Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support

Abstract

This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.

Keywords: Açaí (Euterpe oleracea Martius); Beauveria bassiana; Buriti (Mauritia flexuosa); Fusarium oxysporum; immobilization; lipase; octyl-sepharose; omega-6; omega-9.

Figure 1

Selection of carbon source for lipase production: Total enzyme activity and specific enzyme activity of culture supernatants from B. bassiana (a) and F. oxysporum (b) with different oils and butters. The symbol (⋆) above the vertical bar indicates the difference in significance in relation to other oils, for the studied parameters of interest. The statistics used was One-Way ANOVA followed by Tukey post-hoc with ⋆ p < 0.05. The values are expressed in mean ± SEM, representing n = 3 for each oil. Polyacrylamide electrophoresis (12%) was under semi-denaturing conditions of the supernatant cultures that showed the highest enzymatic activities. (c) and (d) protein electrophoresis (I) and zymogram (II) of B. bassiana supernatant cultures with Tucumã, Cupuaçu, Fish, Buriti and Açaí. (e) and (f) protein electrophoresis (I) and zymogram (II) of the supernatants of F. oxysporum cultures with Tucumã, Bacuri, Fish, Buriti and Açaí. Lane 1: molecular mass. Other specifications are described in Section 3.1 and Section 3.2.

Figure 2

Kinetics immobilization on octyl-sepharose of B. bassiana and F. oxysporum culture supernatants: Yield increase of octyl-sepharose and decrease in the relative activity of the supernatant, expressed as percentage, as a function of the adsorption time, for (a) B. bassiana and (b) F. oxysporum. Aiming to achieve immobilization, 2 g of octyl-sepharose were added to 40 U of crude extract in sodium phosphate buffer 50 mM, pH 7.0, at 4 °C. The 100% of relative activity was considered as 40 U offered to immobilization. Activity was followed by 0.003% p-nitrophenyl-palmitate (pNPP) in McIlvaine buffer. (□) Yield (%) and (■) Supernatant activity (%). The values are expressed in means ± SEM, with n = 3. The SEM is lesser than 1% of the mean value (error bars are not evident, as they lie within the area of the symbol).

Figure 3

Thermal and pH stabilities of B. bassiana lipase: Thermal stability of crude extract (a) and derivative (b). pH stabilities of crude extract (c) and derivative (d). Thermal inactivation was performed in McIlvaine buffer pH 7.0 at the following temperatures; (●) 40 °C; (○) 50 °C; (■) 60 °C; (□) 70 °C and (▲) 80 °C. The pH stability was performed at 40 °C with McIlvaine buffer (pH 3–8) and 200 mM glycine buffer (pH 9 and 10); (●) pH 3.0; (○) pH 4.0; (■) pH 5.0; (□) pH 6.0; (▲) pH 7.0; (△) pH 8.0; (♦) pH 9.0 and (◊) pH 10.0. The lipase activity was obtained by 0.003% pNPP assay and relative activity was expressed according to the initial activity of the derivative prior to incubation at different pH and temperatures at several time intervals. The 100% of relative activity was considered as 940 U of B. bassiana lipase activity immobilized on octyl measured at the beginning of the experiment. Other specifications are described in Section 3.8 and Section 3.9. The symbol (⋆) above an icon indicates the difference in significance between pH/temperatures for the same period of interest. The statistics used was One-Way ANOVA with ⋆ p < 0.05. The values were expressed in mean ± SEM, representing n = 3 for each group. SEM is lesser than 1% of mean value for the experiment set (error bars are not evident, as they lie within the area of the symbol). The statistics to evidence the difference between immobilized and crude extract are described in the text.

Figure 4

Thermal and pH stabilities of F. oxysporum lipase: Thermal stability of crude extract (a) and derivative (b). pH stabilities of crude extract (c) and derivative (d). Thermal inactivation was performed in McIlvaine buffer pH 7.0 at the following temperatures; (●) 30 °C; (○) 40 °C; (■) 50 °C; (□) 60 °C and (▲) 70 °C. The pH stability was performed at 40 °C with McIlvaine buffer (pH 3–8) and 200 mM glycine buffer (pH 9 and 10); (●) pH 3.0; (○) pH 4.0; (■) pH 5.0; (□) pH 6.0; (▲) pH 7.0; (△) pH 8.0; (♦) pH 9.0 and (◊) pH 10.0. Lipase activity was obtained by 0.003% pNPP assay and relative activity was expressed according to the initial activity of the derivative prior to incubation at different pH and temperatures at several time intervals. The 100% of relative activity was considered as 440 U of F. oxysporum lipase activity immobilized on Octyl measured at the beginning of experiment. Other specifications are described in Section 3.8 and Section 3.9. The symbol (⋆) above an icon indicates the difference in significance between pH/temperatures for the same period of interest. The statistics used was One-Way ANOVA with ⋆ p < 0.05. The values were expressed in mean ± SEM, representing n = 3 for each group. SEM is less than 1% of mean value for the experiment set (error bars are not evident, as they lie within the area of the symbol). The statistics to evidence the difference between immobilized and crude extract are described in the text.

Figure 5

Derivative activity and lipase identification: (a) Profiles of B. bassiana and F. oxysporum derivatives in the presence of different concentrations of Triton X-100 (0.05; 0.1; 0.2; 0.3 and 0.4%). The enzymatic activity in the supernatant desorption was monitored by the 0.003% pNPP assay. The 100% of activity was considered as 440 U for Fo and 940 U for Bb. (b) SDS-PAGE for lipase desorption fraction of the supernatant corresponding to the concentration of 0.1% Triton X-100. The SDS-PAGE gel was stained with colloidal Coomassie. The values are expressed in mean ± SEM, with n = 3.

Figure 6

Hydrolysis of Açaí and Buriti oils: Hydrolysis using Bb-octyl derivative and Açaí (a) and Buriti (b) oils. Hydrolysis using Fo-octyl derivative and Açaí (c) and Buriti (d) oils. Concentrations of fatty acids were monitored up to 6 h of hydrolysis by RP-HPLC and were determined according to standard curves of oleic and linoleic acids. Mobile phase: (75/25, v/v) acetonitrile: 10 mM Tris-OH pH 3.0, flow rate: 1 mL.min^-1, injected sample: 100 μL of organic phase at different times of hydrolysis previously diluted in acetonitrile. Oleic acid (●) and linoleic acid (○) produced in the hydrolysis. The values are expressed in mean ± SEM, representing n = 3 for each group. SEM is lesser than 1% of mean value for the experiment set (error bars are not evident, as they lie within the area of the symbol).