Effect of mass and size husk ash adsorbent on acid number and free fatty acid of virgin coconut oil (vco) produced by enzymatic process

Virgin Coconut Oil (VCO) is an oil produced using low temperatures. Efforts to maintain and improve the quality of VCO with papain enzymatic manufacturing method followed by the provision of husk ash adsorbent. The use of papain enzyme as much as 1.5% w/v with an incubation temperature of 40°C 24 hours was effective in reducing acid numbers. The husk ash adsorbent which was treated in an oven at 120°C 2 hours was effective in improving the color quality of the VCO. Variations of husk ash were adsorbent size (60, 80, and 100) mesh and mass (15, 20, and 25) grams. The sample that has been mixed with adsorbent is stirred with a magnetic stirrer at 300 rpm 1 hour then separated by centrifuge at 1000 rpm 1 hour and ends with VCO filtering with filter paper to separate the remaining adsorbent from the oil. Samples were tested for acid number and free fatty acid content. The results show that the smaller the size of the adsorbent and the more the amount added gives a positive effect on decreasing the acid number and free fatty acid content. Production of VCO has low environmental impact because it uses low energy.


Introduction
Virgin coconut oil (VCO) is a vegetable oil derived from coconut milk.VCO has high economic and functional value for health because it is widely used in the fields of medicine, food and cosmetics [1].VCO is said to be good if it meets the standards set, namely based on the National Standardization Agency regarding VCO and the Asean and Pacific Coconut Community where the quality of VCO can be determined from chemical and physical parameters [2].VCO has a weakness in maintaining quality, namely that it is easily damaged when exposed to high temperatures [3].Therefore, manufacturing methods that use low temperatures are used, one of which is the papain enzyme enzymatic method [4].According to [5], the yield increases with the addition of the papain enzyme and the quality is also directly proportional to the function of papain as a protein breaker [6].Papain is a proteolytic enzyme contained in papaya (Carica papaya).Papain is usually traded in the form of a yellowish white powder and must be stored below a temperature of 4°C.The papain enzyme or proteolytic enzyme functions to catalyze the breakdown of peptide, polypeptide and protein bonds using hydrolysis reactions into simpler molecules such as short chain peptides, peptone water and amino acids [4].
Free fatty acids are one of the parameters to determine the quality of VCO.The cause of the increase in free leaching acid is oxidation and the performance of the lipase enzyme contained in the oil [7].The test was carried out using the NaOH acid-base titration method where the test results indicate the amount

T Juwitaningtyas* and A A Vennada
IOP Publishing doi:10.1088/1755-1315/1302/1/012104 2 of hydrolysis that occurred in the oil.The calculation of free fatty acids is seen based on reaching the equivalence point of the OH group in the base used to neutralize H+ in free fatty acids.The test reaches a balance point marked by a change in the color of the sample to pink [8].Hydrolysis of fatty acids in oil comes into contact with water it will cause the formation of glycerol and 3 free fatty acids which can cause rancidity [9].
The adsorption process with rice husk ash occurs due to the surface tension of rice husk ash, the larger the area provided, the more molecules will be absorbed [10].Optimizing the use of husk ash by varying the mass and size of the adsorbent.According to [11] the mass factor of the adsorbent used has an impact on improving oil quality due to the greater the number of particles added, the more VCO will be in contact with the adsorbent, resulting in maximize ion adsorption.Variations in husk ash particle size also have a positive influence on the quality of VCO where the surface area influences the amount of VCO exposed to the adsorbent [12].Husk ash is used as an adsorbent because so far this agricultural waste tends not to be used very much.The existing use is as a dishwasher [7].The choice of husk as an adsorbent compared to other materials is that the surface area of husk ash is 200-250 m2 /g with a particle size of 1-10 µm [13] which is the largest compared to zeolite 48.45 m2 /g and kaolin 120.57m 2 /g [14].
VCO is an agro-industrial product that supports environmental conservation because it uses low energy for processing.Virgin coconut oil, or VCO, is a mechanically produced edible oil and fat with minimal heating and no chemical addition.Old coconut bunches that are fresh are used to make VCO.This method produces VCO that is pure, hard in rancidity, colorless, and low in FFA and peroxide [31]

Materials
The ingredients used in this research include the main ingredient, coconut milk from old coconuts that do not yet have a haustorium.Husk ash, Paya brand commercial papain enzyme, Apollo brand clear plastic, binding rope, distilled water, glacial acetic acid, 95% alcohol, 95% ethanol, 1 M starch, Na2S2O3, chloroform, 1% saturated potassium iodide, PP indicator, standard solution KOH 0.1 N, water, Na2S2O3, filter paper, tissue and labels.

Husk ash preparation.
The husk ash used is varied in size using a sieve sieve (100, 80, and 60) mesh to homogenize and remove impurities [15].The husk ash that passes through the sieve is ovened at a temperature of 120 C ̊ for 2 hours with the aim of reducing the water content in it so that it does not affect the KA in the VCO which is then cooled in a desiccator [7].The husk ash was varied in mass (15, 20, and 25) grams [16].

Sample preparation.
Old coconuts that still have brown skin are grated and ground with a grinder to expand the surface for maximum extraction.Extraction was carried out by adding water 1:1 [17] at a temperature of 60 C ̊ with the aim of making it easier to break up the emulsion [18] 3x squeezed [19] Leave the coconut milk for 1 hour at room temperature in a hanging position so that it is easy to separate [20].Breaking protein bonds with lipids uses the commercial papain enzyme Paya brand [21] with a concentration of 1.5% w/v [5].The harvested VCO will be mixed with 100 ml of husk ash with variations in (100, 80 and 60) mesh and mass (15, 20 and 25) grams.The mixing of husk ash and VCO was assisted by a magnetic stirrer for 1 hour at a speed of 300 rpm and separated by a centrifuge for 1 hour at a speed of 1000 rpm [7].

Acid number and peroxide value analysis.
To determine the fatty acid content, a 10 gram sample of VCO is weighed and placed into a 250 ml Erlenmeyer flask.The Erlenmeyer was covered with alumunium foil then 50 ml of 95% ethanol was added.The sample was shaken 21 with a water bath shaker at 500 C for 15 minutes then 5 drops of PP indicator were added and titrated with a standard 0.1 N KOH solution until pink and did not change color for 30 seconds.The step for determining peroxide levels is that 5 g of the oil sample is weighed in a 250 ml Erlenmeyer.The Erlenmeyer was wrapped in alumunium foil then 12 ml of chloroform was added.18 ml of acetic acid was added then the sample and solvent were shaken until homogeneous.0.5 ml of potassium iodide was added then shaken again.The samples were incubated in a dark place for 1 minute then 30 ml of hot distilled water and 0.5 ml of starch were added.The sample was titrated with 0.1N Na2S2O3 until the blue color disappeared and remained for 30 seconds.

Research design.
The design used was a completely randomized design (CRD) with homogeneous treatment when making samples and then varying the mass and size of the husk ash adsorbent.Statistical data processing was carried out using SPSS, to determine the level of significance of variations in husk ash on the parameters to be tested.Testing of significant data will be continued with the Post Hoc Duncan test with a confidence level of 95%.Sample variations to be tested include A1= 100 mesh, 15 g; A2= 100 mesh, 20 g; A3= 100 mesh, 25g.B1= 80 mesh, 15g; B2= 80 mesh, 20g; B3= 80 mesh, 25g.C1= 60 mesh, 15g; C2= 60 mesh, 20g; C3= 60 mesh, 25g; K= 0 mesh, 0 grams.

Result and discussion
3.1.Acid number.The acid number is the number of free fatty acids contained in the oil and is expressed in mg of base/gram of sample [22].The acid number is formed due to the hydrolysis reaction, namely the formation of glycerol and fatty acids due to the water content in the sample and can be accelerated by the presence of enzymes or other compounds.A decrease in the acid number indicates that the quality of the oil is getting better [23].The acid number test results are shown in Table 1, namely oil samples titrated with KOH as a base.The test results will be considered complete if the colour of the sample shows soft pink, which means that all the acid numbers have been neutralized by the base and bind the dye to the sample so that it changes colour.The number of bases used for titration indicates the number of acids contained in the oil sample, so the acid number is considered the number of bases needed to neutralize 1 g of the oil sample.1, it can be seen that the highest acid number was in the control sample and the provision of variations in husk ash adsorbent had an effect on reducing the acid number.The variation that showed the lowest acid number in the VCO sample given 100 mesh 25 grams of husk ash and 80 mesh 25 grams was 0.365 KOH/g.This shows that the optimal adsorbent reduces the acid number in the sample, namely a variation of 100 mesh to 80 mesh 25 grams.All experimental data meets the requirements (National Standardization Agency, 2008) where the maximum acid value allowed for VCO is <2.0 mg KOH/g.
The addition of a variety of husk ash has been proven to improve the quality of VCO because of the silica content which can reduce the water content [24].The acid content formed due to oxidation due to the presence of water can be reduced or avoided.Research by [23] shows that the silica content used for cooking oil adsorbent with repeated heating can reduce the acid value.The addition of magnesium silica adsorbent mass can increase efficiency in the adsorption process and reduce adsorption capacity.Experiments by contacting the most magnesium silica were able to produce the best acid numbers from other samples.
The process of making VCO using the enzymatic method contributes to the decrease in the acid number in the sample.The more enzymes added to the VCO, the more the acid number will be reduced.This shows that there is a positive influence on improving the quality of VCO produced using the enzymatic method [25].The centrifugation process also plays a role in reducing the acid number.Based on research [26], a stirring time of 25 minutes increases the quality performance of VCO by reducing the acid number compared to stirring < 25 minutes.
Based on statistical analysis using the two way ANOVA test, it shows that the mass factor significantly influences the acid number parameter, namely 0.000 < 0.05, while the size of the adsorbent does not significantly influence the acid number.The results of further testing, namely by Duncan, the adsorbent particle size factor given showed that (60, 80 and 100) mesh was not significantly different from other samples.Meanwhile, variations in adsorbent mass (15, 20 and 25) grams show that samples treated with 25 grams of husk ash are significantly different marked with the letter a and variations of 20 grams and 15 grams show significant differences marked with the letters b and c.When comparing the results of administering 15 grams of husk ash with a control sample without husk ash, the results show that it is marked with the letters c and d.These results show that the addition of 25 grams of husk ash had a significant effect on all the masses added to the sample.

Free fatty acid.
The formation of peroxides is due to oxidation by oxygen of unsaturated or saturated fatty acids which are at the bond of the β carbon atom which will form the final product methyl ketone from ketone acid [27] as well as aldehydes and peroxides [9] where the peroxide number is a parameter used to determine the quality of an oil.The rancid odor produced by oil whose quality has decreased is the result of the level of oil unsaturation or primary oxidation.The results of free fatty acid analysis in Table 2 shows that samples contacted with husk ash with a particle size of 100 mesh had the lowest free fatty acids, namely 0.18%, while the results from 80 and 60 mesh respectively had free fatty acid numbers of 0.24% and 0.26%.These results show that the particle size of the husk ash adsorbent influences and can reduce the levels of free fatty acids, the smaller the particles (100 mesh) indicate the lower the free fatty acids contained in VCO and it increases as the particle size increases.The mass of adsorbent given to the VCO shows the effect that the more husk ash mass added, the more free fatty acids will be reduced, except for samples with a mesh size of 80 grams, husk ash weights of 20 and 15 grams, showing the same results in the test.This can be found out in further tests to find out whether there is a real effect on the mass or not.The control sample without husk ash had a higher free fatty acid value of 0.34%.
When compared with the test results with standards National Standardization Agency, there are samples that exceed the limit due to > 0.2% but meet the standards based on APCC (2009), namely a maximum of 0.5%.According to [6], the high fatty acid content is caused by the high moisture content in the oil so that microorganisms can grow and damage the VCO content.The increase in FFA can be accelerated by the presence of bases, acids, and enzymes.The lipase enzyme contained in oil can accelerate the increase in FFA in direct proportion to storage time.Research by [28] stated that the lipase enzyme which causes oil hydrolysis can be inactivated by hot temperatures.Making samples using the enzymatic method does not use high heat so it is possible that the lipase enzyme which causes high FFA is still active.When compared based on research by [17] showed that the FFA levels of VCO samples made with the papain enzyme were quite high on average, namely 1.18 -1.48%, so other efforts were needed to reduce FFA levels, one of which was using husk ash adsorbents.
The same results were shown from research by [7] regarding the use of husk ash to improve the quality of copra oil.The results of free fatty acid testing for copra oil samples were shown to be in line with the results of research on VCO samples that the highest levels of FFA were in control samples that were not in contact with husk ash.FFA levels decreased from 5 grams to 25 grams of husk ash treatment.Based on research by [8], it shows that the active side of the adsorbent used is directly proportional to the addition of the number of particles.The composite surface area and adsorption efficiency will increase if the mass increases.The effect of adding husk ash on enzymatic VCO free fatty acids is due to hydrogen bonds from carbonyl oxygen contained in the oil which are absorbed by silica grains which contain silanol hydrogen.New bonds are formed from the contact of oil with husk ash by forming hydrogen bonds [16].
Free fatty acids are positive ions contained in oil, where the husk ash adsorbent has hydroxy groups which are able to attract them because they are negative ions [29].Rice husk is an adsorbent containing amorphous silica which, when contacted at room temperature, is able to absorb natural FFA compounds, phospholipids, and also lutein in oil micelles [30].Fatty acid levels affect taste, according to [2], high levels of FFA can cause the oil to taste bad when consumed and reduce the yield.The smaller the free fatty acids contained in the oil, the better the quality of the oil [2].
Based on the two way ANOVA statistical analysis, size and mass of the adsorbent factors significantly influence the testing of free fatty acid levels, shown by a significance value of 0.04 < 0.05.Meanwhile, the relationship between size and mass factors did not have a significant effect on the results of the free fatty acid content test with a significance result of 0.264 > 0.05.The R square results show the good influence of the given factors on free fatty acid levels with a value of 0.872 or 87.2%, where the closer to 100%, the better.

Conclusion
The addition of variations in the mass and size of husk ash to VCO produced using the enzymatic method has a decreasing effect on the acid number and free fatty acid parameters.The acid value of VCO which was not added with husk ash was 0.895 KOH/g, while the sample given husk ash had an acid number of (0.73 -0.36) KOH/g with the best variation being the addition of 100 mesh 25 grams of husk ash and 80 mesh 25 grams.grams.The free fatty acids of the control samples which were not added with adsorbents showed the highest free fatty acid values.The samples with the lowest free fatty acids were found in samples with the addition of 25 grams of 100 mesh, 25 mesh and 80 mesh adsorbents.Statistical tests show that the adsorbent mass has a significant effect on the acid number and free fatty acid.From this research we can conclude that husk ash has contribution to decrease the deterioration factor of oil, in this case is VCO.Production of VCO has low environmental impact because it uses low energy.

Table 1 .
Acid number of VCO with variations of size and mass of adsorbent

Table 2 .
FFA value of VCO with variations of size and mass of adsorbent