# Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Results and Discussion

#### 2.1. Lipid Profile of Oils

^{−1}, 193.4 mgKOH.g

^{−1}, and 186.6 mgKOH.g

^{−1}. The total amount of FAs for each of the oils is therefore 3.49 mmol FAs.g

^{−1}for sunflower oil, 3.45 mmol FAs.g

^{−1}for hempseed oil, and 3.33 mmol FAs.g

^{−1}for linseed oil.

#### 2.2. Determination of Optimum pH and Optimum Temperature of Lipases

#### 2.3. Optimization of the Hydrolysis Rate Using a Central Composite Design

^{2}coefficients of the models are, respectively, for CRL, RML, and PFL 96.21%, 88.43%, and 89.83% for sunflower oil; 93.88%, 89.94%, and 91.99% for hempseed oil; and 92.90%, 91.20%, and 92.42% for linseed oil. The adjusted R

^{2}shown in Table 2 indicates that more than 86.68% of the variability in the hydrolysis rate could be explained by the model. These values of R

^{2}reveal a satisfactory adjustment of the models to the experimental data, so these models can be used for the analysis and prediction of the hydrolysis rate.

^{2}and T

^{2}, meaning both factors have a significant influence on the hydrolysis rate. The interaction term H × T is significant only with CRL action ((a) in Table 3).

^{2}and T

^{2}exhibit a significant influence on the hydrolysis rate. The interaction term H × T is only significant for the RML and PFL action ((b) in Table 3).

^{2}are significant for the action of the three lipases, while the linear term T and the quadratic term T

^{2}have a significant influence on the hydrolysis rate with CRL and PFL action. The interaction term H × T reveals a statistically significant influence only with the RML and PFL action ((c) in Table 3).

#### 2.4. Optimization of the Hydrolysis Reaction Using a Box–Behnken Design

^{2}coefficients of the models are, respectively, 85.22%, 85.72%, and 82.36% for sunflower oil, hempseed oil, and linseed oil with CRL (Table 5).

^{2}shown in Table 5 indicates that more than 76.48% of the variability in the response could be explained by the model. These values of R

^{2}reveal a satisfactory adjustment of the models to the experimental data, so these models can be used for the RSM analysis and the prediction of the hydrolysis rate.

^{2}and E

^{2}, and the interaction term D × E are significant ((a) in Table 6). For hempseed oil, linear terms D and E, quadratic terms D

^{2}and E

^{2}, and the interaction terms D × E and D × O show a significant influence on the hydrolysis rate ((b) in Table 6). For linseed oil, linear terms D, E, and O and the quadratic term O

^{2}are statistically significant ((c) in Table 6).

## 3. Materials and Methods

#### 3.1. Oils and the Determination of Their Composition in Fatty Acids

_{2}(0.7 mL/min); and split ratio: 1:50. The injected volume was 2 µL in automatic mode. The GC-MS quadrupole detector was equipped with the same column as described above. The analyses were conducted on a nonpolar column, and for each sample, reconstructed ionic chromatograms were collected and analyzed. GC conditions were the same as above, and MS conditions were as follows: ionization energy: 70 eV; ion source temperature: 150 °C; and EI-MS spectra obtained over a mass range of 35–350 amu during a scan time of 1 s. The injection volume for the sample was 2 µL in automatic mode.

#### 3.2. Lipases and the Determination of Their Optimum Temperature and pH

#### 3.3. Optimization of the Lipase-Catalyzed Hydrolysis of Vegetable Oils

#### 3.3.1. Optimization of the Hydrolysis Rate Using a Central Composite Design

#### 3.3.2. Optimization of the Hydrolysis Reaction Using a Box–Behnken Design

#### 3.3.3. Statistical Analysis of the Different Experimental Designs

_{0}is the model constant, I is the number of factors (two in the central composite model and three in the Box–Behnken model), β

_{i}is the linear coefficient associated with factor X

_{i}, β

_{ii}is the quadratic coefficient associated with factor X

_{i}, and β

_{ij}is the interaction coefficient between factors X

_{i}and X

_{j}. In order to facilitate the reading and understanding of the results, factors X

_{i}were coded as follows: T for the temperature, H for the pH, D for the reaction duration, E for the enzyme load, and O for the oil/aqueous ratio.

^{2}) analysis. The p-value was checked to find out the significance of all the fitted equation terms at a 5% level of significance.

#### 3.4. Lipids Analysis and Hydrolysis Rate Calculation

^{−1}

_{of oil}(SV

_{0}), was then converted to express the total fatty acid content in mol.g

^{−1}

_{of oil}(SV

_{0}′):

_{0}′ is the total number of moles of FAs per gram of oil (mol.g

^{−1}

_{of oil}), SV

_{0}is the saponification value of the oil (mgKOH.g

^{−1}

_{of oil}), and MW

_{KOH}is the molar weight of KOH (g.mol

^{−1}).

_{s}/m

_{o}corresponds to the number of moles of FAs released after the lipolysis per gram of oil (mol.g

^{−1}

_{of oil}), where n

_{s}is the number of moles of FAs after the lipolysis (mol) and m

_{o}is the weight of oil in the reaction (g).

## 4. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 3.**Contour plots of the hydrolysis rate (%) after lipolysis on sunflower oil (

**a**), hempseed oil (

**b**), and linseed oil (

**c**), as a function of the temperature (T) and pH (H) for CRL, PFL, and RML.

**Figure 4.**Three-dimensional (3D) response surface plots of the hydrolysis rate (%) with CRL, as a function of: (

**I**) the duration reaction (D) and enzyme load (E) for sunflower oil (

**a**) and hempseed oil (

**b**); (

**II**) the duration reaction (D) and oil/aqueous ratio of the mixture (O) for hempseed oil.

**Table 1.**Fatty acid composition of sunflower oil, hempseed oil, and linseed oil after analysis by chromatography with flame ionization detector and mass spectrometry.

Content (%) | |||
---|---|---|---|

Sunflower Oil | Hempseed Oil | Linseed Oil | |

Palmitic acid | 6.4 ± 0.0 | 6.1 ± 0.0 | 5.7 ± 0.1 |

Stearic acid | 3.1 ± 0.0 | 6.6 ± 0.4 | 3.8 ± 0.0 |

Oleic acid | 33.3 ± 0.1 | 4.4 ± 0.4 | 20.6 ± 0.1 |

Linoleic acid | 55.3 ± 0.1 | 56.6 ± 0.1 | 15.5 ± 0.1 |

Linolenic acid | 0.4 ± 0.0 | 19.0 ± 0.2 | 53.0 ± 0.1 |

Others | 1.5 ± 0.0 | 7.3 ± 0.2 | 1.5 ± 0.1 |

**Table 2.**Response surface model analysis of the hydrolysis rate for the action of CRL, RML, and PFL on sunflower, hempseed, and linseed oils; second-order polynomial equations and determination coefficients.

Oil | Lipase | Equation ^{a} | R^{2} | Adjusted R^{2} |
---|---|---|---|---|

Sunflower oil | CRL | Hydrolysis rate (%) = −1599.2 + 49.40 × T + 241.9 × H − 0.5417 × T^{2} − 13.347 × H^{2} − 1.672 × T × H | 96.21% | 95.60% |

RML | Hydrolysis rate (%) = −403.7 + 94.15 × H + 6.44 × T − 6.490 × H^{2} − 0.0813 × T^{2} − 0.098 × H × T | 88.43% | 86.68% | |

PFL | Hydrolysis rate (%) = 75.65 − 17.52 × H + 0.445 × T + 0.8997 × H^{2} − 0.00894 × T^{2} + 0.0311 × H × T | 89.83% | 88.29% | |

Hempseed oil | CRL | Hydrolysis rate (%) = −30.26 + 6.419 × H + 0.6613 × T − 0.4771 × H^{2} − 0.00998 × T^{2} + 0.00557 × H × T | 93.88% | 92.95% |

RML | Hydrolysis rate (%) = −163.4 + 48.78 × H + 0.57 × T − 4.607 × H^{2} − 0.0698 × T^{2} + 0.5861 × H × T | 89.94% | 88.42% | |

PFL | Hydrolysis rate (%) = −127.0 + 37.82 × H + 2.079 × T − 2.350 × H^{2} − 0.01762 × T^{2} − 0.0891 × H × T | 91.99% | 90.77% | |

Linseed oil | CRL | Hydrolysis rate (%) = −109 + 26.2 × H + 9.76 × T − 3.42 × H^{2} − 0.1808 × T + 0.176 × H × T | 92.90% | 90.29% |

RML | Hydrolysis rate (%) = −156.0 + 47.19 × H + 2.31 × T − 3.067 × H^{2} − 0.0051 × T − 0.2735 × H × T | 91.20% | 89.87% | |

PFL | Hydrolysis rate (%) = 225.9 − 22.78 × H − 5.595 × T + 0.732 × H^{2} + 0.06655 × T^{2} + 0.1440 × H × T | 92.42% | 91.27% |

^{a}Factors coded as follows: pH (H) and temperature (T).

**Table 3.**Analysis of variance (ANOVA): response surface model analysis of the hydrolysis rate with CRL, RML, and PFL.

(a) On Sunflower Oil | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} | ||||||||

CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | |

H | 1958.80 | 2196.84 | 395.84 | 1 | 1 | 1 | 141.47 | 78.53 | 159.93 | 0.000 | 0.000 | 0.000 |

T | 2795.60 | 242.73 | 10.95 | 1 | 1 | 1 | 60.44 | 8.68 | 4.42 | 0.000 | 0.006 | 0.043 |

H^{2} | 4124.70 | 4449.73 | 270.28 | 1 | 1 | 1 | 297.91 | 159.06 | 109.20 | 0.000 | 0.000 | 0.000 |

T^{2} | 4246.10 | 436.43 | 16.69 | 1 | 1 | 1 | 306.68 | 15.60 | 6.74 | 0.000 | 0.000 | 0.014 |

H × T | 968.00 | 14.60 | 4.65 | 1 | 1 | 1 | 69.92 | 0.52 | 1.88 | 0.000 | 0.475 | 0.18 |

Lack of fit | 413.10 | 607.59 | 39.21 | 3 | 3 | 3 | 238.58 | 19.25 | 9.23 | 0.000 | 0.000 | 0.000 |

Pure error | 16.20 | 315.61 | 42.47 | 30 | 30 | 30 | ||||||

Total | 11,319.50 | 7978.28 | 802.89 | 38 | 38 | 38 | ||||||

(b) On Hempseed Oil | ||||||||||||

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} | ||||||||

CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | |

H | 16.67 | 87.66 | 805.85 | 1 | 1 | 1 | 173.73 | 7.99 | 111.58 | 0.000 | 0.008 | 0.000 |

T | 4.034 | 246.45 | 42.1 | 1 | 1 | 1 | 42.05 | 22.46 | 5.83 | 0.000 | 0.000 | 0.021 |

H^{2} | 24.05 | 2242.35 | 1844.29 | 1 | 1 | 1 | 250.65 | 204.34 | 255.36 | 0.000 | 0.000 | 0.000 |

T^{2} | 6.57 | 321.40 | 64.83 | 1 | 1 | 1 | 68.50 | 29.29 | 8.98 | 0.000 | 0.000 | 0.005 |

H × T | 0.05 | 521.69 | 38.1 | 1 | 1 | 1 | 0.49 | 47.54 | 5.28 | 0.488 | 0.000 | 0.028 |

Lack of fit | 1.97 | 43.84 | 118.93 | 3 | 3 | 3 | 16.53 | 1.38 | 9.96 | 0.000 | 0.269 | 0.000 |

Pure error | 1.19 | 318.28 | 119.4 | 30 | 30 | 30 | ||||||

Total | 51.72 | 3600.74 | 2974.79 | 38 | 38 | 38 | ||||||

(c) On Linseed Oil | ||||||||||||

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} | ||||||||

CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | |

H | 20.77 | 3204.88 | 3494.26 | 1 | 1 | 1 | 228.40 | 253.33 | 297.82 | 0.000 | 0.000 | 0.000 |

T | 617.30 | 6.66 | 111.68 | 1 | 1 | 1 | 6.89 | 0.53 | 9.52 | 0.017 | 0.473 | 0.004 |

H^{2} | 437.90 | 993.60 | 178.83 | 1 | 1 | 1 | 4.88 | 78.54 | 15.24 | 0.04 | 0.000 | 0.000 |

T^{2} | 763.40 | 1.69 | 924.37 | 1 | 1 | 1 | 8.52 | 0.13 | 78.78 | 0.009 | 0.717 | 0.000 |

H × T | 46.80 | 113.61 | 99.58 | 1 | 1 | 1 | 0.52 | 8.98 | 8.49 | 0.479 | 0.005 | 0.006 |

Lack of fit | 1555.10 | 208.57 | 203.98 | 3 | 3 | 3 | 1.23 | 9.98 | 11.13 | 0.539 | 0.000 | 0 |

Pure error | 148.30 | 208.91 | 183.21 | 30 | 30 | 30 | ||||||

Total | 3589.60 | 4744.26 | 5107.12 | 38 | 38 | 38 |

^{a}Factors coded as follows: pH (H) and temperature (T);

^{b}p-value <0.05 indicates a statistical significance of the factor.

**Table 4.**Predicted and measured hydrolysis rates with optimal pH and temperature for the actions of CRL, RML, and PFL on sunflower, hempseed, and linseed oils.

Sunflower Oil | Hempseed Oil | Linseed Oil | |||||||
---|---|---|---|---|---|---|---|---|---|

CRL | RML | PFL | CRL | RML | PFL | CRL | RML | PFL | |

Optimal pH | 7 | 7 | 5.2 | 7.5 | 7.6 | 7.5 | 6 | 6 | 5.2 |

Optimal temperature | 35 °C | 35 °C | 34 °C | 35 °C | 36 °C | 35 °C | 30 °C | 43 °C | 21 °C |

Predicted hydrolysis rate (%) | 96.2 | 39.8 | 19.7 | 86.7 | 31.5 | 52.8 | 90.6 | 36.3 | 55.4 |

Measured hydrolysis rate (%) | 96.4 ± 1.8 | 38.4 ± 2.3 | 20.2 ± 1.5 | 87.9 ± 3.6 | 40.3 ± 3.2 | 48.7 ± 1.9 | 93.0 ± 3.8 | 39.0 ± 1.5 | 56.1 ± 2.7 |

**Table 5.**Response surface model analysis of the hydrolysis rate for the action of CRL on sunflower, hempseed, and linseed oils; second-order polynomial equations and determination coefficients.

Oil | Equation | R^{2} | Adjusted R^{2} |
---|---|---|---|

Sunflower Oil | Hydrolysis rate (%) = −6.234 + 1.270 × D + 0.003558 × E + 0.1333 × O − 0.0459 × D^{2} − 0.000000 × E^{2} − 0.001375 × O^{2} − 0.000270 × D × E − 0.00652 × D × O − 0.000002 × E × O | 85.22% | 81.43% |

Hempseed Oil | Hydrolysis rate (%) = −87.9 + 37.50 × D + 0.0648 × E + 0.804 × O − 1.698 × D^{2} − 0.000011 × E^{2} − 0.0094 × O^{2} − 0.004911 × D × E + 0.000223 × E × O − 0.1567 × D × O | 85.72% | 82.05% |

Linseed Oil | Hydrolysis rate (%) = −27.7 + 1.06 × D − 0.00701 × E + 6.446 × O + 0.213 × D^{2} + 0.00000 × E − 0.08542 × O^{2} − 0.000728 × D × E - 0.0146 × D × O + 0.000086 × E × O | 82.36% | 76.48% |

(a) On Sunflower Oil | ||||
---|---|---|---|---|

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} |

D | 0.76 | 1 | 6.33 | 0.017 |

E | 5.14 | 1 | 42.63 | 0.000 |

O | 0.01 | 1 | 0.04 | 0.838 |

D^{2} | 7.42 | 1 | 61.46 | 0.000 |

E^{2} | 2.44 | 1 | 20.02 | 0.000 |

O^{2} | 0.31 | 1 | 2.57 | 0.118 |

D × E | 2.05 | 1 | 16.99 | 0.000 |

D × O | 0.17 | 1 | 1.39 | 0.246 |

E × O | 0.34 | 1 | 2.85 | 0.1 |

Lack of fit | 1.87 | 27 | 8.49 | 0.000 |

Pure error | 2.36 | 32 | ||

Total | 22.01 | 44 | ||

(b) On Hempseed Oil | ||||

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} |

D | 7028.95 | 1 | 89.82 | 0.000 |

E | 994.50 | 1 | 12.71 | 0.001 |

O | 247.69 | 1 | 3.17 | 0.084 |

D^{2} | 2585.47 | 1 | 33.04 | 0.000 |

E^{2} | 2001.47 | 1 | 25.58 | 0.000 |

O^{2} | 49.48 | 1 | 0.63 | 0.432 |

D × E | 3152.3 | 1 | 40.28 | 0.000 |

D × O | 597.0 | 1 | 7.63 | 0.009 |

E × O | 161.8 | 1 | 2.07 | 0.159 |

Lack of fit | 1797.8 | 3 | 20.37 | 0.000 |

Pure error | 941.3 | 32 | ||

Total | 19,183.4 | 44 | ||

(c) On Linseed Oil | ||||

Source ^{a} | Sum of Squares | Degree of Freedom | F Value | p-Value ^{b} |

D | 363.65 | 1 | 7.16 | 0.012 |

E | 1425.89 | 1 | 28.06 | 0.000 |

O | 1673.66 | 1 | 32.93 | 0.000 |

D^{2} | 40.66 | 1 | 0.8 | 0.378 |

E^{2} | 0.45 | 1 | 0.01 | 0.926 |

O^{2} | 4092.07 | 1 | 80.52 | 0.000 |

D × E | 69.27 | 1 | 1.36 | 0.251 |

D × O | 5.18 | 1 | 0.1 | 0.752 |

E × O | 24.12 | 1 | 0.47 | 0.496 |

Lack of fit | 1664.57 | 27 | 29.48 | 0.000 |

Pure error | 12.55 | 6 | ||

Total | 9509.25 | 44 |

^{a}Factors coded as follows: duration reaction (D), enzyme load (E), and oil/aqueous ratio of the mixture (O);

^{b}p-value < 0.05 indicates a statistical significance of the factor.

**Table 7.**Predicted and measured hydrolysis rates with optimal conditions of duration reaction, enzyme load, oil/aqueous ratio of the mixture, pH, and temperature for the action of CRL on sunflower, hempseed, and linseed oils.

Oils | CRL | ||
---|---|---|---|

Optimum Conditions of Reaction | Predicted Hydrolysis Rate (%) | Measured Hydrolysis Rate (%) | |

Sunflower oil | 4 h − 1 798 U − 46.5% (oil/aqueous) 35 °C − pH 7 | 96.2 | 96.0 ± 1.7 |

Hempseed oil | 4.5 h − 2 592 U − 49.2% (oil/aqueous) 35 °C − pH 7.5 | 97.6 | 97.2 ± 3.8 |

Linseed oil | 4.5 h − 1 431 U − 38% (oil/aqueous) 30 °C − pH 6 | 96.9 | 91.8 ± 3.2 |

Factor | Lipases | |||||
---|---|---|---|---|---|---|

CRL ^{a}, RML ^{a} | PFL ^{a} | |||||

Coded Factor Levels | Coded Factor Levels | |||||

−1 (Low) | 0 (Middle) | +1 (High) | −1 (Low) | 0 (Middle) | +1 (High) | |

pH | 6 | 7.5 | 9 | 6 | 8 | 10 |

Temperature (°C) | 25 | 32.5 | 40 | 25 | 35 | 45 |

^{a}CRL: Candida rugosa lipase; RML: Rhizomucor miehei lipase; PFL: Pseudomonas fluorescens lipase.

Factors | Coded Factor Levels | ||
---|---|---|---|

−1 (Low) | 0 (Middle) | +1 (High) | |

Reaction duration (h) | 2 | 5 | 8 |

Enzyme load (U) | 800 | 1900 | 3000 |

Oil/aqueous ratio of the mixture (%) | 10 | 35 | 50 |

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**MDPI and ACS Style**

Faillace, E.; Brunini-Bronzini de Caraffa, V.; Mariani, M.; Berti, L.; Maury, J.; Vincenti, S.
Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils. *Int. J. Mol. Sci.* **2023**, *24*, 12274.
https://doi.org/10.3390/ijms241512274

**AMA Style**

Faillace E, Brunini-Bronzini de Caraffa V, Mariani M, Berti L, Maury J, Vincenti S.
Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils. *International Journal of Molecular Sciences*. 2023; 24(15):12274.
https://doi.org/10.3390/ijms241512274

**Chicago/Turabian Style**

Faillace, Eva, Virginie Brunini-Bronzini de Caraffa, Magali Mariani, Liliane Berti, Jacques Maury, and Sophie Vincenti.
2023. "Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils" *International Journal of Molecular Sciences* 24, no. 15: 12274.
https://doi.org/10.3390/ijms241512274