Improvement yield of applewood (Feronia limonia) flavor extract by liquid-liquid extraction with dichloromethane solvent

The use of natural foodstuffs continues to increase along with the increasing awareness of the people to consume safe and nutritious food that has a positive impact on health. Applewood (Feronia limonia) is classified as a rare plant and has a distinctive aroma. In Indonesia, it can be found in Aceh, Rembang, and Bima. Applewood fruit that is perfectly ripe has a distinctive aroma and can give food flavor. Natural flavor has several advantages, in addition to providing flavor to the product, it also has a function for health (Berger, 2009). Natural flavorforming compounds are good for health and extend product shelf life (Ayseli & İpek Ayseli, 2016). The demand for natural flavors by consumers in beverage industry products continues to increase (Zegler, 2012).


Introduction
The use of natural foodstuffs continues to increase along with the increasing awareness of the people to consume safe and nutritious food that has a positive impact on health. Applewood (Feronia limonia) is classified as a rare plant and has a distinctive aroma. In Indonesia, it can be found in Aceh, Rembang, and Bima. Applewood fruit that is perfectly ripe has a distinctive aroma and can give food flavor. Natural flavor has several advantages, in addition to providing flavor to the product, it also has a function for health (Berger, 2009). Natural flavorforming compounds are good for health and extend product shelf life (Ayseli & İpek Ayseli, 2016). The demand for natural flavors by consumers in beverage industry products continues to increase (Zegler, 2012).
Based on the raw material, flavors can be classified into three types: natural flavor, identical natural flavor, and artificial flavor. Natural flavors are compounds that are extracted from ingredients that exist in nature. Natural identical flavors are compounds that are found in nature but are made synthetically or chemically. Artificial flavors are compounds that do not exist in nature and can only be made synthetically (Heath, 1978).
Analysis with Gas Chromatography-Mass Spectrometry-Olfactometry (GC-MS-O) to identify and describe the character impact odorants of applewood fruit flavor, obtained specific aromas, namely fruity, sweet, melon-like, cheese (butter-like), herbaceous (green), and sour aroma. GC-MS successfully identified 75 volatile components of applewood consisting of esters, alcohol, aldehyde, ketones, carboxylic acids, lactones, hydrocarbons, acetals, heterocyclic aromatics, and furans (Apriyantono & Kumara, 2004). Some volatile components are small, less than the detection limit of analytical instruments, but can be detected by the human olfactory (Goff & Klee, 2006). Many factors can affect the composition of volatile components in fruit such as genetics, degree of maturity, environmental conditions, handling after harvest, and storage (El Hadi et al., 2013).
The extraction process is essential because it will determine the extracted components. According to Ormeño et al. (2011), until now, there is no consensus which states that the extraction method is best for extracting a volatile component. Several extraction methods have been developed to obtain flavor components, including using a solvent (maceration), vacuum distillation, Likens-Nickerson distillation, and headspace. The determination of the best method was carried out on the extract by scoring tests by semi-trained panelists.
Some of the flavor extraction techniques are extraction to characterize the flavor. The results obtained were flavor extracts with tiny yield. The research challenge is how to obtain a flavor extract that is similar to real fruit and has a high yield so that it can be applied in industry. The research objective was to select the best extraction technique for AFE to produce high yield and best flavor sensory and chemical.

Selection of panelists
Panelist selection aims to get semi-trained panelists in sensory tests who have a sensitivity to smell scents and select the triangular test method using standard aroma (Setyaningsih et al., 2010). The number of panelists selected was 10 out of 27 panelists who took part in the selection with a score above 75. The samples were tested sequentially. The identification of this standard aroma was carried out three times.

Maceration extraction procedure of AFE
Applewood fruit is broken down, then peel and seeds are separated so that the applewood pulp is obtained. 100g applewood pulp is added with dichloromethane solvent (1:1); glycerin (1:1.5) at a concentration of 25%, 50%, 75%, and 100%; and alcohol solvent (1:4) at a concentration of 50%, 70%, and 96%. The extraction process is carried out for 12-24 hours at 6 hours interval and stirring treatment/without stirring (magnetic stirrer). Furthermore, the solution is filtered with a filter size of 400 mesh. The filtrate was added with anhydrous Na 2 SO 4 to remove water in the sample (dichloromethane and alcohol solvents), followed by evaporating the solvent using a rotary vacuum evaporator (rotavapor). Glycerin is not evaporated because it is thick/sticky. Finally, each extract, namely the DCM extract, glycerin extract (GLY), and alcohol extract (ALC), was analyzed for the sensory scoring value, the absorbance value (190-320nm wavelength spectrophotometer) the analysis of volatile chemical components using GC-MS.

LLE procedure of AFE
Sample 30 ml of alcohol extract (without concentration) and dichloromethane solvent (1:0.5) were extracted for 12 hours (without stirring), stirring for 6 hours, 12 hours, and 18 hours. A decantation process separates the filtrate (dichloromethane) and raffinate (alcohol), then the solvent (dichloromethane) is evaporated using a rotavapor. ALC-DCM extract was analyzed for absorbance values (spectrophotometer wavelength 190-320nm), followed by an analysis of sensory values (similarity, preferences, and QDA), extraction yields, and chemical analysis components using GC-MS and LC-MS/MS.

Statistical analysis
The research data was visualized descriptively, including data tabulation and figure presentation. The statistical test used multivariate analysis with the PCA method on the results of chemical and sensory analysis. According to Setyaningsih et al. (2010), PCA is a multivariate analysis method that can transform original variables into new variables, linear combinations of the original variables called Principal Components. PCA can explain 75%-90% of the data total diversity with only 2-3 main components. Sensory analysis of the sample used a QDA by semi-trained panelists.

Extraction of AFE
The extraction of the non-polar solvent maceration method was chosen as the flavor extraction method of applewood fruit. It has the highest similarity score for fresh fruit, compared to the extraction technique of the Likens Nickerson method, Head Space, Vacuum Distillation. Dichloromethane was chosen as the best solvent compared to diethyl ether, dichloromethane: pentane (1:1), and pentane (Apriyantono & Kumara, 2004). The ratio of the substance to the solvent is 1:1 (Lindawati, 1995;Septiana, 1995;Laarsen & Poll, 1990).
The material, namely Applewood fruit, is not dried and reduced in size as in many extraction studies, because the dried fruit has less aroma (Ardila & Noor, 2020). In this study, the dichloromethane solvent can be used as a control treatment for the standard applewood fruit flavor. Furthermore, other solvent alternatives are sought to obtain an extract product that is ready for use in industry. Glycerin and alcohol solvents were selected to extract the applewood flavor and optimize the solvent concentration and maceration time. The sensory evaluation of GLY extract by semi-trained panelists obtained the highest similarity score for fresh fruit aroma at 50% glycerin concentration with a score of 4.36 ( Figure 1A).
The alcohol extract that has been evaporated by the solvent has a higher score than that without evaporation. The best solvent evaporated alcohol extract was at a 70% concentration, with a score of 5 ( Figure 1B). Optimization of the extraction was carried out with maceration time with 500 rpm stirring for 6-24 hours at 6 hours interval. Qualitative analysis of the extracted volatile components using UV-Vis 190-320nm obtained the highest absorption at 18 hours stirring, namely 3240 ( Figure 1C).

LLE of the ALC extract with dichloromethane solvent
LLE of the alcohol extract with a dichloromethane solvent aims to absorb the flavor of applewood. Evaluation of the grouped based on their groups, then quantified the percentage of compounds, the number of compounds, and the total compounds per group.
Esters dominated by methyl and ethyl series in this study are the same as previous studies (Apriyantono & Kumara, 2004). The aromas that play a role in the applewood of Sri Lanka flavor are butanoic acid, methyl butanoate, and ethyl 3-hydroxyhexanoate in fresh fruit and cream products and overall give a delicious aroma (MacLeod & Pieris, 1981).
The ethyl butyrate component found in applewood fruit is also found in the Baladi mango from Egypt. Ethyl butanoate with a banana-like aroma is the main ester component in the mango (Engel & Tressl, 1983). Butyl acetate has a fruity (pearlike) aroma and is the main ester component of mountain papaya from Chile. The aromas of fruity (pear-like) applewood flavor are components of citronella formate and ethyl octanoate. Esters that play a role in papaya's flavor characteristics are methyl butyrate and ethyl butyrate (Idstein et al., 1985). The butanoic acid component in the applewood flavor gives it a cheesy or butter-like aroma. This butanoic acid component is also found in mangoes and papayas, which give it a rancid butter-like aroma (Yamaguchi et al., 1993). Figure 3A shows that the three extracts' concentration is dominated by the highest carboxylic acid group of ALC-DCM extract, followed by GLY extract and DCM extract; 89%, extraction treatment, namely the extraction time and stirring treatment, was based on the adsorption of volatile compounds at 190-320nm. The lowest absorbance in control was without stirring, and the extraction time was 12 hours, namely 3010, and the highest absorbance was at stirring with an extraction time of 18 hours, which was 3710 ( Figure 2A).
The evaluation of the next extraction is based on the extraction yield. Figure 2B shows the highest yield with stirring extraction time of 12 hours, namely 28.55% (pulp). Several things that must be considered in increasing the yield of LLE are the solvent-feed ratio, extraction time, and stirring speed (Martunus & Helwani, 2006).
The ALC-DCM extract was compared to sensory and volatile compounds with DCM extract and glycerin extract (GLY). The sensory test results for the similarity score and the highest preference score in the DCM extract were 5, and the ALC-DCM extract scored 4.5 and 4.7 ( Figure 2C). The ALC-DCM extract has the potential to be developed because it has a high yield, similarity score, and preference score.

The volatile compound of AFE by GC-MS
In Table 1, it can be seen that as many as 91 volatile chemical compounds were found in AFE with different solvent extraction techniques, namely dichloromethane, glycerin, and alcohol-dichloromethane. Types of chemical compounds are   Amine total (%)*** 0.00 0.00 100.00 ALC-DCM extract has a better flavor quality. In table 1 can be seen it has more ester compounds, namely 28.57%, compared to 13.64% ( Figure 4A). In Figure 4B, it can be seen that the PCA of the natural compounds of the ALC-DCM extract is an ester group compound. In contrast, the DCM extract is an alcoholic and carboxylic acid compound. Figure 4C is an analysis of the total compounds per group. This analysis aims to see to what extent the solvent in maceration effectively attracts the flavor components. As can be seen in table 1, ALC-DCM solvent can produce many esters (58.82%), aldehydes (60%), ketones (50%), and amines (100%). The LLE technique of ALC-DCM extract can increase the top flavor compound compared to the DCM extract, namely ester by 66.67% from 35.29% to 58.82%.
The concentration of ester compounds is 3.38-6.31%, but the ester gives an aroma with high Flavor Dilution (FD) so that it plays a role in the formation of applewood flavor. Ethyl butyrate ester compounds have the highest FD factor of 256, then methyl butyrate with FD factor 64. Compounds of the carboxylic acid group with a low FD of <16 are thought to give nuance to the characteristic flavor of Applewood fruit (Apriyantono & Kumara, 2004).

Analysis of the amount of volatile compounds
The ALC-DCM extract has a class of esters, aldehydes, ketones, and amines higher than other extracts. By comparing the DCM extract as the quality standard for applewood flavor,  Based on PCA the amount of compounds, the marker compounds of ALC-DCM extract is the ester group. Aroma compounds based on sensory analysis, the ALC-DCM extract is apple-like, and vinegar. The ALC-DCM extract had better flavor quality than the DCM extract control because it had more ester compounds, namely 28.57%, compared to 13.64%.
There were 30 organic compounds ALC-DCM extract from LC-MS/MS analysis, 16 compounds with a concentration above 1%. ALC-DCM extract as AFE has the potential to be developed as a food flavor because it has a distinctive aroma and high yield.
compounds that give the applewood fruit a distinctive aroma. In number 8, there is a compound 4-(Allyloxy) -2-methyl-2pentanyl acetate from the ester group contributing to the clary, floral, and fruity aroma of applewood fruit.

Sensory analysis
QDA sensory analysis can assess all sensory properties of a product, the use of limited and trained subjects (Stone, 2004). The QDA sensory analysis results describe 25 specific aromas of the AFE ( Figure 5). The specific aroma of fruity, citrus, vinegar, melon-like, sweet, caramel, wine-like, sour, creamy, strong, powerful, and irregular aroma of rancid and waxy appeared in all three types of extracts. Aroma compounds based on sensory analysis: DCM extract is sour, caramel, sweet, and citrus; ALC-DCM extract is apple-like, and vinegar; GLY extract is strong and fruity.

Conclusions
The graded applewood flavor extraction, namely maceration of alcohol and LLE with dichloromethane solvent (ALC-DCM extract), obtained a high yield of a 28.55% pulp basis. Analysis by GC-MS successfully characterized 91 volatile compounds from AFE. The highest concentrations of volatile compounds in the ALC-DCM extract are the carboxylic acid (89.0%), followed ester (3.41%), heterocyclic aromatic (1.87%), alcohol (1.65%),