Development of simple, scalable protease production from Botrytis cinerea

Rachel A Self,Mark D Harrison,Steve Van Sluyter,Valentino S Te’O

doi:10.1007/s00253-022-11817-1

Rachel A Self, Mark D Harrison + Show 2 more

Open Access

https://doi.org/10.1007/s00253-022-11817-1

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Abstract

Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L−1. Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking.Key points• Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified.• Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion.• Low pH treatment inactivated laccases but not proteases.Graphical abstract

Highlights

Bentonite is a clay that is added to wine after fermentation to adsorb heat-haze forming proteins, including chitinases and thaumatin-like proteins (Waters et al 1996)
Evaluating wild‐type B. cinerea isolates for laccase and protease production
Scoring of the progressive red/brown color change of guaiacol agar during growth of B. cinerea isolates enabled a robust comparison of laccase production between isolates (Fig. 1B), though no isolate that produced both strong levels of proteases and low levels of laccase was identified

Summary

Introduction

Bentonite is a clay that is added to wine after fermentation to adsorb heat-haze forming proteins, including chitinases and thaumatin-like proteins (Waters et al 1996). These proteins come from grapes and aggregate when heated, causing an undesirable haze in the wine (Marangon et al 2009). Alternative techniques for removal of heat-haze forming proteins from wine have been identified, including ultrafiltration (Flores et al 1990), non-bentonite absorbents (Sarmento et al 2000) including zirconium oxide (Marangon et al 2011) and carrageenan (Ratnayake et al 2019), but none has been adopted by commercial winemakers. The combination of a mixture of Aspergillopepsin I and II (proteases) and flash pasteurization successfully heat-stabilized white wine without adverse effects on sensory characteristics (Marangon et al 2012) and at lower operating cost relative to the addition of bentonite (Logan 2015); the

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