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Protozoa population (mL) = 1 1000 n d
0.1 0.065 16 5

T1 = 2000 ppm Ca+1000 ppm P+150 ppm Mn
T2 = 2000 ppm Ca+1500 ppm P+150 ppm Mn
T3 = 2000 ppm Ca+2000 ppm P+150 ppm Mn

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90 79.21a 63.25b 55.36bc
80
70 T2 T3 53.56c
60 MPS T4
50
40
30
20
10

0
T1

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IOP Conference Series: Earth and Environmental Science

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To cite this article: Novirman Jamarun et al 2019 IOP Conf. Ser.: Earth Environ. Sci. 287 012019
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263Dasar Nutrisi Ruminansia (Edisi ke II)

Comparison of in vitro digestibility and rumen fluid
characteristics between the tithonia (Tithonia diversifolia) with
elephant grass (Pennisetum purpureum)

Novirman Jamarun, Roni Pazla, Mardiati Zain and Arief

Faculty of Animal Science, Andalas University, Padang, West Sumatera, Indonesia.
Email: [email protected].

Abstract. The objective of this study was to evaluate the effects of the tithonia (Tithonia

diversifolia) and elephant grass (Pennisetum purpureum) on in vitro digestibility and rumen fluid

characteristics. This research was carried out using a randomized block design with 3 treatments

(the level of tithonia and elephant grass) and 5 replications. The following treatments were

performed: T1 = 100% tithonia; T2 = 100% elephant grass; T3 = 50% tithonia + 50 % elephant

grass. The data were subjected to an analysis of variance (ANOVA), and differences between

the treatment means were t ultiple Range Test (DMRT). The parameters

measured were as follows: dry matter digestibility (DMD) (%), organic matter digestibility

(OMD) (%), pH, volatile fatty acid (VFA) (mM) and NH3. The results revealed that DMD, OMD

and VFA were significantly (P<0.01) increased by the 100 % tithonia (T1), pH were lowest

(P<0.01) in T1. However, T1 and T3 non-significantly affected (P>0.05) NH3 Concentration.

100% tithonia resulted in the best in vitro digestibility and rumen fluid characteristics.

Keywords In vitro, Tithonia diversifolia, Pennisetum purpureum, digestibility, rumen fluid

characteristics.

1. Introduction
Exploration of sources of ruminant animal feed is usually needed for the main work of the world of

nutritional nutrition lately. Production limitations are needed by scientists to find alternatives that can
be used by farmers in their application. One of the feed commodities that can be used as alter native feed
is tithonia plants. There are still few of these plants that are used for ruminant feed because these plants
have been mostly used in agriculture, namely composting with sufficient content such as Nitrogen (N)
and Phosphorus (P) which are quite high [1]. High N and P content is very good for ruminants. High N
content includes high crude protein content, while very high P content is needed by rumen microbes for
growth [2] [3]. Both of these things are needed by the rumen microbes to grow and develop. Rumen
microbes are the main engine of ruminants.

There is a problem in the use of P on the tithonia because P is not available due to being bound to
phytic acid. Phytic acid is the most antinutrient in tithonia [4]. In monogastric livestock the presence of
phytic acid in feed ingredients is a problem because there is no enzyme that can break down phytate in
the digestive tract, so that it will be a threat to livestock health. Ruminants have the advantage of
monogastric in the process of phytic degradation. Rumen microbes are able to produce pytase enzymes
to break P bonds in phytic acid so that p can be available to livestock and become the main mineral
supporting microbial growth [5]. This study aims to evaluate the digestibility and rumen fluids
characteristics of tithonia and elephant grass and evaluate the effect of the combination level of the two
feed ingredients.

264 Dasar Nutrisi Ruminansia (Edisi ke II)

2. Materials and methods
For this study, Analysis of in vitro digestibility and rumen fluid fermentability was conducted in the

Laboratory of Dairy Nutrition of the Bogor Agricultural Institute. Samples of T and EG were collected
around the town of Padang. All samples were finely ground and mixed according to the treatment
combinations. The parameters measured in this study were DM digestibility (DMD), OM digestibility
(OMD), NH3 concentration, total VFA content and pH. The chemical composition of Tithonia
diversifolia and elephant grass is presented in Table 1.

Tabel 1. The chemical composition of Tithonia diversifolia and elephant grass.

Composition (%) Feed Material

Elephant Grass Tithonia diversifolia

Dry Matter 21.23 25.57

Crude Protein 10.88 22.98

Crude Fiber 32.77 18.17

Fat 2.48 4.71

Ash 10.54 15.99

NDF 66.57 61.12

ADF 41.71 40.15

Cellulose 34.18 34.59

Lignin 6.29 4.57

Source: Nutritional Analysis of Ruminant Nutrition Laboratory Faculty of Animal

Husbandry Andalas University (2017).

2.1. In vitro procedure
The in vitro procedure in this study followed that of [6] Rumen fluid was taken from a cow fistula at

Bogor, LIPI. The fermenter tube was filled with 0,5 g of sample and 40 mL of McDougall solution was
added. The tube was placed in a shaker bath at 390C, filled with 10 mL of rumen fluid and shaken with
CO2 for 30 second, the pH was checked (6,5-6,9) and the sample was covered with a ventilated rubber
cap and fermented for 48 h. After 48 h, the rubber cap of the fermenter tube was removed and 2 -3 drops
of HgCl2 were added to kill the microbes. The fermenter tube was centrifuged at 5.000 rpm for 15 min.
The substrate was separated into a precipitate layer at the bottom and a clear supernatant at the top. The
supernatant was removed, the resulting sediment was centrifuged at 5.000 rpm for 15 min and 50 mL of
0,2% pepsin-HCl solution was added. This mixture was then reincubated for 48 h without a rubber cap.
The remaining digested residue was filtered using Whatman filter paper No. 41 (identified by weight)
with the help of a vacuum pump. The precipitate on the filter paper was placed in a porcelain dish that
was placed in a 105EC oven for 24 h. After 24 h, the porcelain cup + filter paper + residue was removed,
inserted into a desiccator and weighed to determine the DM content. Furthermore, the ingredients in the
cup were placed in a kiln or in an electric furnace for 6 h at 450-6000 C and weighed to determine the
amount of organic material. Residues originating from fermentation without feed ingredients were used
as a control. The proximate analysis of materials and residues followed the [7] and [8] procedure.

2.2. Rumen fluid characteristics
The measured rumen fluid characteristics were as follows: Ruminal pH, NH3 N concentration and

VFA content. Fermenter tubes were each filled with 0.5 g of sample and 40 mL of buffer solution and
10 mL of fresh rumen fluid (ratio of 4:1) were added. After the tube received CO2, the tubes were closed
with a rubber ventilator. Tubes were inserted into the fermenter shaker water bath at a temperature of
390 C and incubated for 4 h, followed by analyses of pH, NH3 and VFA. The ruminal pH was measured
using a pH meter. The NH3 -N concentration was measured using the micro diffusion (Conway) method
and the total VFA content was measured using the steam distillation method.

265Dasar Nutrisi Ruminansia (Edisi ke II)

3. Result and discussion
Data on digestibility values and characteristics of rumen fluid treatment are presented in Table 2.

Table 2. Data on digestibility values and characteristics of rumen fluid treatments.

Parameters Treatments

T1 T2 T3

Digestibility (%) 58,56a 55,99b 57,12c

Dry matter (DMD) 55,46a 53,99b 54,72c

Organic Matter (OMD)

Rumen Fluid

Characteristics 6,78a 6,79b 6,80c
125,88a 87,53b 112,15c
pH 22,48ab 20,41a 22,69b
VFA (mM)

NH3 (mM)

Means in the same row with different letter (a,b and c) are significant (P<0,01).

Table 2 shows that the use of 100% tithonia plants (T1) was very significant (P <0.01) increasing
DMD and OMD. The lowest DMD and OMD were obtained on 100% elephant grass (T2). The high
level of DMD and OMD in tithonia plants is due to the tithonia nutrient content better than elephant
grass. This can be seen from the level of protein, the amount of amino acids and mineral content. [9]
reported that tithonia contains a lot of amino acids. [1] added that the tithonia leaves contain high levels
of N, P, K, Ca and Mg minerals. The level of lignin titone is lower when compared to elephant grass.
Amino acids found in tithonia are needed by rumen microbes in their growth, while mineral content
such as P and Mg greatly stimulates the rumen microbes to grow and develop [10]. The development of
rumen bacteria will be positively correlated with an increase in DMD and OMD [11]. Lower lignin in
plants allows more bacteria to degrade feed so that DMD and OMD are higher in this treatment. [11]
reported a positive correlation between decreasing lignin and increasing DMD and OMD. The
combination between tithonia and elephant grass in treatment T3 is better than treatment using elephant
grass (T2). This is due to the contribution of the nutrient content of the tithonia plant to be able to supply
the rumen microbial needs better so that it increases DMD and OMD in this treatment.

The pH value in this study is the pH value that is still normal for the growth and development of
rumen microbes. According to [12] the optimal pH range for cellulose digestion is 6,4-7. If the pH of
the rumen fluid drops to below 6 it will inhibit microbial activity. Furthermore, [13] stated that pH
greater than 7,1 can reduce the microbial population drastically so that the energy produced is low.
Conditions for the rumen microbes to be able to perform activities optimally when the rumen pH is in
normal conditions is 6-6,9 [14]. [15] reported that a mixture of several levels of elephant grass
combination with different tithonia produced a normal pH value of 6,67 -6,68. pH at T1 treatment
showed the lowest pH value. Low pH in this treatment is due to the amount of nutrient content of the
VFA produced. High VFA content will reduce pH. In accordance with the opinion of [16] that the higher
the VFA value, the more other organic acids will be produced so that the rumen fluid pH will be low.

The highest VFA value (P <0.01) is found in T1. VFA describes the amount of carbohydrates
degraded by rumen microbes. The growth and development of rumen microbes is thought to be very
good in T1 due to a better supply of nutrients in the form of amino acids and minerals from tithonia
plants. As reported by [9] that Tithonia contains a lot of essential amino acids for the growth of microbes
such as methionine, leucine, isoleucine and valine. Rumen liquid VFA production can be used as a
benchmark for feed fermentability, where the higher the level of feed fermentability, the greater the
VFA produced [17].

The highest NH3 concentration was produced in T3 treatment but not significantly different (P>
0.05) with T1. The high content of crude protein in the tithonia and the number of amino acids contained
in the tithonia will positively correlate to NH3 concentrations. NH3 concentration is one indicator to
determine feed fermentability which is related to protein digestibility, rumen microbial activity and

266 Dasar Nutrisi Ruminansia (Edisi ke II)

population [18]. NH3 in all three treatments is still within normal limits. [19] states that ammonia
concentration to support rumen microbial growth is 6 mM 21 mM. NH3 and VFA which increase
linearly will form more microbial proteins so that they have a positive effect on livestock digestibility
and productivity.

4. Conclusion
100% tithonia resulted in the best in vitro digestibility and rumen fluid characteristics.

5. References
[1] Jama, B. A., C. A. Palm., R. J. Buresh., A. I. Niang., C. Gachengo., G. Nziguheba., and B.

Amadalo. 2000. Tithonia diversifolia as a Green Manure for Soil Fertility Improvement in
Western Kenya: aReview. Agroforestry Systems. 49: 201-221.
[2] Pazla, R., M. Zain., I, Ryanti and A. Dona. 2018. Supplementation of Minerals (Phosphorus and
Sulfur) and Saccharomyces cerevisiae in a Sheep Diet Based on a Cocoa By-product. Pak. J.
Nutr., 17: 329-335.
[3] Zain, M., J. Rahman and Khasrad. 2014. Effect of Palm Oil by Products on In Vitro Fermentation
and Nutrient Digestibility. Anim. Nutr. Feed Technol., 14: 175-181.
[4] Fasuyi, A. O., F. A. S. Dairo and F. J. Ibitayo. 2010. Ensiling wild sunflower (Tithonia
diversifolia) leaves with sugar cane molasses. Livest. Res. Rural Dev., Vol. 22, No. 3.
[5] Lamid, M. 2012. Karakterisasi Enzim Fitase Asal Bakteri Rumen (Actinobacillus sp dan Bacillus
pumilus) dan Analisis SEM terhadap Perubahan Struktur Permukaan Dedak Padi untuk
Ransum Ayam Broiler. Universitas Airlangga. (Unpublished).
[6] Tilley, J. M and R. A. Terry. 1969. A Two Stage Technique for In Vitro Digestion of Forage
Crops. J Br. Grassland Society 18 (2): 104 111.
[7] AOAC. 1995. Official Methods of Analysis. 15th Edition. Association of Official Analytical
Chemists. Association of Official Analytical Chemists, Washington, D.C.
[8] Van Soest, P. J., J. B. Robertson and B. A. Lewis. 1991. Methods for Dietary Fiber, Neutral
Detergent Fiber and Non-Starch Polysaccharides in Relation to Animal Nutrition. J. Dairy Sci.
74: 3583-3597.
[9] Oluwasola, T. A. and F. A. S. Dairo. 2016. Proximate composition, amino acid profile and some
anti-nutrients of Tithonia diversifolia cut at two different times. African Journal of
Agricultural Research. Vol. 11(38), pp. 3659 -3663.
[10] Febrina, D., N. Jamarun., M. Zain and Khasrad. 2016. The Effects of P, S and Mg
Supplementation of Oil Palm Fronds Fermented by Phanerochayte chrysosporium on Rumen
Fluid Characteristics and Microbial Protein Synthesis. Pakistan Journal of Nutrition 15(3):
299-304.
[11] Jamarun, N., M. Zein, Arief and R. Pazla, 2018. Populations of rumen microbes and the in vitro
digestibility of fermented oil palm fronds in combination with Tithonia (Tithonia diversifolia)
and elephant grass (Pennisetum purpureum). Pak. J. Nutr., 17: 39-45.
[12] Erdman, R. A. 1988. Dietary Buffering Requirements of Lactating Dairy Cows. A Review. J.
Dairy Sci. 71:3246-3246.
[13] Van Soest, R. J. 1982. Nutritional Ecology of the Ruminant Metabolism Chemistry and Forage
and Plant Fiber. Cornell University, Oregon, USA.
[14] Kamra, D. N. 2005. Rumen Microbial Ecosystem. Special Section: Microbial Diversity. Current
Science. 89(1):124-135.
[15] Jamarun, N., Elihasridas., R. Pazla and Fitriyani. 2017. In Vitro nutrients digestibility and rumen
fluid characteristics of the combination Tithonia (Tithonia difersivolia) and napier grass
(Pennisetum purpureum). Proceedings of the 3th Nasional Seminar on Cows and Buffalo,
Oktober 4-5, 2017, Padang, Indonesia.
[16] Pazla, R., N. Jamarun., M. Zain and Arief. 2018. Microbial protein synthesis and in vitro
fermentability of fermented oil palm fronds by Phanerochaete chrysosporium in combination

267Dasar Nutrisi Ruminansia (Edisi ke II)

with tithonia (Tithonia diversifolia) and elephant grass (Pennisetum purpureum). Pak. J.
Nutr.,17: 462-470.
[17] Jamarun, N and M. Zain. 2013. Dasar Nutrisi Ruminansia. Penerbit Jasa Surya. Padang.
[18] Muhktarudin dan Liman. 2006. Penentuan Tingkat Penggunaan Mineral Organik untuk
Memperbaiki Bioproses Rumen pada Kambing secara In Vitro. Jurnal Ilmu-Ilmu Peternakan
Indlrfesia. 8(2): 132-140.
[19] Mc. Donald, P., R. A. Edwards, J. F. D. Greenhalgh and C. A. Morgan. 2010. Animal Nutrition.
7th Edition. Longman. Scientific and Technical John Willey and Sons. Inc. New York.
Acknowlecgments
This research was funded by the Andalas University Profesor Grant 2018 and was part of the Ph.D.
dissertation of Roni Pazla in Animal Nutrition, Faculty of Animal Science, Andalas University.

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2.1. Fermented Oil Palm Fronds (FOPF)

2.2. Rations of ECDG
FOPF are stirred with concentrate consisting of rice bran, soybean meal waste, corn, palm kernel cake,
salt and minerals. Elephant grass and Titonia grasses are mixed according to treatment. Feeding is done
twice a day, which is at 08.00 and 17.00, feed was given according to NRC which is 4% body weightin
the form of dry matter [24]. Drinking water was given ad libitum. The composition of the ingredients in
the treatments ration in this experiment can be seen in tables 1,2 and 3.

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Table 1. Nutritional content of raw materials.

Raw materials

Nutritional content (%) FOPFa EGb Tb Rice Soybean Palm Cornb
branb meal kernel
wasteb cakeb 85.80
91.83 99,10
Dry Matter 72,01 21.23 25.57 87.80 28.40 91,41 07,70
12,36 02,44
Organic matter 91,34 89,46 84,01 90,80 97,67 26,68 03,50
08,23 49,96
Crude protein 08,89 10,88 22,98 10,72 20,11 66,70 36.76
46.10 29.52
Crude fiber 38,59 32,77 18,17 11,60 19,00 43.25 13.20
20.60 07,50
Extract ether 01,27 02,48 04,71 08,73 01,25 81,90
17,29
NDF 66,52 66,57 61,12 55.13 59,28 D
65,40 12
ADF 57.85 41.71 40.15 29.35 26.65 9,2
38.4
Cellulose 37.50 34.18 34.59 15.52 22.93 12
10
Hemicellulose 08.67 24.86 20.97 25.78 32.63 9
8
Lignin 18,35 06,29 04,57 06,90 02,20 1

TDN 61,90 63,48 62,60 66,63 74,61 100,0

Description: a. Laboratory of Dairy Animal Nutrition Faculty of Animal Husbandry IPB (2016) D
b. Laboratory of Ruminant Nutrition Faculty of Animal Husbandry Andalas University (2017) 49.00
88.62
Table 2. Ration composition %. 15.96
20.68
Raw Materials A treatments C 04.39
12 B 12 60.38
FOPF 38,4 19,2 39.64
Elephant Grasa 9,6 12 28,8 31.47
Tithonia 12 28,8 12 20.74
Rice bran 10 19,2 10 07.69
Soybeab meal waste 9 12 9 65.45
Palm Kernel cake 8 10 8 00.94
Corn 1 1 02.44
Salts+Mineral 9
Total 100,0 8 100,0
1

100,0

Table 3. Composition of experimental food ingredients.

food ingredients Treatments (%)

Dry Matter A BC
Organic matter
Crude protein 47.75 48.17 48.58
Crude fiber 90.20
Extract ether 12.48 89.67 89.14
NDF 24.84
ADF 03.75 13.64 14.79
Cellulose 61.95
Hemicellulose 40.08 23.44 22.04
Lignin 31.35
Total Digestible Nutrient 21.86 03.96 04.17
Ca 08.18
P 65.71 61.43 60.90
00.71
01.87 39.94 39.79

31.39 31.43

21.49 21.11

08.02 07.85

65.62 65.53

00.72 00.85

01.90 02.07

276 Dasar Nutrisi Ruminansia (Edisi ke II)

2.3. Milk collection and measurement of milk quality
Livestock maintenance during the study period was carried out in three periods, namely the
period of adaptation, introduction and collection. The adaptation period lasts for 15 days aimed
at adjusting livestock to the experimental ration. The preliminary period lasts for 25 days,
aiming to eliminate the influence of the previous ration. Collecting period is a data collection
period lasting 5 days for fecal sampling and calculation of feed consumption. Collective milk
production begins after the end of the 30-day adaptation period. Sampling for milk quality
testing was carried out for 2 times in the study in 2 different milking times, namely morning
and evening. Determination of the levels of protein, fat, milk lactose using the Milkotester
Master Pro 10211 tool. Phosphorus mineral levels were measured based on Taussky and Shorr
[25].

2.4. Experimental design and statistical analysis
This research was conducted using a completely randomized design (4x4) with 5 replicatios (levels of
FOPF, T and EG as the treatments). The differences between the treatment means were analyzed using

ervals of 5% and 1% Steel and Torrie [26].
The treatments were as follows:

A = 20% FOPF + 16% T + 64% RG
B = 20% FOPF + 32% T + 48% RG
C = 20% FOPF + 48% T + 32% RG
D = 20% FOPF + 64% T + 16% RG

Table 4. The milk quality of ECDG fed with FOPF combined with different levels of titonia and
elephant grass.

Treatment

Parameters A B C D
4.39
Protein (%) 4.03 4.08 4.14
8.23
Fat (%) 7.29 7.61 8.23
4.46
Laktose (%) 4.10 4.15 4.24 0.25c
0.21a 0.23b 0.24b 0.10c
Ca (%)

P (%) 0.08a 0.09b 0.09bc

a-cMeans in the same row with different letters are significantly different (P<0.01)

Judging from protein content, milk protein content of ECDG varied from 4.17-4.56% [27]. Sunarlim et
al. stated that the analysis of ECDG milk protein content was relatively higher at 4.3% for goat milk and
3.8% for cow's milk [28]. According to Afandi the protein content of goat milk ranged from 4.1% to
4.5% [1]. Chaniago and Hartono obtained goat milk protein content of 3.3-4.9% [29], while Adriani
obtained a range of goat milk, the results of which were 3.00-6.90% [30]. These results indicate the
protein content of milk obtained is still within the normal range of goat milkprotein. Protein content of

277Dasar Nutrisi Ruminansia (Edisi ke II)

ECDG milk obtained in this study is higher when compared to the results of Ardiansyah[31] and Rizqan
[32] research which obtained milk protein levels by feeding concentrate based on palm oil waste was
2.95% and 3.68%. Milk protein in this study included the category of premium or best milk protein
based on Thai Agricultural Standards due to> 4% milk protein value[33].

Not different effect (P> 0.05)of treatments A, B, C, and D on milk protein levels caused by crude
protein in all treatments consumed by livestock can still be digested by livestock so that the need for
crude protein to form proteins in milk is still can be fulfilled. The value of crude protein digestibility in
all treatments also differed not significantly (P> 0.05) and the protein quality of the ration given was
also high. Like the opinion raised by Smith et al. which states that high quality protein can be protected
from degradation of rumen microorganisms so that it is more available in the pasca rumen [34]. Then
the protein rations consumed will condense and enter the bloodstream to be converted into blood amino
acids with carbon precursors from non-essential amino acids. Furthermore, amino acids from the blood
will be converted into amino acid deposits and will enter the udder secretory cells and be synthesized
into milk proteins. This is explained by Collier which states that milk protein synthesis comes from
amino acids circulating into the blood as a result of absorption of the digestive tract, body protein
changes and amino acids synthesized epithelial cells gland milk into milk protein[35]. Akers adds that
when there is an increase in milk production, most of the protein or amino acid feed is focused on milk
synthesis so that the milk protein content does not increase [36].

Different types of milk protein treatments A, B, C and D are also caused by genetic factors that
ECDG are used uniformly. The effect of feed on milk protein is relatively small, even though the feed
protein of each treatment is different and increases from treatment A to D. This is in accordance with
the opinion of Le Jaouen which states that variation in milk protein content is relatively small compared
to milk fat content because Milk protein is more influenced by genetic factors than environmental factors
[37].

3.2. fat
The difference in titonia and elephant grass combination levels in each treatment did not affect (P <0.05)
the fat content of goat milk produced. The average fat content of goat milk was 7.84%, with a range of
7.29 - 8.23%. This result is higher than other studies in lactating ECDG which are 6 ± 0.05% Budi [38]
and Arief [39], with palm oil industry based byproducts of food which is 5.24%. Milk fat content
obtained in this study is almost the same as Chania goo and Hartono which is 7.3% [29]. This difference
is caused by differences in the crude fiber content in the ration. Sutardi states that milk fat content is the
most volatile component and is very dependent on the crude fiber content of food[40]. Low coarse food
fiber will produce low acetate, whereas acetate is the main ingredient in the formation of milk fat[41].
The fat content value in this study is quite high due to the high contribution of crude fiber content and
digestibility of crude fiber so that the need for crude fiber to form fat in milk can still be fulfilled and is
able to maintain milk fat. In dairy cattle consumption of crude fiber found in animal feed is very
important and affects the quality of milk, especially milk fat. As explained by Wikantadi the level of
milk fat is strongly influenced by the consumption of crude fiber in the feed given[42].

In addition, consumption of livestock for carbohydrates obtained from the combination of forage
combination FOPF, titonia and elephant grass is still sufficient so that acetic acid produced as a precursor
to form milk fat content is available in large quantities so that it can meet the needs in the formation of
milk fat content. This is supported by Arief' explanation that milk fat content is affected by acetic acid
from forage [39]. Forage eaten by livestock, then undergoes a fermentative process in the rumen by
rumen microbes. The results of the fermentative process are VFA. VFA consists of propionate, acetate
and butyrate. Acetate enters the blood and is converted into fatty acids, then it will enter the secretion
cells of the udder and become milk fat. Milk fat content is influenced by feed because most of the milk
components are synthesized in the udder of a simple substrate derived from feed[39].

278 Dasar Nutrisi Ruminansia (Edisi ke II)

3.3. Milk Lactose
The difference between titonia and elephant grass levels does not affect the lactose content of ECDG
milk. The average lactose of the study milk was 4.34%, with a range of 4.10 to 4.46%. This result is
almost close to some previous researchers, namely goat milk lactose is 5.0% [43] and 4.84% [39].
Lactose content of PE goat milk obtained in this study is still included in the standard levels of goat milk
lactose in the tropics in the opinion of Davendra and Burn which ranges from 3.52% - 6.30% [44].
Lactose milk is one indicator to increase the amount of milk produced, the higher the lactose content in
milk, the higher the absorption capacity of water for milk formation, resulting in increased production
of milk without changing the lactose content in milk [41].

It was not different in fact (P> 0.05) treatments A, B, C, and D due to the digestibility of the fiber
fraction also did not differ significantly, but an increase in titonia levels up to 64% showed the highest
levels of milk lactose. Fiber fraction is a source of carbohydrates. The carbohydrates found in these
feeds will be overhauled by microorganisms into VFA, one of which is propionic acid which is a
precursor in the formation of blood sugar which as the raw material for forming lactose and amino acids
absorbed in the intestine is converted into glucose in the liver through gluconeogenesis, so blood glucose
levels can be maintained. The availability of a substrate in the form of glucose can help in the process
of lactose milk synthesis. This is supported by the opinion of Schmidt41 also stated that glucose is the
main precursor to the formation of lactose milk. In addition, it was added by Leng et al. which states
that 54% of body glucose comes from propionic acid [45]. Concentrate on the four treatments was also
not different in type and number.

The mean of Ca and P of this experimental goat milk was 0.23% and 0.09%. This result is
lower than Arief which is 2.84% and 0.56% [39]. The combination of titonia and elephant grass
levels were very significantly different (P> 0.01) affecting the levels of Ca da P milk. The
highest levels of Ca and P were obtained at Treatment D (Combination of 64% T + 16% RG)
and the lowest in treatment A (16% T + 64% RG). This difference is due to the mineral content
of the ration. The content of Ca and P rations increased from A to D along with the addition of
titonia levels. Ration D gets the highest titonia portion so that it contributes high minerals Ca
and P as well. This is consistent with Jama et al. statement that titonia is rich in minerals such
as Ca and P [20].

Conclusion

Acknowledgments
This research was funded by the Andalas University Professor Grant 2018 and is part of the Ph.D.
dissertation of Roni Pazla in Animal Nutrition, Faculty of Animal Science, Andalas university.

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280 Dasar Nutrisi Ruminansia (Edisi ke II)

281Dasar Nutrisi Ruminansia (Edisi ke II)

Reproduced with permission of copyright owner. Further reproduction
prohibited without permission.

282 Dasar Nutrisi Ruminansia (Edisi ke II)

Volume 21, Number 5, May 2020 ISSN: 1412-033X
Pages: 1833-1838 E-ISSN: 2085-4722
DOI: 10.13057/biodiv/d210509

Effects of supplementation with phosphorus, calcium and manganese
during oil palm frond fermentation by Phanerochaete chrysosporium on

ligninase enzyme activity

RONI PAZLA, NOVIRMAN JAMARUN , FAUZIA AGUSTIN, MARDIATI ZAIN, ARIEF,
NESYA OKTIA CAHYANI

Faculty of Animal Science, Universitas Andalas. Jl. Unand, Campus Limau Manis, Padang 25175, West Sumatra, Indonesia
Tel./fax.: +62-751-71464. email: [email protected], [email protected]

Manuscript received: 15 October 2019. Revision accepted: 7 April 2020.

Abstract. Pazla R, Jamarun N, Agustin F, Zain M, Cahyani NO. 2020. Effects of supplementation with phosphorus, calcium and
manganese during oil palm frond fermentation by Phanerochaete chrysosporium on ligninase enzyme activity. Biodiversitas 21: 1833-
1838. The objective of this study was to evaluate the effects of supplementation with phosphorus (P) in combination with calcium (Ca)
and manganese (Mn) during oil palm frond (OPF) fermentation by Phanerochaete chrysosporium on ligninase enzyme activity and
lignin degradation. This study was carried out using a randomized complete design with 3 treatments (addition of P, Ca and Mn) and 5
replicates. The following treatments were performed: T1 (P 1000 + Ca 2000 + Mn 150 ppm), T2 (P 1500 + Ca 2000 + Mn 150 ppm),
and T3 (P 2000 + Ca 2000 +Mn 150 ppm). The data were subjected to an analysis of variance (ANOVA), and differences between
treatment means were tested using Duncan's multiple range test (DMRT). The parameters measured were as follows: lignin peroxidase
(LiP) activity (U/mL), manganese peroxidase (MnP) activity (U/mL), crude protein (CP) content (%), crude fiber (CF) content (%) and
the decrease in lignin (%). The results revealed a significant increase in LiP activity and CP content and a decrease in the lignin content
(p<0.05) by the addition of P in the T3 treatment. However, the treatment nonsignificantly increased (p>0.05) MnP activity and
significantly decreased (P<0.05) the CF content. In conclusion, supplementation of the OPF fermentation process with P 2000, Ca 2000,
and Mn 150 ppm resulted in the highest ligninase enzyme activity and in decreased lignin content.

Keywords: Calcium, manganese, MnP and LiP, Oil palm frond, Phanerochaete chrysosporium, phosphorus

INTRODUCTION lignin 30.63% (Jamarun et al. 2018). Although the
nutritional content allows the use of OPF as a source of
Livestock ruminants are able to contribute greatly to fiber feed, the OPF contains high amounts of lignin, which
human welfare by providing the most animal protein has limited digestibility (Suryani et al. 2016; Jamarun et al.
through meat and milk. The factors that determine the 2017).
success of the livestock business is the availability of
adequate feed ingredients, which should be available One effort to maximize the use of OPF includes
continuously but may be very difficult to obtain. Under lowering the lignin content. Lignocellulose is a major
those conditions, the need exists for alternative feeds, component of OPF, which consists of lignin, cellulose and
which may come from the utilization of agricultural and hemicellulose (Febrina 2016). Ligninase is a lignin-
plantation wastes, such as palm oil plantation waste breaking enzyme that can degrade lignin. The use of
(Jamarun et al. 2016). microorganisms that produce the enzyme ligninase is
highly recommended because this approach is more
Palm oil waste has the potential to be used as an environmentally friendly (Perez et al. 2002). One such
alternative feed because of its large production and ongoing effort to reduce lignin levels is fermentation using
availability. The area of oil palm plantations in Indonesia in Phanerochaete chrysosporium mold.
2015 reached approximately 11,300,370 Ha, and palm oil
production in West Sumatra in 2015 reached 1,002,920 Phanerochaete chrysosporium is the most efficient
tons, with growth in the production of crude palm oil lignin-degrading fungus (Suparjo 2008). Its growth is
increasing by 8.44% from 2014 to 2015 (BPS 2015) influenced by the availability of minerals in the substrate.
The minerals P, Ca and Mn are added to OPF fermentation
One type of palm oil waste that can be used as an to trigger the growth and extension of the cilia-like
alternative feed is oil palm frond (OPF) waste. It has mycelium (Pasaribu et al. 2003; Suparjo 2010).
nutrients that have potential when the waste is provided as Fermentation of the OPF that was fermented with
fiber feed. The result of the laboratory analysis showed that Phanerochaete chrysosporium with Ca 2000 ppm and Mn
the chemical composition of the OPF is as follows: dry 150 ppm minerals added resulted in a 25.77% decrease in
matter content (DM) 83.96%, organic matter (OM) the lignin (Febrina 2016).
94.23%, CP 3.64%, CF 49.80%, NDF 89.98%, ADF
73.21%, hemicellulose 16.78%, cellulose 41.35%, and Phosphorus (P) is an essential mineral for metabolic
processes and is required by all microbial cells primarily to

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1834 21 (5): 1833-1838, May 2020

maintain the integrity of cell membranes and cell walls, as Enzyme extraction of fermented OPFs
a component of nucleic acids and as part of high-energy Enzyme extraction was initiated with the centrifuge
molecules (ATP, ADP and AMP) (Zain et al. 2010). In
addition, in the cell, phosphorus is present as a were determined according to the method of Gassara et al.
phosphoprotein, phospholipid and nucleic acid compound. (2010): approximately 1 g of the sample was mixed with 50
The addition of the mineral P is expected to trigger the mM phosphate buffer (pH 6.5) at a ratio of 10/1 (v/w) and
development of mold. stirred at a speed of 150 rpm for 1 h. The samples were
then centrifuged at a speed of 7000 xg for 20 minutes. The
The addition of phosphorus to fermentation using resulting supernatant (liquid) was separated and used for
Phanerochaete chrysosporium has not been reported yet. analysis of enzyme activity.
Therefore, based on the description above, a study was
conducted to examine the effect of the mineral P on the Measurement of lignin peroxidase enzyme activity
fermentation of OPF using Phanerochaete chrysosporium, For determination of lignin peroxidase enzyme activity,
with added Ca and Mn minerals, to determine the effects of
these additions on the ligninase enzyme activity and lignin 0.1 mL veratryl alcohol 8 mM, 0.2 mL 50 mM acetate
degradation. buffer pH 3, 0.45 mL distilled water, 0.05 mL 5 mM H2O2,
and 0.2 mL enzyme filtrate were added to a 2 mL tube,
MATERIALS AND METHODS with the reaction being brought to a volume of 1 mL. The
cuvette tube was shaken slowly so that all ingredients were
Experimental site mixed. The enzyme activity reaction was carried out at a
For this study, the fermentation of OPFs and the temperature of 20 ± 1 °C. Absorbance was measured at 0
and 30 minutes at a wavelength ( ) 310 nm.
analysis of enzyme activity was conducted at the
Laboratory of Technology for Feed Industries, Andalas Calculation formula:
University, and the nutrient content analysis was conducted
at the Laboratory of Dairy Cattle Nutrition, Faculty of Where:
Animal Science, Bogor Agricultural University, Indonesia. max : the verified molarity of the veratryl alcohol
(9300 M-1 cm-1)
Materials d : the thickness of the cuvette (cm)
Leaves and palm fronds that have been ground. P.
OD310 : the difference in the absorbance value at
chrysosporium mold obtained from the Microbiology 310 nm
Laboratory of the Faculty of Life Sciences, Bandung
Institute of Technology (ITB), Indonesia. Minerals P, Ca Measurement of activity of manganese peroxidase
and Mn derived from the minerals KH PO4, CaSO .2H O enzymes
and MnSO .2H O. Mineral solution from Brook et al.
(Brook et al. 1969). Potato Dextrose Agar (PDA) culture Two stages of measurement occurred, namely, the
media for the growth of molds (v). Chemicals for measurement following the addition of Mn and the
proximate analysis of the activity of ligninase according to measurement without the addition of Mn.
Van Soest et al. (1991).

Research procedure Measurement upon the addition of Mn
OPFs fermentation For this measurement, 0.1 buffer lactate (pH 4.5, 50

The palm fronds used as the raw material in this study M), 0.1 mL guaiacol (4 mM), 0.2 mL MnSO4, 0.3 mL
were taken from the distal two-thirds of the OPF. The OPF Aqua DesTM (1 mM), 0.1 mL H2O2 (1 mM), and 0.2 mL
substrates were cut, dried and finely milled. The Ca was filtrate enzyme were added to a 2 mL tube for a total
obtained from CaSO4, P was obtained from KHPO4, and volume of 1 mL. The cuvette tube was shaken slowly so
Mn was obtained from MnSO4.H2O. Phanerochaete that all ingredients were mixed. The enzyme activity
chrysosporium was maintained on Potato Dextrose Agar reaction was carried out at a temperature of 20 ± 1 °C.
(PDA) slants at 4 °C, transferred to PDA plates at 37 °C for Absorbance was measured at 0 and 30 minutes at
6 days and subsequently grown on OPFs mixed with rice wavelength ( ) 465 nm.
bran. Equal amounts of OPF leaves and stems were used
and then the Brook mineral solution was added (Brook et Measurement without the addition of Mn
al. 1969). The fermentation process was initiated by adding For this measurement, 0.1 mL buffer lactate (pH 4.5 50
water to the OPFs until the water level reached 70%; then,
the Ca, P or Mn was added, depending on the treatment. M), 0.1 mL guaiacol (4 mM), 0.5 mL distilled water, 0.1
Observations were made every 48 h for 20 days (Febrina mL H2O2 (1 mM), and 0.2 mL filtrate enzyme were added
2016). Samples were then taken for proximate analysis and to a 2-mL tube for a total volume of 1 mL. The cuvette tube
fiber fraction determination. The proximate components was shaken slowly so that all the ingredients were mixed.
were determined as described by AOAC (2010). The The enzyme activity reaction was carried out at a
predominant fibers (NDF, ADF, cellulose, and lignin) were temperature of 20 ± 1°C. Absorbance was measured at 0
determined according to the method of Van Soest et al. (1991). and 30 minutes at wavelength ( ) of 465 nm. The
calculation formula is:

284 Dasar Nutrisi Ruminansia (Edisi ke II)

PAZLA et al. Effects of supplementation during oil palm frond fermentation 1835

Where: supplemented with P mineral in combination with Ca and
max : the molar absorption of the guaiacol (12100 M- Mn is presented in Table 1.
1 cm-1)
Table 1 shows that the highest enzyme activity of
d : the thickness of the cuvette (cm) ligninase (lignin peroxidase and manganese peroxidase) is
found in T3 treatment (P 2000 ppm); the lowest, in the T1
OD465 : the difference in absorbance value at 465 treatment (P 1000 ppm). The enzyme activity of ligninase
increases with the increasing dose of P (1000 ppm to 1500
nm ppm and 2000 ppm). The results of the diversity analysis
show that the P dosage significantly affected (P <0.05) the
The activity of MnP is the activity observed upon the enzyme activity of lignin peroxidase but did not
addition of Mn minus the enzyme activity without the significantly affect (P> 0.05) the activity of the manganese
addition of Mn. peroxidase enzyme.

Experimental design and statistical analysis The high activity of enzyme ligninase in the T3
treatment caused by the P 2000 ppm dose combined with
The study was carried out using a randomized complete Ca 2000 ppm and Mn 150 ppm was the optimum trigger
for the development of the mold hyphae. The increasing
design consist of three treatments with five replicates (the amount of mold enabled enzyme production, which also
increased, thereby increasing the rate at which the mold can
addition of P, Ca and Mn defined the treatments). The work to degrade the cell walls of the substrate. Febrina
(2016) reported that the optimal combination of minerals
differences between the treatment means were analyzed could produce optimal ligninase enzymes as well as break
down the cell wall in the fermentation process. Suparjo and
using multiple range test with a confidence Nelson (2012) stated that the amount of secreted enzymes
depends on the ability of the fungus to penetrate into the
interval of 5% (p<0.05). substrate affected by mycelia and increase mold growth
and production of the lignin-degrading enzymes that can be
The following treatments were carried out: T1 (1000 stimulated by mineral addition. Jamarun et al. (2017)
reported the supplementation of fermented OPFs by
ppm P + 2000 ppm Ca + 150 ppm Mn) (i), T2 ( 1500 ppm Phanerochayte chrysosporium with combination of Ca, P
and Mn provided the highest laccase enzyme activity and
P + 2000 ppm Ca + 150 ppm Mn) (ii), T3 ( 2000 ppm P + in vitro digestibility.

2000 ppm Ca + 150 ppm Mn) (iii). Phosphorus (P) is an essential mineral for metabolic
processes, and the addition of phosphorus from a variety of
Parameters measured previously different sources has been attempted in the
The variables measured in this study were LiP activity, fermentation of oil palm sludge by Aspergillus niger can
help the growth of mold mycelia (Pasaribu et al. 2003). The
MnP activity, the decrease in lignin content, and the addition of phosphorous minerals to deMan, Rogosa and
contents of CP and CF. Sharpe (MRS) medium for the growth of lactic acid
bacteria can lead to increased growth and cell density
RESULTS AND DISCUSSION (Subagiyo et al. 2016). Phosphorus is also a mineral that is
able to increase the activity of rumen microbes, thereby
The analysis of the data for the LiP activity, MnP increasing feed digestibility (Pazla et al. 2018).
activity, decrease in lignin content, and the contents of CP
and CF due to the addition of Ca, P and Mn in the Table 1. The decreased lignin and the contents of CP and CF
fermentation process of OPFs by Phanerochaete because of the addition of minerals in the fermentation of OPFs
chrysosporium is presented in Table 1 and Figure 1. by P. chrysosporium

Phanerochaete chrysosporium is a more complex Parameters T1 Treatments T3
model than the models provided by other strains for use in
the development and understanding of the ligninolytic Decreased lignin (%) 24.33a T2 40.08b
enzyme production system (Singh and Chen 2008). The CP content (%) 7.04a 29.41a 8.89b
resulting ligninolytic enzymes are lignin peroxidase (LiP) CF content (%) 42.95a 8.22ab 38.59b
and manganese peroxidase (MnP). The ligninolytic 40.56ab
peroxidases (LiP and MnP) produced by white-rot fungi
oxidize polymer lignin to aromatic radicals. The final stage Means in the same row with different superscripted letters are
of this process is the formation of a simple compound of significant at p<0.05, CP: Crude protein, CF: Crude fiber.
lignin degradation that enters the mold and synchronizes
into the intracellular catabolic pathway (Martínez et al.
2005). The rate of ligninase enzyme activity from the
fermentation of OPF using Phanerochaete chrysosporium

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1836 21 (5): 1833-1838, May 2020

Figure 1. Enzyme activity of lignin peroxidase and manganese peroxidase because of the addition of minerals in the fermentation of
OPFs by P. chrysosporium

The magnitude of the decrease in the lignin content is fermented substrate biomass (Nelson and Suparjo 2011).
visibly different in T3 with the addition of P mineral at Nelson and Suparjo (2011) stated that the increase in
2000 ppm (40.08%). This result is due to the availability of protein content is due to the bioconversion of sugar into
sufficient P so that the population and growth of protein mycelium or single cell protein; that is, the more
Phanerochaete chrysosporium increase. This increase in that mycelium becomes available due to the growth of the
mold growth will maximize the production and activity of mass, the more nitrogen available to the fungi and the more
the ligninolytic enzymes (LiP and MnP), resulting in proteins being contributed to the fermented substrate
optimal lignin reshuffle. Fadilah et al. (2009) stated that the (Musnandar 2003). In addition, Jonathan et al. (2008) noted
addition of nutrients will increase the rate of lignin that the increase in crude protein content in the
degradation and increase the growth of the fungi. The fermentation process is likely due to the result of the
better the mold growth is supported by the availability of addition of the mold biomass to the fermentation substrate.
minerals, as characterized by spores and mycelium High protein contents provide more Nitrogen (N) for
multiplication, the more LiP and MnP enzymes produced microbial growth. Nearly 80% of rumen microbes require
for the remodeling of the lignin. LiP and MnP are the main N for protein synthesis (Jamarun and Zain 2013). Good
catalysts in the lignolysis process by Phanerochaete microbial growth also improves feed digestibility (Pazla et
chrysosporium mold because they are able to breakdown al. 2018). The growth and development of rumen
the lignin structure. Imsya (2013) added that an increase in microorganisms are highly dependent on the availability of
the production of fungus spore enzymes will increase the nutrients and precursors, such as amino acids, N and
rate at which the mold works in breaking down the cell minerals (Elihasridas 2012).
wall.
The T1 treatment showed the lowest crude protein
The T1 treatment showed the lowest lignin drop. The content. This may be attributed to the nonoptimal growth of
result of lignin decrease in T1 is not much different from mold with supplementation P 1000 ppm so that the amount
the research of Febrina (2016) who showed a decrease in of enzyme produced is low, and the activity is low. Low
lignin of 25.77% in OPF fermentation, which is enzyme activity of LiP and MnP contribute to a lignin
supplemented with Ca and Mn minerals. The slight degradation process that is not optimal. High lignin will
decrease in lignin in this treatment is due to the nonoptimal prevent the reshuffle of nutrients by molds. Suparjo (2008)
growth support of mycelial fungi from the availability of P stated that LiP and MnP are the major enzymes in the
mineral so that the resulting LiP and MnP enzymes are also lignin degradation process because they are able to oxidize
low. The low activity of these enzymes means the process nonphenolic lignin units because of nonphenolic lignin
by which the lignin is changed is not optimal. units constitute approximately 90% of the lignin structure.

The highest CP content was obtained at T3. The Table 1 shows CF content ranging from 38.59% to
increase in the CP content in T3 is due to an increase in the 42.95%. The addition of P mineral to the OPF fermentation
number of mold cells in the mass. The secretion of by Phanerochaete chrysosporium shoots significantly
extracellular enzymes by Phanerochaete chrysosporium affected (P <0.05) the reduction in CF content of the
can also play a role in increasing the protein content of the fermented OPF. The lowest CF content (38.59%) was

286 Dasar Nutrisi Ruminansia (Edisi ke II)