International Journal of Technology 12(3) 661-670 (2021)

Received May 2020 / Revised July 2020 / Accepted March 2021

International Journal of Technology

http://ijtech.eng.ui.ac.id

Efficacy of a Natural Papain-Induced Enzyme on Virgin Coconut Oil-Based

Hand and Body Lotions with Added Orange Peel Extract

Indah Yuniarti1*, Enrico Fendy Sapatra1, Sagitha Fitri Novia1, Hovivah1, Vita Paramita1,
Mohamad Endy Yulianto1
1Chemical Industrial Engineering Technology, Diponegoro University, Semarang 50275, Indonesia

Abstract. The objective of this research was to determine the effect of addition of natural papain
enzyme on virgin coconut oil production and to characterise the physical quality of hand and body
lotions made with virgin coconut oil. A natural papain enzyme was extracted to separate the virgin
coconut oil from blondo and water, and the virgin coconut oil was then made into a hand and body
lotion. The highest yield of virgin coconut oil (13.8±5.30%) was obtained with a 45% papain enzyme
extract. The virgin coconut oil had an acid number of 0.135±0.50%, which fulfilled the SNI
7381:2008 standard for acid numbers. The peroxide number for the virgin coconut oil was
1.16±0.51 meq/kg and in accordance with the SNI 7382:2008 standard for peroxide numbers. The
organoleptic test for hand and body lotion products prepared from the virgin coconut oil extracted
with the papain enzyme was carried out with 32 respondents. The lotion colour had adequate
acceptance by the respondents, who also viewed the lotion texture, homogeneity and humidity as
satisfactory.

Keywords: Body lotion; Vegetable oil; Virgin coconut oil

1. Introduction
For the past two decades, coconut (Cocos nucifera L.) fruit has gained popularity due to
the stability and benefits of the saturated fatty acids in its triglycerides (Satheeshan et al.,
2020). In Indonesia, coconut fruit is typically used in cooking for its good flavour. However,
the value of coconut fruit can be increased by processing it to produce coconut oil.
According to Harimurti et al. (2020), two types of coconut oil can be obtained: ordinary
coconut oil and virgin coconut oil (VCO). Both types have many benefits; for example,
ordinary coconut oil can be processed for use as an alternative fuel to replace petroleum
diesel fuel (Muharam and Putri, 2018), while VCO can be used to maintain skin smoothness
and moisture because of its lauric acid content (Harimurti et al., 2020).
VCO is easy to produce using simple methods. Enzymatic methods are particularly
advantageous because enzymes are nontoxic accelerating agents and are active at low
concentrations. Enzymes are also produced from natural materials, so they are
environmentally friendly (Harimurti et al., 2020). One enzyme that can be used to produce
VCO is papain, a proteolytic sulphydryl protease enzyme found in the fruit sap, stems and
leaves of the papaya plant (Carica papaya L.). The papain enzyme is easy to obtain at
anaffordable price (Setiadi and Hidayah, 2018). In VCO production, papain can help break

*Corresponding author’s email: [email protected], Tel.: +62-85740573276

doi: 10.14716/ijtech.v12i3.4070
662 Efficacy of a Natural Papain-Induced Enzyme on Virgin Coconut Oil-Based
Hand and Body Lotions with Added Orange Peel Extract

the lipoprotein bonds in coconut milk emulsions to release the oil for collection (Silaban et
al., 2014). Papain is therefore a useful active ingredient for producing skin cleansing
creams, especially for the face and for dissolving dead cells that are difficult to physically
detach from the human skin (Soda and Agustini, 2013). VCO can soften and moisturise the
skin, so it can be used as the base material for producing skin lotions. The benefits of hand
and body lotions can be further improved by adding an antioxidant agent to slow down the
production of wrinkles and stop premature aging (Park et al., 2014). Orange peel is a good
antioxidant with more activity than the flesh of the orange fruit (Hegazy and Ibrahium,
2012).
The present research focuses on the novel application of a natural papain extract from
papaya leaves for VCO production. The papain extract was prepared using a maceration
method. Specifically, the effects of the addition of a natural papain extract were examined
on the yield, free fatty acid content and peroxide number of the resulting VCO. In addition,
the effect of adding an orange peel extract on the pH, density and viscosity of a VCO body
lotion was determined.

2. Methods
2.1. Materials
The materials included mature coconuts, tempe yeast and orange peel obtained from a
suburban area in Semarang (Indonesia). A commercial extra virgin coconut oil product was
purchased from CV Ajisaka Kelapa (Malang, Indonesia). Acetic acid-chloroform solution,
Phenol Pthalein (PP) indicator, and buffer solution (pH 4.01 and 7.01) were purchased from
Merck (Darmstadt, Germany). Potassium iodide, 0.1 N Na2S2O3, 0.1 N KOH, distilled water
and 96% alcohol were purchased from CV Indrasari (Semarang, Indonesia).
2.2. Coconut Cream Preparation
A 1000 g sample of mature coconut meat was peeled, shredded and added to warm
water at a ratio of 1:1, followed by filtration through a filter cloth to obtain the coconut milk.
A 1000 mL sample of coconut milk was then allowed to stand at room temperature until it
separated into 2 layers: a skim layer (bottom layer) and the coconut cream. The skim layer
was removed, leaving 800 mL coconut cream. A 600 mL sample of the coconut cream was
then used to make VCO with enzymatic papain and the remaining 200 mL was fermented
with tempe yeast by mixing 3 g of tempe yeast in 5 mL distilled water (Sapatra et al., 2019).
2.3. Preparation of the Papain Extract from Papaya Leaves
Young papaya leaves (25 g) were removed from a papaya tree, washed and chopped
(Ihromilala, 2016). Distilled water was added at a 1:1 ratio to the mass of papaya leaves and
the leaves were blended in a blender. The blended papaya leaves were left at room
temperature for 24 h, with occasional stirring, to extract the papain protein. The papaya
leaf residue was then removed by filtering the extract through a filter cloth (150 mesh).
2.4. Coconut Oil Preparation
Figure 1 shows the coconut oil production protocol based on papaya leaf papain
treatment and on tempe yeast treatment. The prepared papain enzyme extract was added
to 200 mL of coconut cream with varying ratios of enzyme to cream (15, 30 and 45%). The
tempe yeast–based coconut oil was prepared by adding 0.6 g (w/v) of tempe yeast to 200
mL of coconut cream. The mixtures were then left for 24 h to allow formation of 3 layers:
pure coconut oil (upper layer), blondo (middle layer) and water. The pure coconut oil (VCO)
was separated with a separatory funnel (Sapatra et al., 2019).
Yuniarti et al. 663

2.5. Orange Peel Extract Preparation

A 25 g sample of orange peel was dried and ground until smooth and then dissolved in
10 mL ethanol. The mixture was filtered through a filter cloth (150 mesh) to obtain the
orange peel extract.
2.6. Preparation of the Hand and Body Lotion
The lotion was prepared from 5 g cocoa butter, 30 g beeswax and 50 mL VCO, which
were mixed and heated at 50C and then stirred until homogeneous. The orange peel extract
was added to the mixture at a ratio of 3, 5, and 7%. A 3 mL volume of polysorbate 20 was
added as an emulsifier, 0.2 mL methyl paraben was added as a preservative and 5 drops of
perfume were added as a fragrance (Figure 1).

Figure 1 Schematic process showing the preparation of a natural papain enzyme extract from
papaya leaves and its use in the production of virgin coconut oil hand and body lotion with added
orange peel extract

2.7. Determination of the Coconut Oil Yield

The oil yield was determined from the ratio of the weight of the oil produced and the
initial raw material weight (Equation 1).
𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡
𝑌𝑖𝑒𝑙𝑑 = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 100% (1)

2.8. Determination of Peroxide Numbers

A 2.5 g sample of VCO was placed in an Erlenmeyer flask, followed by the addition of
15 mL acetic acid-chloroform (3:2, v/v) and 0.25 mL saturated KI solution. The flask was
sealed and the mixture was shaken several times for 1 min, and then 15 mL distilled water
was added. The mixture was then titrated with 0.1 N Na2S2O3 until a yellow colour formed.
A 0.25 mL volume of 1% starch solution was then added and the mixture was titrated again
664 Efficacy of a Natural Papain-Induced Enzyme on Virgin Coconut Oil-Based
Hand and Body Lotions with Added Orange Peel Extract

until the colour turned blue. The peroxide value was expressed in milliequivalents of
peroxide per 1000 g of sample (Equation 2).
𝑚𝐿 𝑁𝑎2 𝑆2 𝑂3 𝑥 0.1 𝑁 𝑥 1000
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 = (2)
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡

2.9. Determination of Acid Numbers

A 4 g sample of VCO was placed in an Erlenmeyer flask, followed by addition of 10 mL
96% alcohol and PP indicator. The mixture was then titrated with a standardised 0.1 N
NaOH solution until a red colour appeared. The percentage of free fatty acids was
determined using Equation 3. This value is expressed as oleic acid equivalents for most oils
and fats, but coconut oil and palm kernel oil are expressed as lauric acid equivalents and
palm oil is expressed as palmitic acid equivalents. The free fatty acids are expressed as %
FFA or as an acid number.
𝑚𝐿 𝑁𝑎𝑂𝐻 𝑥 0.1 𝑁 𝑥 𝐹𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 100%
%𝐹𝐹𝐴 = (3)
𝑆𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 1000

2.10. Organoleptic Test of the VCO

The organoleptic test was carried out using the scoring test, where a higher number
indicated better VCO quality. The VCO samples were evaluated by asking the panellists to
score how they liked the taste, colour and aroma of each product. The number of samples
was determined by Slovin’s method and expressed in Equation 4 (Ansar et al., 2017):
𝑁
𝑛= (4)
1+𝑛𝑒 2
where N is the population (a student classroom of 48 members), n is the number of samples
and e is error tolerance (0.01).
2.11. Hand and Body Lotion Test
2.11.1. pH testing
Samples (1 g) of lotion containing 3, 5 or 7% orange peel extract were dissolved in 100
mL of distilled water and the pH was determined using a standard pH electrode (Hanna
Digital pH Meter HI 98107, SG, Singapore).
2.11.2. Organoleptic test
The organoleptic test was carried out as a scoring test, where a high number indicated
better quality. The panellists were asked to score how they liked the taste, colour and
aroma of the product. The number of samples was determined by Slovin’s Equation (Ansar
et al., 2017).

3. Results and Discussion

3.1. Chemical Analysis of the VCO
The results for the VCO prepared using tempe yeast or by the papain enzyme extract
were compared with a commercially available market VCO in terms of %yield, %FFA
content, and peroxide number (Table 1).
Table 1 presents the chemical content of VCO with the yield calculated by comparing the oil
produced with the initial coconut milk volume and expressed as a percentage. The VCO
produced with 45% papain enzyme extract had the highest yield (13.8%).
The breakdown of the emulsion in coconut milk can be assisted by proteolytic
enzymes, such as papain, which can catalyse protein breakdown reactions by hydrolysing
peptide bonds and forming simple compounds (Sapatra et al., 2019).
Yuniarti et al. 665

Table 1 Chemical content of virgin coconut oil

Sample De-emulsifier (%) Yield (%) FFA (%) Peroxide Number (meq/kg)
Extract of Papain Enzyme
P1 15 12.0±5.66 0.195±0.01 2.44±0.57
P2 30 12.5±6.36 0.188±0.02 1.78±0.26
P3 45 13.8±5.30 0.135±0.50 1.16±0.51
Tempe Yeast
P4 1.5 10.8±1.06 0.215±0.28 1.80±0.28
Market Purchase
P5 - - 0.198±0.00 5.80±0.85

One technique for extraction of papain enzyme is maceration, which is carried out by
soaking a simplicia powder in a filtered liquid. The liquid penetrates the cell wall and enters
the cell compartment that contains the desired active substance. The active substance
dissolves because of the difference in concentration of the active substance inside and
outside the cell, so the concentrated solution is forced out (Sudarwati and Fernanda, 2019).
The formation of virgin coconut oil by papain treatment is due to the hydrolysis of the
peptide bonds in the coconut cream. The hydrolysis process causes destabilisation of the
emulsion system in the coconut milk, resulting in the release of the oil as virgin coconut oil
(Setiadi and Hidayah, 2018). This causes a faster formation and increased yield of VCO. The
low yield obtained in the present study probably reflects the use of an unpurified, naturally
extracted papain enzyme from the papaya leaf. Paramita et al. focused their studies on non-
commercial enzymes in order to avoid the expense of mass-scale production (Naidu et al.,
2012; Paramita and Yulianto, 2019; Paramita et al. 2019). However, Qiuyu et al., 2009
applied 0.075% papain and obtained an 82.9% yield of VCO. The use of a low temperature
for VCO production (27C) using papain also makes this a green process and
environmentally friendly (Muharam and Putri, 2018; Mulia et al., 2018). By contrast, the
VCO production using tempe yeast fermentation gave the lowest yield due to the influence
of temperature on Rhizopus oligosporus enzyme activity. The optimum temperature for
growth of this fungus is 30–35C (Salehoddin et al., 2020).
The FFA content is important in the final VCO product due to the effects on the taste
and smell of the oil. A high water content in VCO promotes the activity of microorganisms
that can give rise to oil with a high level of fatty acids and further reduce its quality
(Iskandar et al., 2015). The FFA values were calculated based on the colour change from
yellow to purplish-pink while titrating the VCO with NaOH. As shown in Table 1, treatment
with the papain enzyme resulted in lower FFA values compared to the other samples. The
lowest value of 0.135±0.50% was obtained for P3, which was found to have fulfilled the
0.2% maximum acid value required by SNI 7381:2008 for coconut oil and the value
decreased as the enzyme concentration increased. The market VCO had a FFA value of
0.195±0.01%, while the tempe yeast fermentation product had a value of 0.215±0.28% and
these values, according to Male et al. (2014), were unacceptable according to the current
standard (BSN, 2008). The high FFA level was associated with the high water content in the
VCO, which affects its quality, while the high acid numbers were derived from oil hydrolysis
or poor processing.
Peroxide numbers are very important indicators of damage to the oil due to the binding
of oxygen to the double bonds of unsaturated fatty acids. A higher peroxide number
indicates a greater risk of rancidity and the oil, when used in foods, can later cause fat
deposition in blood vessels and decrease the digestibility after consumption (Talbot, 2016).
666 Efficacy of a Natural Papain-Induced Enzyme on Virgin Coconut Oil-Based
Hand and Body Lotions with Added Orange Peel Extract

The P3 sample also had the lowest amount of peroxide, at 1.16 ± 0.51 meq/kg, and this
also met the maximum level of 2.0 meq/kg required by the SNI 7382:2008 standard for
coconut oil. By contrast, P1 did not meet the required standard, as its value was 2.44±0.57
meq/kg due to oxidation and contact with air during titration. The peroxide number tended
to decrease at higher enzyme concentrations due to the higher frequency of attachment of
the enzyme to the substrate, the ease of interaction of the enzyme molecules with other
molecules and the enhanced ability to protect the oil from direct contact with air. The
papain enzyme has high proteolytic power and an ability to reduce direct interactions with
oxygen, and it can degrade protein components and break down the cell walls of the tissues
that contain the coconut milk to separate the oil from water. This results in less water in
the oil and reduces oxidation processes and the peroxide number.
The value for fermentation was also in accordance with the SNI requirements, whereas
the commercial market VCO value, at 5.80±0.85 meq/kg, was not. This meant that the
market VCO was already exhibiting signs of rancidity caused by the formation of peroxides
in the double bonds of several unsaturated fatty acids. The high peroxide number indicated
a lower oil quality (Talbot, 2016).
3.2. Physical Analysis of the VCO
The results for VCO prepared with tempe yeast and papain enzyme and for the market
VCO being for physical characteristics, such as colour, texture and aroma, based on
organoleptic test results from 32 respondents are shown in Figure 2.

Figure 2 Physical analysis of VCO prepared with tempe yeast and papain enzyme or purchased from
a commercial market in terms of colour, texture and aroma

Figure 2 shows the scoring method ranging from 1 to 4 used in the organoleptic tests
to assess the product. A higher number indicates better quality. The colour was determined
as 1 = very turbid, 2 = turbid, 3 = clear, 4 = very clear, while the texture was evaluated as 1
= very runny, 2 = runny, 3 = thick, 4 = very thick and the aroma was classified as 1 = very
strong rancid aroma, 2 = rancid aroma, 3 = good aroma, 4 = very good aroma. The product
samples tested included the VCO produced with tempe yeast (A), VCO produced with
papain enzyme (B) or commercial VCO purchased from a market (C). The VCO produced
with tempe yeast had a clear colour with a slightly runny texture, the product produced
with papain enzyme had a cloudy colour with a slightly runny texture, and the commercial
product had a clear colour with a thick texture. None of the VCO products had a rancid
aroma.
The watery texture observed in the oil produced with tempe yeast and papain enzyme
was caused by the imperfect separation process between the oil and water using a
separatory funnel, while the murky colour observed with the papain enzyme was due to the
Yuniarti et al. 667

yellowish-green oil-soluble pigments (chlorophyll and carotene) contained in the papaya

leaf (Soleha et al., 2018).
3.3. Chemical Analysis of the Hand and Body Lotion
The results for the hand and body lotion prepared with 45% papain enzyme and
addition of 3, 5 and 7% orange peel extract, the lotion prepared with tempe yeast VCO, and
the lotion prepared with market purchased VCO were analysed for their pH, density and
viscosity, as shown in Table 2. The best pH, with a value close to neutral, was recorded for
P2, which is the papain enzyme VCO lotion +5% orange peel extract. The average pH value
for all the samples ranged between 5.65±0.35 and 6.70±0.28, and values in this range are
considered to be within the allowable limits (pH 4.5 to 8) according to SNI 16-4399-1996
for skin moisturisers. The best pH for the skin varies between pH 4 and 7, indicating that all
the samples tested here were safe to use as a moisturising lotions. Sumbayak and Diana,
(2018) found that lotions with lower pH caused dry skin and irritation by damaging the
skin epidermis.

Table 2 Chemical characteristics of hand and body lotion

Orange peel pH Density Viscosity
Sample
extract (%) (-) (gr/cm³) (mPa·s)
Papain enzymatic 45 % of VCO production with
P1 3 5.65±0.35 1.020±0.026 479.9681±1.672
P2 5 6.70±0.28 0.982±0.023 427.917±20.685
P3 7 5.90±0.14 0.962±0.091 355.3996±66.856
Tempe yeast fermentation
P4 - 6.25±0.07 0.993±0.007 452.0624±64.932
Market Package
P5 - 5.75±0.21 1.005±0.046 465.7556±56.806

The density was measured to determine the stability of the emulsion products, as an
inappropriate ratio between the dispersing and dispersed phase usually reduces the
stability (Kurniawan, 2012). The average density was lower for the samples with orange
peel extract than without orange peel extract, and this lower density is associated with the
increase in the volume of the lotion due to the added extracts (Tumbelaka et al., 2019). The
values ranged from 0.962±0.091 to 1.020±0.026 gr/cm³ and these were within the 0.95–
1.05 gr/cm³ required by standard SNI 16-4399-1996.
The average viscosity was also lower for the samples with orange peel extract than for
the others, and the value decreased as the concentration of papaya leaf extract increased.
The viscosity also affected the density, so a higher value caused a greater density of the
product (Tumbelaka et al., 2019).
3.4. Physical Characteristics of the Hand and Body Lotion
The hand and body lotion prepared with 3, 5 and 7% orange peel extract was compared
with the market VCO for the physical characteristics of colour, texture, homogeneity and
moisture (Figure 3).
The results did not differ appreciably for the different samples. For example, sample
(A) was yellow and (B) was yellowish-green, while (C) was also yellow; however, they were
all thick textured, homogenous and very humid. The different colour recorded in sample
(B) was caused by the addition of papaya leaf extract that contained yellowish-green
pigments (chlorophyll and carotene), which are soluble in oil (Soleha et al., 2018).
668 Efficacy of a Natural Papain-Induced Enzyme on Virgin Coconut Oil-Based
Hand and Body Lotions with Added Orange Peel Extract

5,00

Average Grade Points

4,00
Colour
3,00
Texture
(-) 2,00
Homogeneity
1,00
Moisture
0,00
Tempe Yeast Papain enzyme Market packaging

Figure 3 The physical analysis of hand and body lotion by organoleptic testing in terms of colour,
texture, homogeneity and moisture

The organoleptic tests for hand and body lotion products prepared from papain
enzyme-treated VCO carried out with 32 respondents confirmed that the texture,
homogeneity and moisture were acceptable. As shown in Figure 3, the texture, homogeneity
and moisture scores were 3.19, 3.22 and 3.75, respectively, based on a maximum score of 5
for each characteristic, whereas the score for colour was 1.7, indicating adequate
acceptance.
Rini et al. focused their study on skin lotion from VCO containing black tea extract as a
skin protector and natural dye. They showed organoleptic test scores of 4.0 for colour, 4.3
for odour, 4.5 for appearance, and 4.5 for adhesiveness. Black tea extract contains
polyphenol compounds, which have antioxidant activity and have attractive colours,
making tea suitable as a natural dye for cosmetic ingredients (Rini et al., 2020).
Skin contamination is one of the likeliest risks with potential health consequences for
people with occupational exposure to various hazards. Many of the skin care products that
are available on the market are based on chemical formulations that are harsh, irritating
and even toxic. This underlines the need to identify and develop a research protocol for
natural products. In this context, the topical body lotion formulation developed in this study
is significant for its potential to reduce the danger to human health, while also lessening
pollution in the environment. The VCO, which constitutes a major proportion of the
formulation, itself acts as a preservative, thereby increasing the shelf life of body lotion and
protecting it from microbial degradation. The body lotion developed in the present study is
free from skin irritancy, has a long shelf life and is quite stable and safe (Satheeshan et al.,
2020).

4. Conclusions
The highest yield of VCO, at 13.8±5.30%, was obtained using a 45% papain enzyme
extract. The resulting VCO had an acid number of 0.135±0.50%, which fulfilled the SNI
7381:2008 standard for acid numbers, and a peroxide number of 1.16±0.51 meq/kg,
which was also in accordance with the SNI 7382:2008 standard for peroxide numbers.
Organoleptic tests conducted by 32 respondents on the hand and body lotion products
made from VCO prepared using the papain enzyme extract showed that the colour had
an adequate acceptance, while the texture, homogeneity and humidity were viewed as
satisfactory. Further studies should focus on the isolation process for papain.

Acknowledgements
This scientific research was partly supported by the Ministry of Higher Education,
Indonesia [No. 176-23/UN7.5.1/PG/2016].
Yuniarti et al. 669

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