Calcium soap from palm fatty acid distillate

for ruminant feed: Analysis of antioxidant

Cite as: AIP Conference Proceedings 2085, 020031 (2019); https://doi.org/10.1063/1.5095009
Published Online: 21 March 2019

Lienda A. Handojo, Antonius Indarto, Dian Shofinita, Muhammad R. Saadi, Dea Yulistia, and Fathinah I.
Hasyyati

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© 2019 Author(s).
Calcium Soap from Palm Fatty Acid Distillate for Ruminant
Feed: Analysis of Antioxidant

Lienda A. Handojoa), Antonius Indartob), Dian Shofinitac), Muhammad R. Saadi,

Dea Yulistia and Fathinah I. Hasyyati

Department of Chemical Engineering, Institut Teknologi Bandung, Jl. Ganesha 10, 40132 Bandung, Indonesia
a)Corresponding author: lienda@che.itb.ac.id
b)antonius.indarto@che.itb.ac.id
c)shofi@che.itb.ac.id

Abstract. Dairy cows often get metabolic disorders and low milk productions during lactating periods because the
animals don’t have adequate feed supply and use only the food reserves from their body. Supplementary fat is known to
provide sufficient energy but creating problem with fiber digestion in the rumen. The calcium salt of fatty acids is being
used to solve the problem. The mixture of such soaps with cattle’s regular feed can increase the yield, the number of fats,
and the gross energy in cattle's milk. Palm fatty acid distillate (PFAD) can serve the fatty acids needed for calcium soap
production, also have the role as the natural source of antioxidants for the products as it has several bioactive compounds
including vitamin E (mainly as tocotrienols), phytosterols, and squalene. Those components are believed to have the
good impact for cows, and it has a good oxidation resistance. This research studied the possibility of those antioxidants
for being derived to the products after the reaction occurred by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, a
widely and commonly-used spectrophotometric method to measure the radical scavenging activity. By using ethanol, the
half maximal inhibitory concentration (IC50) of PFAD and calcium soap are 60.4 µg/ml and 487.6 µg/ml respectively,
while using methanol the results are 49.9 µg/ml for PFAD and 256.6 µg/ml for the soap. There must be some
antioxidants in the product after the reaction, despite the small number. The loss of antioxidants might be caused by
several factors, one of them is the reaction temperature as antioxidants are thermally sensitive.

INTRODUCTION
Dairy cows often experience metabolic disorders and low milk productions as they use the food reserves from
their body during the lactating periods. The conditions where they are suffering from malnutrition is because
inadequate energy intake from the feed [1]. The most appropriate solution for this problem is to maximize energy
intake by increasing the energy density of cattle feed.
Giving excess grain as cattle feed can increase energy density but also accelerate the fermentation process, will
lead to acidosis conditions which adversely affect the condition of the cows’ rumen [2] and reduce the concentration
of milk fat. Giving crude fat supplements can also increase energy density but high fat levels can cause a decrease in
fiber digestibility and a decrease in the percentage of milk fat, depending on the amount and type of fat given [3].
To avoid these adverse effects, giving salt from long chain fatty acids as cattle feed is a good alternative [4].
Long-chain saturated or unsaturated fatty acids in the form of free fatty acids have more influence on fermentation
in cows’ rumen than given in the form of calcium salts (calcium soap) [5]. Another plus is the increase in reserves of
unsaturated fatty acids (polyunsaturated fatty acids) in the cows’ milk glands which will benefit consumers with
heart problems [6, 7]. The addition of calcium soap as a dairy cattle feed mixture can increase milk acquisition, fat
acquisition in milk, and crude energy from cows’ milk [8].

The 11th Regional Conference on Chemical Engineering (RCChE 2018)

AIP Conf. Proc. 2085, 020031-1–020031-5; https://doi.org/10.1063/1.5095009
Published by AIP Publishing. 978-0-7354-1815-8/$30.00

020031-1
 The raw material suitable for making cattle supplements for calcium soap is Palm fatty acid distillate (PFAD). In
addition to high free fatty acid content, PFAD also contains phytochemical compounds in the form of vitamin E,
phytosterol, and squalene hydrocarbons [9]. Vitamin E is an antioxidant that has an important role in the
maintenance of cellular membranes, immunity, and reproduction of dairy cows [10].
Antioxidants are electron-giving compounds (electron donors) that can inhibit oxidation reactions, by binding
free radicals and highly reactive molecules. Antioxidants act to counteract free radicals in the body so that they can
fight oxidative damage and also inhibit the oxidation process of fats or oils so that they have a preservative function
[11]. Antioxidants have the ability to neutralize free radicals without being free radicals themselves [12].
Vitamin E is not degraded by ruminant microorganisms. The recommended level of vitamin E supplementation
is 0.8 IU/ kg body weight (around 20 IU/kg-dry matter intake (DMI)) for nursing cows. Increased supply of vitamin
E to the cell membrane can improve immune function by protecting neutrophils from oxidative damage after the
destruction of intracellular bacteria that are swallowed [10].
This study is intended to determine the potential for antioxidant which is still contained in calcium soap
produced from the PFAD saponification reaction process. Determination of antioxidant activity was carried out
using the DPPH method which was then compared with PFAD as raw material. DPPH (1,1-diphenyl-2-
picrylhydrazyl) is a stable free radical compound indicated by the presence of reserve electron delocalization in the
whole molecule to prevent the molecule from being dimerized, in contrast to most other free radicals. Delocalization
also causes a dark violet color, characterized by absorption band in ethanol solution centered at around 520 nm [13].
When a DPPH solution is mixed with a substance that can donate hydrogen atoms (in this case antioxidants), DPPH
will be reduced marked by loss of violet color. These properties cause DPPH to be superior and commonly used for
the analysis of antioxidant activity using spectrophotometry.

MATERIAL AND METHODS

Materials
There are two samples in this research, PFAD and calcium soaps. The PFAD obtained from PT Tunas Baru
Lampung Tbk, a crude palm oil refining plant in Sidoarjo, East Java, Indonesia. The calcium soap was obtained by
reacting the mentioned PFAD with calcium oxide (CaO) from Padalarang. Vitamin E was used as the sample’s
comparison, a commercial one with concentration of 11000 IU. The other materials were methanol (pro analysis
grade) and 2,2-diphenyl-1-picrylhydrazyl (C18H12N5O6, Sigma Aldrich), which were used for the DPPH assay.

Methods
The sample was dissolved in a solvent with a weight ratio of 3:1 (solvent to sample). Ethanol and methanol were
used as the solvent. The sample was stirred and heated at 65-70 °C in two hours. The extracted sample was diluted
into a 1000 µg/ml standard solution which was used to make other solutions in a different, ranged concentration.
DPPH solution prepared by dissolving the DPPH to get a concentration of 90 µM, then it was added to each sample
solution in 20% v/v of the sample. The control solution was the DPPH solution without the presence of the sample
solution. The samples incubated for about 30 minutes, considered as the time needed for the antioxidants react with
free radical. Then the absorbance of those solutions was measured by the spectrophotometer (Genesis 10 UV-Vis) in
the working wave of 517 nm.
Inhibition percentage is calculated from the difference between the absorbance of the control solution with the
absorbance of samples at various concentrations, or simply defined by Equation (1).

(1)
I is the inhibition percentage
A0 is the absorbance of control solution
Ac is the absorbance of samples in certain concentration

The inhibition percentage in concentration variation was plotted on the graph to get a linear equation and the IC50
value was approximated by that equation. The IC50 was used as the comparison of antioxidant activity among the
samples.

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 RESULTS AND DISCUSSION
PFAD is in a golden yellow semi-solid form at room temperature and melts into the gold liquid on heating.
PFAD for the most part composed of free fatty acid (>80%) with palmitic and oleic acid as the main components,
the remains are glycerides and unsaponifiable matters such as vitamin E, squalene, and sterols [13]. PFAD is highly
soluble in the organic solvent, different from calcium soap which is not soluble in organic solvent nor water.
Calcium soap is in powder when the unsaponified matters (antioxidants) are extracted in alcohol.
The absorbance of each kind of samples checked in the different range of concentration because based on
preliminary experiments, they have different antioxidant activity. The range adjusted so as the inhibition percentage
isn't far too low, or even more than 100%. It becomes important as the aim of this measurement is to get a linear
correlation between the inhibition and concentration of the sample. Table 1 shows the inhibition percentage of each
sample by using ethanol as the solvent.
TABLE 1. Inhibition percentage of samples
Concentration Control’s Sample’s Inhibition
Sample
(µg/ml) Absorbance Absorbance (%)
20 0.177 42.94
40 0.173 46.24
PFAD 60 0.253 0.169 49.70
80 0.165 53.33
100 0.161 57.14
350 0.197 28.43
400 0.187 35.29
Calcium soap 450 0.253 0.177 42.94
500 0.167 51.50
550 0.157 61.15

The plot between inhibition percentage and the sample’s concentration bring in the equation as shown in Figure
1 which is used to determine the value of IC50. The coefficients of determination (R2) from both graphs show that
there is a significant correlation between the inhibition percentage and the sample’s concentration, noticed by their
values (>0.995).

70 70
60 60
50 50
Inhibition (%)

y = 0.1775x + 39.222 y = 0.1633x - 29.618

Inhibition (%)

40 R² = 0.9992 40 R² = 0.9955
30 30
20 20
10 10
0 0
0 20 40 60 80 100 120 0 100 200 300 400 500 600
Concentration (µg/ml) Concentration (µg/ml)
(a) (b)
FIGURE 1. The relation between inhibition percentage and sample’s concentration of (a) PFAD, and (b) calcium soap by using
ethanol as the solvent

The IC50 value shows how much is the antioxidant potential in the sample to inhibit half of the total radical
activity caused by the addition of DPPH. From the linear equation of Figure 1, the IC50 of PFAD and calcium soap is
60.4 µg/ml and 487.6 µg/ml respectively. PFAD has greater radical scavenging activity than calcium soap samples
because to do 50% radical inhibition, it takes less PFAD (60.4 µg/ml) compared to the amount of calcium soap
needed for doing the same inhibition (487.6 µg/ml). When compared between PFAD as raw material and calcium
soap as a product, there is a significant difference in radical scavenging activity which reflects the number of total

020031-3
antioxidants, wherein calcium soap there is less antioxidant compared to PFAD. Based on antioxidant activity
classification [14] shown in Table 2, PFAD has active antioxidants while calcium soap has weak antioxidants.

TABLE 2. Classification of antioxidant activity by DPPH assay

Intensity IC50 Value (µg/ml)
Highly active <50
Active 50-100
Moderate 101-250
Weak 250-500
Inactive >500

Based on mass balance calculations, the antioxidant content in 60.4 µg/ml PFAD should be equivalent to the one
at 72.7 µg/ml calcium soap if all the antioxidants from PFAD maintained in the product. The loss of antioxidant can
be caused by several factors. First, there is a possibility that some antioxidants from PFAD lost due to the
exothermic reaction [15], so there aren't many antioxidants left in the product. Bioactive compounds especially
vitamin E, phytosterol, and squalene are known to be extremely sensitive to temperature [16]. The process of
making soap that takes place at 60-100 °C [17] is suspected to be the reason for the reduced amount of antioxidants.
Second, the process of heating and stirring the calcium soap in ethanol during the analysis of antioxidant causing the
powder to agglomerate and harden, so the diffusion of the antioxidant component to the solvent is suspected to be
inhibited. Figure 2 shows the extraction process of samples.

(a) (b)
FIGURE 2. The extraction results of (a) PFAD, and (b) calcium soap by using ethanol as the solvent

Methanol is used to see whether there are physical or chemical differences in the extraction process and IC50
values. There is no physical difference in the soap extraction process by using methanol as a solvent.
Agglomerations are still formed as shown in Figure 2(b), but the extract solution of calcium soap using methanol has
a smaller IC50 value than ethanol solvents, as well as the IC50 value of PFAD. Table 3 shows the IC50 value by using
different solvents.

TABLE 3. The IC50 value of samples by using ethanol and methanol

IC50 (µg/ml)
Sample
Ethanol Methanol
PFAD 60.4 49.9
Calcium soap 487.6 265.6

DPPH test on vitamin E samples (11.000 IU) is done to get a comparison of antioxidant activity in vitamin E
with PFAD and calcium soap. Vitamin E samples have IC50 values of 26.02 µg/ml, classified as compound with
very strong antioxidant content (<50 µg/ml) [14]. When compared to the IC50 value with methanol, antioxidant
activity in PFAD and calcium soap is about 1.9 times and 10.2 times weaker than vitamin E sample respectively.
Vitamin E requirement for lactating dairy cow is 360 IU/day or equal to 241.2 mg α-tocopherol/day according to
United States Pharmacopeia (USP) conversion factors [10]. Calcium soap has antioxidant 10.2 weaker than vitamin

020031-4
E sample, which means equal to approximately 1077 IU of vitamin E. Thus, giving calcium soap as dairy cow’s
daily supplement can fulfill the daily intake of vitamin E.

CONCLUSIONS
PFAD is one of the suitable materials for making calcium soap as feed supplements. Besides its free fatty acid
content, PFAD also rich in phytochemical compounds such as vitamin E, phytosterol, and squalene which can give
health benefit to the dairy cattle. However, antioxidants are thermally sensitive and exothermic reaction can reduce
the concentration in the product. Following the antioxidant extraction characteristics, the use of solvents other than
methanol and ethanol could be evaluated to obtain the best performance. Further next study is necessary to be
conducted to ensure that the antioxidant doesn’t degraded during reaction. Moreover, the product of the experiment,
following the value of IC50, is already meet the requirement of daily vitamin E needs for cattle.

ACKNOWLEDGMENTS
This research is supported by BPDPKS Indonesia financially and PT Tunas Baru Lampung Tbk in providing
PFAD.

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