Studies on Fermentative Extraction of Antioxidants from Unripe Areca Nut

Abstract

The pericarp of Areca catechu L., commonly known as areca nut, contains a wealth of valuable phenolic compounds. The presence of the psychoactive agent Arecoline can make it challenging to utilize these compounds. To address this issue, current research work was aimed at isolating a microorganism that can grow on areca nut and facilitate the extraction of valuable phenolic compounds. Two organisms, a mold and yeast, were isolated from the microbial mat growing on the stored areca extract leachate surface. The mold was identified as Rhizopus oryzae MW538932 and yeast as Geotrichum cucujoidarum MW538971. Based on HPLC-MS/MS analysis and MetFrag search engine, major components in chogaru (unripe areca nut extract) were identified as Arecatannin B1 and Procyanidin B4/B5 which were being used by the mold and yeast as their primary carbon sources. However, the mold was found to be more efficient at utilizing these compounds than the yeast, which led to it being selected for further research. Shake flask studies indicated that the isolate, Rhizopus oryzae MW538932 mold was converting Arecatannin B1 to Procyanidin B, which was then further converted to Catechin and Protocatechuic acid (PCA). Similarly, Procyanidin B4/B5 was converted to Catechin and PCA. In order to maximise the extraction of phenolic compounds, the isolated mold Rhizopus oryzae MW538932 was employed for the solid-state fermentation of unripe areca nut (6–7 months’ maturity) powder. Supplementary nutrients (carbon and nitrogen sources) for the media and the solvents for the extraction of phenolic compounds from the fermented medium were optimised. The optimised process could produce an extract having a total phenolic content of 186.03 ± 2.50 mg gallic acid equivalent and total flavonoid content of 139.70 ± 2.00 mg catechin equivalent per gram of the sample. UHPLC–MS/MS studies and HPLC analysis showed the presence of plethora of phenolic compounds and the absence of Arecoline and other alkaloids. The media for the fermentative extraction of phenolic components by the SmF process was optimized using the Plackett-Burman design and RSM. The optimised process could produce an extract having a total phenolic content of 146.06 ± 0.75 mg gallic acid equivalent and a total flavonoid content of 53.74 ± 0.83 mg catechin equivalent per gram of the sample. UHPLC-MS/MS studies and HPLC analysis revealed a wide range of phenolic compounds found exclusively in fermented extracts and decreased Arecoline content by 28.57%. A comparative study of the antioxidant properties of the extracts produced by SSF and SmF showed that fermented extracts had better antioxidant potential than unfermented extracts. The light brown colored lyophilized powder free from arecoline, obtained by the fermentative extraction of unripe areca nuts by SSF was analyzed for its efficacy in imparting oxidative stability to the fish oil. The total phenolic and flavonoid contents of the fermented extract were 10.41 ± 0.81 mg gallic acid equivalent and 7.58 ± 1.01 mg catechin equivalent per mg of the powder respectively. The DPPH radical scavenging activity and FRAP were 0.23 ± 0.01 and 0.064 ± 0.02 mmol Trolox equivalent per mg, respectively. UHPLC-MS/MS analysis revealed the presence of more than 45 different phenolic compounds. It exhibited superior efficacy in imparting oxidative stability to the n-3 PUFA containing fish oil in 50 days of storage studies. Its performance was comparable to BHT for the initial 25 days of storage. This could be a new addition to well-proven herbal extracts such as rosemary extract and green tea extracts for food application.