Identification of acetic acid levels in ecoenzyme from organic materials of orange peel with acid-base titration method

This study aimed to elucidate the comparison of the levels of acetic acid and the pH of the ecoenzymes from organic matter variations of sweet and sour orange peels. The acetic acid is an organic compound containing a carboxylic acid group which is also widely applied in the health and industrial fields. Preparation of acetic acid from substrate products containing ethanol which can be obtained from fruit ingredients including oranges. One of the uses and management of organic waste, including vegetables and fruit, is by making ecoenzymes from fruit peels with a mixture of water and sugar. Fruit peels that can be used as organic ecoenzyme ingredients vary widely, so the content in ecoenzymes becomes very diverse and tends to be unstable. This type of research is descriptive. This study consisted of three stages, namely the manufacture of ecoenzymes from pasaman orange peel, gunung omeh, kaffir lime and lime in 7 variations (for 100 days), measurement of acetic acid levels and pH in the ecoenyzme samples for 14 days and 100 days. Acetic acid levels were determined using the acid-base titration method. The results of this study showed that the highest levels of acetic acid ecoenzyme in the 100-day fermentation were 5.53% in the sour orange group, while the lowest in the mixed ecoenzyme was 3.32%. The most acidic ecoenzyme pH was at 100 days of fermentation (pH ±3.5).


Introduction
Acetic acid or vinegar is an organic compound that contains a carboxylic acid group, which is known as a sour taste and aroma in foods as well as organic acids [1].Organic acids are common components found in foods and beverages that have an important role in the product characteristics of taste and aroma.Fruits including organic acid products also contain acetic acid including oranges and pineapples.
One way to manage organic waste is by making ecoenzymes.Ecoenzyme is the result of fermentation of organic materials such as skins of fruits and vegetables, sugar, and water [2,3].Ecoenzyme was first introduced by Dr. Rosukon Poompanvong is a co-founder of the Thai Organic Agriculture Association [4].Dr. Rosukon Poompanvong focuses on researching the processing of useless kitchen waste into IOP Publishing doi:10.1088/1755-1315/1312/1/012055 2 useful and environmentally friendly enzymes that have many benefits such as plant fertilizers, river cleaners, and can be used by farmers to repel plant pests [4][5][6][7].The manufacture of ecoenzyme comes from simple ingredients, namely sugar, waste or organic waste and water by using a composition ratio of 1 (molasses): 3 (Substrate): 10 (Water) through a fermentation process [4,5].
The process of overhauling organic matter can take place under aerobic and anaerobic conditions or multi fermentation [1,8].The production of acetic acid involves two stages of fermentation, namely anaerobic and aerobic.Anaerobic fermentation produces alcohol, while aerobic fermentation to convert alcohol into acetic acid is used by bacteria.The fermentation process in the ecoenzyme lasts approximately 100 days.Possible ecoenzyme fermentation processes include multifermentation.Ecoenzyme will produce a few alcohol and organic acids.The content of organic acids includes acetic acid, lactic acid, malic acid, oxalic acid, and citric acid [3,[9][10].The fermentation process is an attempt by bacteria to obtain energy from carbohydrates under anaerobic and aerobic conditions, with byproducts in the form of alcohol or acetic acid [11].This depends on the type of microorganism.
Based on data from the Central Statistics Agency (2020) Siamese citrus fruit ranks first for fruit production in West Sumatra, which is 145,033.40tons.The level of citrus consumption in West Sumatra ranks first as much as 0.104 kg per capita per week.The level of orange consumption increased by 5.60% from 2017 (Central Bureau of Statistics, 2019).These data indicate the high level of consumption and interest of the people of West Sumatra towards citrus fruits, so it has the potential to process the remaining citrus peels into ecoenzymes as alcohol and acetic acid products that can be utilized.Based on this background, this research was conducted to determine the levels of acetic acid and alcohol in this ecoenzyme.Therefore, by measuring the levels of acetic acid and alcohol using the acid-base titration method, data on the levels of acetic acid and alcohol are obtained which are useful for increasing the value of ecoenzymes and providing wider benefits and impacts for the environment globally as well as from the health sector and especially industry.

Materials and Methods
This research is descriptive research conducted in June -December 2021 at the Laboratory of Biochemistry, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang.
The tools that will be used in this research are Erlemeyer, beaker, measuring cup, ecoenzyme liquid storage bottle, analytical balance, pH meter, funnel, stirrer glass, dropper pipette, filter paper, aluminum foil, plastic, and gloves.The tools used for acid-base titration are 50 ml burette, stative, and burette clamp.While the materials that will be used in this research are ecoenzymes in orange peel, pineapple peel, mint leaves, aquadest, molasses, 0.1 N NaOH, 1% phenolphthalein (PP) indicator.

Ecoenzyme Production
The process of making ecoenzyme is that the organic material or substrate that will be used comes from orange peel, molasses and air in a ratio of 3:1:10.Where 3 liters of distilled water is needed, 900g of organic material and 300g of molasses.Then fermented for 100 days.The forms of variation provided are: As for the form of a combination of organic ingredients of orange peel given: The organic materials that will be used consist of various orange peels, namely Pasaman Oranges (M1), Gunung Omeh Oranges (M2), Limes (A1), Prut Oranges (A2) which will be made into seven different combinations of orange peel organic materials in duplicate..

Ecoenzyme Harvest
Ecoenzyme is harvested after going through the fermentation stage for 14 and 100 days.In the dregs of organic materials, the organic materials are separated from the ecoenzyme using a filter.Then the ecoenzyme is put into the jerry cans that have been provided so that they can be used later.

Sample Measurement Preparation
Ecoenzyme is concentrated at 3000 rpm, 4°C for 10 minutes.The supernatan obtained is then collected to be further used to measure levels of acetic acid and alcohol (10).

Solution Preparation
2.4.1.Titrant NaOH 0,1 N. NaOH solution will be made by dissolving 4 grams of NaOH granules in 10 ml of aquades, after which it is diluted with a akuades in a 1 L measuring pumpkin to the limit mark on the measuring gourd.

2.4.2.
Phenolftalein Indicator 1 %.Weighing 1 gram pp powder is then diluted with a akuades in a 100 ml measuring pumpkin to the limit mark.

Method
Measurement of acetic acid levels in ecoenzyme is done by titration, each ecoenzyme sample is taken as much as 2 ml and then transferred into the erlenmeyer.Before being titrated first diluted by adding 8 ml of a akuades, then given a PP indicator of 1% as much as 2 drops, the sample is ready to be titrated with NaOH 0.1 N. Titration until there is a color change right at the equivalent point at pH 8.0-9.6.

Calculation of Acetic Acid with Acid Titration Method
To calculate acetic acid levels, the following equation is used (12).

Data analysis
Data on the identification of acetic acid levels in the ecoenzyme from the organic material of a combination of sweet and sour orange peel with sweet and single sour orange peel on the 14th and 100th day of fermentation using the acid base titration method will be analyzed descriptively.Data will be presented in the form of tables and graphs.

Result and Discussion
This research is intended to identify ecoenzymes from organic orange peel materials.Variations in organic materials have different acetic acid contents.The results of acetic acid levels were obtained based on measurements by the acid-base titration method and calculations.The following acetic acid levels for each sample are presented in Figure 1.
Concentration of Acetic Acid = Vol NaOH X N NaOH X BE Acetic Acid X DF (   1000) Measurement of the acetic acid content showed that the acetic acid content in the ecoenzyme from the organic material combined sweet and sour orange peel with sweet orange peel and single acid at the 14th and 100th day of fermentation using the acid base titration method, there were differences in each combination of organic materials.The highest level of acetic acid was found in ecoenzyme A2 (Kaffir lime) 100 days at 5.53%.Meanwhile, the lowest acetic acid content was found in ecoenzyme M1, M2, A1, A2 (Mixture) 14 days at 3.32%.
Acetic acid is an organic compound that contains a colourless carboxylic acid group, has a pungent odour, has a sour taste and is soluble in water, alcohol, glycerol, ether.Acetic acid is produced through fermentation, the application of acetic acid is very wide in the industrial and food sectors.The ongoing fermentation process causes the sucrose contained in the sap to turn into alcohol and then become acetic acid.Acetic acid is obtained from the fermentation process of food ingredients such as fruit and vegetables which contain large amounts of carbohydrates.In this study, fruit was used, namely oranges, and fermented using molasses sugar [13].
Acetic acid fermentation is based on two principles, namely alcoholic and acetic acid fermentation.The initial stage of the fermentation process is to produce alcohol, to break down sugar into alcohol.The length of fermentation depends on the type of substrate, the initial sugar content, and the desired final alcohol content.The alcohol content influences the course of the subsequent acetic acid fermentation process.This is in accordance with the statement [14] which states that the fermentation process begins with the breakdown of sucrose into glucose and fructose by yeast activity.Glucose is then converted into alcohol and CO₂ which reacts with water to form carbonic acid.The ethanol formed is oxidized by Acetobacter as the main bacteria in the culture to acetaldehyde and then to acetic acid [14][15].
Making acetic acid from a substrate product containing ethanol which can be obtained from fruit.Making acetic acid can be done synthetically or chemically and microbiologically or by aerobic fermentation, but it is widely used because it is cheaper, more practical and the risk of failure is relatively small.In acetic acid fermentation from a liquid substrate, namely alcoholic fermentation.Alcohol fermentation is carried out if the ingredients used are rich in sugar but do not contain alcohol [16].
The method that can be used to measure acetic acid levels is the titration method.There are several types of titration methods, including acid-base titration, argentometric titration and complexometric titration, which is used in this research using the acid-base titration method.Titration is a process or method of volumetric analysis where the concentration of a titrant or standard solution is first known when it is dropped with a burette into another solution which can react until it reaches the equivalence point or end point, meaning that the concentration of the substance to be determined is called a titrant and is usually placed in an Erlenmeyer flask.while a substance whose concentration is known is called a titter and is usually placed in a burette.Both titters and titrants are in solution form [17]. Acid-base titration is an example of volumetric analysis, the method used for the solution is called a titrant and is released from a gelatine device called a burette.If the solution being tested is basic then the titrant must be acidic and if the solution is acidic then the titrant must be basic [18].This method is considered quite accurate to date in titration experiments, so the conventional titration method is used today [13].
The performance of the conventional titration method in determining the concentration of acetic acid is carried out using acid-base titration.In this study, measurements used a titrant, namely NaOH 0.1 N and the indicator used phenolphthalein (PP).The colour change of the indicator is in a pH interval which is expressed as a pH trajectory [18,19].The indicator in the acid-base titration test is a substance that can change colour according to the pH of the solution being identified and can determine the acid-base properties of a test solution in a solution.The synthetic indicator that is usually used for acid-base titrations is phenolphthalein or called the PP indicator, as the comparison is the PP indicator, the end point of the titration is obtained when 0.1 N NaOH is added, namely when the colour changes from colourless to pink.In the research, because the sample is coloured, the titration process until the colour change occurs right at the equivalent point at pH 8.0-9.6.The colour changes that occur are caused using acetic acid which can function to reduce the acidic atmosphere in the indicator, so that the titration takes place more quickly.In addition, the colour change process can occur through a balance in the molecular and ionic forms of the indicator compound itself [20].The measurement results showed that the highest level of acetic acid was found in ecoenzyme A2 (Jeruk Prut) 100 days, 5.53%.Meanwhile, the lowest acetic acid content was found in ecoenzyme M1, M2, A1, A2 (Mixture) 14 days 3.32%.This is in accordance with research [18] that the tendency of ecoenzyme solutions produced from organic materials in the form of fruit to produce acidic chemical parameters with a low pH value means that the higher the organic acid content, the lower the pH value because kaffir limes have the most sour taste among other oranges, in this study the ecoenzyme has the pH value low, namely (pH ± 3.5) as a result of the high organic acid content such as acetic acid or citric acid.The citric acid in limes and kaffir limes, which both have a sour taste, also has properties like acetic acid, namely the properties of a weak acid with a pH of 2 [21].
In this research, there are differences because they are influenced by factors that influence the fermentation process, namely the type of organic material, temperature, temperature, environment, and factors during production such as air contamination so that the fermentation process is not optimal and the length of fermentation also influences the results of alcohol and acetic acid levels.This is in accordance with research [22] The fermentation results of both types of pineapple and papaya show that the ecoenzyme produced is acidic with a low pH.Besides that, the length of fermentation time and the use of molasses affect the pH and TDS levels.Therefore, to further utilize ecoenzyme technology, it is deemed necessary to develop research by combining various organic materials and following time variations.On research [8] also explained that the longer the fermentation time, the more acid produced will increase.The longer the fermentation time and the more Acetobacter xylinum bacteria used, the higher the viscosity of the acetic acid produced, this indicates that the length of fermentation time and the number of bacteria greatly influence the viscosity of acetic acid [1].On the other hand, one of the important inputs in processing organic materials into ecoenzymes is the influence of fermentation time, where parameter values except pH will decrease over fermentation time due to degradation of organic materials by microorganisms in the enzyme solution [23].
Making acetic acid from organic waste, namely dry pineapple skin, has the advantage of a faster fermentation process so that in terms of time it is more efficient [22], but the product [1] yield is small, compared to semi-wet and wet pineapple skin, the fermentation process is slower but the final product yields more ecoenzyme.This is in accordance with the statement The higher the air temperature used for drying, the greater the moisture content of the material, so the time required for drying will be faster [24].

Conclusion
Based on the results of research that measured acetic acid levels in the ecoenzyme of organic ingredients in a combination of sweet and sour orange peels with single sweet and sour orange peels during 100 days of fermentation using the acid-base titration method, there were differences in each combination of organic ingredients in the combination of orange peels.measurement of acetic acid levels and pH in the ecoenyzme samples for 14 days and 100 days.Acetic acid levels were determined using the acidbase titration method.The results of this study showed that the highest levels of acetic acid ecoenzyme in the 100-day fermentation were 5.53% in the sour orange group.while the lowest in the mixed ecoenzyme was 3.32%.The most acidic ecoenzyme pH was at 100 days of fermentation (pH ±3.5).

Tabel 1 .
Variations of Organic Orange Peel Ingredients.