The influence of Saccharomyces cerevisiae enzyme ratio on preparation virgin coconut oil for candidate in-house reference materials

Transcription

1 The influence of Saccharomyces cerevisiae enzyme ratio on preparation virgin coconut oil for candidate in-house reference materials Yuli Rohyami, Rafika Debby Anjani, and Napthalina Putri Purwanti Citation: AIP Conference Proceedings 1823, (2017); View online: View Table of Contents: Published by the American Institute of Physics Articles you may be interested in Phenolic removal using phenylamine modified montmorillonite AIP Conference Proceedings 1823, (2017); / An analysis of learning process based on scientific approach in physical chemsitry experiment AIP Conference Proceedings 1823, (2017); / Kinetic study of hydrolysis of coconut fiber into glucose AIP Conference Proceedings 1823, (2017); / The effect of coconut oil and palm oil as substituted oils to cocoa butter on chocolate bar texture and melting point AIP Conference Proceedings 1840, (2017); / Gas chromatography Mass spectrometry analysis and antibacterial activity of Cinnamomum burmanii essential oil to Staphylococcus aureus and Escherichia coli by gaseous contact AIP Conference Proceedings 1823, (2017); / Optimization of the liquid biofertilizer production in batch fermentation with by-product from MSG AIP Conference Proceedings 1823, (2017); /

2 The Influence of Saccharomyces cerevisiaeenzyme Ratio on Preparation Virgin Coconut Oil for Candidate in-house Reference Materials Yuli Rohyami 1,a), Rafika Debby Anjani 1), and NapthalinaPutri Purwanti 1) 1 (Diploma of Analytical Chemistry, Islamic University of Indonesia Jl. Kaliurang Km. 14.4, Besi Sleman Yogyakarta 55584) a) Corresponding author: rohyami@uii.ac.id Abstract. Virgin coconut oil is an excellent product which has result of oil processing business opportunities in the international market. Standardization of virgin coconut oil necessary to satisfy the requirements industry needs. This research is expected as procedure preparation of reference materials. Preparation of virgin coconut oil by Sacharomycescerevisiaeenzyme. Based on the results of this study concluded that the ratio of Saccharomyces cerevisiae can affect the yield of virgin coconut oil produced. The preparation of virgin coconut oil enzymatically using a variety of mass ratio of to 0.006% is obtained yield average of 12.40%. The optimum separation of virgin coconut oil on the use of enzymes with a mass ratio of 0.002%. The average water content at a ratio of 0.002% is 0.04 % with a value of uncertainty is 0.005%. The average iodine number in virgin coconut oil produced is ,1974 grams of iodine per 100 grams of oil and optimum iodine number is obtained from the manufacturing process virgin coconut oil with a ratio of 0.006% Saccharomyces cerevisiae. Sacharomycescerevisiae with a ratio of 0.002% results virgin coconut oil with acid number %. The peroxide value of virgin coconut oil between to milliequivalent per kilograms. Organoleptic test results and test chemical parameters can be used as the test data that can be developed in prototype preparation of candidate in-house reference material in the testing standards of quality virgin coconut oil. INTRODUCTION Virgin coconut oil (VCO) according to the Indonesian National Standard is the oil obtained from the fruit flesh of coconut (Cocosnucifera L.) old fresh and processed squeezed with or without the addition of water, without heating or with heating less not more than 60 C and safe for human consumption [1]. Virgin coconut oil contains fatty acids that are beneficial for health. The fatty acids contained in virgin coconut oil are caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, Olet acid, linoleic acid and linolenic acid. The main content of fatty acids in virgin coconut oil is lauric acid ranged from 45.1 to 53.2%, myristic acid from 16.8 to 21%, palmitic 7.5 to 10.5%, stearic acid from 2.0 to 4.0 %, and oleic acid from 5.0 to 10.0% [1]. Method for production of virgin coconut oil has been developed by various technologies. Extraction of virgin coconut oil can be done in several ways, including by means of fermentation, centrifugation, salting and ultrasonification. Virgin coconut oil extraction is carried out with the addition of yeast, such as Saccharomyces cerevisiae and papain. Yeast will produce enzymes to break the bonds of fatty acids and proteins that can be separated from coconut milk. Fermentation can be done primarily Saccharomyces cereviseae use fishing techniques and the addition of acid in the production of virgin coconut oil [2] or hydrolase enzyme from papain [3]. Virgin coconut oil separation is performed by using fishing methods using pure virgin coconut oil so it can be separated from the coconut cream.separation of virgin coconut oil by centrifugation using a centrifugal force so as to sever the bond of the fatty acid-protein in milk. Preparation of virgin coconut oil can be made by adding calcium salts such as calcium carbonate. The calcium salt serves as an emulsifier so that water and virgin coconut oil can be International Conference on Chemistry, Chemical Process and Engineering (IC3PE) 2017 AIP Conf. Proc. 1823, ; doi: / Published by AIP Publishing /$

3 separated well [4]. Separation of virgin coconut oil can also be done by using ultrasonic waves to break down proteins so that after undergoing a process of centrifugation [5]. This study was conducted to obtain virgin coconut oil by enzymatic process using the enzyme Saccharomyces cerevisiae [1] for candidate in-house reference materials. Research will be conducted using the same method without having done using extraction by virgin coconut oil and addition of acid. This process is expected to improve the efficiency of high-quality and better quality standards through organoleptic test and chemical test. This research is expected to contribute to the Development of the procedures for producing virgin oil coconut enzymatically to obtain standardized products that can meet the needs of the food industry, cosmetics and pharmaceuticals. This process is also recommended for the preparation of candidate reference material standards in product testing and development of virgin coconut oil product chemical testing methods virgin coconut oil. Reference materials are an important tool in release a number of aspects of measurement quality and are used for method validation, calibration, estimation of measurement uncertainty, training and for internal quality control and external quality assurance (proficiency testing) purposes [6].The reference material has an important role in validating the accuracy of the data. Reference materials used for some the purpose is to facilitate the accurate testing of the whole system testing during the development or application of an analytical method for determining whether a method of being in control during routine use, for establishing traceability to a reference value in-house control material or as a sample inter-laboratory comparison for assessment. Preparation of candidate in-house reference material useful for ensure quality standards of virgin coconut oil. This research is done to optimize the extraction of virgin coconut oil enzymatically. The study was conducted to determine the effect of the ratio Saccharomyces cerevisiae for the preparation of virgin coconut oil and determine the standard parameters by uncertainty value. The virgin coconut oil provided have been in-house reference material for standardization of the product. MATERIALS AND METHODS Materials Materials used in this study are coconut, enzyme Saccharomyces cerevisiae, ethanol (C 2 H 5 OH), phenol ftalein, sodium hydroxide (NaOH), chloroform, glacial acetic acid (CH 3 COOH), potassium iodide (KI), sodium thiosulfate (Na 2 S 2 O 3 ), indicators starch, cyclohexane, iodine, calcium hypochlorite, filter paper and distilled water. The chemicals used for testing is analytical grade purchased by Merck. Preparation of Virgin Coconut Oil Virgin coconut oil is made from the old coconut fruits. Cleaned and scraped coconut fruit. One kilogram of oil extracted by using 1000 ml of warm water while squeezed in order to obtain thick coconut cream. The coconut transferred into a separating funnel and allowed to stand to obtain a thick layer. Clearer layers separated. Thick layer coupled with Saccharomyces cerevisiae with variations from 0.01 to 0.06 g. The mixture was allowed to stand for 24 hours so that virgin coconut oil is then separated. Virgin coconut oil that is produced is filtered and stored in a sealed container. Organoleptic Analysis Organoleptic test was conducted to identify the smell, taste, and color Coconut virgin oil produced by using the senses of smell, taste and sight. The smell virgin oil Coconut identified by shaking examples Coconut virgin oil in sealed bottles and then close the bottle is opened. Test the smell was observed at a distance of 5 cm from nose then whisked into the nose to recognize smells. Analyses were performed by three panelists. If the typical smell of coconut oil fresh and not rancid then the results are otherwise normal, if a foreign smell then declared not normal. Taste test conducted by pouring the sample into a spoon and perceived by the tongue. The taste test by three panelists. If it feels distinctive flavor of coconut oil, the otherwise normal, and if there is a foreign taste then declared not normal. Test made by observing the color of the eyes by three panelists. If the oil is not visible virgin Coconut other color or pale yellow, the result is otherwise normal. If the results look the other colors then declared not normal

4 Determination of Water Content Certainly prepared porcelain dish and porcelain cups in a clean and dry. Porcelain dish are heated in an oven at 105 o C for 1 hour and let cool in the desiccator to the temperature equal to room temperature. The cup was weighed to constant weight and weigh example virgin coconut oil and heat it in the oven at 105 o C for 1 hour. Results cooled in desiccator until its temperature is equal to room temperature. Weight to constant weight and determine the water content by loss of weight after the heating process.the water content is determined using the Eq. 1. Water content (%) = ( ) 100% (1) w 1 w 2 : weight of sample before heated : weight of sample after heated Determination of Iodine Value Three gram sample of virgin coconut oil was weighed and put in erlenmeyerflask.samples added with 15 ml of a mixture of cyclohexane: acetic acid, 1: 1 v/v) to dissolve the fat. The samples was added with 25 ml Wijs using erlenmeyer then closed, allowed to stand for 1 to 2 hours in a dark room and then added with 10 ml of 20% potassium iodide solution and 100 ml of distilled water. Erlenmeyer closed and shaken immediately and then titrated with standard sodium thiosulfate solution using starch indicator.the iodine value is determined using the Eq. 2. Iodine value = ( ).., (2) V 0 V 1 N m : blank titration volume : volume titration of the sample : the normality of standard solution of sodium thiosulfate : weight of sample Wijs solution made from 13 grams of iodine dissolved in 1 liter of concentrated acetic acid and chlorine gas powered (no exaggeration), so that a number of chlorine-bound iodine that is equivalent to 3.6 g of chlorine needed. Adequacy of chlorine examined using erlenmeyer containing acetic acid solution was weighed before and after flowing chlorine gas or by observing the color changes from dark brown to yellowish brown. Wijs solution is put in a bottle and kept in a dark place at a temperature less than 30 C. The20% of potassium iodide solution was prepared by dissolving 20 g of potassium iodide in 100 ml of distilled water. The 0.1N of sodium thiosulfate by diluting the standard solution of sodium thiosulfate 1 N. Preparation of standard solution of sodium thiosulfate made by weighing 248 milligrams sodium thiosulfate, diluted with CO 2 -free distilled water and put in a 1 liter flask and then fill impressions. The solution was diluted to 100 ml 1 N sodium thiosulfate solution to a 1 liter flask and impressions flask up to the mark line with CO 2 -free distilled water. Standardization of sodium thiosulfate is performed using potassium bromate. Determination of Free Fatty Acids Five grams sample is weighed carefully and then put in a 250 ml erlenmeyer, coupled with 95% neutral ethanol and 3 to 5 drops of indicator phenolphthalein and titrated with a standard solution of sodium hydroxide 0.1 N solution of 95% neutral alcohol made by entering the 95% alcohol as much as necessary into erlenmeyer, spilled a few drops phenolphthalein indicator is then titrated with a standard solution of sodium hydroxide 0.1 M. Free fatty acids number is determined using the Eq.3... Free fattyacids number = (3). V : volume of sodium hydroxide solution N : the normality of standard solution of sodium hydroxide m : weight of sample 200 : molecular weight of lauric acid Sodium hydroxide solution prepared from Sorensen solution or sodium hydroxide solution 50%. Making the 50% solution of sodium hydroxide was prepared by dissolving 100 g of sodium hydroxide in CO 2 -free distilled water of 100 ml. Making a standard solution of sodium hydroxide 0.1N is done with pipette 5.26 ml of 50%

5 sodium equivalent to 19 N in a 1000 ml volumetric flask and matched to mark the line with CO 2 -free distilled water. Normality of the solution matched with a solution of a primary standard. Standardization is done using oxalic acid. The CO 2 -free distilled water prepared by boiling distilled water for 20 minutes, then cooled in a container equipped with a protection device in the form of CO 2 absorber tube containing a mixture of sodium hydroxide and calcium oxide. Determination of Peroxide Value As much as 0.3 to 5 grams of sample is inserted into the erlenmeyer then added with 10 ml of chloroform and shaken so homogeneous. The sample solution added with 15 ml of glacial acetic acid and 1 ml of saturated potassium iodide solution is then closed tightly. The solution was shaken 5 minutes in a dark room at a temperature of C. The solution was added with 75 ml of distilled water and shaken vigorously. The solution was titrated with a standard solution of 0.02 N sodium thiosulfate with starch indicator solution. Preparation the standard solution of sodium thiosulfate 1N done by weighing 246 milligrams of sodium thiosulfate penta-hydrate, dissolved in one liter flask with CO 2 -free distilled water. The 0.02 N sodium thiosulfate solution made by dissolving 20 ml of sodium thiosulfate 0.1 N put into 100 ml volumetric flask and then fill with CO 2 -free distilled water.peroxyde value is determined using the Eq. 4. Peroxide number = ( ) (4) V 0 : blank titration volume V 1 : volume titration of sample N : normality of sodium thiosulfate m : weight of sample RESULT AND DISCUSSION Preparation of Virgin Coconut Oil Preparation of virgin coconut oil made from coconut fruit selection process. Coconut fruit obtained from coconut farmers. Coconut been a really old to get a percentage optimal results. Coconut fruit is separated from the coconut shell is then washed thoroughly to prevent contamination with impurities components and prevent microbiological contamination that would affect the quality standards of virgin coconut oil. Shredded coconut using coconut grater machines. Coconut cream made using comparisons with grated coconut and warm water 1: 1 to obtain coconut milk cream is really thick.coconut milk is extracted in addition to containing a liquid cytoplasm and oil globules also contain other components making up coconut meat including cellulose, phospholipids, sugars, protein and solids size is very small. The preparation of virgin coconut oil is done with the addition of microbes Saccharomyces cerevisiae as producer hydrolase enzymes to break the bond between the fatty acid to the protein in milk cream that helps the process of separation of virgin coconut oil [2,8]. The ratio of enzyme Saccharomyces cerevisiae do with variations from 0.01 to 0.06%. The incubation process is carried out for 24 hours so that virgin coconut oil is then separated. The yield is determined by the weight ratio of virgin coconut oil produced by the weight of the coconut is used as raw material for the manufacture of virgin coconut oil.the enzyme concentration may affect the virgin coconut oil is produced, where the higher the concentration of hydrolase enzymes are used, the higher the randemen results. Table 1 shows that the preparation of virgin coconut oil enzymatically using a variety of mass ratio of to 0.006% is obtained yield average of 12.40%. The optimum separation of virgin coconut oil on the use of enzymes with a mass ratio of 0.002%. The higher the yield of the addition of the enzyme produced virgin coconut oil is increasing. This is because the higher the enzyme added to a growing number of peptide bonds in proteins that envelop coconut oil can be hydrolyzed, because the enzyme papain is a proteolytic enzyme that can hydrolyze the peptide bond. The higher the temperature of incubation, protein hydrolysis reaction speed more quickly so that oil can be released and the yield will be higher [8]

6 TABLE 1. The yield of virgin coconut oilon the ratio of Saccharomyces cerevisiae No The ratio of Sacharomycescereviceae Yield % 13.11% % 14.83% % 12.20% % 10.92% % 12.46% % 10.90% Average 12.40% Hydrolase enzyme from Saccharomyces cerevisiae working effectively at room temperature. The higher the concentration, the higher yield of virgin coconut oil produced. Saccharomyces cerevisiae be able to produce proteolytic enzymes that be able to hydrolyze the protein that surrounds the fat globules. The higher the concentration of Saccharomyces cerevisiae added, the more the proteolytic enzyme produced and the more fat is freed from coconut milk. The fermentation process will continue to produce water and acetic acid. Acetic acid has the ability to break the fat-protein, therefore, the more fat is separated from the protein. Acetic acid production can minimize the use of chemicals, so in the process of making virgin coconut oil without the addition of acid. The results obtained at 45 to 50% [2,8]in contrast with the results obtained optimum results 14.83%. The process of making virgin coconut oil which is carried out using extraction techniques so that when the process of incubation is added virgin coconut oil to extract oil optimally. The results are calculated from the number of pure virgin coconut oil which is used for extraction process virgin coconut oil to be separated. The result of virgin coconut oil that is obtained is determined by the process of preparing coconut milk. The extraction of coconut milk with warm water be able to affect the results manually extract coconut milk. The preparation coconut cream can be done manually by repeated leaching process so as to provide optimal results. The virgin coconut oil produced is influenced by the separation process virgin coconut oil from coconut milk cream after the incubation process. Virgin coconut oil trapped in a creamy coconut milk and separation process is required. Separation of the mixed layer can be done by extraction techniques with pure virgin coconut oil or virgin coconut oil have been produced. This separation technique has not been done and be able to be recommendations on the process of making virgin coconut oil without the process of fishing. The inducement techniques is only done on the process of separation or extraction of virgin coconut oil from coconut milk. Organoleptic Analysis Organoleptic test is useful to determine the state of virgin coconut oil which includes smell, taste and color. Quality requirements in the Indonesian National Standard for virgin coconut oil is determined from the typical smell of coconut oil and fresh and not found a rancid smell and other odors. Virgin coconut oil is normally colorless or pale yellow and has a distinctive taste of coconut oil. The organoleptic tests performed by three panelists to obtain the conclusion of the test in accordance with quality requirements. The results of organoleptic tests show that virgin coconut oil produced in the manufacture of virgin coconut oil using enzymes ratio Saccharomyces cerevisiae to 0.006% results in accordance with the Indonesian National Standard. Data organoleptic test results are presented in Table 2 shows that virgin coconut oil quality requirements have been met. The test results in TABLE 2.illustrates that the process of making virgin coconut oil by using enzymes Saccharomyces cerevisiae with a variety of ratios without using the process of fishing and the addition of acid can provide results of pure coconut oil or virgin coconut oil which has a normal and typical smell of fresh coconut. Virgin coconut oil produced is not found to rancidity. The third panelist to test the organoleptic parameters were normal states that virgin coconut oil can be said to meet the quality requirements.organoleptic test results of three panelists also stated that the ratio of Saccharomyces cerevisiae not affect the taste of virgin coconut oil produced. Virgin coconut oil obtained has a normal flavor typical of fresh coconut oil so that it can meet the quality requirements in the Indonesian National Standard. Taste virgin coconut oil to determine the quality and provide an overview of the quality of the products and information related to the presence and absence of contaminant microorganisms and chemical contaminants. Quality virgin coconut oil that will be free from contamination of

7 microorganisms and can last up to 12 months. Quality virgin coconut oil can be seen from the change in taste that indicate deterioration and decomposition of chemical and microbiological so as to reduce the taste of fresh coconut oil. The existence of chemical oxidation process can alter the taste virgin coconut oil. TABLE 2. The results of organoleptic testing for virgin coconut oil No The ratio of Saccharomyces Panelists Smell Taste Colour cereviceae % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal % The first of panelist Normal Normal Normal The second of panelist Normal Normal Normal The third of panelist Normal Normal Normal Virgin coconut oil quality requirements of Indonesian National Standard is based on the color of virgin coconut oil. Virgin coconut oil is normally colorless or pale yellow. The results of organoleptic tests showed that the three panelists stated that virgin coconut oil produced has normal color, which is colorless. Therefore, the color parameters in according with quality requirements virgin coconut oil. Determination of Water Content Determination of water content in coconut oil is very important to estimate the oil resistance. The water content in coconut oil greatly affects the quality of coconut oil is made by fermentation enzymes, oil has water level will tend to have a short shelf life.according to Table 3 shows that the average water content in virgin coconut oil is %. These test results indicate that the quality requirements in the Indonesian National Standard No. been met, ie less than 0.2%. Use of Saccharomyces cereviceaebe able to produce virgin coconut oil produced. The separation process virgin coconut oil from coconut milk cream without the fishing could take place so that the water level is very low. The water content in the product virgin coconut oil be able to determine the quality requirements because it is closely related to the process of oil damage. The presence of water in virgin coconut oil allows the microorganisms can flourish so that damage will occur as a result of oil contamination of microorganism. Water content are parameters that affect the level of oil resistance to damage. The presence of a number of water in oil or fat can lead to a hydrolysis reaction. Oils or fats will be converted into free fatty acids and glycerol. This reaction will produce the taste and smell of rancid oil. Table 5.shows that the lowest water content is in the process of making virgin coconut oil with Saccharomyces cerevisiae ratio of 0.002%. In generally, the water content in virgin coconut oil is still below 0.2% so it be able to found to comply with quality requirements. The average water content at a ratio of 0.002% is %

8 TABLE 3. The water content of virgin coconut oil No The ratio of Saccharomyces cereviceae(%) Water content (%) Uncertainty (%) The higher the addition of hydrolase enzymes of Saccharomyces cerevisiae in the fermentation process can increase the water [2]. This is because many enzyme concentration, the higher the virgin coconut oil separate, but the higher the water during the process of releasing the enzyme produces acetic acid, and the more protein dissolved. When the enzyme glucose perform hydrolysis reaction will occur process water discharge. The water content is also influenced by the longer the fermentation process it will produce more acid fermented. The resulting acid may alter the conditions of acidity (ph) reached the protein isoelectric point. When fermentation time increasing the ph will back away from the ph of the isoelectric point of the protein back so late. The increase in solubility of proteins and enzymes produced Saccharomyces cerevisiae hydrolase may result in increased amount of water in virgin coconut oil produced. W 1 W 2 balance balance oven Standard deviation Water content Temperature Repeatability FIGURE 1. Fishbone diagram on the estimation of measurement uncertainty for the determination of water content by gravimetric method Determination of Uncertainty for Water Content Determination of measurement uncertainty estimation is done by identifying the sources of uncertainty in the determination of water content by gravimetric method. Figure 1 shows the fishbone diagram the determination of water content measurement uncertainty.estimation of measurement uncertainty calculations are presented in TABLE 5. Standard uncertainty of analytical balance is determined using Eq. 5. m = Standard uncertainty ofthe oven is determined using Eq. 6. m = Standard uncertainty ofrepeatability is determined using Eq. 7. m = The combined uncertainty is determined by Eq. 8. U = %H O x ( m ) + ( m ) + ( m ) + ( m ) (8) (5) (6) (7)

9 The expanded uncertainty is determined by Eq. 9.(11) U = U c. k (9) Uncertainty is a parameter that describes the distribution of a quantitative value of the test results which indicate the nature of traceability of measurement results. The results of measurements of water content in the use of 0.005% Saccharomyces cerevisiae has the smallest measurement uncertainty estimation, ie %. The largest contributor to measurement uncertainty comes from repeatability, with an average contributed 63.85%. TABLE 4. The sources and calculation of uncertainties in determination of water content The ratio of Saccharomyces Sources of Standard Relative standard Value Unit cereviceae(%) uncertainty uncertainty uncertainty W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 C 5.58 x x 10-3 Precision x x 10-4 Combined uncertainty Expanded uncertainty W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 C 5.58 x x 10-3 Precision x x 10-5 Combined uncertainty Expanded uncertainty W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 C 5.58 x x 10-3 Precision x x 10-3 Combined uncertainty Expanded uncertainty W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 C 5.58 x x 10-3 Precision x x 10-8 Combined uncertainty Expanded uncertainty 0.01 W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 C 5.58 x x 10-3 Precision x x 10-3 Combined uncertainty Expanded uncertainty W g 1.15 x ,79 x 10-6 W g 1.15 x ,79 x Temperature 1 o C 5.58 x x 10-3 Precision x x 10-2 Combined uncertainty Expanded uncertainty 0.01 Determination of Iodine Value Unsaturation of oil is known from iodine number. Unsaturated fatty acids in oils and fats capable of absorbing iodine and the amount of saturated forming compounds [9]. The large amount of iodine absorbed indicates the number of double bonds or unsaturated. Testing iodine number performed using redox titration using the Wijs method. The virgin coconut oil is reacted with iodine mono-chloride in a mixture of acetic acid and cyclohexane

10 TABLE 5. The iodine number of virgin coconut oil No The ratio of Saccharomyces The number of iodine Uncertainty cereviceae (gram iodine per 100 grams oil) (gram iodine per 100 grams oil) TABLE 6.show that the iodine number of virgin coconut oil. The iodine number indicates the number of double bonds contained in the oil and when it reacts with iodine to form saturated compounds [10]. The iodine number obtained in virgin coconut oil enzymatically with Saccharomyces cereviceae that has not lasted optimally. There is still a possibility that the oil contains unsaturated fatty acids both free and bound that has not been released and they form bonds with proteins. TABLE 6.shows that the highest iodine number in virgin coconut oilproduced is ,1974grams of iodine per 100 grams of oil and optimum iodine number is obtained from the manufacturing process virgin coconut oil with a ratio of 0.006% Saccharomyces cerevisiae. The higher the ratio of the mass of hydrolase enzymes Saccharomyces cerevisiae the more effective the hydrolysis reaction between the fatty acid binding protein that the number of iodine becomes larger. The increase in iodine number is not due to increased bond on oil, but because it has been the separation of the non-oil compounds containing a double bond such as polymers, proteins, hydrocarbons, glycosides and -carotene [9]. Determination of Uncertainty for Iodine Value Determination of measurement uncertainty estimation is done by identifying the sources of uncertainty in the determination of iodine value by titrimetric method. FIGURE 2.showsthe fishbone diagram the determination of iodine value measurement uncertainty and the estimation of measurement uncertainty calculations are presented in TABLE 7. The coefficient of thermal expansion V 0 V 1 The coefficient of thermal expansion Equivalent weight Mr I 2 Iodine Value Purity KBrO 3 MrKBrO 3 mkbro 3 N Na 2 S 2 O 3 Bias The coefficient of thermal expansion VNa 2 S 2 O 3 m Repeatability Standard deviation FIGURE 2. Fishbone diagram on the estimation of measurement uncertainty for the determination of iodine value Standard uncertainty of volume is determined using Eq. 10. m = (m V ) + (m T ) + (m P ) (10) m = V. m. β : coefficient of thermal expansion Standard uncertainty of analytical balance is determined using Eq. 11. m = (11)

11 TABLE 6.The sources and calculation of uncertainties in determination of iodine value The ratio of Saccharomyces Sources of Standard Relative standard Value Unit cereviceae(%) uncertainty uncertainty uncertainty V ml x 10-4 V ml x 10-5 Mass g x M Iodine g/mol x Repeatability x 10-4 NNa 2 S 2 O 3 N x x 10-3 Combined uncertainty Expanded uncertainty V ml x 10-4 V ml x 10-3 Mass g x 10-6 M Iodine g/mol x Repeatability x 10-6 NNa 2 S 2 O 3 N x x 10-3 Combined uncertainty Expanded uncertainty V ml x 10-4 V ml x 10-6 Mass g x M Iodine g/mol x Repeatability x 10-5 NNa 2 S 2 O 3 N x x 10-3 Combined uncertainty Expanded uncertainty V ml x 10-4 V ml x 10-6 Mass g x M Iodine g/mol x Repeatability x 10-5 NNa 2 S 2 O 3 N x x 10-3 Combined uncertainty Expanded uncertainty V ml x 10-4 V ml x 10-7 Mass g x 10-6 M Iodine g/mol x Repeatability x 10-6 NNa 2 S 2 O N x x 10-3 Combined uncertainty Expanded uncertainty V ml x 10-4 V ml x 10-6 Mass g x 10-6 M Iodine g/mol x Repeatability x 10-5 NNa 2 S 2 O 3 N x x 10-3 Combined uncertainty Expanded uncertainty

12 Standard uncertainty of concentration of sodium thiosulfate is determined using Eq. 12. m = (m V ) + (m m ) + (m PKBrO3 ) + (m MrKBrO3 ) (12) Standard uncertainty of equivalent weight is determined using Eq. 13. m = (m 2Ar I2/n ) (13) Standard uncertainty ofrepeatability is determined using Eq. 14. m = The combined uncertainty is determined by Eq. 15. U = x ( m ) + ( m ) + ( m ) + ( m ) + ( m ) (15) The expanded uncertainty is determined by Eq. 16. (11) U = U c. k (16) Determination of Free Fatty Acids TABLE 8.show thatthe use of the ratio of enzyme Saccharomyces cerevisiae 0.002% virgin coconut oil product provides the most highest. Sacharomycescerevisiae with a ratio of 0.002% results virgin coconut oil with acid number % under below 2% from standard. The ratio of Saccharomyces cerevisiae enzyme can affect the levels of free fatty acids in virgin coconut oil produced. The higher the ratio of hydrolase enzymes produced from Saccharomyces cerevisiae can lead to the formation of acetic acid and water that could cause the hydrolysis of fats into fatty acids. TABLE 7.Free fatty acids content of virgin coconut oil No The ratio of Saccharomyces cereviceae Free fatty acid (%) Uncertainty (%) The higher the ratio of Saccharomyces cerevisiae, the higher the free fatty acids. This is because the more the yeast, the higher the moisture content of virgin coconut oil. The presence of water, the oil can be hydrolyzed into glycerol and fatty acids. The presence of water in the oil will occur hydrolysis process oil into free fatty acids and glycerol to free fatty acids generated higher. The higher the concentration of enzyme and incubation period, the higher the free fatty acid in virgin coconut oil produced [8]. This is due to virgin coconut oil still contains water so still allow the breakdown of triglycerides into fatty acids and glycerol. The higher the concentration of hydrolase enzymes can cause the hydrolysis reaction of oil or fat will turn into free fatty acids and glycerol. Hydrolysis reaction that can result in damage to the oil or fat occurs because there is a water in oil or fat. Determination of Uncertainty for Free Fatty Acids Determination of measurement uncertainty estimation is done by identifying the sources of uncertainty in the determination of iodine value by titrimetric method. Figure 3 shows the fishbone diagram the determination of free fatty acids value measurement uncertainty and the estimation of measurement uncertainty calculations are presented in TABLE 9. Standard uncertainty of concentration of sodium hydroxide is determined using Eq. 17. m = N x (m V ) + (m mkhp ) + (m pkhp ) + (m MrKHP ) (17) The combined uncertainty is determined by Eq. 18. U = %FFA x ( m ) + ( m ) + ( m ) + ( m ) + ( m ) (18) (14)

13 TABLE 8.The sources and calculation of uncertainties in determination of free fatty acids The ratio of Saccharomyces Sources of Standard Relative standard Value Unit cereviceae(%) uncertainty uncertainty uncertainty V ml x 10-3 mass g x M g/mol x Repeatability x 10-4 N of NaOH N x Combined uncertainty Expanded uncertainty V ml x 10-3 mass g x M g/mol x 10-9 Repeatability x 10-3 N of NaOH N x 10-1 Combined uncertainty Expanded uncertainty V ml x 10-4 mass g x M g/mol x 10-9 Repeatability x 10-4 N of NaOH N x 10-1 Combined uncertainty Expanded uncertainty V ml x 10-3 mass g x M g/mol x 10-9 Repeatability x 10-3 N of NaOH N x 10-1 Combined uncertainty Expanded uncertainty V ml x 10-3 mass g x M g/mol x 10-9 Repeatability x 10-3 N of NaOH N x 10-1 Combined uncertainty Expanded uncertainty V ml x 10-3 mass g x M g/mol x 10-9 Repeatability x 10-4 N of NaOH N x 10-1 Combined uncertainty Expanded uncertainty Determination of Peroxide Value Base on TABLE 10.the peroxide value of virgin coconut oil between to milliequivalent per kilograms. The parameter of peroxide value just below 2% for the standard. Determination of the peroxide used as an index of the amount of fat or oil that has undergone oxidation. The peroxide number is very important to assess the level of oxidation of virgin coconut oil produced. Virgin coconut oil which contains acidunsaturated fatty acid can be oxidized by oxygen to produce a peroxide compound. One of the parameters a decrease

14 in the quality of virgin coconut oil is a peroxide. Measurement of peroxide value is basically used to measure levels of peroxide and hydro-peroxide formed in the early stages of fat oxidation reactions. V The coefficient of thermal expansion Equivalent weight Mr lauric acid Free fatty acids P KHP Mr KHP m KHP N NaOH Bias The coefficient of thermal expansion VNaOH m Repeatability Standard deviation FIGURE 3. Fishbone diagram on the estimation of measurement uncertainty for the determination of free fatty acids TABLE 9. The peroxide value of virgin coconut oil No The ratio of Saccharomyces The peroxide value Uncertainty cereviceae (milli-equivalent per kilograms) (milli-equivalent per kilograms) V Repeatability The coefficient of thermal expansion The coefficient of thermal expansion Purity KBrO 3 Bias MrKBrO 3 VNa mkbro 2 S 2 O 3 3 The coefficient of thermal expansion N Na 2 S 2 O 3 Mass Peroxide Value FIGURE 4. Fishbone diagram on the estimation of measurement uncertainty for the determination of peroxide value

15 TABLE 10.The sources and calculation of uncertainties in determination of peroxide value The ratio of Saccharomyces Sources of Standard Relative standard Value Unit cereviceae(%) uncertainty uncertainty uncertainty V ml x 10-4 Mass g x Repeatability x 10-4 NNa 2 S 2 O N x 10-4 Combined uncertainty Expanded uncertainty V ml x 10-3 Mass g x Repeatability x 10-3 NNa 2 S 2 O N x 10-4 Combined uncertainty Expanded uncertainty V ml x 10-3 Mass g E Repeatability x 10-3 NNa 2 S 2 O N x 10-4 Combined uncertainty Expanded uncertainty V ml x 10-3 Mass g x Repeatability x 10-3 NNa 2 S 2 O 3 N x 10-4 Combined uncertainty Expanded uncertainty V ml E-04 Mass g x Repeatability x 10-3 NNa 2 S 2 O N x 10-4 Combined uncertainty Expanded uncertainty V ml x 10-3 Mass g x Repeatability x 10-3 NNa 2 S 2 O N x 10-4 Combined uncertainty Expanded uncertainty The high peroxide value indicates the fat or oil has undergone oxidation, but at a lower number is not always indicate the condition of oxidation is still early. Low peroxide values may be caused by a new peroxide formation reaction rate less than the rate of reaction of degradation into other compounds. Peroxide compounds readily degradable and react with other substances. The presence of fat oxidation reaction by oxygen occurs spontaneously when fatty material is left in contact with air, while the speed of the process depends on the type of fat oxidation and storage conditions.peroxide could speed up the process rancid odor and flavor that is undesirable in foodstuffs. If the amount of peroxide over 100 milli-equivalents per kilogram of oil would be highly toxic and has an unpleasant smell. The increase in peroxide is an indicator that the oil will become rancid. The hydrolase enzyme be able to affect the amount of peroxide value virgin coconut oil. The higher the concentration of hydrolase enzymes, the peroxide value on virgin coconut oil produced is increasing [8]. This is due to the higher concentration of hydrolase enzymes and incubation temperatures will increase the reaction rate of hydrolysis of proteins in coconut cream, so that the process of separating oil from coconut cream occurs faster. The more quickly the oil separation process, it will be sooner oil direct contact with oxygen, thus the reaction of free radical formation which is then converted into the hydro-peroxide will increase

16 Saccharomyces cerevisiae have the longer the fermentation, the peroxide virgin coconut oil produced higher. This is the acid produced the more so as the solubility of the protein increases. Soluble protein binds water. The presence of water in oil can hydrolyze the oil resulting in termination of the carbon chain of the oil to form free fatty acids. Interrupted carbon chains will bind with oxygen to peroxide oil increases. Determination of Uncertainty for Peroxide Value Determination of measurement uncertainty estimation is done by identifying the sources of uncertainty in the determination of peroxide value by titrimetric method. Figure 3 shows the fishbone diagram the determination of free fatty acids value measurement uncertainty and the estimation of measurement uncertainty calculations are presented in TABLE 11. CONCLUSION Based on the results of this study concluded that the ratio of Saccharomyces cerevisiae can affect the yield of virgin coconut oil produced. The preparation of virgin coconut oil enzymatically using a variety of mass ratio of to 0.006% is obtained yield average of 12.40%. The optimum separation of virgin coconut oil on the use of enzymes with a mass ratio of 0.002%. The average water content at a ratio of 0.002% is % with a value of uncertainty is 0.005%. The average iodine number in virgin coconut oil produced is ,1974grams of iodine per 100 grams of oil and optimum iodine number is obtained from the manufacturing process virgin coconut oil with a ratio of 0.006% Saccharomyces cerevisiae. Sacharomycescerevisiae with a ratio of 0.002% results virgin coconut oil with acid number %.The peroxide value of virgin coconut oil between to milli-equivalent per kilograms. Organoleptic test results and test chemical parameters can be used as the test data that can be developed in prototype preparation of candidate in-house reference materials in the testing standards of quality virgin coconut oil. REFERENCES 1. National Standardization Agency,Virgin Coconut Oil (VCO)ISO 7381 (National Standardization Agency, Jakarta, 2008), pp R. Aditya, H. Rusmarilin and L.N. Limbon, Journal of Food and Agricultural Engineering51-57(2014). 3. S. Wiharti, Jariyah and Y. Purnomo, Journal of Agricultural Engineering (2007). 4. Susilowati, Journal of Chemical Engineering (2009). 5. N.R. Fatwatun, K. Chusna and Pramundono, Journal Chemical Enineering and Industry (2013). 6. ISO/Guide 30:2015, Reference materials-selected terms and definitions,pp. 1-6 (2015). 7. S. Raharjo and M. Dwiyuni, Journal Indonesian of Agricultural Technology71-78(2004). 8. S. Winarti, Jariyah and Y. Purnomo, Journal of Agricultural Technology (2007). 9. A.M. Efendi, Winarni andsumarni, Indonesian Journal of Chem. Science1-6(2012). 10. N. M. Suaniti, M. Manurung and N. Hartasiwi, Journal of Chem (2014). 11. K. Birch,Measurement Good Practice Guide- Estimating Uncertainties in Testing(Addison-Wesley Publishing Company Inc., London, 2001),pp IUPAC Commission on Atomic Weights and Isotopic Abundances,Pure Appl. Chem.75, (2003)