Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)

International Journal of Current Research in

Biosciences and Plant Biology
Volume 4 ● Number 1 (January-2017) ● ISSN: 2349-8080 (Online)
Journal homepage: www.ijcrbp.com

Original Research Article

doi: http://dx.doi.org/10.20546/ijcrbp.2017.401.003

In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus
flabellifer L.) Husk Fermented with EM-4 for Beef Cattle Feed
Dwi Nur Aini Dahlan and Khusnul Khotimah*
Lecturer Staff, Teacher Training Science Program, Biology, Samarinda 75251, East Kalimantan, Indonesia*Corresponding author.

Abstract

Article Info

Beef cattle livestock production that affects domestic meat needs is highly determined bymeeting feed needs. However, the production is often hampered by continuity and feed
cost issues. The husk of Asian Palmyra palm contains high nutrient and can be used asalternative feed. The husk is first fermented with EM-4 to improve its nutrient content.
Husk’s potency as livestock feed can be determined by measuring its nutrient,digestibility, and total digestible nutrient TDN value in vitro. This study aimed todetermine the effect of in vitro treatment using Asian Palmyra palm fermented with EM-4
on its nutrient content (dry matter-DM, organic matter-OM, crude protein-CP, crude fiberCF, and crude fat) when it is fed to beef cattle. This experimental quantitative study used
Complete Randomized Design (CRD) with 5 treatments and 3 replications. Treatment wasdifferenced by the concentration of EM-4 as fermenter on the husk of Asian Palmyra
palm, i.e. 0% (PO); 0.1% (P1); 1% (P2); 5% (P3); and 10% (P4). Parameters observed on
nutrient content were among other dry matter (DM), organic matter (OM), crude protein(CP), crude fiber (CF), and crude fat following Association of Official Analytical
Chemists-AOAC (1980). To determine the influence, proximate analysis and statisticalanalysis using One Way ANOVA were employed, followed with 5% Least Significant
Difference (LSD) test when significant influence was found. Study result indicated thatusing husk waste from Asian Palmyra palm (P<0.05) significantly affects nutrient content(DM, OM, CP, CF, and crude fat). Average 1% EM-4 in the husk of Asian Palmyra palmis already capable of improving nutrient content (DM, OM, CP, CF, and crude fat).

Accepted: 23 December 2016
Available Online: 06 January 2017

Introduction
Cattle livestock, particularly beef cattle, is one of meatproducing resources which have high economic valueand high importance in community (Mustofa et al.,2015). Main issues affecting cattle livestock production
are among other continuity and feed cost. Therefore, it isnecessary to carry out efforts to make feed cost, such as

Keywords
Asian Palmyra palm
Proximate analysis
Ruminants

agriculture waste, efficient. Rahmanto (2011) stated thatwaste use is one of the alternatives to improve theeconomic value of the waste. Waste use as livestock
feed has previously been performed by Parulin (2009)
who used oil palm fronds and industrial waste. It is
presumed that Asian Palmyra palm which has close
relationship with oil palm also is a potential cattle
livestock feed. According to Assessment Institute for

D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed

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Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)

Agricultural Technology (BPTP) in ISBN 978-9793112-32-9 (2010), livestock feed materials usuallycontain nutrients comprise of water, minerals, proteins,fats, carbohydrates, and vitamins. Hariyanto (2012)
reported that ruminant nutrient provides stronger
foundation in developing feed formula to meet livestock
need in accordance with their genetic potential andphysiological status to survive and normally reproduce.
Nutrient need increases during growth and fattening
phases. According to Assessment Institute for
Agricultural Technology (BPTP) in ISBN 978-9793112-32-9 (2010), beef cattle feed during fattening
phase (7 months) requires nutrient content with
DM>88%, CP>12%, crude fat< 6%, CF 12-17%, TDN >64%, and ash < 10%. In addition, Wahyudi and
Bambang (2002) stated that in dry condition the husk of
Asian Palmyra palm contains 5.942% cellulose, 0.144%
water, and 5.268% ash. However, it is necessary to havemore detailed information on the content of Asian
Palmyra palm to improve its nutrient. In addition,
chopping and grinding combined with biologicaltreatment through fermentation are also necessary.
Livestock feed fermentation is carried out to meet 3
purposes, i.e. to preserve feed to make it stays in goodcondition when needed, to improve the nutrient value ofthe material, and to improve digestibility (Zhang et al.,2012). According to Javaid and Bajwa (2011), EM-4 is a
mix of fermentation bacteria from genus Lactobacillus,
Actinomycetes, photosynthetic bacteria and yeast. EM-4
in livestock feed will improve the composition of
microorganisms that can help feeds easier to digest by
livestock.
Basing on the ideas above, it is necessary to carry out
study on the potency of Asian Palmyra palm’s husk
waste as livestock feed after being treated with andwithout EM-4. One of the methods used to determine
the nutrient content of Asian Palmyra palm’s husk waste
is proximate analysis following AOAC (1980).
Materials and methods
Fermentation of Asian Palmyra palm
The husk of Asian Palmyra palm was chopped to obtain
1-5 cm as the smallest size prior to being put in 60°C
oven for 14 hours. A total of 100 grams husk wasweighed and put into plastic bag. EM-4 was then put inaccordance with the treatments and distilled water of

30% of sample weight was added. The mix was stirred
evenly prior to incubation for 7 days.
Retrieving rumen fluid
Boiled water was put into water flask and discarded.
Syringe hose was then put into rumen through modified
cattle stomach and immediately put into the empty flask.
The fluid was then filtered using filter cloth prior mixing
with buffer solution.
Proximate analysis following AOAC (1980)
Determining DM concentration: Porcelain dish washeated in 105°C oven for 1 hr prior to being put inexsiccator for 1 hr and weighed (A g). The dish was thenadded with 5 g sample and re-weighed (B g) prior tobeing put in exsiccator for 1 hour and re-weighing (C g).

Determining OM concentration: Porcelain dish was put
into 550°C furnace for 4 hrs prior to being put inexsiccator for 1 hr and re-weighing (D g).

OM = 100% - ash
Determining CP concentration: Greaseproof paper was
weighed (A g) and 3 g sample was put onto the paper
prior to re-weighing (B g). Sample was then put into
Kjeldahl flask and 1.4 g catalysator (Kjeldahl table) andboiling stone were added. A total of 5 mL concentratedhydrogen peroxide with dispenser was then added prior
to destruction until clear green. A total of 60 mL
distilled water (divided into four) was then added prior
to stirring and being put into Erlenmeyer flask. A totalof 25 mL 40% NaOH was then added into theErlenmeyer flask prior to distillation with beaker glass
containing 25 mL 0.1 N H2SO4 and max 3 drops
indicator as the receiving end. The beaker glass was thentitrated using 0.1 N NaOH until the solution color
become clear and the titration volume was measured (CmL). Blank solution was made by performing titrationon the beaker glass containing 25 mL H2SO4 and max 3
drops indicator prior to measurement of the titration
volume (D g).

D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed

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Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)

Data analysis

Determining crude fiber, CF: Grease proof paper was
weighed (A g) and 1 g sample was put onto the paper
prior to re-weighing (B g). Sample was then put intomeasuring cylinder for CF analysis and 50 mL 0.3 N
H2SO4 was added using measuring cylinder prior toboiling for 30 minutes. A total of 25 mL 1.5 N NaOH
was then quickly added prior to boiling for 25 minutes.
A total of 0.5 g EDTA was then quickly added prior to
re-boiling for 25 minutes. Sand was first put in filtration
dish and measuring cylinder was taken out from boiling
equipment prior to rinsing using hot distilled water until
all solution were in filtration dish. A total of 50 mL 0.3
N HCl was then added and allowed to stay for 1 minuteprior to being sucked using vacuum pump.
Acetone was then added and allowed to stay for 1
minute prior to being sucked until dry. The sample wasthen put in 140°C oven for 1.5 hr prior to being put inexsiccation for 1 hr and weighing (C g). The sample wasput in 550°C furnace for 2 hrs prior to being put again in
exsiccator for 1 hr and weighing (D g).

Determining crude fat: Measuring cylinder, containing
2-3 boiling stones, was put into 105°C oven for 1 hrprior to being put in exsiccator for 1 hr. Ash-free filter
paper was weighed (A g) and 3-5 g sample was addedprior to re-weighing (B g). Sample was then wrappedusing the filter paper prior to being put in porcelain dish.
Measuring cylinder special for fat analysis was taken out
from exsiccator and weighed (C g) prior to filling beakerglass with 50 mL n-hexane using measuring cylinder.
The cylinder and porcelain tool were then installed onto
Goldfish extraction apparatus and extraction was
performed for 4 hrs. Porcelain tool was taken out withflask special to collect hexane until only small amountof hexane left in the beaker glass. Measuring cylinder
containing fats was then put into 80°C vacuum oven and
air was sucked from the oven for 1.5 hr. Measuringcylinder was then put into exsiccator for 1 hr and reweighed (D g).

This experimental laboratories study used 5x3 Complete
Randomized Design (CRD) (5 treatments and 3
replications), followed with 5% Least Significant
Difference (LSD) test when significant influence wasfound.
Results and discussion
Effect of EM-4 treatment on the dry matters (DM)and organic matters (OM) of the husk of Asian
Palmyra palm
Study result indicates that treatment using EM-4
significantly influenced the DM of the husk (p<0.05).
According to 5% LSD test, there was no significant
difference between control added with 0.1% EM-4 andadded with 1% EM-4. However, highly significant
difference was found between treatment with 5% EM-4
and 10% EM-4 (Table 1).
Table 1. Summary of 5% LSD test on the effect of EM-4 on
the dry matters (DM) of Asian Palmyra palm’s husk.
Treatment
Mean ± SD
Notation
P0 0%90.05% ± 0.51003
a
PI 0.1%
91.1% ± 1.07258
ab
PII 1%
91.455% ± 0.36116
abc
PIII 5%92.32% ± 1.17843
bc
PIV 10%92.66% ± 0.05132
C
Note: Figures with same letter indicate no significant
difference according to 5% LSD test.

Fig. 1: Mean Dry Matters (DM) content.

The DM of the husk increased along with increasing
EM-4 dosage (Fig. 1). The fourth treatment (10%D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed

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Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)EM-4), as the highest dosage, showed the highest DM(92.66%), followed by the third (5% EM-4), second (1%EM-4), and first (0.1% EM-4) treatments with DM
content of 92.32%, 91.45%, and 91.1%, respectively.
According to Assessment Institute for Agricultural
Technology (BPTP; ISBN 978-979-3112-32-9; 2010),
beef cattle feed during fattening phase (7 months)
requires nutrient content with 88% DM, meaning feed
without EM-4 is capable of meeting cattle’s need.
Decrease in water content not only capable of increasing
the DM of the husk but also influencing Organic Matters(OM) where increase in DM decreases OM.

OM decreased because it is the main source for energy,
growth, and cellular repair for important microorganism
such as mold, yeast, and Lactobacillus in EM-4 (Smithet al., 1978). Arief et al. (2008) stated that nutrient
source from OM added with high microorganism counts
lead to high microorganism activity. According to Dewi
et al. (2006), in addition to water in the growth media,
mold also requires other components such OM, carbon
source, nitrogen source and growth factor.

According to statistic test on Organic Matters (OM),significant influence due to EM-4 treatment (p>0.05) was
found, and 5% LSD test showed that there was a highly
significant difference between treatment and control. OM
content decreased along with increasing EM-4 dosage(Table 2). The highest OM content (94.74%) belonged to
0 treatment (control), followed by first (0.1% EM-4),
second (1% EM-4), third (5% EM-4), and fourth (10%EM-4) treatments with OM content of 93.54%, 91.26%,
83.9%, and 76.54%, respectively (Fig. 2).

According to this study and statistic test on crude protein(CP), significant influence due to EM-4 treatment(p<0.05) on CP content of the husk was found, and 5%
LSD test showed that significant difference was yet tofind between 0.1% EM-4 treatment and control.
However, highly significant difference was foundbetween control and 1%, 5%, and 10% EM-4 treatments(Table 3).

Table 2. Summary of 5% LSD test on the effect of EM-4 on
the organic matters (OM) of Asian Palmyra palm’s husk.
Treatment
Mean ± SD
Notation
P0 0%94.75% ± 0.09238
a
PI 0.1%93.54% ± 0.18930
b
PII 1%
91.26% ± 0.36364
c
PIII 5%83.91% ± 2.32987
cd
PIV 10%76.54% ± 1.93412
D
Note: Figures with same letter indicate no significant
difference according to 5% LSD test.

Effect of EM-4 treatment on the crude protein (CP)of the husk of Asian Palmyra palm

Table 3. Summary of 5% LSD test on the effect of EM-4 on
the crude protein (CP) of Asian Palmyra palm’s husk.
Treatment
Mean ± SD
Notation
P0 0%5.95% ± 1.10693
a
PI 0.1%
8.28% ± 1.59800
a
PII 1%
10.25% ± 0.62554
b
PIII 5%13.04% ± 1.57068
c
PIV 10%16.9% ± 0.8167
D
Note: Figures with same letter indicate no significant
difference according to 5% LSD test.

Fig. 3: Mean Crude Protein (CP) content.

Fig. 2: Mean Organic Matters (OM) content.

CP content increased along with increasing EM-4
dosage (Fig. 3). The lowest CP content (5.95%)

D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed

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Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)

belonged to 0 treatment (control), followed by first(0.1% EM-4), second (1% EM-4), third (5% EM-4), andfourth (10% EM-4) treatments with CP content of
8.28%, 10.25%, 13.04%, and 16.9%, respectively.
According to Assessment Institute for Agricultural
Technology (BPTP; ISBN 978-979-3112-32-9; 2010),
beef cattle feed during fattening phase (7 months)
requires nutrient content with 12.7% CP, meaning 5%
EM-4 treatment is capable of meeting cattle’s need.
CP content increased presumably due to othernutrients decreasing, particularly carbohydrates whichwere used by the microbes to grow and develop such
as Lactobacillus in EM-4 which played important role
fermenting organic matters (OM) to produce lactateacid. Lactobacillus growth is highly supported byother contents in the growth media such as water,where the microbe is made up of a single protein(Mathews et al., 2000). According to Mirwandhono
et al. (2006), increasing CP is presumably because ofmold.
Duringmold
fermentation
process,
carbohydrates (nitrogen-free extract) are broken down
into glucose units. Glucose is needed by mold as
precursor for formation of pyruvate acid that willproduce ATP. In addition, glucose also needed ascarbon source for microbes in EM-4.
Effect of EM-4 treatment on the crude fiber (CF) of
the husk of Asian Palmyra palm
According to this study and statistic test on crude fiber(CP), significant influence due to EM-4 treatment(p<0.05) on CF content of the husk was found, and 5%
LSD test showed that all EM-4 treatments (0.1% to10%) already showed highly significant difference with
control (Table 4).
CF content mostly increased along with increasing EM4 dosage as shown by 0 treatment control) with CF of
23.53%, followed by increase on first treatment (0.1%
EM-4) (28.94%) and second treatment (1% EM-4)(30.14%). Such increases were followed by slightdecrease on third treatment (5% EM-4) (28.94%) andthe highest CF content on fourth treatment (10% EM-4)(31.47%) (Fig. 4).
According to Assessment Institute for Agricultural
Technology (BPTP) in ISBN 978-979-3112-32-9 (2010),
beef cattle feed during fattening phase (7 months) requires
18.4% CF, meaning even without EM-4 treatment the feedis already capable of meeting cattle’s need.

Table 4. Summary of 5% LSD test on the effect of EM-4 on
the crude fiber (CF) of Asian Palmyra palm’s husk.
Treatment
Mean ± SD
Notation
P0 0%23.53% ± 0.10504
a
PI 0.1%29.68% ± 0.16862
b
PII 1%
30.14% ± 0.4041
bc
PIII 5%28.94% ± 1.21540
c
PIV 10%31.47% ± 0.11533d
Note: Figures with same letter indicate no significant
difference according to 5% LSD test.

Fig. 4: Mean Crude Fiber (CF) content.

Effect of EM-4 treatment on the crude fat of the husk
of Asian Palmyra palm
According to the last proximate analysis on
measurement data of Crude Fat of the husk along withstatistic test, significant influence due to EM-4 treatment(p>0.05) on Crude Fat was not found. In addition, 5%
LSD test showed that significant difference was yet tofind between control and 0.1% and 1% EM-4
treatments. However, highly significant difference wasfound between control and 5%and 10% EM-4 treatments(Table 5).
The highest crude fat content was found in 0 treatment(control), i.e. 1.04%, followed by first (0.1% EM-4),
second (1% EM-4), third (5% EM-4), and fourth (10%EM-4) treatments with crude fat content of 1.02%,
1.01%, 0.92%, and 0.91%, respectively (Fig. 5).
According to Assessment Institute for Agricultural
Technology (BPTP; ISBN 978-979-3112-32-9; 2010),
beef cattle feed during fattening phase (7 months)
requires nutrient content with 3% Crude Fat, meaning
feed without EM-4 treatment is capable of meeting
cattle’s need. From all the data obtained, it is known that
increasing EM-4 dosage decreases crude fat content.

D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed

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Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19 (2017)

Sari et al. (2015) stated that decreasing Crude Fat ispresumably because of fat breakdown by yeast’s
lipolytic enzyme. Pratiwi et al. (2008) stated that during
fermentation process, fat in the husk of Asian Palmyra
palm decreases due to fat degradation into fatty acids bylipase enzyme that naturally presents in feed materials or
is naturally produced by microorganisms growing infermented feed materials such as lipolytic bacteria (e.g.
Pseudomonas, Alcaligenes, Serratia and Micrococcus).
Such bacteria also belong to halophilic group. Fats arebroke down into volatile and non-volatile fatty acids thatwill produce aroma and taste.
Table 5. Summary of 5% LSD test on the effect of EM-4 on
the crude fat of Asian Palmyra palm’s husk
Treatment
Mean ± SD
Notation
P0 0%1.04% ± 0.01528
a
PI 0.1%
1.02% ± 0.04933
ab
PII 1%
1.01% ± 0.07000
abc
PIII 5%
0.92% ± 0.01528
bc
PIV 10%0.91% ± 0.08505
c
Note: Figures with same letter indicate no significant
difference according to 5% LSD test.

Fig. 5: Mean Dry Matters (DM) content.

Conclusion
Non-fermented husk of Asian Palmyra palm can be used
as 7 month beef cattle feed during fattening phase withbody weight of around 100-150 kg. The use of the husk
fermented with EM-4 (p<0.5) significantly influenced
nutrient contents (DM, OM, CP, CF, and crude fat).
Asian Palmyra palm husk treated with 1% EM-4 wasaveragely already capable of improving the husk
nutrient contents (DM, OM, CP, CF and crude fat).

Conflict of interest statement
Authors declare that they have no conflict of interest.
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How to cite this article:
Dahlan, D.N.A., Khotimah, K., 2017. In vitro evaluation of nutrient content of Asian Palmyra palm’s (Borassus
flabellifer L.) husk fermented with EM-4 for beef cattle feed. Int. J. Curr. Res. Biosci. Plant Biol. 4(1), 13-19.
doi: http://dx.doi.org/10.20546/ijcrbp.2017.401.003

D. N. A. Dahlan and K. Khotimah (2017) / In Vitro Evaluation of Nutrient Content of Asian Palmyra Palm’s (Borassus flabellifer L.)
Husk Fermented with EM-4 for Beef Cattle Feed19