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hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 472
JOURNALOFADVANCEDVETERINARYANDANIMALRESEARCH
ISSN2311-7710(Electronic)
hp://doi.org/10.5455/javar.2018.e301December 2018
A periodical of the Network for the Veterinarians of Bangladesh (BDvetNET)VOL5,NO.4,PAGES472–480
ORIGINALARTICLE
Eect of two-step fermentaon by Chrysonilia crassa and Bacillus sublis on nutrional
values and anoxidave properes of agro-industrial by-products as poultry feed
ingredients
SugihartoSugiharto,IsroliIsroli,TurriniYudiar,EndangWidiastu,HannyIndratWahyuni,TriAgusSartono
DepartmentofAnimalScience,FacultyofAnimalandAgriculturalSciences,DiponegoroUniversity,Semarang,Indonesia
Correspondence Sugiharto Sugiharto sgh_undip@yahoo.co.id Department ofAnimalScience, FacultyofAnimal and Agricultural
Sciences,DiponegoroUniversity,Semarang,Indonesia.
How to cite:SugihartoS,IsroliI,YudiarT,WidiastuE,WahyuniHI,SartonoTA.Eectoftwo-stepfermentaonbyChrysoniliacrassa
andBacillussublisonnutrionalvaluesandanoxidaveproperesofagro-industrialby-productsaspoultry feedingredients.JAdv
VetAnimRes2018;5(4):472–80.
ABSTRACT
Objecve:Thiscurrentstudywassubjectedtoinvesgatetheinuenceoftwo-stagefermenta-
onbyChrysonilia crassaandBacillus sublisonnutrionalvaluesandanoxidaveproperes
ofagro-industrialby-products.
Materials and methods:Two-stage fermentaon with Ch. crassa (inoculatedin advance; sin-
gle-stepfermentaon) andB. sublis(inoculated later;two-stepfermentaon)wasconducted
onagro-industrialby-products,i.e.,bananapeelmeals,cassavapulp,andricebran.ThepHmea-
surement,microbialenumeraon,proximate,andanoxidantanalyseswereconductedfollowing
4-and2-daysaerobicincubaonwithCh. crassaandB. sublis,respecvely.
Results: The pH of banana peels and cassava pulp increased with Ch. crassa-fermentaon,
but then decreased following B. sublis-fermentaon. Chrysonilia crassa-fermentaon did not
change,but B. sublis-fermentaondecreasedpHofricebran.Thenumberoflaccacidbacteria
washigherintwo-stagethaninsingle-stagefermentedby-products.Crudeproteinandfatwere
higherinfermentedthaninunfermentedbananapeels.Crudeproteinwashigherinsingle- and
two-stage fermented, while fat higher in single-stage fermentedthan in unfermented cassava
pulp.Crudefatandashcontentsincreasedwithfermentaoninricebran.Single-stagefermenta-
onincreasedsomeofaminoacidscontentsinbananapeelsandcassavapulp.Theconcentraon
ofpolyphenols,tannins,andanoxidantpotenalof bananapeels reducedwithfermentaon.
Totalpolyphenolsandtanninswerehigher,whereasanoxidantacvitywaslowerinfermented
thaninunfermentedcassavapulp.Totalpolyphenols,tannins,andanoxidantacvitywerelower
intwo-stagethaninsingle-stagefermentedandunfermentedricebran.
Conclusion: Single-stage fermentaon with Ch. crassa improved nutrional characteriscs of
agro-industrialby-products.
ARTICLE HISTORY
ReceivedOctober08,2018
Revised:November08,2018
AcceptedNovember10,2018
PublishedDecember02,2018
KEYWORDS
Agro-industrialby-product;banana
peels;cassavapulp;ricebran;
fermentaon
Introducon
In response to the increase in feed price, nutritionists are
now searching for alternative feedstuffs for poultry. Agro-
industrial by-products have long been used as alternative
feed ingredients in poultry ration as they are abundantly
available throughout the year. However, the inclusion of
such by-products into poultry ration is often limited by
of antinutritional factors [1,2]. It has widely been known
that fermentation could be an easy technique to improve
the nutritive values of agro-industrial by-products.
contents of agro-industrial waste, so that can be incor-
porated in poultry ration at higher proportions [2,3]. In
addition to the improved nutritional characteristics, fer-
mentation may produce functional properties, such as
antioxidants, which are essential for poultry health [2].
Fermentation has commonly been carried out either
with single- or two-stage fermentation method, depend-
ing on the purpose of fermentation [4,5]. With respect to
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hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 473
nutritional improvement, two-stage fermentation may,
however, favor better nutritional characteristics in the
fermented products, relative to single-stage fermentation
[4,6,7]. Indeed, two-phase fermentation with Rhizopus spp.
and B. subtilis resulted in higher protein content, when
compared with fermentation using B. subtilis alone [7]. In
another work, two-step fermentation with B. subtilis and
Enterococcus faecium could increase crude protein content,
amino acids, ash and total phosphorus. Such fermentation
phytate in maize-soybean meal based feed [8]. Likewise,
recent study showed that two-stage fermentation resulted
in higher level of polyphenols in the vinegars generated
from cornelian cherry, when compared with that produced
through single-stage fermentation [5]. Owing to the afore-
mentioned studies, two-stage fermentation could be the
preferred method to increase the nutritive and functional
values of by-products derived from agro-industries.
Application of two-stage fermentation is generally sub-
jected to gain the merits of both microorganisms used as
starter cultures [7]. Shi et al. [8] used B. subtilis
fermentation starter to decrease antinutritional factors,
while E. faecium was used in the second step for produc-
ing acids as well as lowering the pH values of feed. In the
earlier preliminary study, we did fermentation on rice
bran using Ch. crassa, a fungus isolated from intestine of
the Indonesian indigenous chicken. The fungus lowered
fat contents of rice bran [9]. In another studies, fermen-
tation with B. subtilis increased the content of solvable
sugars, solvable proteins, crude protein, and crude fat in
soya bean hull [7,10]. Taking these into consideration,
fermenting in advance with Ch. crassa followed with B.
subtilis was, therefore, expected to decrease and increase
-
dustrial waste. It has been shown from the previous study
that some agro-industrial by-products possess antioxida-
tive properties, which may promote the healthy growth of
poultry [2]. Considering the antioxidative features of Ch.
crassa [9] and the antioxidant-enhancing effect of B. sub-
tilis [11,12], two-step fermentation with Ch. crassa and B.
subtilis was, therefore, subjected not only to improve the
nutritional qualities, but also to increase the antioxidant
potentials of the agro-industrial waste. The objectives of
the current work were to evaluate the impact of two-stage
fermentation by the fungus Ch. crassa and later by B. sub-
tilis on nutritional values and antioxidative properties of
agro-industrial by-products as poultry feed ingredients.
Materials and Methods
Fermentaon procedures
The isolate of Ch. crassa was obtained from the stock
culture of fungi kept at 4°C. The isolate was rejuvenated
on potato dextrose agar (PDA; Merck KGaA, Darmstadt,
Germany) containing chloramphenicol. After 48 h aerobic
incubation (at 38°C), the mycelia of fungi were dislodged
and diluted in sterile distilled water (100 ml). The afore-
mentioned suspension (inoculum) was subsequently inoc-
ulated to 500 gm of sterilized (using autoclave at 121°C
for 15 min) agro-industrial by-products (i.e., cassava pulp,
banana peel meals, and rice bran). For each inoculation, the
inoculum was standardized to contain ca. 1 × 1012 cfu/ml
of Ch. crassa. To obtain the water content of ca. 40% in the
substrates during solid-state fermentation, a 400, 300, and
100 ml of sterilized water was incorporated into the cas-
sava pulp, banana peel meals, and rice bran, respectively.
Following the 4 days of aerobic incubation at room tem-
perature, sample (100 gm, “as is”) from each culture was
collected for pH measurement, microbial enumeration,
proximate, and antioxidant analysis. The rest of the fer-
mented agro-industrial by-products were then inoculated
with B. subtilis (1 mg/gm inoculum containing minimum
1010 spores/gm; PT. Bayer Indonesia, Jakarta, Indonesia)
and aerobically fermented for 2 days at room temperature.
Samples from every culture were eventually obtained for
the analysis. The experiment was conducted using tripli-
cate replications.
Measurement of parameters and data analysis
The value of pH of each sample was determined with pH
meter (Eutech EcoTestr pH 1, Thermo Fisher, Singapore).
The population of coliform in every sample was counted
on MacConkey agar (Merck KGaA) as red colonies. The
coliform enumeration was conducted after 24 h aerobic
incubation at 38°C. The number of lactic acid bacteria
(LAB) was determined on deMan, Rogosa, and Sharpe
(MRS; Merck KGaA) agar. The bacteria were enumerated
after 48 h anaerobic incubation at 38°C. The number of
yeast was counted on PDA (Merck KGaA) containing chlor-
amphenicol. The enumeration was performed after 48 h
aerobic incubation at 38°C. The lowest dilution applied for
the microbial counting was 1:10,000.
The chemical characteristics of each by-product were
assessed following the procedures/proximate analysis [13].
The content of amino acid in each product was measured
according to a standard ultra-performance liquid chroma-
tography procedure [14]. The antioxidant activities of the
agro-industrial by-products were determined by the 2,
2-diphenylpicrylhydrazyl (DPPH) test according to Wu et al.
[15]. The assay was preceded by the preparation of the sam-
ple extracts by dissolving the sample of each by-product (10
gm) in methanol (100 ml). Ultrasonication for 30 min and
maceration for 3 days were then conducted. Homogenization
for 30 min was conducted every day (during the period of
maceration) with magnetic stirrer. Subsequently, evapora-
tion of the homogenate was done using rotary vacuum evap-
orator (50°C, 100 rpm, Sigma-Aldrich, St. Louis, MO) until
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 474
the volume of the homogenate was 25 ml. To test the free
radical scavenging activity, the homogenate (0.5 ml) was
diluted in DPPH solution (3 ml) and incubated at room tem-
perature for 30 min. The absorbances of the solution were
then measured at 515 nm using spectrophotometer. The
DPPH assays were performed in triplicates. Total polyphe-
nols in the agro-industrial by-products were determined
according to Folin-Ciocalteu method [16]. The same homog-
enate (0.5 ml) as prepared above was mixed with distilled
water (8 ml), Folin-Ciocalteu reagent (0.5 ml; Merck KGaA)
and sodium carbonate (1 ml; Merck KGaA). After 30 min
incubation at room temperature, the spectrophotometer
was employed to measure (at 765 nm) the absorbance of
the mixture. Gallic acid was employed to plot the standard
curve. The test was run in triplicate. The content of tannins
in the agro-industrial by-products was assayed colorimetri-
cally, and Folin-Denis reagent was used to estimate the tan-
nins content in the samples [17]. Along with distilled water
(8 ml) and sodium carbonate (1 ml, Na2CO3, Merck KGaA),
Folin-Denis reagent (0.5 ml, Merck KGaA) was added to the
homogenate (0.5 ml). The solution was subsequently incu-
bated for 30 min at room temperature, and eventually the
measurement of the absorbance was conducted at 760 nm.
Tannic acid solution (Sigma-Aldrich) was used to prepare
the standard curve. Each sample was assayed triplically.
Data on pH, proximate compositions, amino acids, and
antioxidant properties of each by-product were subjected
to analysis of variance. Duncan’s multiple range test was
further conducted to evaluate the variance among group
means. Data on microbial populations were analyzed by
t-test to contrast the group means between single- and
two-stage fermented by-products. A substantial level of
p < 0.05 was implemented.
Results
The pH values and populations of microbes in the
agro-industrial by-products are presented in Table 1. The
pH values of banana peels and cassava pulp increased
(p < 0.05) after fermentation with Ch. crassa
but then decreased to the values as of the unfermented
by-products when followed by B. subtilis-fermentation.
Different from these two by-products, the pH value of rice
bran did not change (p > 0.05) after Ch. crassa-fermen-
tation, but decreased following the fermentation with
B. subtilis. The numbers of coliform, yeast, and LAB were
not detected in all by-products (sterilized by-products)
prior to fermentation. No substantial difference in coli-
form bacteria populations was observed in the by-prod-
by-products, the number of LAB was higher (p < 0.05) in
two-stage than in single-stage fermented by-products.
The data on chemical compositions of the fermented
agro-industrial by-products are shown in Table 2. The
crude protein and fat contents were higher (p < 0.05) in
single- and two-stage fermented than in unfermented
banana peel meals. In cassava pulp, crude protein was
Table 1. pHandmicrobialpopulaonsinthefermentedagro-industrialby-products.
Items Unfermented
by-product
Single-stage
fermented by-product
Two-stage fermented
by-product SE p value
Bananapeels
pH 6.60b7.73a7.10b0.15 0.01
Coliform(logcfu/gm) ND 5.91 5.94 0.26 0.93
Yeast(logcfu/gm) ND 7.92 >8.26 0.34 0.42
LAB(logcfu/gm) ND 7.96b>10.3a0.30 0.02
Cassavapulp
pH 4.93b6.67a5.20b0.23 <0.01
Coliform(logcfu/gm) ND 4.36 5.32 0.48 0.93
Yeast(logcfu/gm) ND 7.49 >8.26 0.39 0.42
LAB(logcfu/gm) ND 5.33b>10.2a0.44 0.02
Ricebran
pH 6.37a6.10a5.33b0.25 0.02
Coliform(logcfu/gm) ND 5.33 4.81 0.38 0.93
Yeast(logcfu/gm) ND 7.38 >8.26 0.13 0.42
LAB(logcfu/gm) ND 7.52b>10.2a0.18 0.02
a,bValueswithdierentleerswithinthesamerowandtypeofagro-industrialby-productsaresignicantlydierent(p<0.05).
Thesymbol“>”indicatesthatsomeobservaonsfromwhichthemeanwascalculatedhadvaluesabovedeteconlevels.Whenthecolonies
couldnotbeenumeratedontheplates,the deteconlevelwasappliedandusedtomakethecalculaons.Hence,therealmeanvalueisabove
thanthatreported.
ND(notdetected)indicatesthatsomeobservaonshadvaluesbelowdeteconlevels.
LAB=laccacidbacteria.
SE=standarderror.
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 475
substantially higher (p < 0.05) in single- and two-stage fer-
mented than that in unfermented product. The single-stage
fermented cassava pulp contained higher (p < 0.05) crude
fat than unfermented cassava pulp, but such variation was
-
sava pulp. Single-stage fermented cassava pulp had higher
(p < 0.05) ash content than two-stage fermented and unfer-
mented cassava pulp. There was a tendency (p = 0.08) that
both single- and two-stage fermentation increased the
content of crude protein in rice bran, while crude fat and
ash contents notably increased (p < 0.05) with the fermen-
p = 0.08) to decrease in the
fermented as compared with unfermented rice bran. In
-
fer between single- and two-stage fermented by-products,
except for the ash content of cassava pulp.
The data of amino acid contents in the fermented
agro-industrial by-products are presented in Table 3. First-
stage fermentation of banana peel meals using Ch. crassa
increased (p < 0.05) the contents of L-alanine, L-aspartate
acid, L-isoleucine, L-leucine, L-lysine HCl, L-threonine,
L-tyrosine, and L-valine in banana peel meals. However,
the subsequent fermentation (second-stage fermentation)
with B. subtilis tended to lower the amino acid contents in
banana peels. Compared with unfermented cassava pulp,
single-stage fermented cassava pulp contained higher
(p < 0.05) levels of glycine and L-threonine, but the con-
centrations of these amino acids were not different
as compared with two-stage fermented cassava pulp.
L-serine content was greater (p < 0.05) in single-stage fer-
mented cassava pulp than in unfermented and two-stage
fermented cassava pulp. With regard to rice bran, single-
p > 0.05) the
amino acid contents of the product.
Data on total polyphenols, total tannins, and free radical
(DPPH) removing activity are presented in Table 4. Total
polyphenols, total tannins, and antioxidant potential of
banana peels decreased (p < 0.05) following the fermen-
tation. The concentrations of total polyphenols and total
tannins were greater (p < 0.05) in single- and two-stage
fermented than in unfermented cassava pulp. Yet, the anti-
oxidant activity was weaker (p < 0.05) in the fermented
than in unfermented cassava pulp. In rice bran, total poly-
phenols, tannins, and free radical scavenging capacity were
less (p < 0.05) in two-stage fermented rice bran, when
compared with single-stage fermented and unfermented
rice bran.
Discussion
-
tation with Ch. crassa increased the pH values of banana
peels and cassava pulp. The reason for these increased pH
values was not clear, but such increase in pH was also previ-
ously reported in soybean koji fermented with Aspergillus
oryzae S. [18]. The latter authors further reported that the
increase in enzyme production seemed to be responsible
for the increased pH value in the fungal fermented prod-
ucts. Moreover, Liang et al. [19] revealed that the increased
production of extracellular protein in the fermentation
substrate (due to metabolic activity of fungi) may also
lead to the increased pH value in the fermented products.
Table 2. Chemicalcomposions(as-drybasis)ofthefermentedagro-industrialby-products.
Items (%) Unfermented
by-product
Single-stage
fermented by-product
Two-stage fermented
by-product SE p value
Bananapeels
Crudeprotein 7.99b8.94a9.06a0.09 <0.01
Crudefat 4.23b5.96a6.72a0.47 0.02
Crudeber 18.0 15.9 15.0 0.93 0.15
Ash 12.9 13.1 12.6 0.57 0.80
Cassavapulp
Crudeprotein 2.12b2.33a2.32a0.04 0.01
Crudefat 0.33b1.10a0.88ab 0.17 0.04
Crudeber 11.2 8.59 10.6 0.80 0.11
Ash 3.29b3.75a3.46b0.05 <0.01
Ricebran
Crudeprotein 10.9 11.9 12.2 0.35 0.08
Crudefat 1.92b9.04a8.73a0.62 <0.01
Crudeber 10.3 8.67 9.05 0.44 0.08
Ash 9.14b10.2a10.5a0.28 0.03
a,bValueswithdierentleerswithinthesamerowandtypeofagro-industrialby-productsaresignicantlydierent(p<0.05).SE=standard
error.
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 476
Our inference should, however, be noted with caution as
Ch. crassa-fermentation did not change the pH value of
rice bran in the present study. In this regard, the nature of
substrates used for fermentation seemed to determine the
change in pH value of the Ch. crassa-fermented products.
Note that each substrate may have divergent buffering
capacities [8]. In all agro-industrial by-products, pH values
were lower in two-stage fermented than in single-stage
fermented by-products. The greater number of LAB in
two-stage fermented than in single-stage fermented
by-products was most likely to induce the decreased pH in
two-stage fermented in the present study. Concomitant to
Table 3. Aminoacidcontentsinthefermentedagro-industrialby-products.
Items (mg/kg) Unfermented
by-product
Single-stage fermented
by-product
Two-stage fermented
by-product SE p value
Bananapeels
Glycine 2.61 5.19 4.06 0.79 0.09
L-Alanine 2.5b5.89a3.99ab 0.79 0.03
L-Arginine 2.3 5.15 3.05 0.89 0.09
L-Aspartateacid 3.32b8.13a6.28ab 1.21 0.04
L-Glutamicacid 3.56 11.7 7.12 2.21 0.06
L-Phenylalanine 2.44 4.95 3.47 0.76 0.09
L-Hisdine 0.71 1.91 1.26 0.33 0.07
L-Isoleucine 1.63b3.75a2.64ab 0.51 0.03
L-Leucine 2.78b6.67a4.62ab 0.99 0.04
L-LysineHCl 0.98b4.69a2.19ab 0.94 0.04
L-Proline 2.47 4.23 3.15 0.46 0.05
L-Serine 1.93 4.46 3.58 0.73 0.07
L-Threonine 1.94b4.01a3.34ab 0.51 0.03
L-Tyrosine 1.1b2.33a1.83ab 0.31 0.04
L-Valine 2.42b5.13a3.25ab 0.69 0.04
Cassavapulp
Glycine 0.82b1.07a0.92ab 0.06 0.03
L-Alanine 1.00 1.19 1.03 0.08 0.20
L-Arginine 0.77 0.92 0.87 0.05 0.18
L-Aspartateacid 1.39 1.63 1.45 0.10 0.24
L-Glutamicacid 1.52 1.80 1.71 0.12 0.25
L-Phenylalanine 0.86 0.99 0.86 0.06 0.22
L-Hisdine 0.33 0.40 0.38 0.02 0.07
L-Isoleucine 0.56 0.63 0.56 0.04 0.41
L-Leucine 1.00 1.09 1.04 0.12 0.88
L-LysineHCl 1.21 1.36 1.20 0.09 0.38
L-Proline 0.91 1.11 1.03 0.10 0.43
L-Serine 1.10b1.40a1.10b0.07 0.01
L-Threonine 0.89b1.12a1.03ab 0.06 0.04
L-Tyrosine 0.30 0.24 0.24 0.02 0.06
L-Valine 0.90 1.01 0.89 0.06 0.35
Ricebran
Glycine 5.97 6.45 5.33 0.45 0.26
L-Alanine 5.7 6.09 5.01 0.71 0.58
L-Arginine 7.35 6.64 5.15 0.65 0.08
L-Aspartateacid 8.29 8.33 7.38 0.55 0.41
L-Glutamicacid 15 13.5 11.6 1.17 0.14
L-Phenylalanine 4.96 5.95 4.77 0.49 0.23
L-Hisdine 2.58 2.66 2.04 0.22 0.13
L-Isoleucine 3.62 4.31 3.51 0.33 0.21
L-Leucine 7.34 7.87 6.31 0.63 0.24
L-LysineHCl 6.26 5.85 4.8 0.57 0.21
L-Proline 4.47 4.51 3.79 0.33 0.25
L-Serine 5.02 5.54 4.26 0.39 0.09
L-Threonine 4.17 4.65 3.8 0.3 0.16
L-Tyrosine 2.91 2.88 2.28 0.21 0.09
L-Valine 5.39 5.87 4.7 0.45 0.22
a,bValueswithdierentleerswithinthesamerowandtypeofagro-industrialby-productsaresignicantlydierent(p<0.05).
SE=standarderror.
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 477
[8] showed that fermentation using
B. subtilis and E. faecium produced higher population of
LAB as well as lactic acid resulting in lower pH value in
the fermented products. With regard to coliform bacteria,
the presence of such pathogenic bacteria in the fermented
products seemed due to be the presence of free sugars in
the by-products that may promote the proliferation of the
bacteria [20].
It was apparent in our present study that fermentation
especially using Ch. crassa elevated the contents of raw
protein and fat in the substrates. Earlier work showed that
fermentations using Aspergillus niger and Trichoderma
pseudokoningii were capable of increasing the protein
content in cassava root [21] and cassava residue [22],
respectively. According to Liang et al. [19], the increased
production of extracellular protein by the fungus may be
responsible for the increased protein content of the fer-
mented products. Bayitse et al. [22] further suggested
that the capability of the fungus to produce enzymes to
degrade amylum/starch and non-starch polysaccharides
to monosaccharides that are conveniently processed to
protein may also be the reason for the protein-enhanc-
ing capacity of the fungal starters. With regard to fat, fer-
mentation increased the content of fat in the by-products.
Sukma et al. [23] previously showed that the fermentation
with the fungus R. oryzae increased fat content of rice
bran. The latter authors suggested that the increased fat
content in the fermented products may be associated with
the increased biomass of the fungi. Note that cell wall and
plasma membrane of the fungi are generally composed of
fat (phospolipid and lipoprotein). In cassava pulp and rice
bran, ash content was increased by the fungal fermenta-
tion. In accordance with this, fermentation with R. oryzae
increased ash content of rice bran in the study of Sukma
et al. [23]. This increase seemed to be associated with the
increased fungal populations in the substrates, as cell wall
of fungi was rich in minerals [24]. In this current study,
decreased with the fungal fermentation. Although not sig-
of banana peel meals and cassava pulp following the fungal
being fermented with R. oryzae [23] and Ch. crassa [9]. The
of the by-products was not exactly known, but the fungus
simpler carbohydrates [23].
In general, the proximate compositions did not differ
between the single- and two-step fermented by-products,
except for the lower ash content in two- than in single-step
fermented by-products. This circumstance seemed to indi-
cate the incapability of B. subtilis in improving the nutrient
composition of the by-products. Our present result was in
concomitant with Kanghae et al. [25] showing the absent
B. subtilis on the proximate
composition of soybean. In contrast, Wongputtisin et al.
[10] revealed an improvement effect of B. subtilis on the
proximate compositions in soybean hulls. The rationale for
differences in nature of substrates and strains of B. subti-
lis used as a fermentation starter as well as the conditions
during fermentation may affect the nutritional composi-
tions of the fermented products.
Data in our present study showed that fermentation with
Ch. crassa was capable of increasing the contents of both
Table 4. Totalpolyphenols,totaltannins,andDPPHradicalscavengingacvity(IC50)ofthefermentedagro-industrialby-products.
Items Unfermented
by-product
Single-stage fermented
by-product
Two-stage fermented
by-product SE p value
Bananapeels
Totalpolyphenols(mg/gm) 5.14a1.56b1.64b0.09 <0.01
Totaltannins(mg/gm) 4.34a1.32b1.16b0.09 <0.01
IC50(ppm)1479.2b3964a3793a448 <0.01
Cassavapulp
Totalpolyphenols(mg/gm) 0.39b1.21a1.94a0.23 0.01
Totaltannins(mg/gm) 0.15b1.09a1.83a0.22 0.01
IC50(ppm)13110c5571a4361b237 <0.01
Ricebran
Totalpolyphenols(mg/gm) 2.91a3.37a1.80b0.18 <0.01
Totaltannins(mg/gm) 2.53a3.12a1.60b0.18 <0.01
IC50(ppm)11139b875.7b1869a138 0.01
a,b,cValueswithdierentleerswithinthesamerowandtypeofagro-industrialby-productsaresignicantlydierent(p<0.05).
1IC50isconsideredastheconcentraonoftheDPPHradicalswerescavengedby50%.AlowerIC50valueimpliesahigherofDPPHradical
scavengingacvity.
SE=standarderror.
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 478
essential (L-leucine, L-isoleucine, L-lysine HCl, L-threonine,
and L-valine) and non-essential amino acids (L-alanine,
L-aspartate acid, and L-tyrosine) in banana peel meals. In
[26] showed
that fermentation with the fungus Rhizopus oligosporus
resulted in higher amino acids in buckwheat. Bujang and
Taib [27] further documented that R. oligosporus-fermen-
tation increased the contents of amino acids (both essen-
tial and non-essential) in soybean, garbanzo bean, as well
as groundnut. The proteolytic activity of protease enzyme
decomposition of protein to amino acids resulting in higher
free amino acids in the fermented products [27]. Former
-
lyzing the long-chain carbohydrates to produce protein-rich
biomass [28]. Owing that the fungal biomass is rich in amino
acids [29], the production of biomass during fungal fermen-
tation may also be attributable to the elevated amino acid
levels in the fermented stuffs. Study by Sarkar et al. [30] as
well as Song et al. [31] noticed that the fermentation using
Bacillus sp., L. plantarum, and B. lactis were able to enhance
free amino acids in soybean, respectively. Different from the
above studies, further (two-stage) fermentation using B.
subtilis did not increase the content of amino acids in the
fermented banana peels in the current study. The reason
for the absent impact of B. subtilis-fermentation on amino
acid contents was not exactly known, but the increased pop-
ulation of yeast in the substrates during the fermentation
process may attenuate the potential of Bacillus to increase
amino acid content in the substrates. Indeed, Song et al. [31]
reported that fermentation using Saccharomyces cerevisae
resulted in reduced amino acid contents in soybean meal.
In the very earlier study by Majumdar and Bose [32], it was
shown that amino acids may be utilized by B. subtilis during
the growth and the production of secondary metabolites
(such as antibiotic). In this regard, certain amino acids may
be used by B. subtilis resulting in lack-increased amino acids
contents in the two-stage fermented banana peel meals. In
Ch. crassa resulted
in increased amino acids glycine, L-serine, and L-threonine.
Following the second-stage fermentation, the concentra-
while the concentration of L-serine decreased from that of
[7] noticed that the changes in amino acids contents of sub-
strates were not constant during the fermentation process.
The duration of fermentation seems to be one of the factors
affecting the content of amino acids. Bujang and Taib [27]
revealed that R. oligosporus-fermentation for 24 h elevated,
whereas extended fermentation for 30 h decreased amino
acid contents in soybean, garbanzo bean, and ground-
nut. Such prolonged fermentation may deplete nutrients,
increase yeast population, and change the fermentation
conditions, which in turn reduce the amino acids produc-
tion [27]. With regard particularly to the decreased L-serine
level in cassava pulp following the second stage fermenta-
tion, the exact reason for such condition remains unclear. It
has been reported by Zhang et al. [33] that Escherichia coli
was capable of utilizing L-serine to produce pyruvate. In this
regard, the numerical increased coliform bacteria popula-
tion after second stage fermentation seemed, therefore, to
be responsible for the reduced L-serine content in the two-
stage fermented cassava pulp. Unlike the other by-products,
there were no substantial changes in amino acids in rice
be presented regarding the latter condition, but the dif-
ferent substrates of fermentation may affect the nature of
protein that can be decomposed to free amino acids.
It was shown in this work that the fermentation
resulted in reduction in total polyphenols, tannins, and
antioxidant activity of banana peel meals. In most studies,
fermentation was attributed to the increased antioxidant
components and antioxidant potential of the substrates
[34]
workers may not be elucidated clearly, but one possibil-
ity could be that the prolonged period of fermentation
decreased antioxidant compounds and antioxidant activity
of the substrates. Adetuyi and Ibrahim [35] reported that
fermentation (for 24 h) of okra (Abelmoschus esculentus)
seeds increased the contents of total phenols, vitamin C,
-
idant compounds and antioxidant activity of okra seeds
decreased after fermentation for 72 and 120 h. In the study
[36], phenolic compounds in red cabbages
increased until day 7 of fermentation using L. plantarum
and L. acidophilus, but gradually decreased with the time
of fermentation. Amarasinghe et al. [37] suggested that the
reduced antioxidant components may be associated with
the utilization of such compounds by the microorganisms
during fermentation. In our case, fermentation of banana
peel meals for 4 days by the fungus Ch. crassa and then 2
days by B. subtilis seemed to promote the use of polyphe-
nols and tannins by the microbes resulting in decreased
concentration of the anti-oxidative compounds and hence
antioxidant activity of banana peel meals. The extended
fermentation may also prolong the exposure of antioxidant
components to oxidation [37] and increase the diffusion of
phenolic compounds out of the substrates [35], resulting
in lower antioxidant properties of the substrates. In this
context, it is crucial to investigate the most appropriate
fermentation time to improve the nutritional values with-
out inducing deleterious effect on the antioxidant proper-
ties of agro-industrial by-products.
Unlike banana peels, fermentation with Ch. crassa and
then B. subtilis increased total polyphenols and tannins in
cassava pulp. This was in accordance with that of noticed
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 479
by Hur et al. [34]. They suggested that fermentation
increases the release of antioxidant components (from
their complex bindings) and the production/synthesis of
antioxidant compounds. In this study, the increased poly-
phenols and tannins was, however, not accompanied by
the increased antioxidant activity in cassava pulp. In the
study using kombucha, Amarasinghe et al. [37] pointed
out that the antioxidant capacity of the substrate was not
always determined by the concentration of phenolic com-
pounds, but several metabolites produced during fermen-
tation may have substantial effect on the antioxidant activ-
ity instead. In our study, other antioxidative compounds
(not investigated in the current study) may be decreased
by fermentation and hence, reduced the antioxidant activ-
ity of cassava pulp. With regard to rice bran, our current
data showed that single-stage fermentation did not change
the antioxidant compounds and antioxidant activity of the
substrate. Unexpectedly, the two-step fermentation using
B. subtilis reduced phenols and tannins contents, and anti-
oxidant potential of rice bran. In accordance with this pres-
of total polyphenols, tannins, and antioxidant activity of
herbal medicine waste following the fermentation using B.
subtilis for 4 days [38]. Concomitant result was also noted
by Yoon et al. [39], in which B. subtilis KU3- fermentation
alleviated antioxidative activity of rice bran. The reason for
the reduced antioxidant properties in the rice bran was not
clearly known, but B. subtilis seemed to degrade phenolic
compounds and tannins in the substrate during fermen-
tation resulting in reduced antioxidant activity. Our infer-
ence was supported by the facts regarding the capability of
B. subtilis in degrading phenols [40] and tannins [41].
Conclusion
It can be concluded that the single-stage fermentation
using Ch. crassa produced better nutritional characteristics
of agro-industrial by-products, when compared with the
two-stage fermentation (Ch. crassa inoculated in advance
and B. subtilis inoculated later). Single-stage fermentation
with Ch. crassa seems, therefore, to be more practical to
produce poultry feed from the agro-industrial by-products.
Conict of Interest
Authors’ Contribuon
Sugiharto Sugiharto planned, carried out the experiment,
and prepared the article. Turrini Yudiarti, Endang Widiastuti,
Hanny Indrat Wahyuni, and Tri Agus Sartono carried out
the in vivo experiment and corrected the article and II con-
ducted the statistical analysis and revised the article.
References
[1] Khempaka S, Molee W, Guillaume M. Dried cassava pulp as an alter-
native feedstuff for broilers: Effect on growth performance, carcass
traits, digestive organs, and nutrient digestibility. J Appl Poult Res
2009; 18:487–93; https://doi.org/10.3382/japr.2008-00124
[2] Sugiharto S, Yudiarti T, Isroli I, Widiastuti E. The potential of trop-
ical agro-industrial by-products as a functional feed for poultry.
Iran J Appl Anim Sci 2018; 8:375–85.
[3] Khempaka S, Thongkratok R, Okrathok S, Molee W. An evaluation
of cassava pulp feedstuff fermented with A. oryzae, on growth per-
formance, nutrient digestibility and carcass quality of broilers. J
Poult Sci 2014; 51:71–9; https://doi.org/10.2141/jpsa.0130022
[4] Chen CC, Shih YC, Chiou PWS, Yu V. Evaluating nutritional quality
of single stage- and two stage-fermented soybean meal. Asian-
Australas J Anim Sci 2010; 23:598–606; https://doi.org/10.5713/
ajas.2010.90341
[5] Kawa-Rygielska J, Adamenko K, Kucharska AZ, Piórecki N. Bioactive
compounds in cornelian cherry vinegars. Molecules 2018; 23:379;
https://doi.org/10.3390/molecules23020379
[6] Padonou SW, Nielsen DS, Akissoe NH, Hounhouigan JD, Nago
MC, Jakobsen M. Development of starter culture for improved
processing of Lafun, an African fermented cassava food
product. J Appl Microbiol 2010; 109:1402–10; https://doi.
org/10.1111/j.1365-2672.2010.04769.x
[7] Weng TM, Chen MT. Effect of two-step fermentation by Rhizopus
oligosporus and Bacillus subtilis on protein of fermented soybean.
Food Sci Technol Res 2011; 17:393–400. https://doi.org/10.3136/
fstr.17.393
[8] Shi C, Zhang Y, Lu Z, Wang Y. Solid-state fermentation of corn-soy-
bean meal mixed feed with Bacillus subtilis and Enterococcus
faecium for degrading antinutritional factors and enhancing
nutritional value. J Anim Sci Biotechnol 2017; 8:50; https://doi.
org/10.1186/s40104-017-0184-2
[9] Sugiharto S, Yudiarti T, Isroli I, Widiastuti E, Putra FD. Effect of
dietary supplementation with Rhizopus oryzae or Chrysonilia
crassa
population, and carcass traits in broilers exposed to heat stress.
Arch Anim Breed 2017; 60:347–56; https://doi.org/10.5194/
aab-60-347-2017
[10] Wongputtisin P, Khanongnuch C, Kongbuntad W, Niamsup P,
Lumyong S, Sarkar PK. Use of Bacillus subtilis isolates from Tua-
nao towards nutritional improvement of soya bean hull for mono-
gastric feed application. Lett Appl Microbiol 2014; 59:328–33;
https://doi.org/10.1111/lam.12279
[11] Juan MY, Chou CC. Enhancement of antioxidant activity, total phe-
-
mentation with Bacillus subtilis BCRC 14715. Food Microbiol 2010;
27:586–91; https://doi.org/10.1016/j.fm.2009.11.002
[12] Sanjukta S, Rai AK, Muhammed A, Jeyaram K, Talukdar NC.
Enhancement of antioxidant properties of two soybean varieties
of Sikkim Himalayan region by proteolytic Bacillus subtilis fermen-
tation. J Funct Foods 2015; 14:650–8; https://doi.org/10.1016/j.
jff.2015.02.033
chemists. Washington DC, USA, 1995.
validation for determination of amino acids in feed by UPLC.
Accredit Qual Assur 2017; 22:247; https://doi.org/10.1007/
s00769-017-1281-9
[15] Wu N, Fu K, Fu YJ, Zu YG, Chang FR, Chen YH, et al. Antioxidant
activities of extracts and main components of pigeonpea [Cajanus
cajan (L.) Millsp.] leaves. Molecules 2009; 14:1032–43; https://
doi.org/10.3390/molecules14031032
[16] Orak HH. Total antioxidant activities, phenolics, anthocyanin, poly-
phenoloxidase activities and its correlation of some important red
wine grape varieties which are grown in Turkey. EJPAU 2006; 9:18.
hp://bdvets.org/javar/ Sugiharto et al./ J. Adv. Vet. Anim. Res., 5(4): 472–480, December 2018 480
http://www.ejpau.media.pl/volume9/issue1/art-18.html
(Accessed 16 September 2015).
[17] Chanwitheesuk A, Teerawutgulrag A, Rakariyatham N. Screening
of antioxidant activity and antioxidant compounds of some edi-
ble plants of Thailand. Food Chem 2005; 92:491–7; https://doi.
org/10.1016/j.foodchem.2004.07.035
[18] Chancharoonpong C, Hsieh PC, Sheu SC. Enzyme production and
growth of Aspergillus oryzae S. on soybean koji fermentation.
APCBEE Procedia 2012; 2:57–61; https://doi.org/10.1016/j.
apcbee.2012.06.011
[19] Liang Y, Pan L, Lin Y. Analysis of extracellular proteins of Aspergillus
oryzae grown on soy sauce koji. Biosci Biotechnol Biochem 2009;
73:192–5; https://doi.org/10.1271/bbb.80500
[20] Satari B, Karimi K, Taherzadeh MJ, Zamani A. Co-production of fun-
gal biomass derived constituents and ethanol from citrus wastes
free sugars without auxiliary nutrients in airlift bioreactor. Int J
Mol Sci 2016; 17:302; https://doi.org/10.3390/ijms17030302
[21] Ramin M, Yaakub H, Alimon AR, Jelan ZA. Effects of fungal treat-
ment on the in vitro degradation of cassava. Livest Res Rural Dev
2011; 23:7; http://www.lrrd.org/lrrd23/7/rami23145.htm
[22] Bayitse R, Hou X, Laryea G, Bjerre AB. Protein enrichment
of cassava residue using Trichoderma pseudokoningii (ATCC
26801). AMB Express. 2015; 5:80; https://doi.org/10.1186/
s13568-015-0166-8
[23] Sukma A, Jos B, Sumardiono S. Kinetic of biomass growth and pro-
tein formation on rice bran fermentation using Rhizopus oryzae.
MATEC Web Conf 2018; 156:01023; https://doi.org/10.1051/
matecconf/201815601023
[24] Oliveira M. dos S, Feddern V, Kupski L, Cipolatti EP, Badiale-Furlong
E, de Souza-Soares LA. Physico-chemical characterization of fer-
-
masa del salvado de arroz fermentado. CyTA-J Food 2010; 8:229–
36; https://doi.org/10.1080/19476330903450274
[25] Kanghae A, Eungwanichayapant PD, Chueamchaitrakun P,
Nitsawang S, Chukeatirote E. Proximate analysis of the fermented
soybean prepared by Bacillus subtilis and Rhizopus oligosporus.
The 26th Annual meeting of the Thai Society for Biotechnology and
International Conference, Mae Fah Lunag University, Chiang Rai,
26–29 Nov, 2014.
[26] Wronkowska M, Christa K, Ciska E, Soral-Smietana M. Chemical
characteristics and sensory evaluation of raw and roasted buck-
wheat groats fermented by Rhizopus oligosporus. J Food Qual 2015;
38:130–8; https://doi.org/10.1111/jfq.12127
[27] Bujang A, Taib NA. Changes on amino acids content in soybean,
garbanzo bean and groundnut during pre-treatments and tempe
making. Sains Malaysiana. 2014; 43:551–557.
[28] Filho PFS, Zamani A, Taherzadeh MJ. Edible protein production by
Valor 2018; 1–10; https://doi.org/10.1007/s12649-018-0265-2
with potential as aqua-feed ingredients. Sustainability 2018;
10:3296; https://doi.org/10.3390/su10093296
[30] Sarkar PK, Jones LJ, Craven GS, Somerset SM, Palmer C. Amino acid
59:69–75; https://doi.org/10.1016/S0308-8146(96)00118-5
[31] Song YS, Frias J, Martinez-Villaluenga C, Vidal-Valdeverde C,
Gonzalez de Mejia E. Immunoreactivity reduction of soybean meal
by fermentation, effect on amino acid composition and antigenic-
ity of commercial soy products. Food Chem 2008; 108:571–81;
https://doi.org/10.1016/j.foodchem.2007.11.013
[32] Majumdar SK, Bose SK. Utilization of amino acids by
Bacillus subtilis during growth and antibiotic produc-
tion. Biochim Biophys Acta 1958; 29:509–13; https://doi.
org/10.1016/0006-3002(58)90006-4
interferes with one-carbon metabolism and cell wall synthesis in
Escherichia coli K-12. J Bacteriol 2010; 192:5515–25; https://doi.
org/10.1128/JB.00748-10
[34] Hur SJ, Lee SY, Kim YC, Choi I, Kim GB. Effect of fermentation on
the antioxidant activity in plant-based foods. Food Chem 2014;
160:346–56; https://doi.org/10.1016/j.foodchem.2014.03.112
[35] Adetuyi FO, Ibrahim TA. Effect of fermentation time on the pheno-
okra (Abelmoschus esculentus) seeds. Niger Food J 2014; 32:128–
37; https://doi.org/10.1016/S0189-7241(15)30128-4
I. Improvement of antioxidant activities in red cabbage sprouts by
lactic acid bacterial fermentation. Food Biotechnol 2013; 27:279–
302; https://doi.org/10.1080/08905436.2013.836709
[37] Amarasinghe H, Weerakkody NS, Waisundara VY. Evaluation of
physicochemical properties and antioxidant activities of kombu-
cha “Tea Fungus” during extended periods of fermentation. Food
Sci Nutr 2018; 6:659–65; https://doi.org/10.1002/fsn3.605
[38] Sugiharto S, Yudiarti T, Isroli I, Widiastuti E, Wahyuni HI, Suprijatna
E. The potential of Bacillus strains isolated from the rumen content
of dairy cows as natural antibacterial and antioxidant agents for
broilers. J Indones Trop Anim Agric 2018; 43:115–23; https://doi.
org/10.14710/jitaa.43.2.115-123
[39] Yoon HJ, Lee KA, Lee JH, Jin HJ, Kim HJ, Kim KT, et al. Effect of fer-
mentation by Bacillus subtilis on antioxidant and cytotoxic activ-
ities of black rice bran. Int J Food Sci Technol 2015; 50:612–18;
https://doi.org/10.1111/ijfs.12693
[40] Nair CI, Jayachandran K, Shashidhar S. Biodegradation of phenol.
Afr J Biotechnol 2008; 7:4951–8.
[41] Ilori MO, Adebusoye SA, Amund OO, Oyetoran BO. A study of tannic
acid degradation by soil bacteria. Pak J Biol Sci 2007; 10:3224–7;
https://doi.org/10.3923/pjbs.2007.3224.3227
