentation soybean husk started from 1 to 4
(Bad, Less good, Good and Excellent) where
greater score indicated good fermentation [17].
In the scoring media of fermentation soybean
husk is shown in table 1 below.
Table 1. Physical Assessment Fermentation
Score
Lumps (%)
Scent (%)
Water
Steam
(%)
Hyphae
(%)
1
<10
>10
<10
<10
2
<10 - <25
>10 - <25
<10 - <25
<10 - <25
3
>25 - <40
>25 - <40
>25 - <40
>25 - <40
4
>40
<40
>40
>40
Notes: Lumps (Soft <10, Few <10-<25%, Some>25-<40%, A lot
of >40%), Scents (No <10%, Slight >10-25<%, Normal >25-
<40%, Strong <40%), Water steam (Dry <10%, Normal >10-
<25%, Less >25-<40%, Moist >40%), Hyphae (No <10%, Few
>10-<25%, Several >25-<40%, Many >40%).
Chemical analysis
The parameters observed in this research in-
clude chemical compounds contained in fer-
mented soybean husk before and after being
fermented through proximate test (moisture
content, ash content, crude protein, crude fat,
and crude fiber. The proximate analysis which
was tested with the analysis of water content at
a temperature of 105°C by using the oven for 6
hours, while the protein analysis was analyzed by
using Kjeldahl method. Simultaneously, the fat
was also analyzed by using soxhlet and petr o-
leum ether in order to dissolve the fat. Crude
fiber was assessed by using a solution of
H2SO4 and NaOH as the solvent and the analyza-
tion of the ash was carried out by using a furnace
with a temperature of 600°C for 2 hours [18].
Statistical analysis
Statistical analysis used the analysis of va-
riance (ANOVA). ANOVA was used to test the
effect of the treatment and then further used the
least significant difference (LSD) test at the level
of 5%. In the other hand, the data of organoleptic
test were analyzed descriptively qualitative.
RESULTS AND DISCUSSION
Successful Rate Fermentation
Based on the results, the best fermentation
time was on the 4th day. The physical observation
of the fermentation soybean husk includes tex-
ture, aroma, moisture, and the formed hyphae.
The data scoring of fermentation soybean husk
can be seen in Figure 1 below:
Figure 1. The average scoring value of physical assess-
ment time fermentation containing ■ 6 days
■ 4 days ■ 2 days fermentation soybean husk.
The high scoring value indicated that the fer-
mentation process has been going very well. The
best result of the scoring occurred on the 4th
day. On the 4th day gives the highest score based
on physical characteristics lumps, scent, water
steam, and the formed hyphae.
Moreover, the texture or lumps of the fer-
mented soybean husk indicated the best result
on the 4th day compared to other days. Fermen-
tation causes the changing nature of the feed
J.Exp. Life Sci. Vol. 6 No. 1, 2016 ISSN. 2087-2852
E-ISSN. 2338-1655
54
The Influence of Time Duration in Soybean Husk Fermentation
(Ihtifazhuddin et al.)
material including the texture; it is as a result of
the content cleavage in the foodstuff caused by
microorganisms that are in there [19]. Establish-
ment of a texture is also influenced by water con-
tent, fat content, type and amount of carbohy-
drate food products [20].
Another thing is that in the 2nd and 4th day,
the aroma of the fermentation soybean husk
showed the best fragrance that is slightly sour
and fragrant but it created an acid aroma and an
odor of ammonia on the 6th day. Good fermenta-
tion has a sour and fragrant aroma [21]. Effect on
the hydrolysis time increased levels of NH3 (am-
monia) giving rise to acid aroma [22]. Feed given
additional fermentation may be associated with
aroma and flavor that can affect the appetite of
the animal [23].
In the fermentation with A. niger, water
steam was formed on the 4 and 6th day because
of an exothermic reaction when the process of
organic material cleavage occurred. Fermenta-
tion process will produce CO2 and heat as a result
of the organic material breakdown [24]. The wa-
ter content also affected the growth of mold and
dynamics that occur during the process ensilase
because water is required for the synthesis of
protoplasm microorganisms and dissolved organ-
ic compounds [25].
Based on the results of the 2nd day, there
were some very heavy hyphae throughout the
fermentation media, and besides that, several
spores grew well in some of the fermentation
points. On the 6th day, hyphae began to decrease
because the spores grew very much. Previous
research also demonstrated that Coconut oil cake
and palm kernel oil cake that were fermented
with A. niger would have hyphae by 90% and
spores by 10% on the edge of the spores on the
4th day [26]. Hyphae thrive but few spores that
grow on the fourth day, so that the material is
more easily digested and utilized because the
spores increase fiber content material [5].
Improvement Nutrient Content
The results of the chemical analysis can be
seen in Table 2 below. Based on the results of
proximate treatment C (fermented for 4 days)
are considered better, it can be seen from the
decline in crude fiber contained in the largest C
treatment is 13%. This observation is not as good
as the research of leaves lamtoro fermented with
A. niger crude fiber decreased by 46.61% for 3
days [5]. The decrease in crude fiber occurred
due to A. niger has three essential enzymes
needed for celluloses [9]. The increased crude
fiber during the final phase of fermentation vege-
table waste was due to the utilization of the nu-
trients provided by the mold and then the reduc-
tion can be attributed to breaking the non-starch
polysaccharide for mold protein [27]. Crude fiber
is part of a carbohydrate that difficult to digest by
the digestion of fish, the higher the fiber in the
diet then lowers the energy [28]. High crude fiber
will give a sense of satiety because of the compo-
sition of complex carbohydrates that stops the
appetite that caused a decline in food consump-
tion [29]. The fiber content is too high will sup-
press growth [30].
Based on the proximate result of an increase
in the value of the protein with the length of
fermentation time, testing crude protein fer-
mented soybean husk is the highest by a long
period of 6 days at 17.02%. Vegetable waste and
fermentation by using A. niger S14 for 8 days
increased the protein by 38% [27]. Increased
protein content after the fermentation process
probably derived from A. niger which has synthe-
sized the urease enzyme to break urea into am-
monia and CO2. This ammonia was then used for
the mold to form amino acids (protein) [31].
In the 6th day fermentation, higher A. niger
content was given and it resulted in fat degrada-
tion. It happened because the mold has achieved
an exponential growth [12]. The decreased fat in
fermented palm kernel oil cake flour occurred
due to the conversion of fat into a single protein
biomass [32]. Lipase enzyme produced by fungi
greatly affects crude fat content after fermenta-
tion substrate because the enzyme lipase will
remodel fat to be used by fungi as an energy
source [33]. Microbial lipases have been used as
a catalyst in producing oleochemicals-based
products include fats or oils such as triglycerides
modified low-calorie [34], so even though the
resulting reduced oils or fats rich in EPA and DHA
[35].
Increased NFE in the 4th day fermentation due
to increased glucose as a result of fermentation
of A. niger hydrolyze cellulose. Glucose levels
continued to rise from 8 up to 64 hours, but de-
clined to 72 in rice straw fermentation using A.
niger [36]. Aspergillus niger also produce ß-
glucosidase enzyme that is strong that this en-
zyme serves to accelerate the conversion of cel-
lobiose widened glucose [37]. Carbohydrates are
used as energy source of non-protein replacing
protein as an energy source. If the feed shortage
of non-protein energy then the fish will use a
portion of the protein to insufficient energy
needs [38]. Feed containing carbohydrates (NFE)
J.Exp. Life Sci. Vol. 6 No. 1, 2016 ISSN. 2087-2852
E-ISSN. 2338-1655
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The Influence of Time Duration in Soybean Husk Fermentation
(Ikhwan et al.)
exact to reduce the use of protein as an energy
source known as protein sparring effect [39].
In the trials, the dry matter loss was about 7%
in the present study. 20% dry matter loss after 96
h of fermentation of mixed oil cakes with A. niger
616 [40]. In a similar study with wheat bran, has
also reported significant (p < 0.05) reduction in
dry matter throughout the fermentation period
for wheat bran using A. niger S14, suggesting
utilization of nutrients present in the substrate
by fungi for its growth and metabolic activities
[41]. Feed given additional fermentation may be
associated with aroma and flavor that can affect
the appetite of animal and an additional
fermentation in feed also provides an additional
element of essential amino acids [23].
Table 2. The results of the fermentation soybean husk proximate analysis
Table 2. Result of observation towards chemical composition including dry content, ash, protein, fat, crude fiber and NFE in
different time fermentation soybean husk towards A. (0 days), B. (2 days), C. (4 days) and D. (6 days). Values are means±SEM of
three replicates (n=3); means in the same row followed by the same superscript letter are not significantly different (DMRT,
p>0.05). *NFE (Nitrogen Free Extract) = 100-Protein-Fat-Fiber-Ash.
CONCLUSION
The results show that 4 days fermentation of
soybean husk using Aspergillus niger is successful
gives the highest score based on physical charac-
teristics texture, aroma, moisture, and the
formed hyphae and chemical analysis result the
best time fermentation is 4 days the most effec-
tive treatment for decrease in crude fiber and
increase in NFE contained in the largest on 4 days
fermented soybean husk by Aspergillus niger
with a long time 4 days. All of these changes en-
hance the value of the soybean husk as an animal
feed, including aquafeed.
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Composition
Treatment
A
B
C
D
Dry content (%)
87,34±1,56a
82,75±0,58b
83,21±0,57b
82.12±0,39b
Ash (%)
4,14±0,30a
3,67±0,37a
3,91±0,31a
3.84±0,70a
Protein (%)
14,32±0,64a
15.13±0,05a
15,55±0,11a
17.02±0,18b
Fat (%)
2,32±0,43a
2,13±0,09a
2,03±0,14a
2.03±0,1a
Crude fiber (%)
38,35±0,65d
37,52±0,28c
33,45±0,39a
35.72±0,30b
NFE (%)*
40,87±0,78a
41.55±0,73a
45,06±0,67b
41.39±0,71a
J.Exp. Life Sci. Vol. 6 No. 1, 2016 ISSN. 2087-2852
E-ISSN. 2338-1655
56
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