The effect of papain enzyme concentration on the yield and clarity level of VCO

VCO can be produced by the enzymatic method, which does not involve excessive heating, thereby preventing the degradation of essential compounds. One thing that needs to be considered in the enzymatic production of VCO is the enzyme storage conditions. Enzyme stability during storage can be maintained using maltodextrin coatings, but the production of enzymatic VCO is often not accompanied by the use of coatings. Therefore this study aimed to determine the effect of enzyme concentration and the use of maltodextrin coatings on the yield and quality of the resulting VCO. This research method goes through two stages, in the first stage uses a completely randomized design (CRD), which was arranged factorially consisting of enzyme concentration factors (0.5%, 1.0%, and 1.5% (w/v)). The second stage uses CRD to use a maltodextrin coating (without enzymes, enzymes, enzymes with a maltodextrin coating). The results obtained in this study were that the best enzyme concentration treatment to produce VCO was obtained at a concentration of 0.5% with the use of maltodextrin coatings based on a yield of 20.5% and a degree of clarity of 86.73. This study concludes that the treatment using enzymes with coatings is considered the most effective because of the role of coatings in maintaining the stability of enzymes during storage.


Introduction
Coconut (Cocos nucifera L.) is one of the results of the plantation sector, which is abundant in Indonesia.Based on statistical data from the Indonesian Ministry of Agriculture (2021) coconut production in Indonesia in 2020 will reach 2,811,954 tons.One of the provinces with a high production rate is South Sulawesi, with an estimated production rate in 2021 of 68,734 tonnes.Coconut is a very potential commodity because almost all parts of the coconut plant have economic value, especially the part of the coconut meat, which is widely used as raw material for making coconut oil [1].Making oil from raw coconut meat materials is due to the thick white layer containing an average of 28% vegetable oil sources [2].The high oil content in coconut meat has the potential as a raw material in the manufacture of virgin coconut oil or Virgin Coconut Oil (VCO).
Several methods can be used in VCO production, including centrifugation, heating, fermentation, and enzymatic methods [3].The enzymatic method is a very profitable VCO production method because enzymes are non-toxic and specific catalyzing agents and are produced from natural ingredients, so they are environmentally friendly [4].In addition, according to [5], the enzymatic method is considered the correct method for VCO production because this method does not use excessive heating so that essential compounds are not damaged.Making VCO using proteolytic enzymes will speed up the breakdown of lipoproteins covering the oil, which will cause the oil to bind to each other so that pure coconut oil or VCO is obtained [6].
One of the proteolytic enzymes that can be used is the enzyme papain.The papain enzyme can be obtained from papaya fruit because the papain enzyme content reaches 50% [7].Some advantages of the papain enzyme are that it is more resistant to temperature treatment or has the highest heat resistance, which ranges from 50-65°C, a more comprehensive pH range of around 5-7, and is purer than other proteolytic enzymes [8].In addition, [7] mentioned that papain enzymes can be active at low concentrations.One thing that needs to be considered in the enzymatic production of VCO is the enzyme storage conditions.Enzyme stability can be affected by temperature, pH, light, and inhibitors or inhibitory substances [9].During storage, maltodextrin coating can be used to protect enzymes' active sites from external influences by protecting their active sites.Maltodextrin is easily soluble in water [10], therefore it is the right coating for the papain enzyme because it will be applied to the substrate of coconut cream which is an oil-in-water emulsion.
Several studies on making VCO with enzymatic methods include using proteolytic enzymes from pineapple humps [11,12]; and pineapple extract as a source of bromelain enzymes [13].Furthermore, pieces of young papaya were used [11,14] as a source of the papain enzyme.However, this study used an uncoated enzyme source as an agent that could maintain the stability of the enzyme during storage.Based on this, this research was conducted to determine the effect of enzyme concentration and the use of maltodextrin coatings on the yield and quality of the resulting VCO.The purpose of this study was to determine the effect of using enzyme concentrations with and without coating on the yield of the resulting VCO.

Research procedure 2.2.1 Preparation coated with crude papain from papaya sap.
The production of the papain enzyme begins with cleaning the papaya fruit of dirt and dust, then tapping the papaya latex.Tapping is done with a tapping knife with a notch depth of 1-2 mm.The sap obtained is accommodated in a beak NaCl) 0.3% with a ratio of 1:4 (v/v) and homogenized with a homogenizer to form a white emulsion.er.After that, the gum is mixed with an activating solution sodium chloride (After that, it was filtered using a funnel to separate the filtrate and residue.The residue is then dried in the oven vacuum at 55°C, then crushed and filtered to obtain powder or crude papain.Crude Good quality papain is marked with a yellowish white color.After that, a coating in the form of maltodextrin was added in a ratio of 2:1 with crude papain.(Modified from [15]).

Coconut cream extraction.
Preparation is carried out by selecting fresh old coconuts, then proceeding to open the coir, breaking the shell, separating the shell from the fruit flesh followed by stripping the testa using a knife.After that, the pulp is washed and grated.Grated coconut is added to water in a total ratio of 1:1 (w/v.)After that, it is put into the machine spinner then filtered to extract coconut milk.Coconut dregs squeezed coconut milk is put back into the machines pinner which was repeated 3 times.After that, the extracts were collected and stirred, then the coconut milk extract was put in a transparent jar for ± 2 hours until the coconut cream and skim were separated.Cream is taken as a manufacturing material Virgin Coconut Oil (VCO) (Modified from [6]).

Making Virgin Coconut Oil (VCO).
Making Virgin Coconut Oil (VCO) begins by putting 100 ml of coconut cream into a beaker.After that, 0.5%, 1.0%, and 1.5% (w/v) papain enzymes were added, then the mixture of coconut milk and papain enzymes was stirred until homogeneous.Then the beaker was closed and incubated for 24 hours at 30 °C until 3 layers were formed, namely blondo at the top, oil in the middle, and water at the bottom.After 3 layers are formed, the water in the lower layer is separated usingsyringe, then oil and blondo are separated by filtering using filter paper (Modified from [6]).

Research design
The research design consisted of 2 stages, namely in the first stage using a Completely Randomized Design (CRD), which was arranged factorially consisting of enzyme concentration factors (0.5%, 1.0%, and 1.5% (w/v) ).The second stage uses CRD with the factor of using a maltodextrin coating (without enzymes, enzymes, enzymes with a malrodextrin coating).Each treatment was repeated 2 times.The data obtained were analyzed by analysis of variance orAnalysis of Variance (ANOVA).If there is a difference, continue with the testDuncan Multiple Range Test (DMRT).Data processing uses SPSS 16.0 and Microsoft Excel 2019.

Observation parameters 2.4.1 Yield Virgin Coconut Oil (VCO).
Determination of VCO yield was done by measuring the volume of the cream before incubation, then measuring the volume Virgin Coconut Oil (VCO) produced after incubation, then entered into the following equation [16].
2.4.2.Degrees of clarity.Determination of the degree of clarity (Laga et al., 2019) begins with preparing a VCO sample and aquadest as a blank.After that, the transmittance measurement was carried out on the sample using a spectrophotometer with a wavelength of 395 nm.The transmittance value is recorded as a degree of clarity.

Determination of enzyme concentration
Preliminary research was carried out by treating the enzyme concentration (%) of 0.5; 1.0; and 1.5 with parameters of yield and degree of clarity.

Yield (%).
Yield Virgin Coconut Oil (VCO) is the percentage of the amount of VCO produced per unit of coconut cream used [2].The results of observations from the use of enzyme concentrations (%) were 0.5; 1.0; and 1.5 obtained yield data (%) in the range of 22.45-22.7.The results of the ANOVA statistical test showed that the concentration of the enzyme treatment was 0.5; 1.0; and 1.5% had no significant effect (p>0.05) on the resulting VCO yield.The effect of enzyme concentration on VCO yield can be seen in Figure 1.Based on Figure 1, the use of enzyme concentration (%) is 0.5; 1.0; and 1.5 was not significantly different between levels of VCO yields.This is presumably because the enzyme is already saturated with the substrate or or all the active sites of the enzyme are filled with the substrate [15], so that the addition of the enzyme no longer makes a significant difference to the yield of the resulting VCO.These results indicate that the treatment with an enzyme concentration of 0.5% is the most efficient treatment to use because it is the lowest enzyme concentration.

Degree of clarity (%).
The degree of clarity is a parameter resulting from the percentage of transmittance (%T) or the amount of light that passes through the oil The transmittance percentage indicates that the higher the %T value, the clearer the VCO produced [17].The results of observations from the use of enzyme concentrations (%) were 0.5; 1.0; and 1.5 obtained data degree of clarity (%) in the range of 78.93-86.73.The results of the ANOVA test showed that the treatment of enzyme concentration had no significant effect (p>0.05) on the degree of clarity of VCO.The effect of enzyme concentration on the degree of clarity of VCO can be seen in Figure 2. Based on Figure 08, the use of enzyme concentration (%) is 0.5; 1.0; and 1.5 was not significantly different between levels on the degree of clarity of VCO.These results indicated that the treatment with an enzyme concentration of 0.5% was considered the most efficient to use because it was the lowest enzyme concentration, but did not provide a significant difference with the enzyme concentration of 1.0 and 1.5% to the degree of clarity of VCO.

Yield (%).
Yield Virgin Coconut Oil (VCO) is the percentage of the amount of VCO produced per unit of coconut cream used [2].The results of observing the effect of using enzymes with and without coatings in Appendix 3a, obtained yield data (%) in the range 14.5-21.The results of the ANOVA statistical test showed that there was a significant effect (p <0.05) in the treatment of using enzymes with and without maltodextrin coating on the yield (%) of VCO produced.The effect of using enzymes with and without maltodextrin coating on VCO yields can be seen in Figure 3. Figure 3 shows that there is a significant difference between the treatment without enzymes and the use of enzymes without coating and enzymes using coatings after Duncan's further testing.These results indicate that the addition of enzymes with or without the use of coatings can produce a more optimal yield than without the addition of enzymes.This is because the papain enzyme is included in the proteolytic enzymes which can cause breakdown of the lipoprotein bonds that surround the oil so that the yield or VCO produced is more optimal than without the addition of This is supported by the statement [18], that the protease enzyme in the form of the papain enzyme functions in breaking lipoprotein bonds in fat emulsions.In addition, the addition of maltodextrin did not affect the performance of the papain enzyme.Based on this, the use of enzymes with coatings is considered the most effective because of the role of coatings in maintaining the stability of enzymes during storage.

Conclusion
The conclusions obtained from this study are: 1. Enzyme concentration treatment to produce the best VCO was obtained at a concentration of 0.5% with the use of maltodextrin coating based on the yield of 20.5% and a degree of clarity of 86.73. 2. The treatment of using enzymes with coatings is considered the most effective because of the role of coatings in maintaining the stability of enzymes during storage.

Figure 2 .
Figure 2. Effect of enzyme concentration on the degree of clarity of VCO.

Figure 3 .
Figure 3.The effect of using enzymes with and without maltodextrin coatings on the yield (%) of VCO.