Determination of Perillic Acid in Bioconversion Supernatants by Gas Chromatography

Abstract

Perillic acid can be obtained from microbial oxidation of the exocyclic methyl group of limonene. Due to the pharmacological potential of such a metabolite, the biotransformation processes leading to its synthesis have been approached in recent studies. A robust analytical method is needed to assess the performance of such studies. An analytical method was developed and validated to determine perillic acid in the supernatants of a yeast-induced bioconversion of limonene, involving gas chromatography (GC) and an acid-induced precipitation during the sample preparation. GC analysis was performed using a column with polyethylene glycol as stationary phase (HP-Innowax) which resulted in higher loads and better peak shape. The sample preparation involved the supernatant initial filtration and precipitation with 0.6 M HCl followed by centrifugation and dissolution in ethyl acetate. GC analysis conditions were oven from 50˚C to 250˚C at 20˚C·min −1 , and then held 5 min (total runtime 15 min). Injector was set at 280˚C, and detector at 300˚C. Helium was the carrier gas at 1 ml·min ; Repeatability of 2.1% RSD. Thus, a complete methodology to determine perillic acid in a bioconversion supernatant was developed and validated. This overall approach may be useful for bioconversions of monoterpenes by other microorganisms that metabolize limonene.

Fungal biotransformation of limonene and pinene as a biotechnological approach for production of aroma compounds

“…Hydroxylation at the 7-position for production of Perillyl derivatives Whilst limonene enantiomers is a cheap and widely available chemical, its oxidised derivatives are relatively expensive.The biotransformation process permits the production of a hydroxymethylated monoterpene analog, for example perillyl derivatives, from a monoterpene analog having a terminal methyl group, for example limonene enantiomers, which is substantially free from other hydroxylation products (Maróstica and Pastore 2007b;Bicas, Fontanille and Larroche 2008;Carvalho et al 2017). Speci cally the allylic oxidation reaction by fungal biocatalysts to the methyl group (hydroxylation at 7-position) obtained different aroma compounds such perillyl alcohol (CAS 536-59-4), perillyl aldehyde (CAS 2111-75-3), and perillyl acid (CAS 23635-14-5) (Duetz et al 2003;Trytek and Fiedurek 2005a;Carvalho et al 2017).…”

“…Research and development on the biotransformation of limonene has continued to draw much attention on a wide variety of conversion products, such as perillic compounds in signi cant amounts, which could be more valuable to the elds of cosmetics, food ingredients and pharmaceutical industries (Lerin et al 2010). Among the main bioconversion products, perillyl alcohol is of particular importance due to its high bulk prices that range from US$300-600/kg (Duetz et al 2003;Carvalho et al 2017). It has chemopreventive properties that include treatment of cancers of the liver, mammary and lungs and, as such, suggests great market potential (Castellanos, Villamil and López 2007).…”

“…A number of reports in the literature demonstrate that the epoxidation pathway starts with an attack to the 1,2-double bond of limonene by the enzyme limonene 1,2-monooxygenase, thus generating limonene-1,2-epoxide Limonene-1,2-epoxide hydrolase catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol, which is an important aroma compound used in the food industry. Subsequent oxidations in limonene-1,2-diol gave rise to the compounds 1-hydroxy-2-oxolimonene, this molecule spontaneously rearranging obtained 3-isopropenyl-6-oxoheptanoate and 3-isopropenyl-6oxoheptanyl-CoA (Maróstica and Pastore 2007; Bicas, Fontanille and Larroche 2008;Carvalho et al 2017).…”