Prolyl endopeptidase from Aspergillus niger immobilized on a food-grade carrier for the production of gluten-reduced beer
Introduction
Beer is an alcoholic beverage consumed throughout the world, with an average annual consumption of about 74 kg/capita in Europe and 86 kg/capita in Northern America (Colen & Swinnen, 2016). As reported by Barth-Haas (2013), the European beer market is second to the U.S. beer market, with a production volume of 545 million hectolitres. People affected by celiac disease and non-celiac gluten sensitivity represent about 6% of the global population (Carroccio et al., 2012; Genetics Home Reference, 2015). Individuals with celiac disease must adhere to a gluten-free (GF) diet. Therefore, they can safely drink beer surrogates which differ significantly from authentic (barley-based) beer in terms of aroma and taste. The Codex Alimentarius Standard and the EU-regulation 828/2014 established a gluten concentration below 20 mg/kg for GF food (Knorr, Wieser, & Koehler, 2016; Shan, 2002), whereas the range of gluten content in brewed barley malt beers may vary from <10 mg/kg in some industrial lager beer to 4000 mg/kg in Weissbier (Fanari et al., 2017, 2018; Guerdrum & Bamforth, 2011; Hager, Taylor, Waters, & Arendt, 2014; Van Landschoot, 2011; Watson, Decloedt, Vanderputten, & Van Landschoot, 2018).
In the last decade, the demand for high quality GF foods, including GF beer, has increased strongly and their production has been becoming an important socio-economic issue. Despite the fact that beer is not an essential part of human nutrition, the availability of safe, healthy and tasty GF beer would appreciably improve peoples’ well-being and perception of a normal social life (Hager et al., 2014). In this scenario, the global GF beer market is expected to grow at a Compound Annual Growth Rate (CAGR) greater than 40% over the period 2017–2023.
The main strategies for producing GF beer are: i) the use of naturally GF grains (i.e. rice, corn, sorghum, millet) or pseudocereals (i.e. quinoa, buckwheat, amaranth) as raw materials (Wiedemair, Ramoner, & Huck, 2019), thus obtaining beer surrogates; ii) the precipitation of proteins using tannins, silica gel or PVPP (Benìtez, Acquisgrana, Peruchena, Sosa, & Lozano, 2016; Dostalek, Hochel, Mendez, Hernando, & Gabrovska, 2006; Hager et al., 2014; Watson, Vanderputten, Van Landschoot, & Decloedt, 2019); iii) the degradation of gluten during the brewing process by enzymatic treatments (Arendt & Zannini, 2013; Dostalek et al., 2006; Guerdrum & Bamforth, 2012; Hager et al., 2014; Watson et al., 2019). Recently, a controlled hydrocavitation-assisted brewing technique has been applied for gluten reduction in beer (Albanese, Ciriminna, Meneguzzo, & Pagliaro, 2017), as well as the use of silica gel during several beer brewing stages (Benìtez et al., 2016). Prolyl endopeptidase from Aspergillus niger (AN-PEP), currently used in the brewing industry for haze prevention, breaks down the proline-rich prolamin fraction of gluten. This exogenous enzyme has been successfully applied, in free form, for the production of GF beer from conventional malts (Akeroyd et al., 2016; Watson et al., 2019) without negatively impacting foam stability (Di Ghionno, Marconi, Sileoni, De Francesco, & Perretti, 2017; Guerdrum & Bamforth, 2012).
It is recognized that free enzymes usually have poor stability under process conditions (i.e., pH, temperature, and natural inhibitors in food matrix) and they cannot be recovered/reused. As reported in literature, enzyme immobilization, obtained by fixing enzyme on an appropriate carrier, is believed to provide an excellent base for improving the environmental tolerance and the operational stability of the biocatalyst in food matrix (Tang, Peng, Li, Meng, & Liu, 2018). In addition, enzyme immobilization on solid supports could allow its reuse for many cycles, also in continuous processes, avoiding protein contamination of the product (Gardossi et al., 2008).
Recently, Zhao et al. (2017) covalently immobilized AN-PEP on nonporous silica nanoparticles functionalized with amino groups, proving its efficiency in preventing chill-haze formation in beer. Despite these results, no studies have yet been carried out applying immobilized AN-PEP in a continuous bioreactor for the reduction of gluten content in beer. Among continuous bioreactors, fluidized bed reactor (FBR), is especially recommended when the substrates are viscous or contain suspended particles (Gòmez et al., 2007). In this reactor, the flow of substrate keeps the immobilized enzyme particles in a fluidized state, thus obtaining a high catalytic surface area. FBRs have widespread application in the food industry [i.e. for apple juice clarification (Diano et al., 2008), for the production of fructo-oligosaccharides and invert sugars (Lorenzoni et al., 2015), for lactose hydrolysis (Roy & Gupta, 2003), for the flavour enhancement of beverages (Gueguen, Chemardin, Pien, Arnaud, & Galzy, 1997) and for controlling malolactic fermentation in wine (Cappannella et al., 2016)].
Therefore, in this study AN-PEP has been covalently immobilized on a food-grade carrier and its environmental tolerance (temperature and pH), as well as its catalytic properties have been investigated toward a synthetic peptide substrate in synthetic beer. Afterwards, the biocatalyst was used for the first time in a continuous FBR for the production of GF conventional barley malt beer.
Section snippets
Materials
Brewers Clarex® (lot No. 817786501, DSM, Delft, The Netherlands), a commercial proline-specific peptidase preparation from Aspergillus niger (AN-PEP), was kindly given by IMCD Italia SpA (Milan, Italy). The chromogenic para-nitroanilide (pNA) peptide Z-Gly-Pro-pNA was purchased from Bachem (Bubendorf, Switzerland) and used as synthetic substrate for protease activity. Beads as carriers for AN-PEP immobilization were produced using chitosan powder from Aspergillus niger (lot No. 12121611L4,
AN-PEP immobilization
A first experiment was carried out in order to determine the effect of the protein concentration in the immobilization solution (ranging from 0.1 to 4.6 mgBSAeq/mL) on the protein loaded, as well as on the specific activity of immobilized AN-PEP.
Data in Fig. 1 show that protein loading significantly increased when higher initial protein concentrations were used, until reaching the maximum value (about 7 mgBSAeq/gchitosan using an immobilization solution at 3 mgBSAeq/mL). The further increase of
Conclusions
The production of high quality gluten-reduced beer remains a challenging problem, which is worthy of serious exploration in order to find better useful approaches to be applied in brewing.
In this study, we have demonstrated that the enzyme prolyl endopeptidase from Aspergillus niger (AN-PEP), currently used in the brewing industry, could be applied for the industrial production of GF beer also in immobilized form. The continuous treatment of beer in a fluidized bed reactor (FBR), containing the
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgement
This work was supported by BioEnBi project “Biotecnologie enzimatiche innovative per processi di chiarifica sostenibili nel settore birrario”, funded by Lazio Region (Italy) in the context of Progetti Gruppi di Ricerca, LazioInnova 2018–2020.
We are particularly grateful to Free Lions Brewery (Viterbo, Italy) which kindly supplied the Pilsner-type beer.
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