Philippine Journal of Science 150 (2): 377-389, April 2021 ISSN 0031 - 7683 Date Received: 20 Jul 2020

Acetic Acid as Value-added Product from Pesticide-free Banana and Peels

Dominica DM. Dacera, Jennifer P. Fronteras*, Daisic D. Bello, and Kathleen Joy L. Delos Santos

University of the Philippines Mindanao Tugbok District, Davao City 8022 Philippines

Wastes from banana and pineapple peels pose increasing disposal and pollution problems as they represent a large fraction of the fruit. One mitigating measure to address this concern is the conversion of these wastes into high-value products. This study explored the potential of producing from banana and pineapple peels through fermentation. Physicochemical characterization showed an initial sugar content of 12.60% for banana peel and 11.61% for pineapple peel, thus indicating their potential for conversion to acetic acid. Further, the pesticide residue analysis in the peels revealed that organochlorines, organophosphorus, and pyrethroids are way below the maximum residue limit (MRL) values set by the Bureau of Philippine Standards (BPS), Joint FAO/WHO Codex Alimentarius Commission (CAC), and the European Commission (EC) Regulation No. 396/2005, which enhanced their suitability as raw material for use in fermentation. Processing the peels to achieve various sugar concentrations of 15% (15°Brix), 20% (20°Brix), and 25% (25°Brix), and the subsequent addition of Saccharomyces cerevisiae allowed the peels to undergo anaerobic fermentation to produce ethanol. The maximum amount of ethanol obtained at a temperature of 26.7 °C and pH of 3.63 was 10.81% v/v (Day 10) from banana peels and 10.60% v/v (Day 10) from pineapple peels at 27 °C and pH of 3.27, both from 20% (20°Brix) sugar concentration. Aerobic fermentation of the extract with aceti converted ethanol to acetic acid. The maximum amount of acetic acid produced, which was from 20% sugar solution at 27.4 °C and pH of 3.59, was 4.56% for banana peels after 16 d while that of pineapple was obtained after 18 d of fermentation at 28 °C and pH of 3.34. For both banana and pineapple peels, no significant differences in the amount of acetic acid produced from three different sugar concentration were observed. The acetic acid produced from banana and pineapple peels can be explored further for potential industrial applications.

Keywords: acetic acid, Acetobacter aceti, alcohol fermentation, fruit peel, pesticide contamination, Saccharomyces cerevisiae

INTRODUCTION discarded as wastes. These wastes could be considered valuable by-products if there were appropriate technical Fruit processing wastes are those end products of various means and if the value of the subsequent products were to fruit processing industries that have not been recycled or exceed the cost of reprocessing (UNIDO 2015). used for other purposes. They are the non-product flows of raw materials whose economic values are less than the For many fruit processing plants, a large fraction of the cost of collection and recovery for reuse and, therefore, solid waste comes from the separation of the desired fruit constituents from undesired ones in the early stages *Corresponding Author: [email protected]

377 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels of processing. The undesirable constituents include The common diseases for banana crops are the “black trimmings, peels, pits, seeds, and pulp (Ammar 2014). Sigatoka,” which is a fungal leaf spot disease and “bunchy Peels from banana, for instance, constitute up to 30% top” disease that affects the banana fruit and foliage of the ripe fruit and in Davao, about 79,000 metric tons and is caused by a single-strand DNA virus (Banana of banana wastes are produced annually (BAS 2013). Planters 2013). In , the common diseases and Further, considering the Philippine setting, data from pests in pineapple are black rot, brown rot, infestation of the Philippine Statistics Authority (PSA 2018) show that nematodes, toy beetle, mealybug, scales, mites, weeds, and pineapple wastes can reach up to 130,000 metric tons even rodents (The Pineapple Technical Committee 2010). annually since about 75% of the fruit consists of peels, Pesticides in fruits are classified into different families – crown, and core. These wastes pose increasing disposal including organophosphate, carbamate, organochlorine, and potential severe pollution problems and represent and pyrethroid. These pesticides should not exceed the a loss of valuable biomass and nutrients. Currently, in MRL of the pesticide residue established by the CAC at Davao, wastes from fruit processing factories are disposed the point of entry into a country or (b) at the point of entry of in landfills or sent to wastes processing facilities for into trade channels within a country (FAO 2017). The use treatment at PhP 0.50/ kg of waste, which entails an of pesticides during production often leads to the presence additional cost to the factories. For instance, for the fruit of pesticide residues in fruits after harvest. These residues processing factory where pineapple and banana peels were might be present in peels, which would be of concern if collected, about 9,000 kg/d of wastes are produced, which utilized as a value-added food product. would cost them PhP 4,500.00/d or about PhP 90,000.00/ mo for disposal. The general objective of this study was to investigate the potential of banana and pineapple peels to produce a value- Disposal of solid wastes in landfills is becoming added product in the form of acetic acid. Specifically, less favorable due to the generation of foul odors as the study aimed to determine the physicochemical communities expand and reside in proximity to fruit characteristics of the fruit peels, determine possible processing plants. Leaching of undesirable constituents pesticide contamination in the peels, and produce acetic such as soluble organics into the soil and groundwater is acid from peel extracts with varying sugar concentrations. also an important concern where pollution of groundwater used by communities can occur, which can migrate into nearby streams (Ammar 2014; Hawkins 2009). MATERIALS AND METHODS Besides their pollution and hazard aspects, in many cases, fruit processing wastes might have a potential for conversion into useful products of higher value as a by- Sample Collection product, raw material for other industries, or for use as Banana and pineapple peels were collected from a animal feeds. In this study, the potential for producing fruit processing company located in Toril, Davao City, acetic acid from fruit processing wastes was explored. Philippines. The samples were immediately transported Acetic acid is used in many industrial processes for the to the College of Science and Mathematics, University of production of substrates; chemical compounds such the Philippines Mindanao, and stored in the chiller until as acetic anhydride, ester, vinyl acetate monomer, and processing for fermentation. Approximately 2 kg of peels vinegar; and many other polymeric materials. It is also was sent to a third-party laboratory for pesticide analysis. widely used for etching metals, as a solvent in chemical On the other hand, another 1 kg of peels was stored in an laboratory analysis, fabric dyeing, production of nylon, ultralow freezer at –80 °C for analysis of physicochemical leather tanning, additive, or flavoring in food canning properties. The rest of the peels was used for substrate and medicines. Moreover, acetic acid has also been preparation for acetic acid production. recognized as a non-selective contact herbicide, especially for broadleaf weeds and weed grasses. Characterization of Fruit Peels A major concern in fruit processing wastes utilization, Proximate composition . Determination of moisture, however, is pesticide contamination. Fruits are attacked crude ash, crude fat, crude fiber, crude protein, and total by pests and diseases during production and storage, carbohydrate content was done using the official methods leading to damages that reduce the quality and yield. In of the Association of Official Analytical Chemists (AOAC order to reduce the loss and maintain the quality of fruits, 2000). All determinations were done using three replicates pesticides are used to destroy pests and prevent diseases. per fruit peel. Banana crops are pesticide-intensive since they are grown Volatile solids. The amount of volatile solids was in the tropics with a warm and humid environment and are determined using the method of the American Public prone to infestations of insect pests and fungal diseases. Health Association (APHA 2005).

378 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

Total sugar content . The total sugar content of banana and 510-nm wavelength against a reagent blank. The starch pineapple peels was determined using the anthrone method content was then calculated using Megazyme Mega- (McCready et al. 1950; Yoshida et al. 1972). About 0.05 CalcTM, as shown in Equation 1: g of sample was weighed in a test tube, after which 5 mL of hot 80% ethanol was added. The mixture was mixed (1) through a vortex mixer then allowed to stand for 10 min with occasional mixing. The sample was then centrifuged at 3000 rpm for 5 min and the supernatant liquid was where ΔA is the absorbance (reaction) read against the decanted into a 100-mL volumetric flask. The extraction reagent blank, F is 100 μg glucose/ absorbance of 100 μg process was repeated and the supernatant liquids were glucose, FV is the final volume of the extract in mL, and pooled. The resulting solution was diluted to the mark W is the weight of the sample in mg. and mixed thoroughly. Several standard glucose solutions (10, 20, 40, and 60 μg/mL) were prepared to constitute Pesticide Analysis of Fruit Peels a standard curve. In the quantification of sugar, 0.1 mL The analysis of pesticide residues present in the peels aliquot of every solution (sample and standard solutions) was outsourced to a third-party laboratory. A total of was pipetted into a test tube containing 0.9 mL distilled 2 kg of each fruit peel was sent to be analyzed for the water. Three (3) mL of 0.2% anthrone reagent was then amount of organochlorines, organophosphates, and added to the mixture, after which it was mixed, and the test pyrethroids. The amount of pesticide residues was tube was covered with a glass marble. It was then heated in determined by gas chromatography using an electron a boiling water bath for 10 min and cooled in an ice bath. capture detector. The active compounds determined for The solutions were allowed to stand for at least 1 hr, then organochlorines were aldrin, α-benzene hexachloride the absorbance of each solution was read at a wavelength (BHC), β-BHC, δ-BHC, γ-BHC (lindane), α-chlordane, of 630 nm using a UV-Vis spectrophotometer (Microplate γ-chlordane, 4,4’-dichlorodiphenyldichloroethane Reader Stat Fax 4200). The concentration of sugar in the (DDD), 4,4’-dichlorodiphenyldichloroethylene (DDE), sample was extrapolated from the standard curve. 4,4’-dichlorodiphenyltrichloroethane (DDT), dieldrin, Total starch content. The total starch content was endosulfan I, endosulfan II, endosulfan sulfate, endrin, analyzed using a Megazyme Total Starch Assay Kit®, endrin aldehyde, endrin ketone, heptachlor, heptachlor which is based on the amyloglucosidase/α-amylase epoxide (B), and methoxychlor. The active compounds method. Fruit peels were dried at 60 °C for 24 h and determined for organophosphorus were dichlorvos, milled to pass through a 0.5-mm screen. About 100 mg mevinphos, demeton-S, ethoprophos, phorate, demeton-O, of the milled sample was weighed into a glass test tube. diazinone, methyl parathion, ronnel, fenthion, chlorpyrifos, Aqueous ethanol (0.2 mL, 80% v/v) was added to wet trichloronate, merphos, tetrachlorvinphos, tokuthion, the sample and aid dispersion. The tube was then stirred fensulfothion, sulprofos, coumaphos, and tribufos. The on a vortex mixer and was immediately added with 3 active compounds determined for pyrethroids were mL of thermostable α-amylase (diluted with 100-mM tefluthrin, transfluthrin, anthraquinone, allethrin, resmethrin, sodium acetate buffer pH 5.0 at a ratio of 1:30). The tetramethrin, bifenthrin, phenothrin, λ-cyhalothrin, cis- sample was incubated in a boiling water bath for 6 min permethrin, trans-permethrin, cyfluthrin, cypermethrin, with vigorous stirring after the 2nd, 4th, and 6th min flucythrinate, fenvalerate, tau-fluvalinate, and deltamethrin. to ensure complete homogeneity. Afterward, the tube was placed in a water bath at 50 °C. Subsequently, 0.1 Laboratory-scale Production of Acetic Acid mL of amyloglucosidase was added to the tube then Substrate preparation . The preparation of peel was done mixed and incubated at 50 °C for 30 min. The mixture following the method of Elijah and Etukudo (2016) with was transferred into a 100-mL volumetric flask and the slight modifications. For each fruit, about 5 kg of peels volume was adjusted using distilled water. The solution were washed thoroughly in water and drained for 30 min. was then transferred to a tube and centrifuged at 3000 The peels were then cut, homogenized in a Waring blender, rpm for 10 min. Duplicate aliquots of approximately 0.1 and boiled for 20 min. The boiled mixture was cooled to mL of the filtrate were then transferred to glass test tubes. room temperature and filtered using a muslin-cheese cloth. Three (3) mL of glucose oxidase/ peroxidase (GOPOD) An additional amount of peels was added per fruit in order reagent was added to each tube and incubated at 50 °C to produce three set-ups of the liquid extract with varying for 20 min. D-Glucose standard (0.1 mL) and 0.1 mL sugar concentrations of 15, 20, and 25%. The extracts were distilled water were also added with 3 mL of GOPOD then sterilized using an autoclave at a pressure of 15 psi and incubated as with the test samples. The absorbance for 15 min. The residue was analyzed for total sugar, pH, for each sample and the D-glucose control was read using and total soluble solids (TSS). a UV-VIS spectrophotometer (UV-1700 Shimadzu) at

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Starter culture preparation . The yeast starter culture, to increase the available oxygen. Changes in temperature, Saccharomyces cerevisiae, was prepared by inoculating pH, and percent acetic acid were monitored daily. a loop full of isolated yeast in 20 mL of 10% sucrose solution for 1 h at 30 °C. Similarly, a loop full of Analysis of Monitoring Parameters Acetobacter aceti was inoculated into glucose yeast (GY) The pH of the mixture was determined using a pH meter while broth (D-glucose 100 gL–1; yeast extract 10 gL–1). The the TSS in the extract and fermented liquid were measured as culture was incubated on a rotary shaker at 30 °C and 120 °Brix with a handheld refractometer. The measurements were rpm for 24 h before adding to the fruit extract. made in triplicate for each sample. The amount of acetic acid Alcohol fermentation . Twenty (20) mL of yeast (S. expressed in terms of percentage by mass was determined cerevisiae) culture was inoculated into 2.5 L of fruit by titrating 5 mL of the sample with a standard 0.1N NaOH extract contained in a 12-L sterile glass container. The solution and calculated using Equation 3: glass container consisted of two holes – one for sample collection and the other for aeration purposes. The (3) fermentation broth was initially aerated for 2 d. During this period, the hole was covered with a small screen to prevent the entry of contaminants. After the 2-d aeration, the glass Statistical Analysis container was properly sealed and corked to prevent the The results of the analyses were reported as mean ± entry of both contaminants and air. Fermentation was standard deviation of the triplicates. The data for banana allowed to proceed at ambient temperature until the and pineapple peel characterization were analyzed for maximum alcohol content was obtained. Parameters significant differences using a one-sample t-test. On the like alcohol content, temperature, pH, and TSS of the other hand, the data for acetic acid fermentation were fermented fruit extract were monitored daily. The alcohol subjected to analysis of variance and the means were content was estimated using Equation 2 that involved the compared using Duncan’s multiple range test at a 5% conversion of °Brix to specific gravity, then to percent level of significance. alcohol using measurements obtained before and after the fermentation process (Lee 2015): % Alcohol (v/v) = (Original Specific RESULTS AND DISCUSSION Gravity − Final Specific Gravity x 131.25 (2)

Acetic acid production. In a 15-L sterile glass container, Physicochemical Characteristics of Banana Peels Acetobacter aceti was added to the fermented extract at a Based on the Banana Ripening Stage Chart (Munasinghe ratio of 2.5 L: 5 mL (clarified fermented extract: acetic acid 2013), the peels collected were considered to be in the bacterial suspension). The mixture was then incubated at third stage of ripening as characterized by the presence ambient temperature until the maximum acetic acid content of more yellow than green color in the peel. was obtained. The set-up for acetic acid fermentation consisted of glass containers with stainless-steel stirring Table 1 shows the physicochemical properties of banana propeller and precision air pump with an air output of peels. The analysis revealed that the peels contained a 8,500 cm3/min connected to a stainless-steel nipple pipe relatively high amount of moisture (12.75%), which is comparable to the result of Abubakar et al. (2016). The

Table 1. Physicochemical characteristics of banana peels. Composition (%) This study Abubakar et al . (2016) Happi Emaga et al . (2006) Moisture 12.745 ± 0.830 13.49 ± 0.17 8.7 ± 0.1 Ash 7.365 ± 0.128 9.83 ± 0.06 9.6 ± 0.2 Fat 11.950 ± 0.601 23.93 ± 0.68 3.8 ± 0.1 Fiber 7.228 ± 0.817 14.83 ± 0.28 43.2± 0.5 Protein 0.664 ± 0.108 5.53 ± 0.11 6.3 ± 0.1 Volatile solids 84.216 ± 0.243 ND ND Sugar content 12.60 ± 1.58 ND ND Starch content 14.02 ± 0.22 ND 11.1 ± 0.1 ND – not determined

380 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels ash content was also high (7.365%) for a plant-based The average starch content of banana peels was 14.02 ± material, and this signifies the possible presence of high 0.22%, which is comparable to that obtained by Happi amounts of minerals in the peels. Anhwange (2008) Emaga et al. (2006). Banana such as Cavendish tends to stated that banana peels can be a good source of minerals have low starch content and, as the fruit matures/ripens, such as potassium, manganese, and calcium. The fat starch decreases further as it is broken down to simple content of banana peels as determined in this study was sugars (Happi Emaga et al. 2007). just half of that obtained by Abubakar et al. (2016). This may be due to differences in the variety of the samples Alcohol Fermentation and Acetic Acid Production of used. The fiber content was significantly lower at 7.23% Banana Peels than that obtained by Happi Emaga et al. (2006). The Figure 1 shows the changes in the TSS and alcohol content protein content of banana peels was found to be 0.664%, of the mixture as the fermentation period progressed. an amount lower than those obtained by Abubakar et al. Although TSS also includes other soluble solids present, (2016) and Happi Emaga et al. (2006). This, however, it is a widely accepted estimate of the amount of sugar is comparable to the 0.9% protein content obtained by present in the fermentation medium. As can be seen in Anhwange (2008). The difference in values obtained may Figure 1, TSS showed a gradual decrease until reaching be due to the difference in the variety of samples used. the final values of 6.07, 8.37, and 13.40°Brix at the end The amount of volatile solids in banana peel was very of fermentation for 15, 20, and 25% sugar concentration, high at 84.216%. Scott and Ma (2004) stated that food respectively. The decrease in TSS can be explained by wastes typically have high ratios of volatile solids. The the fact that sugars are continuously being consumed as latter gives an approximation of the amount of organic a substrate during fermentation. Conversely, the alcohol matter present, which is suitable for biological treatments content showed an increasing trend where the optimal such as anaerobic digestion (Peces et al. 2014). amount reached 10.12% (Day 11) at a temperature of 27 The average concentration of sugar in banana peels °C and pH of 3.83, 10.81% (Day 10) at 26.7 °C and pH of determined to be 12.60 ± 1.58% was brought about 3.64, and 9.06% (Day 10) at 27.9 °C and pH of 3.34 for by the conversion of starch in the peel to sugar during 15, 20, and 25% sugar concentrations, respectively. The maturation of the fruit. The Cavendish banana peels used decrease in TSS and increase in alcohol content indicate in this study were classified under stage three of ripening, that sugar was being converted into alcohol, specifically thus implying the onset of starch conversion to sugar. ethanol. This conversion was initiated by the yeast added The presence of sugar in the peel makes it a suitable raw to the medium. Carbon dioxide gas was liberated along material for fermentation to other high-value products like the process as a by-product (Benazir and Mishra 2015), alcohol and organic acids. as shown in Equation 4:

Figure 1. Alcoholic fermentation of banana peels at different sugar concentrations (B1 = 15%, B2 = 20%, B3 = 25%).

381 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

the amount of acetic acid produced. Hence, in future (4) fermentation experiments, a 15% sugar solution can be used as a substrate for fermentation. The amount of acetic After obtaining the maximum alcohol concentration, acid obtained from the fermentation of banana peels Acetobacter aceti was then added to the fermentation conforms to the 4–6% v/v range reported by Morales et medium. The latter facilitates the conversion of alcohol to al (2002). This indicates that the fermentation conditions acetic acid. Shown in Figure 2 are the changes in the acetic used in this study represent a suitable condition for A. acid content of the medium as the fermentation progressed. aceti to be capable of oxidizing ethanol to acetic acid. Results showed a gradual increase in the amount of acetic acid ranging from 0.66–4.52% for 15% sugar Physicochemical Characteristics of Pineapple Peels concentration, 0.98–4.56% for 20% sugar concentration, The pineapple samples used in this study were observed and 0.72–4.50% for 25% sugar concentration. The to be completely ripe. This level of ripeness is ideal as increase in acetic acid content indicates the conversion the fruit contains high levels of fermentable sugar (Joy of ethanol to acetic acid (Cheryan 2009), as shown in and Rajuva 2016). Equation 5: Table 2 shows that pineapple peels contain 85.64% (5) moisture, which is comparable to the values obtained by Morais et al. (2017) and Abdullah and Mat (2008). The The maximum amounts of acetic acid obtained were slight differences could be accorded to varying ripening 4.52% (Day 17) at 28.5 °C and pH of 3.53, 4.56% (Day 16) stages of pineapples used in the studies. On the other at 27.4 °C and pH of 3.59, and 4.50% (Day 18) at 27.8 °C hand, the ash content was found to be 3.58%, indicating and pH of 3.27 for 15, 20, and 25% sugar concentrations, a significant presence of minerals in the peels – which respectively. In terms of yield, these values are equivalent can be presumed to be calcium, magnesium, potassium, to 12.48, 10.85, and 10.43 g acetic acid/ kg peels for sodium, copper, iron, manganese, and zinc (Hossain et 15, 20, and 25% sugar concentrations, respectively. al. 2015). The crude fat content of pineapple peels was Statistical analysis revealed that these amounts did not found to be only 1.21%. The low-fat content of the peel differ significantly from each other. Hence, it can be said makes it suitable as a possible ingredient to simulate and that for economic reasons, fermentation can be carried replace fat in some food products. Selani et al. (2016) out for 16 d. Furthermore, the concentration of sugar used pineapple peels as a fat replacer in low-fat beef in the fermentation medium did not significantly affect burgers and observed that this resulted in a healthier

Figure 2. Acetic acid production of banana peel at different sugar concentrations (B1 = 15%, B2 = 20%, B3 = 25%). Per concentration, columns with common letters are not significantly different at P < 0.05.

382 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

Table 2. Physicochemical characteristics of pineapple peels. Composition (%) This study Abdullah and Mat (2008) Morais et al . (2017) Moisture 85.64 ± 0.69 87.50 82.7 Ash 3.58 ± 0.48 04.05 05.1 Fat 1.21 ± 0.02 00.15 07.3 Fiber 8.94 ± 0.41 10.57 15.9 Protein 0.481 ± 0.105 5.18 7.3 Volatile solids 96.52 ± 0.50 ND ND Sugar content 11.91 ± 0.90 ND ND Starch content 1.28 ± 0.06 ND ND ND – not determined product with reduced cholesterol content and improved increasing trend where the maximum amounts of 10.57% nutritional quality. The crude fiber content of pineapple (Day 10) at 27 °C and pH of 3.98, 10.60% (Day 10) at peel was found to be 8.94%, which suggests that the peel 27 °C and pH of 3, and 9.77% (Day 10) at 27.9 °C and can be a great source of fiber when incorporated into food pH of 3.53 were achieved for 15, 20, and 25% sugar products. The protein content was found to be 0.48%, concentrations, respectively. The decrease in TSS and which is compared to the results of Morais et al. (2017) the corresponding increase in alcohol content indicate and Abdullah and Mat (2008). Fruits in general are not that sugar is being converted to ethanol. This conversion potential sources of protein. However, the peels can still was initiated by the yeast, S. cerevisiae added in the be utilized as feed despite low amounts of crude protein. fermentation medium. Pineapple peel was combined with pineapple pulp residue After obtaining the maximum alcohol concentration, A. and was processed using submerged liquid fermentation aceti was then added to the fermentation medium. Changes in the study of, which resulted in higher protein content in acetic acid content are presented in Figure 4. Results in the pulp and peel mixture. This supports the statement showed a gradual increase in the amount of acetic acid of Adrizal et al. (2017) that pineapple peels must be ranging from 0.64–4.52 % for the setup with 15% sugar processed to enhance nutritional quality before using as concentration, 0.92–4.56% acetic acid for 20% sugar, and animal feed. The volatile solids for pineapple peels were 0.84–4.56% for 25% sugar concentration. This increase in found to be very high at 96.52%. This implies the high acetic acid content indicates the conversion of ethanol to potential of pineapple peel as a suitable substrate for acetic acid by A. aceti. The maximum amounts of acetic metabolic processes such as fermentation that requires acid obtained were 4.52% (Day 19) at 28 °C and pH of sugar as raw material. 3.83, 4.56% (Day 18) at 28 °C and pH of 3.34, and 4.56% The total sugar content of pineapple peels was 11.91 ± (Day 19) at 27 °C and pH of 3.53 for 15, 20, and 25% 0.90%. This is higher than the value obtained by Saraswaty sugar concentrations, respectively. In terms of yield, these et al. (2017). The high sugar content implies that pineapple values are equivalent to 18.18, 13.94, and 11.62 g acetic peel is a good substrate for fermentation. acid/ kg peels for 15, 20, and 25% sugar concentrations, respectively. However, statistical analysis revealed that no On the other hand, the starch content of pineapple peels was significant differences exist among these values. Hence, only 1.28 ± 0.06%. This value is anticipated as the pineapple future fermentations of pineapple peel can be conveniently fruit was already mature and ripe. This implies that starch carried out for 18 d at 20% sugar solution. The maximum has broken down into smaller carbohydrates such as sugar acetic acid obtained conforms to the 4–6% v/v range by amylase enzymes (Happi Emaga et al. 2007). reported by Morales et al. (2002).

Alcohol Fermentation and Acetic Acid Production of Pesticide Residue Determination for Banana and Pineapple Peels Pineapple Peels Figure 3 shows the changes in the TSS and alcohol content Appendix Tables I and II show the amount of pesticide of the pineapple peel extract as the fermentation period residues in banana and pineapple peels as determined by progressed. TSS showed a gradual decrease until reaching a third-party laboratory. The MRL values are also shown a final value of 6.03, 8.17, and 13.37°Brix at the end of as obtained from the BPS (2015), Joint FAO/WHO fermentation for 15, 20, and 25% sugar concentrations, CAC (2020), and EC Regulation No. 396/2005. Results respectively. Conversely, the alcohol content showed an

383 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

Figure 3. Alcohol fermentation of pineapple peels at different sugar concentrations (P1 = 15%, P2 = 20%, P3 = 25%).

Figure 4. Acetic acid fermentation of pineapple peel at different sugar concentrations (P1 = 15%, P2 = 20%, P3 = 25%). Per concentration, columns with common letters are not significantly different at P < 0.05. showed that the amount of pesticides in banana and CONCLUSION pineapple peels were below the limit of detection of the gas chromatography instrument used. Accordingly, values Physicochemical characterization of banana and pineapple were way below the MRL. These results imply that the peels showed a relatively high sugar content, thus utilization of pineapple and banana peels for conversion indicating their potential for conversion to other high- to acetic acid does not pose a threat of possible pesticide value products such as acetic acid. Further, analysis for contamination. Information like this is valuable as the possible pesticide contamination in the peels revealed presence of pesticides in the raw material could depreciate that organochlorines, organophosphorus, and pyrethroids the versatility of the latter for conversion to other high- are way below the MRL values set by the BPS (2015), value products due to the inherent hazards and tendency Joint FAO/WHO CAC (2020), and EC Regulation for the accumulation of these pesticides both to humans No. 396/2005, thereby reinforcing their suitability for and the environment. use as raw material for fermentation. The peels were

384 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels processed accordingly in order to achieve various sugar Nutr 1(6): 25–27. concentrations of 15, 20, and 25%. Anaerobic and aerobic ADRIZAL NN, HERYANDI Y, AMIZAR R, MAHATA fermentation was then done by the sequential addition ME. 2017. Evaluation of Pineapple [Ananas comosus of S. cerevisiae and Acetobacter aceti. The maximum (L.) Merr] Waste Fermented Using Different Local concentrations of alcohol from banana peels amounted Solutions as Poultry Feed. Pakistan J to 10.12% (Day 11) at a temperature of 27 °C and pH of Nutr 16: 84–89. 3.83 from 15% sugar concentration, 10.81% (Day 10) at 26.7 °C and pH of 3.64 from 20% sugar concentration, AMMAR ASM. 2014. Food Processing Wastes: and 9.06% (Day 10) at 27.9 °C and pH of 3.34 from 25% Characteristics, Treatments and Utilization Review. sugar concentration. This led to the production of acetic Journal of Agricultural and Veterinary Sciences 7(1): acid at concentrations of 4.52% (Day 17) at 28.5 °C and 71-84. pH of 3.53, 4.56% (Day 16) at 27.4 °C and pH of 3.59, ANHWANGE B. 2008. Chemical Composition of Musa and 4.50% (Day 18) at 27.8 °C and pH of 3.27 for the sapientum (Banana) Peels. J Food Technol 6: 263–266. three sugar concentrations, respectively. For pineapple peels, the maximum alcohol concentrations obtained [AOAC] Association of Official Analytical Chemists. were 10.57% (Day 10) at 27 °C and pH of 3.98 from 2000. Official Methods of Analysis (17th ed.). 15% sugar concentration, 10.60% (Day 10) at 27 °C Arlington, VA. and pH of 3 from 20% sugar concentration, and 9.77% [APHA] American Public Health Association. 2005.

(Day 10) at 27.9 °C and pH of 3.53 from 25% sugar Standard Methods for Examination of Water and concentration. This eventually resulted in conversion Wastewater, 21st ed. Washington, DC. to acetic acid at concentrations of 4.52% (Day 19) at 28 °C and pH of 3.83, 4.56% (Day 18) at 28 °C and pH of BANANA PLANTERS. 2013. Banana Cultivation 3.34, and 4.56% (Day 19) at 27 °C and pH of 3.53 for Guide. Retrieved on 26 Oct 2017 from http://www. the three sugar concentrations, respectively. For both bananaplanters.com/site/banana-cultivation-guide/ banana and pineapple peels, no significant differences [BAS] Bureau of Agriculture Statistics. 2013. Selected were observed in terms of the amount of acetic acid Statistics on Agriculture. Quezon City, Philippines. produced from the fruit peel extracts with 15, 20, and 25% sugar solutions. BENAZIR F, MISHRA AA. 2015. Optimization of Process Parameter for the Production of Vinegar from Banana Peel and Coconut Water. Int J Sci Eng Technol 3: 7. ACKNOWLEDGMENTS [BPS] Bureau of Philippine Standards. 2015. Pesticide This study was funded by the Department of Science and residues in banana: Maximum Residue Limits (MRLs). Technology–Philippine Council for Industry, Energy, and Retrieved on 14 Nov 2017 from http://www.bafs. Emerging Technology Research and Development. We are da.gov.ph/images/Approved_Philippine_Standards/ also grateful to the University of the Philippines Mindanao PNS-BAFS161-2015PesticideResiduesinBananaM for the invaluable support to this research. RLs.pdf [CAC] Joint FAO/WHO Codex Alimentarius Commission. 2020. EU Pesticide Database. Retrieved on 24 Feb 2020 from https://ec.europa.eu/food/plant/pesticides/ NOTES ON APPENDICES eu-pesticides-database/public/?event=pesticide. The complete appendices section of the study is accessible residue.selection&language=EN at http://philjournsci.dost.gov.ph CHERYAN M. 2009. Acetic acid production. Appl Microbiol 1: 13–17. ELIJAH AI, ETUKUDO MP. 2016. Quality Evaluation REFERENCES of Vinegar Produced from Banana Peel Using Saccharomyces cerevisiae and Acetobacter aceti ABDULLAH A, MAT H. 2008. Characterisation of Solid Isolated from Pal Wine Dreg. Nigerian Journal of and Liquid Pineapple Waste. Reaktor 12: 48–52. Agriculture, Food and Environment 12(4): 205–211. ABUBAKAR US, YUSUF KM SAFIYANU I, [FAO] Food and Agriculture Organization. 2017. FI 0327 ABDULLAHI S, SAIDU SR, ABDU GT, INDEE – Banana. Retrieved on 10 Nov 2017 from http://www. AM. 2016. Proximate and mineral composition of fao.org/fao-who-codexalimentarius/codex-texts/dbs/ corn cob, banana and plantain peels. Int J Food Sci

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pestres/commodities-detail/en/?lang=en&c_id=131 experimental aging in wood. Journal of Agricultural Food Chemistry 50: 3173–3178. GUERRA NB, STAMFIRD TMM, DE MEDEIROS RB, DE FREITAS CP, MAIA SR, CAVALCANTE ML. MUNASINGHE PM. 2013. Banana Packaging. Retrieved 1986. Protein Enhancement of Pineapple Waste for on 17 May 2018 from https://www.slideshare.net/ Animal Feeds Bioconversion of Vegetable, Animal, mmpmm/banana-packaging and Industrial Wastes by Means of Fungi Mycelia in [OJEU] Official Journal of the European Union. 2014. an Artifical Rumen. Food Nutr Bull 8: 73–82. EU Pesticides MRLs: Pesticides EU-MRLs Regulation HAPPI EMAGA T, ANDRIANAIVO RH, WATHELET (EC) No. 396/2005. Retrieved on 10 May 2018 from B, TCHANGO JT, PAQUOT M. 2006. Effects of https://www.centrallabthai.com/web/uploadfiles/pdf/ the stage of maturation and varieties on the chemical MRLs/EU/Vegetables&Fruits/EU_MRLs_Bananas. composition of banana and plantain peels. Food pdf Chemistry 103(2007): 590–600. PECES M, ASTALS S, MATA-ALVAREZ J. 2014. HAPPI EMAGA T, ROBERT C, RONKART SN, Assessing total and volatile solids in municipal solid WATHELET B, PAQUOT M. 2007. Dietary fibre wastes samples. Environmental Technology 35(24): components and pectin chemical features of peels 3041–3046. during ripening in banana and plantain varieties. [PSA] Philippine Statistics Authority. 2018. Fruit Supply Bioresource Technology 99(2008): 4346–4354. Utilization Account by Commodity, Year and Item, HAWKINS G. 2009. Characterization of Fruit and 2012–2017. Vegetable Wastes for Energy Production. Biochemical SARASWATY V, RISDIA C, PRIMADONA I, Engineering Journal. p. 78–83. ANDRYANI R, ANDAYANI DGS, MOZEF T. HOSSAIN F, AKHTAR S, ANWAR M. 2015. Nutritional 2017. Pineapple peel wastes as a potential source of Value and Medicinal Benefits of Pineapple. Int J Nutr antioxidant compounds. Earth Environ Sci 60: 1–4. Food Sci 4: 84–88. SCOTT N, MA J. 2004. A Guideline for Co-Digestion JOY PP, RAJUVA R. 2016. Harvesting and postharvest of Food Wastes in Farm-based Anaerobic Digesters. handling of pineapple. Pineapple Research Station, Retrieved on 15 Nov 2018 from http://www. Kerala, India. p. 1–23. manuremanagement.cornell.edu/Pages/General_Docs/ Fact_Sheets/Codigestion_factsheet.pdf LEE TK. 2015. The SAGE Encyclopedia of Alcohol: Social, Cultural, and Historical Perspectives. Newbury SELANI MM, SHIRADO GAN, MARGIOTTA GB, Park, CA: SAGE Publications, Inc. p. 59–61. RASERA ML, MARABESI AC, PIEDADE SMS, CONTRERAS-CASTILLO CJ, CANNIATTI- MCCREADY RM, GUGGOLZ J, SILVIERA V, OWENS BRAZACA SG. 2016. Pineapple by-product and HS. 1950. Determination of Starch and Amylose in canola oil as partial fat replacers in low-fat beef burger : Vegetables. Analytical Chemistry 22(9): 1156–1158. effects on oxidative stability , cholesterol content and MEGAZYME. 2017. Total Starch Assay Procedure. fatty acid pro file. Meat Sci 115: 9–15. Amyloglucosidase/α-Amylase Method. Bray, Ireland. THE PINEAPPLE TECHNICAL COMMITTEE. 2010. MORAIS DR, ROTTA EM, SARGI SC, BONAFE The Philippine Recommends for Pineapple, Second EG, SUZUKI RM, SOUZA NE, MATSUSHITA M, ed. PCAARD-DOST, Los Baños, Laguna. VISENTAINER JV. 2017. Proximate composition, [UNIDO] United Nations Industrial Development mineral contents and fatty acid composition of the Organization. 2015. Small-scale Fruit and Vegetable different parts and dried peels of tropical fruits Processing and Products: Production Methods, cultivated in Brazil. J Braz Chem Soc 28: 308–318. Equipment and Quality Assurance Practices. Vienna. MORALES ML, TESFAYE W, GARCIA-PARRILLA YOSHIDA S, FORNO DA, COCK JH, GOMEZ KA. MC, CASAS JA, TRONCOSO AM. 2002. Evaluation 1972. Laboratory manual for physiological studies of of the aroma profile of sherry wine vinegars during an rice. The International Rice Research Institute, Los Banos, Phiilippines.

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APPENDICES

Table I. Pesticide analysis of banana peels. Type of pesticide Amount, mg/kg Limit of detection, mg/kg MRL, mg/kg Organochlorine Aldrin < 0.0006 0.0006 0.01*** alpha-BHC < 0.0007 0.0007 0.01*** beta-BHC < 0.0006 0.0006 0.01*** delta-BHC < 0.0008 0.0008 0.01*** gamma-BHC (lindane) < 0.0007 0.0007 0.01*** alpha-chlordane < 0.0006 0.0006 0.01*** gamma-chlordane < 0.0006 0.0006 0.01*** 4,4’-DDD < 0.0007 0.0007 0.05*** 4,4’-DDE < 0.0006 0.0006 0.05*** 4,4’-DDT < 0.0008 0.0008 0.05*** Dieldrin < 0.0006 0.0006 0.01*** Endosulfan I < 0.0006 0.0006 0.05*** Endosulfan II < 0.0006 0.0006 0.05*** Endosulfan sulfate < 0.001 0.001 0.05*** Endrin < 0.0006 0.0006 0.01*** Endrin aldehyde < 0.0006 0.0006 0.01*** Endrin ketone < 0.0007 0.0007 0.01*** Heptachlor < 0.0006 0.0006 0.01* Heptachlor epoxide (B) < 0.0006 0.0006 0.01*** Methoxychlor < 0.001 0.001 0.01*** Organophosphorus Dichlorvos < 0.03 0.03 0.01*** Mevinphos < 0.03 0.03 0.01*** Demeton-S < 0.03 0.03 0.01*** Ethoprophos < 0.02 0.02 0.02** Phorate < 0.03 0.03 0.01*** Demeton-O < 0.03 0.03 0.01*** Diazinone < 0.02 0.02 0.01*** Methyl parathion < 0.03 0.03 0.01*** Ronnel < 0.02 0.02 0.5*** Fenthion < 0.03 0.03 0.01*** Chlorpyrifos < 0.02 0.02 4.0*** Trichloronate < 0.003 0.003 No ADI*** Merphos < 0.03 0.03 0.01*** For animal products Tetrachlorvinphos < 0.03 0.03 only*** Tokuthion < 0.003 0.003 0.02** Fensulfothion < 0.03 0.03 0.01*** Sulprofos < 0.02 0.02 –*** Coumaphos < 0.03 0.03 –***

387 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

Type of pesticide Amount, mg/kg Limit of detection, mg/kg MRL, mg/kg Tribufos < 0.02 0.02 –*** Pyrethroids Tefluthrin < 0.002 0.002 0.05*** Transfluthrin < 0.002 0.002 0.01*** Anthraquinone < 0.01 0.01 0.01*** Allethrin < 0.005 0.005 0.01*** Resmethrin < 0.01 0.01 0.01*** Tetramethrin < 0.05 0.05 –*** Bifenthrin < 0.003 0.003 0.1*** Phenothrin < 0.01 0.01 0.02*** lambda-Cyhalothrin < 0.002 0.002 0.15*** cis-Permethrin < 0.005 0.005 5.00** trans-Permethrin < 0.005 0.005 5.00** Cyfluthrin < 0.003 0.003 0.02* Cypermethrin < 0.005 0.005 0.05*** Flucythrinate < 0.005 0.005 0.01*** Fenvalerate < 0.003 0.003 0.02*** tau-Fluvalinate < 0.005 0.005 0.01*** Deltamethrin < 0.002 0.002 0.01*** ADI – acceptable daily intake Values obtained from: *PNS (2015) **CAC (2020) ***EC Regulation No. 396/2005

Table II. Pesticide analysis of pineapple peels. Type of pesticide Amount, mg/kg Limit of Detection, mg/kg MRL, mg/kg Organochlorine Aldrin < 0.0006 0.0006 0.01*** alpha-BHC < 0.0007 0.0007 0.01*** beta-BHC < 0.0006 0.0006 0.01*** delta-BHC < 0.0008 0.0008 0.01*** gamma-BHC (lindane) < 0.0007 0.0007 0.01*** alpha-chlordane < 0.0006 0.0006 0.01*** gamma-chlordane < 0.0006 0.0006 0.01*** 4,4’-DDD < 0.0007 0.0007 0.05*** 4,4’-DDE < 0.0006 0.0006 0.05*** 4,4’-DDT < 0.0008 0.0008 0.05*** Dieldrin < 0.0006 0.0006 0.01*** Endosulfan I < 0.0006 0.0006 0.05*** Endosulfan II < 0.0006 0.0006 0.05*** Endosulfan sulfate < 0.001 0.001 0.05*** Endrin < 0.0006 0.0006 0.01*** Endrin aldehyde < 0.0006 0.0006 0.01*** Endrin ketone < 0.0007 0.0007 0.01*** Heptachlor < 0.0006 0.0006 0.01*

388 Philippine Journal of Science Dacera et al.: Acetic Acid from Vol. 150 No. 2, April 2021 Banana and Pineapple Peels

Type of pesticide Amount, mg/kg Limit of Detection, mg/kg MRL, mg/kg Heptachlor epoxide (B) < 0.0006 0.0006 0.01*** Methoxychlor < 0.001 0.001 0.01*** Organophosphorus Dichlorvos < 0.03 0.03 0.01*** Mevinphos < 0.03 0.03 0.01*** Demeton-S < 0.03 0.03 0.01*** Ethoprophos < 0.02 0.02 0.02*** Phorate < 0.03 0.03 0.01*** Demeton-O < 0.03 0.03 0.01*** Diazinone < 0.02 0.02 0.3*** Methyl parathion < 0.03 0.03 0.01*** Ronnel < 0.02 0.02 0.5*** Fenthion < 0.03 0.03 0.01*** Chlorpyrifos < 0.02 0.02 0.01*** Trichloronate < 0.003 0.003 No ADI*** Merphos < 0.03 0.03 0.01*** For animal products Tetrachlorvinphos < 0.03 0.03 only*** Tokuthion < 0.003 0.003 0.02*** Fensulfothion < 0.03 0.03 0.01*** Sulprofos < 0.02 0.02 –*** Coumaphos < 0.03 0.03 –*** Tribufos < 0.02 0.02 –*** Pyrethroids Tefluthrin < 0.002 0.002 0.01*** Transfluthrin < 0.002 0.002 0.01*** Anthraquinone < 0.01 0.01 0.01*** Allethrin < 0.005 0.005 0.01*** Resmethrin < 0.01 0.01 0.01*** Tetramethrin < 0.05 0.05 –*** Bifenthrin < 0.003 0.003 0.01*** Phenothrin < 0.01 0.01 0.02*** lambda-Cyhalothrin < 0.002 0.002 0.01*** cis-Permethrin < 0.005 0.005 5.00** trans-Permethrin < 0.005 0.005 5.00** Cyfluthrin < 0.003 0.003 0.02* Cypermethrin < 0.005 0.005 0.05*** Flucythrinate < 0.005 0.005 0.01*** Fenvalerate < 0.003 0.003 0.02*** tau-Fluvalinate < 0.005 0.005 0.01*** Deltamethrin < 0.002 0.002 0.01*** ADI – acceptable daily intake Values obtained from: *PNS (2015) **CAC (2020) ***EC Regulation No. 396/2005

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