Synthesis of hexyl butyrate (apple and citrus aroma) by Candida rugosa lipase immobilized on Diaion HP-20 using the Box-Behnken design

Abstract

This study aims at the synthesis of hexyl butyrate by Candida rugosa lipase (CRL) immobilized on Diaion HP 20. The lipase load used was 28.7 ± 2.1 mg/g (mg of lipase/g of support), whose hydrolytic activity was 132.0 ± 2.5 U/g. To obtain the maximum production of hexyl butyrate, the Box-Behnken design statistical planning was used, having as independent variables; biocatalyst concentration, temperature and acid:alcohol molar ratio and ester conversion as a dependent variable at 60, 180 and 480 min. For 60 min, 90.8% conversion was obtained at 47.25 ºC, 1:1.4 molar ratio and 17.65% of biocatalyst; 180 min, 94.5% conversion at 59.5 ºC, 1:2 molar ratio and 15.8% biocatalyst; 480 min, 95.01% conversion at 47.0 ºC, 1:2 molar ratio and 16.9% biocatalyst. CRL-Diaion HP 20 retained 60% of its initial activity after ten cycles of reactions showing potential for industrial use. The ester produced was identified by gas chromatography analyses.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-022-01200-1.

Introduction

The process of globalization and modernization has directly contributed to the increase in demand for flavorings (Strojnik et al., 2019). This growth moved the global flavor market in 2016 by around US$9.2 billion, with the prospect of reaching US$12.8 billion by 2023 (Dos Santos et al., 2020; Strojnik et al., 2019). Currently, the growing flavor market is adapted by consumers who are increasingly looking for natural products, which directly impacts the process of obtaining these aromas (Chang et al., 2013; Dos Santos et al., 2020; Strojnik et al., 2019).

Hexyl butyrate is a short-chain ester with an apple peel aroma, citrus fruits and fresh flavor profiles, and is used in the formulation of products in the food, personal care and cosmetics sector (Vasilescu et al., 2019). This class of compounds can be obtained by chemical synthesis or by extraction with organic solvents from natural materials. Commercially, the latter is requested by consumers, however, due to the high cost of solvents and the low yield of extraction, this type of procedure increases the cost of obtaining aroma compounds (Gawas et al., 2018). Chemical synthesis, in turn, is performed using strong acids and p-toluenesulfonic as catalysts, leading, in addition to the product of interest, to by-products without great industrial acceptance, and sometimes with toxic characteristics (Gawas et al., 2018). Alternatively, chemical synthesis processes, in the production of aromas using biological catalysts, enzymes, under mild conditions, reduce the production of by-products and meet the market interest for being produced through a green chemistry (Gawas et al., 2018; Vasilescu et al., 2019).

Thus, the search for technologies in the synthesis of esters, enzymatically, in industrial processes has increased, aiming; establish standards in the quality of production, reduce energy consumption and environmental impacts of the processes (Fernandez-Lorente et al., 2008), made possible, mainly, by the use of immobilized enzymes, with the conservation of almost all industrially important properties (Mateo et al., 2007). In this context, lipases (triacylglycerol acyl hydrolases EC 3.1.1.3) have been the most widely applied enzymes in the production of aroma esters. Brito et al. (2020) used type II pig pancreas lipase immobilized on activated charcoal from peach palm sheath in the synthesis of isoamyl acetate (banana odor).

Microbial lipases stand out in the industrial sector mainly due to their high catalytic activity over a wide range of temperatures and pHs (De Menezes et al., 2021; Melani et al., 2019). Lipases from yeasts have a relevant prominence in food applications, emphasizing the lipase from Candida rugosa due to its great biocatalytic performance, both in terms of activity and enantioselectivity (Domínguez de María et al., 2006). Sarno and Luliano (2020) used lipase from Candida rugosa immobilized on magnetic nano-hybrid graphene hydrophobic support coated with oleic acid G_Fe₃O₄/Ag in the synthesis of liquid wax esters with up to 94% conversion. Dos Santos et al. (2020) carried out the study of the performance of three microbial lipases immobilized on Diaion HP-20 hydrophobic support and obtained the Candida rugosa lipase with greater efficiency, presenting a conversion of 89.1% in the synthesis of hexyl butyrate, and conserving up to 93% from its catalytic activity to its sixth reuse cycle.

Thus, the immobilization of lipases on solid supports proves to be an important tool in the activity and stability of the biocatalyst (Dos Santos et al., 2020), allows an easy recovery of the biocatalyst, as the enzyme will remain insoluble in the medium (Okura et al., 2020). Different protocols have been used to immobilize lipases, such as physical adsorption on hydrophobic and hydrophilic supports (Dos Santos et al., 2020; Ferreira et al., 2018) and covalent binding on previously activated supports (Mendes et al., 2014). The enzymatic synthesis of aroma esters is preferably carried out in an organic medium (Sá et al., 2017), under these conditions a strong enzyme/support interaction is not required and physical adsorption is the most advantageous method, as it is low cost and allows easy reuse of the support after multiple cycles (Bassi et al., 2016).

The enzyme production reactions of esters have been optimized by applying experimental designs by evaluating relevant factors such as reaction temperature, agitation, concentration of biocatalyst, molar ratio of starting materials, among other factors (Taghizadeh, et al., 2021). The use of a Box-Behnken design is widely used in the optimization of systems with three variables and three levels in which extreme experimental conditions are not tested. This type of statistical planning is characterized by relating the variables through mathematical and statistical techniques, in which the variables are classified as dependent or independent (Toprakçi et al., 2022).

The objective of this work was to optimize the concentration, temperature and agitation parameters with the aid of the Box-Behnken design matrix in the synthesis of hexyl butyrate (apple and citrus aroma) by lipase from Candida rugosa immobilized in Diaion HP-20.

Materials and methods

Materials

Diaion HP-20, support with specific mass of 1.09 g/cm³, particle size of 300 to 700 μm, specific surface area of 500 m²/g, and average pore size of 170 Å (Technical support information) was acquired from Supelco (Bellefonte, PA, USA). Candida rugosa lipase (CRL − 27.4 mg protein/g crude extract powder) was purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). This lipase was used in this study without prior treatment. Butyric acid, hexanol and bovine serum albumin (BSA) were also purchased from Sigma Aldrich (St. Louis, MO, USA). Gum Arabic was purchased from Synth® (São Paulo, SP, Brazil). Carbonell’s low acid olive oil was purchased from the local market (Itabuna, BA, Brazil). All other chemical reagents and organic solvents were analytical grade supplied by Synth (São Paulo, SP, Brazil).

Immobilizationof Candida rugosa lipase on Diaion HP-20

Immobilization via physical adsorption was performed and adapted according to the methodology described by Mendes et al. (2012). 19 g of Diaion HP 20 beads were submerged in 475 mL of ethanol (95%) for 24 h at 25 °C under static conditions. After this period, the support was filtered in a vacuum Buchner funnel and washed with distilled water. In parallel, 361 mL of enzyme solution (5 mM sodium phosphate buffer pH 7.0) was prepared, containing Candida rugosa lipase (protein load of 40 mg/g of support) and centrifuged at 1254×g at 25 °C for 10 min. In 500 mL Duran flasks, the pre-treated support and the prepared enzyme solution were mixed and kept in mechanical agitation in a QUIMIS orbital (Diadema, Brazil) at 200 rpm, 25 ºC for 12 h. The immobilization process was monitored by removing aliquots of the initial and final supernatant to determine the concentration of immobilized protein using the methodology proposed by Bradford (1976). At the end, the prepared CRL-Diaion HP 20 was filtered in a vacuum Buchner funnel, washed with distilled water (volume ratio 1:5) and stored at 4 °C for 24 h before use.

Determination of hydrolytic activity

The hydrolytic activity of lipase in the form of crude extract in powder or immobilized on Diaion HP-20 spheres was determined by the method of hydrolysis of the olive oil emulsion (Alves et al., 2017; Carvalho et al., 2017). An international unit (U) of hydrolytic activity was defined as the mass of enzyme required to release 1 mol of fatty acids (FFA) per minute of reaction under the experimental conditions (100 mM sodium phosphate buffer pH 7.0; 37 ºC and stirring 200 rpm mechanics).

The hydrolytic activity was measured through Eq. 1 below:

$$HA\left(U,g^{-1}\right)=\frac{\left(V_s,-,V_c\right)\times M\times {10}^3}{t\times m}$$ 1

where HA was the hydrolytic activity of the immobilized lipase (U/g), VS is the volume of NaOH solution used to titrate the sample (mL), VC is the volume of NaOH solution used to titrate the control sample (mL), M is the concentration of NaOH solution (mol/L), t is the reaction time (min) and m is the mass of immobilized lipase (g).

Immobilized lipase concentration

Protein concentration was determined by the Bradford method (Bradford, 1976), based on binding the Coomassie Brilliant Blue G-250 dye to the protein and monitoring the evolution of absorbance at 595 nm. Bovine serum albumin (BSA) was used as the standard protein. Equation 2 was used to determine the concentration of protein immobilized on the support.

$$PI\left(m,g,/,g\right)=\frac{V(C_o-C_f)}{m}$$ 2

where: PI: Immobilized protein (mg/g); V: Volume of solution (mL), CO: Initial Protein Concentration (mg/mL); CF: Final Protein Concentration (mg/mL) and M: mass of the immobilized support (g).

Application of CRL - Diaion HP20

As shown in Fig. 1 (Supplementary Material), the synthesis of hexyl butyrate was carried out in closed Duran flasks (25 mL) containing 6 mL of reaction medium prepared in the proportion of butyric acid and n-hexanol (1:1) (500 mM in heptane medium). The reactions were started by adding CRL-Diaion HP 20 to the reaction system, keeping the stirring speed fixed in an orbital shaker (200 rpm) (QUIMIS, Diadema, SP, Brazil) with temperature control (30, 45 and 60 °C). Aliquots of 200 µL were periodically withdrawn and diluted in an ethanol:acetone mixture (1:1) for quantification by titration of residual butyric acid. The titration was performed using 30 mM NaOH solution and phenolphthalein as an indicator. All experiments were performed in triplicate and the conversion was measured through acid consumption, calculated using Eq. 3 (Alves et al., 2017; Dos Santos et al., 2020).

$$Conversion\left(\%\right)=\left(\frac{A_{\mathit{initial}}-A_{\mathit{final}}}{A_{\mathit{inital}}}\right)\times 100$$ 3

where: A_initial and A_final is the initial and final fatty acid concentration in the reaction medium (mM).

Optimization of the esterification reaction

The esterification reaction was optimized using the Box-Behnken design (BBD) with the independent variables reaction temperature, biocatalyst concentration and molar ratio (acid:alcohol) and the dependent variable was the ester conversion (%), which was calculated from the consumption of acid in the reaction medium (De Menezes et al., 2022). The levels of the variables studied were determined according to previous studies for the enzymatic production of industrial esters (Mendoza-Ortiz et al., 2020). For modelling and analysis of the BBD, the software Statistica v.12 (Statsoft, USA) was used. In the experimental matrix, the coded values of all parameters vary in three levels (− 1, 0, 1) (Table 1), consisting of 12 experiments and 3 repetitions of the central point, totaling 15 experiments. Selected statistical design (BBD) is classified as a second-order rotating or quasi-rotating experiment inspired by incomplete three-level factorial designs (De Menezes et al., 2020).

Table 1.

Experimental matrix of Box-Behnken design with real values (in parentheses) and coded and Observed and predicted values results applied to optimize the synthesis of hexyl butyrate ester by CRL – Diaion HP 20 in 60, 180 and 480 min

	Experimental matrix			Observed and predicted results
	Variables			60 min		180 min		480 min
Exp.	A	B	C	Conversionexp. (%)a	Conversionpredict (%)	Conversionexp (%)a	Conversionpredict (%)	Conversionexp (%)a	Conversionpredict (%)
1	− 1 (30)	− 1 (1)	0 (1:1)	08.20 ± 0.19	9.97	08.22 ± 1.56	14.31	37.14 ± 2.04	40.06
2	1 (60)	− 1 (1)	0 (1:1)	28.10 ± 0.35	29.79	24.29 ± 1.03	24.57	40.00 ± 2.54	37.71
3	− 1 (30)	1 (19)	0 (1:1)	85.71 ± 0.10	84.02	84.29 ± 0.78	84.01	80.00 ± 1.74	82.29
4	1 (60)	1 (19)	0 (1:1)	82.86 ± 0.11	81.09	81.43 ± 0.96	75.34	84.29 ± 2.02	81.37
5	− 1 (30)	0 (10)	− 1 (1:2)	52.30 ± 0.04	54.09	86.96 ± 1.03	79.58	92.75 ± 1.79	83.80
6	1 (60)	0 (10)	− 1 (1:2)	67.60 ± 0.21	69.47	91.30 ± 2.34	89.74	88.41 ± 0.67	84.67
7	− 1 (30)	0 (10)	1 (2:1)	44.60 ± 0.64	42.73	53.96 ± 0.89	55.52	43.17 ± 0.82	46.91
8	1 (60)	0 (10)	1 (2:1)	46.04 ± 1.58	44.25	39.57 ± 2.04	46.95	33.81 ± 1.92	42.76
9	0 (45)	− 1 (1)	− 1 (1:2)	13.04 ± 0.07	9.48	14.50 ± 1.93	15.79	28.98 ± 2.05	35.01
10	0 (45)	1 (19)	− 1 (1:2)	81.16 ± 1.67	81.06	89.86 ± 1.47	97.52	94.20 ± 2.33	100.86
11	0 (45)	− 1 (1)	1 (2:1)	00.00 ± 0.00	0.10	11.51 ± 0.96	3.86	25.18 ± 1.89	18.52
12	0 (45)	1 (19)	1 (2:1)	50.30 ± 0.53	53.86	43.88 ± 1.65	42.60	44.60 ± 0.07	38.57
13	0 (45)	0 (10)	0 (1:1)	70.00 ± 0.79	71.43	80.50 ± 1.35	80.83	82.86 ± 1.03	81.91
14	0 (45)	0 (10)	0 (1:1)	72.86 ± 1.02	71.43	81.20 ± 0.86	80.83	81.43 ± 0.81	81.91
15	0 (45)	0 (10)	0 (1:1)	71.43 ± 0.98	71.43	80.80 ± 1.07	80.83	81.43 ± 0.05	81.91

Variables A Temperature (ºC), B CRL – Diaion HP 20 (% m/v), C Molar ratio (acid:alcohol)

^aData are shown as ± mean standard deviation of three replicates

The significance of the model was verified by Fisher’s test (F-value) through analysis of variance (ANOVA) and the quality of statistical adjustment was verified through the coefficient of determination (R²). The validation of the selected model was performed by running the experiments in triplicate under the optimal conditions provided, followed by comparison with the previous response.

Reuse of CRL - Diaion HP20

The reuse of the CRL – Diaion HP20 bicatalyst was determined by measuring its ability to catalyze consecutive cycles of hexyl butyrate ester synthesis. The CRL – Diaion HP20 was submitted to the same conditions mentioned in section Application of CRL - Diaion HP20, applying the optimal conditions presented in Table 2 for the times of 60, 180 and 480 min, respectively. Thus applying the optimized experimental conditions of temperature 47.25, 59.5 and 47.0 ºC; CRL – Diaion HP20 17.65, 15.8 and 16.9%; molar ratio (acid:alcohol) 1:1.4, 1:2, 1.2 for the respective times 60, 180 and 480 min. Ten cycles were performed for the respective times 60, 180 and 480 min. At the end of each batch, the prepared heterogeneous biocatalyst (CRL – Diaion HP 20) was removed from the reaction medium by filtration and washed with cold hexane at 4 ºC to remove the product and starting materials in its microenvironment and a new cycle was started. Acid conversion was determined at the end of each cycle (Bassi et al., 2016; Dos Santos et al., 2020).

Table 2.

Model proposed by the response surface methodology with coded and real optimal points (in parentheses) for 60, 180 and 480 min

Time (min)	Temperature (ºC)	CRL – Diaion HP 20 (% m/v)	Molar ratio (acid:alcohol)	Pred. %	Obs. %
60	0.16 (47.25)	0.86 (17.65)	− 0.42 (1:1.4)	87.15	90.86
180	0.97 (59.5)	0.66 (15.8)	− 1 (1:2)	100.00	94.50
480	0.15 (47.0)	0.88 (16.9)	− 1 (1:2)	100.00	95.01

Analysis by GC-FID

The synthesis of the hexyl butyrate ester was analyzed in gas chromatography compared to the standard, using GC-2010 Plus - SHIMADZU (kyoto - Japan) - equipped with a flame ionization detector (FID), with a Restek capillary column (0. 25 mm in diameter, 0.25 μm in length). The column and FID temperatures were 260 and 250 oC, respectively. The initial temperature was 100 °C in 1 min to 170 °C in 2 min with r = 5, then the temperature remained constant at 170 °C. Retention time for hexyl butyrate was 5.7 min, and run time was 17 min.

Results and discussion

Obtaining the CRL – Diaion HP 20

The profile of the catalytic properties of Candida rugosa lipase immobilized on the prepared Diaion HP 20 support (CRL – Diaion HP 20) was first determined from an initial load offered of 40 mg per gram of support. The physical adsorption process was performed by the immobilization method promoting better enzyme dispersion (Lage et al., 2016; Mendoza-Ortiz et al., 2020). As immobilization parameters, the immobilized protein (PI) on the Diaion HP 20 support was 28.7 ± 2.1 mg/g, this was due to the support adsorbing a high concentration of protein and its surface area being high (Dos Santos et al., 2020). The hydrolytic activity on the immobilized support showed 132.0 ± 2.5 U/g, and the specific activity 4.59 ± 0.8 U/mg. Comparing these results, the specific activity shows a decrease, an expected value due to the diffusion limitation effect, in which there is a decrease in the access of some immobilized lipases inside the pores present in the support (Bassi et al., 2016; Alves et al., 2017; Dos Santos et al., 2020).

Application of CRL – Diaion HP 20 in the aroma ester synthesis

The BBD matrix presented in Table 1 was applied at three different times (60, 180 and 480 min) to optimize the enzymatic production of hexyl butyrate and the results obtained are reported in Table 1. Through analysis of variance (Table 3), the adequacy and efficiency of the model were calculated. The capability of the model was evaluated by determining the correlation coefficient (R²), in which the model explained 99.5, 97.8 and 96.1% of the experimental data for 60, 180 and 480 min, respectively.

Table 3.

Analysis of variance to fit the quadratic model with 95% confidence level to optimize the synthesis of hexyl butyrate in 60, 180 and 480 min by CRL – Diaion HP.

Source of variation	Sum of squares	Degree of freedom	Medium square	F calc.	F tab.
60 min
Model	11037.51	9	1226.4	111.83	4.772
Residue	54.83	5	10.97
Pure error	4.09	2	2.045
Total	11092.3	14
R2	0.995
R2-adj	0.986
180 min
Model	13681.21	9	1520.13	24.61	4.772
Residue	308.88	5	61.78
Pure error	0.25	2	0.123
Total	13990.1	14
R2	0.978
R2-adj	0.938
480 min
Model	9307.63	9	1034.18	13.65	4.772
Residue	378.82	5	75.76
Pure error	1.36	2	0.68
Total	9686.44	14
R2	0.961
R2-adj	0.890

The conversion variation occurred between 8.2 ± 0.19% (experiment 1) to 85.7 ± 0.1% (experiment 3) in 60 min, 8.2 ± 1.56% (experiment 1) to 91.3 ± 2.34% (experiment 6) in 180 min and 25.1 ± 1.89% (experiment 11) to 94.2 ± 2.33% (experiment 10) in 480 min (Table 1). The predicted and experimental percentage conversion values were similar, demonstrating that the Box-Behnken design successfully described the relationship between independent variables and response.

Significance could also be verified by Fisher’s F test, since the calculated F values were considerably higher than the tabulated F values (Table 3).

Based on the Pareto chart (Fig. 2a, b and c) (Supplementary Material) for 60, 180 and 480 min of reaction, respectively, it is possible to observe that the temperature variable had little significance in the three reaction times, showing that the CRL – Diaion HP 20 has high catalytic activity in the temperature range evaluated in this study (30–60 ºC). On the other hand, the variables: concentration of CRL – Diaion HP 20 and molar ratio (acid:alcohol) were significant in all reaction times studied (Fig. 2) in the production of aroma ester, mainly, the variable concentration of CRL – Diaion HP 20.

Increasing the CRL – Diaion HP 20 concentration in the medium increases the initial rate and conversion, concomitantly reducing the reaction time (Cao et al., 2021; Gawas et al., 2018; Mendoza-Ortiz et al., 2020). The positive correlations observed in the Pareto chart (Fig. 2) for the biocatalyst and the molar ratio suggest that the increase in the amount of these variables directly influences the increase in the response. While, as, the amount of variables; biocatalyst and molar ratio decrease, there will be a decrease in the percentage of ester synthesis (Bassi et al., 2016).

Thus, with the aid of the Pareto chart (Fig. 2) (Supplementary Material), the terms that did not show significance at 95% (P > 0.5) were removed from the model. The adequacy of the model was approved by means of ANOVA and the response surfaces (Fig. 3) (Supplementary Material) for 60, 180 and 480 min of reaction, respectively, were obtained expressing the effects of the independent variables (reaction temperature, concentration of biocatalyst and molar ratio (acid:alcohol) on the conversion percentage.

Through the response surfaces (Fig. 3), together with the Eqs. 4, 5 and 6, which express the relationship between the independent variables coded to predict a response, the maximum conversion points of hexyl butyrate at 60, 180 and 480 min (Table 2).

$$\text{X}=\text{71}.\text{43}+\text{31}.\text{34}B+\text{4}.\text{33}{A}^{\text{2}}-\text{2}.\text{89}{A}^{\text{2}}+\text{19}.\text{29}B-\text{9}.0\text{9}AB-\text{17}.\text{97}A{B}^{\text{2}}-\text{5}.\text{75}AC$$ 4

$$\text{X}=\text{8}0.\text{83}+0.\text{39}A-\text{1}.\text{63}{A}^{\text{2}}+\text{3}0.\text{11}B-\text{29}.\text{64}{B}^{\text{2}}-\text{4}.\text{73}AB-\text{4}.\text{68}C-\text{1}0.\text{74}B$$ 5

$$\text{X}=\text{81}.\text{91}+\text{21}.\text{47}B-\text{18}.\text{92}{B}^{\text{2}}-\text{19}.\text{69}A-\text{14}.\text{74}{A}^{\text{2}}+\text{11}.\text{45}AC$$ 6

Validation of aroma ester synthesis

To validate the model, experiments in triplicate of the optimal points obtained were performed under the same operating conditions (Table 2). The conversion values obtained were 90.0, 94.5 e 95.0% for 60, 180 e 480 min of reaction, respectively. Productivity values for esterification reactions (60, 180 e 480 min) in the concentration of 500 mM of butanoic acid were determined as 1.26, 0.49 e 0.18 mmol/min.gbiocat, respectively. These values demonstrate that, although the highest percentage of ester production was in 480 min, the best productivity was in 60 min, being more interesting from an industrial point of view (Chang et al., 2013; Mendoza-Ortiz et al., 2020).

To validate the model, experiments in triplicate of the optimal points obtained were performed under the same operating conditions (Table 2). The conversion values obtained were 90.0, 94.5 and 95.0% for 60, 180 and 480 min of reaction, respectively. Productivity values for esterification reactions (60, 180 and 480 min) at 500 mM butylic acid concentration were determined as 1.26, 0.49 and 0.18 mmol/min.gbiocat, respectively. These values demonstrate that although the highest percentage of ester production was in 480 min, the best productivity was in 60 min, which is more interesting from an industrial point of view (Chang et al., 2013; Mendoza-Ortiz et al., 2020).

Based on Table 2, it can be seen that the data presented is equivalent to an average recovery of approximately 93% between the theoretical value and the experimental value, confirming the data obtained and validating, experimentally, the proposed model for the synthesis of aroma esters.

CRL reuse – diaion HP 20

The reuse of biocatalysts in industries favors cost reduction and makes the process viable (Cao et al., 2021; Dos Santos et al., 2020; Otari et al., 2020). Tests were carried out to determine the ability of CRL-Diaion HP 20 to be reused in successive esterification of hexyl butyrate. The reactions were carried out under optimal experimental conditions obtained through Eqs. 4, 5 and 6 and presented in Table 2. Figure 4 (Supplementary Material) shows the variation of the relative activity of CRL – Diaion HP 20 after ten successive cycles. At 60 min, 90.86% conversion was observed and was defined as 100% relative activity. At the end of the tenth cycle, the CRL – Diaion HP 20 still retained 59.55% of the initial activity. At 180 min, the CRL – Diaion HP 20 obtained 94.5% conversion in the first cycle and in the tenth cycle it still retained 44.63% of the initial activity and finally, at 480 min, the initial conversion was 95.01% and at the end of the tenth cycle maintained 25.99% of initial activity. The retention capacity of the biocatalyst dropped considerably after the tenth cycle. The decline in catalytic activity over 10 reaction cycles may have occurred in the washing step, which allows accelerating possible desorption and/or inhibition of enzyme molecules on the support (Lage et al., 2016; Rezania et al., 2021). The results obtained in successive cycles suggest a strong interaction of the CRL with the surface of the Diaion HP 20 support (Bassi et al., 2016; Dos Santos et al., 2020), indicating that the CRL – Diaion HP 20 is sufficiently stable and can be reused in the synthesis of aroma esters. In addition, in the present study, a chemometric statistical tool is used to aid in the synthesis of esters, and it is observed that the chemometric application maximized the conversion values, thus, this pioneering study may encourage the scientific community in further studies for the development of low-cost reusable biocatalysts.

Chromatographic analysis of the aroma compound

The aroma ester synthesis by CRL – Diaion HP 20 was confirmed by comparative chromatographic analyzes with the respective standards: butanoic acid, hexanol and hexyl butyrate.

Samples were subjected to analysis using column and FID temperatures at 260 °C and 250 °C, respectively, with 17 min run.

The GC-FID chromatograms show peaks with retention time at 9.7, 4.8 and 5.7 min for the respective standards; butanoic acid, hexanol and hexyl butyrate respectively (Fig. 5) (Supplementary Material) and 5.7 min for the ester synthesized by CRL – Diaion HP 20, thus characterizing the effectiveness of esterification by CRL immobilized in Diaion HP 20.

The optimization of the synthesis of hexyl butyrate (apple and citrus aroma) catalyzed by lipase from Candida rugosa immobilized on Diaion HP 20 (CRL – Diaion HP 20) support showed good conversion values (90.8, 94.5 and 95.01%) for 60, 180 and 480 min, respectively. CRL – Diaion HP 20 retained 60% of its initial activity after 10 successive reaction cycles, showing potential for industrial use. The synthesis of hexyl butyrate was confirmed by chromatographic analysis (GC-FID).

Electronic supplementary material

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Declarations

Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.