CONICET | Buscador de Institutos y Recursos Humanos

The process of using enzymes to extract plant biomolecules that are trapped in a complex biological matrix is referred to as "enzyme-assisted extraction." This method facilitates the release of phenolic compounds by the action of hydrolases, which reduces the complexity of the plant matrix found in agro-industrial residues. The type of hydrolase used varies depending on the residue in question, with options including cellulases, amylases, xylanases, pectinases, and others. While a commercial enzyme cocktail can be directly added to the biological matrix, this approach can be cost-prohibitive. A more feasible alternative involves using hydrolytic enzymes induced by solid-state fermentation (SSF) with fungi and agro-industrial waste as substrates. An excellent example of this is the extraction of antioxidants (AO) from grape pomace (GP), a residue from winemaking that contains a high load of polyphenols with antioxidant properties. An effective approach to valorizing the abundant waste generated by the winemaking industry is through the recovery of antioxidants (AO) for commercial applications. While some AO can be easily extracted using aqueous or hydroalcoholic media, others remain trapped in the plant matrix, making their recovery challenging. Tannase is a particularly interesting enzyme for this purpose, as it can hydrolyze the most inaccessible tannins to yield gallic acid (GA), an organic acid with high commercial value due to its antioxidant properties highly demanded by pharmaceutical, cosmetic, and food industries. However, tannase is a less common enzyme to induce in fungal culture extracts compared to the aforementioned hydrolases. In this study, we employed grape pomace (GP) alone or supplemented with flour mill waste (FMW), a residue from the flour industry, as enzyme-inducing substrates using solid-state fermentation (SSF) to produce enzymes. Our objectives were, i) to identify the enzymatic profile present in the extracts obtained by fermenting the Neocosmospora lichenicola fungus on agro-industrial substrates, ii) to optimize the composition and culture conditions to maximize enzyme production for each agro-industrial substrate tested, and iii) to evaluate the ability of the optimized enzymatic extract to liberate GA from tannins of GP. To achieve our goal, we first characterized the enzymatic profiles induced by various substrates using solid-state fermentation with N. lichenicola. We also tested different fermentation conditions, substrate residues, and GP/FMW ratios, as well as different humidity levels in the presence and absence of tannic acid (TA), a potent inducer of tannase expression. All cultures were incubated at 30°C for 6 days, and enzymatic activity measurements were recorded throughout. For the optimization of cultivation conditions, we repeated the tests using the highest humidity condition (1:4) and GP, FMW, and a combination of both as solid substrates for SSF. We monitored enzymatic activity levels during the 6-day incubation period at 30°C. The most efficient combination for tannase and pectinase production was found to be FMW + GP + 2% TA. We observed the highest levels of tannase production on days 4 and 5, and the highest levels of pectinase production on days 5 and 6. Cellulase production remained consistent across all culture conditions and substrates tested, with no significant variations observed. Interestingly, GP alone proved to be a good inducer for both cellulase and tannase production. Notably, no fungal growth was observed in the GP + TA 2% condition. Total polyphenol content was determined using the Folin-Ciocalteu method, while antioxidant capacity was measured using the TEAC method. In both cases, a clear peak of antioxidant activity was observed in the condition with the highest humidity, presence of TA 2%, and presence of tannase enzyme. The FMW + GP + TA 2% condition yielded the highest levels of total phenols and antioxidant activity on the fourth day of culture, followed by a decline. This behavior can be correlated with the amount of GA produced during that period. HPLC analysis revealed a 26% conversion rate of initial tannic acid to gallic acid on day 4. In conclusion, the results of this study demonstrate that enzyme-assisted extraction using hydrolytic enzymes can efficiently recover antioxidants under optimized fermentation conditions. This method is cost-effective and has low water demand, while the remaining residue after fermentation is highly degraded, making it a circular process with a triple impact on social, economic, and environmental factors. Overall, these findings suggest that enzyme-assisted extraction can be a promising approach for valorizing agro-industrial waste and reducing waste disposal.

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