Virginie Brunini-Bronzini de Caraffa

Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the growing demand for natural GLVs with high added value, the use of biocatalytic processes appears as a relevant application. In such processes, vegetable oils are bioconverted into GLVs. First, the triacylglycerols of the oils are hydrolyzed by a lipase. Then, the free polyunsaturated fatty acids are converted by a lipoxygenase. Finally, volatile C6 or C9 aldehydes and 9- or 12-oxoacids are produced with a hydroperoxide lyase. Optimization of each biocatalytic step must be achieved to consider a scale-up. In this study, three oils (sunflower, hempseed, and linseed oils) and three lipases (Candida rugosa, Pseudomonas fluorescens, and Rhizomucor miehei lipases) have been tested to optimize the first step of the process. The experimental design and response surface methodology (RSM) were used to determine the optimal hydrolysis conditions for each oil. Five factors were considered, i.e., pH, temperature, reaction duration, enzyme load, and oil/aqueous ratio of the reaction mixture. Candida rugosa lipase was selected as the most efficient enzyme to achieve conversion of 96 ± 1.7%, 97.2 ± 3.8%, and 91.8 ± 3.2%, respectively, for sunflower, hempseed, and linseed oils under the defined optimized reaction conditions.

The chemical variability and the in vitro anti-inflammatory activity of the leaf essential oil from Ivorian Isolona dewevrei were investigated for the first time. Forty-seven oil samples were analyzed using a combination of CC, GC(RI), GC-MS and ¹³C-NMR, thus leading to the identification of 113 constituents (90.8–98.9%). As the main components varied drastically from sample to sample, the 47 oil compositions were submitted to hierarchical cluster and principal components analyses. Three distinct groups, each divided into two subgroups, were evidenced. Subgroup I−A was dominated by (Z)-β-ocimene, β-eudesmol, germacrene D and (E)-β-ocimene, while (10βH)-1β,8β-oxido-cadina-4-ene, santalenone, trans-α-bergamotene and trans-β-bergamotene were the main compounds of Subgroup I−B. The prevalent constituents of Subgroup II−A were germacrene B, (E)-β-caryophyllene, (5αH,10βMe)-6,12-oxido-elema-1,3,6,11(12)-tetraene and γ-elemene. Subgroup II−B displayed germacrene B, germacrene D and (Z)-β-ocimene as the majority compounds. Germacrene D was the most abundant constituent of Group III, followed in Subgroup III−A by (E)-β-caryophyllene, (10βH)-1β,8β-oxido-cadina-4-ene, germacrene D-8-one, and then in Subgroup III−B by (Z)-β-ocimene and (E)-β-ocimene. The observed qualitative and quantitative chemical variability was probably due to combined factors, mostly phenology and season, then harvest site to a lesser extent. The lipoxygenase inhibition by a leaf oil sample was also evaluated. The oil IC₅₀ (0.020 ± 0.005 mg/mL) was slightly higher than the non-competitive lipoxygenase inhibitor NDGA IC₅₀ (0.013 ± 0.003 mg/mL), suggesting a significant in vitro anti-inflammatory potential.

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R² coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L⁻¹_{of culture} of the soluble enzyme was produced in microliter plates and 21,920 U L⁻¹_{of culture} in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported.

In higher plants, the lipoxygenase enzymatic pathway combined actions of several enzymes to convert lipid substrates into signaling and defense molecules called phytooxylipins including short chain volatile aldehydes, alcohols, and esters, known as green leaf volatiles (GLVs). GLVs are synthesized from C18:2 and C18:3 fatty acids that are oxygenated by lipoxygenase (LOX) to form corresponding hydroperoxides, then the action of hydroperoxide lyase (HPL) produces C6 or C9 aldehydes that can undergo isomerization, dehydrogenation, and esterification. GLVs are commonly used as flavors to confer a fresh green odor of vegetable to perfumes, cosmetics, and food products. Given the increasing demand in these natural flavors, biocatalytic processes using the LOX pathway reactions constitute an interesting application. Vegetable oils, chosen for their lipid profile are converted in natural GLVs with high added value. This review describes the enzymatic reactions of GLVs biosynthesis in the plant, as well as the structural and functional properties of the enzymes involved. The various stages of the biocatalytic production processes are approached from the lipid substrate to the corresponding aldehyde or alcoholic aromas, as well as the biotechnological improvements to enhance the production potential of the enzymatic catalysts.