Production of free fatty acids by enzymatic hydrolysis of residual frying oil using non-commercial lipases from Aspergillus nigerAbstract
Lipases are enzymes that have an important role in the industry for their wide use, giving rise to a great interest in industrial bioprocesses due to their versatility. One of the applications is the enzymatic hydrolysis of waste oils. This work consists of evaluating the production of lipases using several concentrations of residual frying oil (RFO) and different pHs, through ANOVA analysis. The production of free fatty acids was carried out by hydrolysis of RFO using noncommercial lipases produced in the previous step. The production of lipase enzymes was favored at pH 2.0, with an oil concentration of 1.5% (v/v) for a period of 24 hours, resulting in an enzyme activity of 141.90 U. mL-1 . The highest result of enzymatic hydrolysis of RFO was 20.70% in 40 minutes. This conversion was favored by increasing the concentration of the enzymatic extract. In this study, it was possible to obtain free fatty acids (FFAs) using a noncommercial enzyme and waste oil as an environmentally correct and low-cost alternative.
Key words
enzymatic hydrolysis; free fatty acids; residual frying oil; lipases
INTRODUCTION
In recent years, the processes of transformation of oils and fats into free fatty acids have acquired a great commercial interest due to the different functionalities present in their chemical structure and obtaining products of high added value (Hares Júnior 2017).
Vegetable oils are commonly used in frying processes due to their practicality and the less time it takes to prepare food in people’s routines (Muniz & Silva 2018 ). In the constitution of these oils, triglycerides are the majority compounds in the constitution, with approximately 95% in its composition and, in smaller quantities, monoacylglycerols, diacylglycerols, and free fatty acids, among others (Focking 2017). The heating process, for a prolonged period, produces a large amount of waste oil that is often disposed of inappropriately and, in large part is not reusable (Linganiso et al. 2022).
As a result of the frying process, residual frying oils (RFOs) form a mixture of triglycerides and fatty acids, contaminated by some by-products during the frying process such as free fatty acids (FFAs), heterocycles, Maillard reaction products, and trace metal originating from absorbents and food leaching (Khodadadi et al. 2020).
Through enzymatic hydrolysis, it is possible to mitigate the impacts generated by this type of oil. Since this is used in many processes in the modification of oils and fats (Steinke 2018, Bastos 2020). The result of this modification is the formation of free fatty acids (FFA), monoacylglycerols (MAG), diacylglycerols (DAG), and glycerol, used as raw materials for a diversity of industrial applications (Hares Júnior 2017).
The conventional process of hydrolysis of oils and fats requires the use of high temperatures (250°C) and pressures (4.82 MPa) to obtain a 98 % conversion of free fatty acids (Ferreira et al. 2019, Focking 2017). Enzymatic hydrolysis has become an alternative to traditional methods, employed in solvent-free systems. The reactions in biocatalytic systems occur between liquid and aqueous phases (Hares Júnior 2017).
Enzymes have been used in several industrial applications due to their characteristics, among them, high catalytic conversion, specificity, and accelerating chemical reactions without the formation of contaminants or unwanted products (Heck 2021, Lima et al. 2019). With these advantages, the use of enzymes as biocatalysts has become frequent in various industrial sectors (Hares Júnior 2017).
Lipase enzymes catalyze the hydrolysis and synthesis of triacylglycerols from glycerol esters and long-chain fatty acids (oils) (Lima et al. 2019). In addition, they can also catalyze reactions such as esterification, interesterification, acidolysis, alcoholysis, and aminolysis (Chandra et al. 2020). These enzymes can be found in various sources in nature, such as animals, plants, and microbial (Centurión 2017, Lima et al. 2019). Several applications include organic synthesis, hydrolysis, and modification of oils and fats, also, flavor enhancement in food processing, in the definition of racemic mixtures, and chemical analysis (Rigo et al. 2021).
The present work aims to evaluate the production of free fatty acids (FFAs) by hydrolysis of residual frying oil (RFO) using non-commercial lipases from Aspergillus niger. At first, lipases were produced in submerged fermentation. Then, used in the enzymatic reactions with RFO.
MATERIALS AND METHODS
The residual frying soybean oil (RFO) used in this research was acquired in a pastry shop in the market of the Conjunto Eduardo Gomes in the City of São Cristóvão-SE (10.928° S, 37.119° W). All the work was developed in the Laboratório de Biotecnologia Ambiental (LABAM) of the Universidade Federal de Sergipe (UFS) (10.928°S, 37.119° W). Aspergillus niger was obtained at LABAM, Universidade Federal de Sergipe, São Cristóvão, Brazil. These microorganisms were isolated on potato dextrose agar (PDA) medium in tilted tubes.
Production of the crude lipase extract (CLE)
The production of the enzyme extract (EE) was performed by adapting the methodology suggested by (Dos Santos et al. 2023). After performing the optimization assays, the broth was separated by centrifugation and the supernatant containing the extract showed activity of 141.90 U. mL-1 after 24 hours of fermentation. Then, the CLE was used in the hydrolysis of RFO to determine the best condition.
Characterization of residual frying oil (RFO)
Physical-chemical characterization
The RFO was filtered to remove the suspended solids resulting from the food frying process, and the following parameters were determined: acidity index (Zenevicz et al. 2016); free fatty acid content (Ferreira et al. 2019); electrical conductivity and pH; density and dynamic viscosity at 30.0°C WEAST 1978.
Chemical composition of RFO before hydrolysis
For detection of the fatty acid methyl esters present in the sample it was used gas chromatography with a flame ionization detector by GC-FID method Shimadzu (2010) The analysis was performed in a Shimadzu gas chromatograph model 2010 PLUS equipped with a flame ionization detector. A Supelco wax column (30 m x 0.25 mm ID, 0.25μm film thickness) was used for the separation of the sample components. The gas flow rate was 1.2 mL. min-1 for carrier gas (He), 30 mL. min-1 for o make-up ( N2 ), and 30 and 300 mL. min-1 for H2 and synthetic flame air, respectively.
The chemical composition analysis was performed after the sample was submitted to transesterification of the fatty acids present in the vegetable oil with methanol (Figure 1):
All derivatization steps were performed under stirring and heating with reflux at 40 °C. The organic phase was collected, and a solution was prepared (200 μL sample + 100 mg . L-1 methyl tricosanoate (internal standard)) and completed to 1 mL with hexane, for subsequent injection into the chromatograph. The injections were performed in triplicate and the identification of esters was performed by the retention time of the analysis of a standard solution of fatty acid methyl esters (SSFAE), at a concentration of approximately 100 mg . L-1 , under the same conditions of analysis of the samples.
The heating ramp programming of the chromatograph oven was: 80 °C (1 min) - 10 °C. min-1 - 210 °C (1 min) - 5 °C. min-1 - 230 °C (10 min). A volume of 1 μL was injected automatically in split mode (1:20), detector and injector temperatures 230 °C, and total analysis time 29 min.
Production of free fatty acids (FFAs)
The production of fatty acids was obtained by enzymatic hydrolysis according to Costa et al. (2020) with adaptations. The enzymatic hydrolysis reactions of used residual frying oil (RFO) were performed in a Fisaton shaker fixing the shaking speed at 900 rpm, temperature of 25°C, and pH 2.0. In this procedure, 100 mL of reaction mixture composed only of RFO, distilled water, and the enzymatic crude extract was used. The amounts used in each methodology were: RFO/water (15 - 45 % v/v) and enzyme crude extract (1.74 - 4.84 U. mL-1 of the reaction mixture). The response variable is the percentage of enzyme hydrolysis over time obtained between 40 min. A total of eleven experiments were carried out in a randomized manner. The independent variables of the experimental design and the factorial planning matrix are presented in Tables I and II. The results were analyzed with a confidence level of 95% using the software Statistica 12.0. The tests were performed in duplicate.
To monitor the production of free fatty acids (FFAs), 1 g samples were taken at determined time intervals and analyzed by titration. The reactions were stopped by adding 10 mL of acetone: ethyl alcohol 1:1 solution for each g of the sample. Then, 3 drops of phenolphthalein were added, and the mixture was titrated with 20 mMol. L-1 standard sodium hydroxide solution. All reactions were performed in triplicate (Costa et al. 2020).
The calculation of the percentage of enzymatic hydrolysis was performed as described by Costa et al. (2020) using Equation 1:
Where: V NaOH: volume of sodium hydroxide solution spent in the titration; M NaOH: Molarity of NaOH; M a: mass of sample used in the titration; F o/A: Fraction of oil in water; EH: enzymatic hydrolysis.
Chemical composition of RFO after hydrolysis
Analysis of RFO by gas chromatography with flame ionization detector by GC-FID method Shimadzu (2010).
RESULTS AND DISCUSSIONS
Characterization of the residual frying oil (RFO)
Table III shows the results of the physical-chemical characterization of the RFO used in this study.
According to the results (Table III), it can be seen that RFO presents free fatty acid content and acidity index consistent with oils resulting from the frying process where some molecules are broken down into smaller molecules due to prolonged exposure to high temperatures. These results are lower than those found by Costa et al. 2020 who obtained 3.97 % n mass of free fatty acids (FFAs), acid value of 7.9 mg KOH. g -1 , and 34.95 cP of viscosity at 40°C. These properties are important when one wants to evaluate the quality of the oil used in research, as well as the level of applicability in enzymatic hydrolysis using a non-commercial and non-purified enzyme.
Chemical composition of RFO before hydrolysis
Table IV shows the fatty acid composition of the RFO sample after gas chromatography analysis. Also, compare the fatty acid profile for similar RFO samples used in other works.
It is observed that the results found for saturated and monounsaturated fatty acids were lower than the results found by (Martinelli et al. 2022 and Costa et al. 2020). For the polyunsaturated fatty acids, the RFO sample presented values similar to those obtained by Costa et al. (2020). The results were found to suggest the possibility of elongation and conversion of acids according to (Martinelli et al. 2022).
Production of free fatty acids (FFAs)
Table V shows the results obtained for the percentage of enzymatic hydrolysis after performing the pre-established trials of experimental composite factorial planning central 2 3 .
Percentage of enzymatic hydrolysis of residual frying oil (RFO) for the central compound factorial experimental planning 2³.
From the results obtained in Table V, the highest result of enzymatic hydrolysis found was 20.70% for trial 3 and the lowest was 4.51% for trial 10. Some factors may be related to these low values of hydrolysis, such as agitation speed, pH, time, temperature, and the degree of purity of the enzyme used. Costa et al. (2020) investigated the hydrolysis of residual frying oil under conditions of 20% (m/m) of mass fraction and an enzyme concentration of 4.4 U. g-1 of the enzyme of Geotrichum candidum and obtained 98.36% of enzymatic hydrolysis. Machado (2017) in his study of hydroesterification of macauba oil as a potential for biodiesel production, obtained 77% in the hydrolysis step, presenting 93% of hydrolysate using a commercial enzyme of the species Candida rugosa (LipomodTM 34p). The conditions used were: T=40°C; pH= 7.0 (with sodium phosphate buffer and gum Arabic emulsifying agent); with a mass ratio of 1:3 (33.3% m/m of oil) and 8 hours of reaction. According to the results found by Costa et al. (2020) and Machado (2017), it is possible to verify coherence in the results of enzymatic hydrolysis found in this research.
The mixer was able to maintain the homogeneous reaction mixture at a speed of 900 rpm, which increased the interactions between the enzyme and the RFO. In other words, the emulsion formed allowed the oil in the solution to be hydrolyzed by the enzyme. These effects of increase and decrease of conversion into free fatty acids occur due to the interactions of the oil and aqueous phase (i.e., as these interactions increase, the higher the conversions into FFAs will be).
Although there was a decrease in hydrolysis, probably due to the little interaction of the enzyme with the RFO (assay 9), variation of hydrolysis due to external factors, such as temperature oscillation of the thermal bath used (30.0±5.0°C) and interactions of the enzyme with the oily phase by increasing the amount of oil in the emulsion (assay 8). From the results presented in Table V, it is concluded that the enzymatic hydrolysis was favored by increasing the concentration of the enzymatic extract (assays 3 and 6), that is, the greater the amount of enzyme in the reaction medium, the greater the conversion into free fatty acids (FFAs). Still, by increasing the amount of water disposed of in the medium, for example, tests 1 and 4. Machado (2017) affirms that the increase in the amount of water in the reaction increases the enzymatic activity due to a better enzymatic conformation and a larger interface area.
The methodology tested served to evaluate the influence of some parameters studied by (Costa et al. 2020, Marotti et al. 2017, Machado 2017), such as agitation speed, reaction time, pH, temperature, and specificity of the enzyme. In this research, some of the parameters studied, such as pH 2, stood out in comparison to these authors as many used pH ranges between 4.0 and 8.0 and commercial and purified enzymes (Costa et al. 2020, Machado 2017). The use of acid pH in processes like this avoids the saponification reactions arising from the strong base ions released in the medium.
The results obtained (Table V) demonstrated that hydrolysis is limited at high oil concentrations due to the occurrence of the coalescence phenomenon, which causes a grouping of several oil particles to form large oil droplets, which drastically reduces the interface area and, consequently, the reaction speed.
It should be noted that, among the conditions studied in this work, the best result of enzymatic catalysis was composed of 15% (v/v) of RFO, 4.84 U. mL-1 of the concentration of the enzymatic crude extract and distilled water, for a hydrolysis reaction without the addition of emulsifiers, solvents, buffer solution or the use of ultrasound bath.
Based on the results obtained from the experimental planning, the best hydrolysis percentage assay was selected for the enzyme kinetics analysis (Figure 2). The Figure 2 shows the hydrolysis kinetics of RFO using the enzyme produced using 15 % v/v of the oil and 4.84 U. mL-1 of enzyme concentration.
Kinetics of enzymatic hydrolysis of RFO using non-commercial and unpurified enzyme up to 60 minutes.
Through the enzyme kinetic curve shown in Figure 2 it is possible to observe growth in the percentage of hydrolysis in the first 40 minutes and, after this period, there is a decay due to the decrease in solubility of the oil phase in the medium. As well as it can occur the inhibition of the enzyme with activity losses due to the formation of a water layer around the enzyme, thus, the organic reagents and products with low solubility in water will have difficulty to the active sites of enzymes (Machado 2017).
Also, a hydrolysis degree higher than 20% in 40 minutes was obtained using 15% (v/v) of residual frying oil concentration, and a maximum level of enzyme concentration. In this research, the enzymatic extract produced had specificity for saturated and unsaturated fatty acids present in the RFO. Thus, they were able to hydrolyze ester bonds formed by palmitic, stearic, oleic, and linoleic acids, the latter two being the main components of commercial soybean oil.
Figure 3 shows the amount in terms of the percentage of fatty acids before and after hydrolysis of the oil.
Percentage of FFAs of RFO in before hydrolysis and post-hydrolysis performed in a gas chromatograph with flame ionization detector (GC - FID).
In Figure 3, it was observed using gas chromatography (GC-FID) the presence of approximately 70% and 90% of fatty acids, respectively, before and after the hydrolysis of RFO. It is concluded that hydrolysis of the triglycerides and diglycerides present in the RFO occurred, according to the results presented in the enzymatic kinetics (Figure 2).
Analysis of the composition of the residual frying oil (RFO) before and after hydrolysis
After performing the gas chromatography analysis by GC - FID method Shimazu (2010) fatty acids present in the RFO sample were identified (Figure 4). The values obtained in the composition of free fatty acids (FFAs) were considered as 100% the sum of the partial percentage of the components present.
A higher chemical composition is noted for linoleic acid (C18:2), followed by oleic (C18:1), stearic (C18:0), and palmitic (C16:0) acids. The results obtained suggest that the enzyme showed preferential hydrolysis of the acids of the triglyceride molecule in the following order: linoleic acid > oleic acid > stearic acid > palmitic acid.
Costa et al. (2020) studied the hydrolysis of RFO and obtained approximately 52% linoleic acid (C18:2), 26% oleic acid (C18:1), 17% palmitic acid (C16:0), and 3% stearic acid (18:0). Marotti et al. 2017 obtained after hydrolysis 2.1% lauric acid (C12:0), 0.8% myristic acid (C14:0), 7.8% palmitic acid (C16:0), 49% oleic acid (C18:1) and 13.6% linoleic acid (C18:2). This shows that the conversion depends on the specificity of the lipase and the characteristics of the oil used.
CONCLUSIONS
In the enzymatic hydrolysis of RFO the best results in the conversion into free fatty acids (FFAs) were obtained at 900 rpm. The hydrolysis was favored by increasing the concentration of the enzymatic extract, of 4.84 U. mL-1 , and a concentration of residual oil of 15.0 % v/v, with a result of 20.70 % in 40 minutes of reaction. These results indicate the use of the enzymatic crude extract as a potential biocatalyst for FFAs production using vegetable oils containing a high concentration of unsaturated fatty acids in their composition. Moreover, the produced lipase did not require the use of chemicals, promoting a hydrolysis reaction without the addition of emulsifiers, solvents, buffer solution, or the use of an ultrasound bath.
ACKNOWLEDGMENTS
The authors express their gratitude to the Universidade Federal de Sergipe and Departamento de Engenharia Química (DEQ)/ Laboratório de biotecnologia Ambiental (LABAM) for their research support.
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