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Parchami, M., Ferreira, J., and Taherzadeh, M. (2021). "Brewing process development by integration of edible filamentous fungi to upgrade the quality of Brewer’s spent grain (BSG)," BioResources 16(1), 1686-1701.

Abstract

Brewer’s spent grain (BSG) is the main solid by-product of the brewing sector. High moisture and nutrient-rich content render BSG easily perishable, leading to waste generation and environmental impacts. BSG has narrow applications in both feed and food sectors due to its composition including high fiber and low protein. Therefore, a processing strategy leading to the nutritional valorization of BSG could widen its applications. In this study, submerged cultivation of edible filamentous fungi (Aspergillus oryzae, Neurospora intermedia, and Rhizopus delemar) was introduced as a strategy to enhance the protein content of BSG. The growth of all strains in BSG increased the protein content of the fermented BSG. The highest increase of protein content (from 22.6% to 34.6%), was obtained by cultivation using A. oryzae and medium supplementation. The protein content increase was followed by a decrease in the content of polysaccharides (up to ca. 50%), namely starch, glucan, xylan, and arabinan. The addition of cellulase resulted in enhanced ethanol production from BSG but led to lower concentration of recovered solids. In conclusion, simple processing of BSG using edible filamentous fungi can lead to quality improvement of BSG, providing potential economic and environmental benefits to the brewing sector.


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Brewing Process Development by Integration of Edible Filamentous Fungi to Upgrade the Quality of Brewer’s Spent Grain (BSG)

Mohsen Parchami,* Jorge A. Ferreira, and Mohammad J. Taherzadeh

Brewer’s spent grain (BSG) is the main solid by-product of the brewing sector. High moisture and nutrient-rich content render BSG easily perishable, leading to waste generation and environmental impacts. BSG has narrow applications in both feed and food sectors due to its composition including high fiber and low protein. Therefore, a processing strategy leading to the nutritional valorization of BSG could widen its applications. In this study, submerged cultivation of edible filamentous fungi (Aspergillus oryzaeNeurospora intermedia, and Rhizopus delemar) was introduced as a strategy to enhance the protein content of BSG. The growth of all strains in BSG increased the protein content of the fermented BSG. The highest increase of protein content (from 22.6% to 34.6%), was obtained by cultivation using A. oryzae and medium supplementation. The protein content increase was followed by a decrease in the content of polysaccharides (up to ca. 50%), namely starch, glucan, xylan, and arabinan. The addition of cellulase resulted in enhanced ethanol production from BSG but led to lower concentration of recovered solids. In conclusion, simple processing of BSG using edible filamentous fungi can lead to quality improvement of BSG, providing potential economic and environmental benefits to the brewing sector.

Keywords: Edible filamentous fungi; Brewer’s spent grain; Protein recovery; Submerged cultivation

Contact information: Swedish Centre for Resource Recovery, University of Borås, 50190 Borås, Sweden;

* Corresponding author: mohsen.parchami@hb.se

GRAPHICAL ABSTRACT

INTRODUCTION

One sector in the food industry that retains huge market value is the brewing industry. In 2018, 188 billion liters of beer (made from malt and excluding beer containing ≤ 0.5% v/v alcohol) were manufactured worldwide with the revenue of 504 billion Euro. China was the leading producer, accounting for approximately 20% of the global production; the USA followed with 11% of the global production (Barth-Haas Group 2019).

The main raw material for beer production is barley, which, before brewing, undergoes malting steps for enzyme activation. The malt is then milled, transferred to the mash tun, and mixed with water and adjuncts. During mashing, enzymatic hydrolysis originates a sweet liquid called wort. Brewer’s spent grain (BSG) is the solid fraction collected after the filtration of wort (Fig. 1) (Hardwick 1995; Mussatto et al. 2006). BSG is mainly barley grain husk mixed with (testa) seed coat and pericarp layers, and depending on the adjuncts, parts of other grains can be found. The main components of BSG are proteins, cellulosic and non-cellulosic polysaccharides, lignin, lipids, vitamins, and minerals (Mussatto 2014; Lynch et al. 2016). The brewing process originates other by-products such as spent hops and spent yeast, but BSGs is the most abundant by-product of brewing, accounting for 85% of total by-products and 30% of the initial malt weight (Townsley 1979; Mussatto et al. 2006). Considering the average generation of 20 kg wet BSG (70 to 80% moisture) for every 100 liters of beer (Steiner et al. 2015), the global amount of BSG generated in 2018 was approximately 37.6 million metric tons. Due to high moisture and nutrient-rich content, BSG is highly susceptible to biological deterioration, leading to waste generation and environmental impacts (El‐Shafey et al. 2004).

Fig. 1. An overview of the beer production process

Currently, BSG encounters limited applications in animal feed, due to low digestibility, rendering landfilling the most common disposal route. Recently proposed valorization routes have included the incorporation of BSG in food products with potential health benefits (Mussatto 2014). There are a number of studies on the direct application of BSG as a cheap enrichment source for the nutritional fortification of food products such as bread, cookies, cakes (Townsley 1979; Waters et al. 2012; Fărcaş et al. 2014), and egg pasta (Cappa and Alamprese 2017). Nonetheless, the use of BSG is also limited in food systems (about 5 to 10%) due to undesired changes in the final product properties such as taste, texture, and color (Mussatto et al. 2006). Production of phenolic compounds (Moreira et al. 2012; Spinelli et al. 2016; Guido and Moreira 2017), arabinoxylans (Coelho et al. 2014; Vieira et al. 2014), absorbents (Chiang et al. 1992), and paper (Ishiwaki et al. 2000), or energy recovery through combustion and pyrolysis (Anal 2018), are other approaches proposed for valorization of BSG.

Upgrading the nutritional profile of BSG could lead to higher incorporation in both feed and food systems, and the use of edible filamentous fungi for conversion of cellulosic and non-cellulosic polysaccharides into nutritious biomass and protein could achieve this goal. Increasing population and changing diets support the need of alternative protein sources for animal feed and meat replacers, respectively.

Filamentous fungi share low substrate specificity and wide range of potential value-added products (Ferreira et al. 2016). Different filamentous fungal species, such as Aspergillus spp., Rhizopus spp., and Neurospora spp., are used for the production of native East Asian foods and beverages (e.g., tempe, oncom, miso, syoyu, and sake) (Karimi et al. 2019). Consequently, various species are categorized as generally regarded as safe (GRAS) microorganisms, indicating their suitability for the valorization of wastes into food and feed products. Their high protein content with essential and non-essential amino acids, low fat, and cholesterol-free content make filamentous fungi a nutritive source of food and feed. Moreover, filamentous fungi secrete a large number of different enzymes and secondary metabolites, with great importance in different food and feed processing industries, making them advantageous for food and feed applications (Ghorai et al. 2009). Nutritional valorization of BSG has been investigated on low-moisture solid-state fermentation, which requires a difficult scale-up (Cooray and Chen 2018; Ibarruri et al. 2019; Tan et al. 2019; Gmoser et al. 2020). Another strategy is needed to cope with the cumbersome amounts of BSG available.

This study upgraded BSG using submerged cultivation by edible filamentous fungi, a strategy currently applied at the industrial scale (Ferreira et al. 2016). The effects of fungal strain, medium supplementation, and cellulase addition on the amounts of recovered solids and protein content were investigated.

EXPERIMENTAL

Substrate

The BSG was obtained from Göteborgs Nya Bryggeri AB (Gothenburg, Sweden). The collected wet BSG was air-dried at room temperature for three days, followed by storage in zip-sealed bags at room temperature.

Filamentous Fungi

Three filamentous fungi, namely Aspergillus oryzae var. oryzae CBS 819.72 (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands), Neurospora intermedia CBS 131.92, and Rhizopus delemar CBS 145940, isolated originally from tané koji used for sake making (CBS strain database), fermented peanuts for making Oncom (CBS strain database), and leaves of Tectona grandis used to produce tempe (CBS strain database), respectively, were used in this study. The strains were kept on potato dextrose agar (PDA) plates containing 20 g/L glucose, 15 g/L agar, and 4 g/L potato infusion powder. New PDA plates were prepared by flooding pre-grown plates with 20 mL of sterile distilled water, and a disposable plastic L-shape spreader was used to bring the spores into solution. New plates were inoculated with 100 μL of spore solution, incubated at 30 °C for four days, and stored at 4 °C.

Cultivation in Shake-Flasks

Submerged fungal cultivations were performed in batch mode using 250 mL cotton-plugged Erlenmeyer flasks containing 100 mL of medium. The composition of the medium for each cultivation condition can be found in Table 1. The salt and BSG/yeast extract solutions were autoclaved at 121 °C for 20 min separately before cultivation. For all conditions, the pH was adjusted to 5.5 ± 0.1 using 6.0 M NaOH except for the cultivation without the addition of nutrients (initial pH = 5.2). For the cultivation with addition of cellulase cocktail, 10 FPU Cellic Ctec3/g glucan (Novozymes, Copenhagen, Denmark) was added to the flask prior to inoculation with spore solution. The enzyme activity was 234 FPU/mL. The flasks were inoculated with 2 mL of spore solution, as presented in Table 1. The cultivations were carried out in a water bath with shaking at 125 rpm and at 35 °C for three days. During the cultivation period, samples of 1 mL were taken every 12 h and centrifuged for 10 min at 14,000 rpm. The supernatant was filtered through a syringe filter of 0.2 μm pore size and stored at −20 °C until high-performance liquid chromatography (HPLC) analysis.

Analytical Methods

After cultivation, the solids (fungal biomass entangled with undigested BSG components) were recovered by sieving using a fine mesh (1 mm2 pore size), washed with distilled water until a clear effluent was obtained, and oven-dried at 70 °C until they reached a constant weight. The total solids, structural carbohydrates, and total lignin of both initial and cultivation-derived samples were measured according to the National Renewable Energy Laboratory (NREL) method for the determination of structural carbohydrates and lignin in biomass (Sluiter et al. 2008).

The crude protein content of BSG and of recovered solids from the cultivations was determined according to the Kjeldahl method using an InKjel P digestor and a behrotest® S1 distillation unit (Behr Labor‐Technik, Düsseldorf, Germany). In the first step, 20 mL of 98% H2SO4, antifoam, and KT1 tablets (Thompson & Capper Ltd, Runcorn, UK) were added to 0.5 ± 0.0 g material and digested for 100 min at 100% power (where 10 min were needed for heating the digestion block). Afterwards, the digested solutions were neutralized with 32% NaOH solution and distilled for 5 min; the distillation vapor was collected in 50 mL of 4% H3BO4. Finally, the condensates were titrated with 0.1 M HCl until the pH reached 4.6. For conversion of nitrogen to protein, a factor of 6.25 was used (Magomya et al. 2014), whereas the percentage of protein content increment of BSG following fungal cultivation was calculated according to Eq. 1,

where X is the increase in protein content of the BSG, Xbiomass is the protein content of the biomass and XBSG is the protein content of the BSG.

The liquid samples from cultivation and structural carbohydrate and lignin analysis were analyzed using a HPLC system (Waters 2695, Waters Corporation, Milford, MA, USA). A hydrogen-ion based ion exchange column (Aminex HPX87-H, BioRad Laboratories, München, Germany) at 60 °C and with 0.6 mL/min 5 mM H2SO4 as eluent was used for the analysis of glucose, sugars other than glucose, and ethanol.

Table 1. Medium Recipes and Spore Concentrations Used at the Various Conditions Studied for Cultivation of Three Edible Filamentous Fungi

In addition, a lead (II)-based column (Aminex HPX-87P, Bio-Rad) at 85 °C and 0.6 mL/min ultrapure water was used for the analysis of xylose, galactose, arabinose, and mannose. An ultraviolet (UV) absorbance detector (Waters 2487), operating at 210 nm wavelength, was used in series with a refractive index (RI) detector (Waters 2414).

Statistical Analysis

All experiments and analyses were performed in duplicate. The statistical analysis of the data was carried out using MINITAB® 17 (Minitab Ltd., Coventry, UK). The error bars and intervals reported in the text, tables, and graphs represent two times the standard deviation. The one-way ANOVA (analysis of variance) with a confidence interval of 95% was used for analyzing the results and pairwise comparisons were carried out according to Tukey’s test.

RESULTS AND DISCUSSION

Biological quality upgrading of BSG for feed application is a promising method for reducing the negative environmental impact of the brewing industry. Although BSG is a food-grade byproduct rich in nutrients with great potential for production of food products, most of the research attention has been directed to the production of chemicals. Hence, this study investigated the nutritional valorization of BSG through submerged cultivation of edible filamentous fungi. The industrial applicability of the process was considered during the process design. The submerged cultivation was chosen as the cultivation mode because it is a similar to the technology used in the beer industry and is easier to scale up than solid-state fermentation. Moreover, the proposed process was kept very simple, at a minimum number of stages, providing a higher chance of integration into an already established brewery. The submerged cultivation of three edible filamentous fungi, namely A. oryzaeN. intermedia, and R. delemar, are reported in this study. In addition to fungal strain, the effect of the addition of nutrients and cellulase on the concentration of recovered solids and respective protein content were evaluated. Finally, a discussion is provided on the implications of the proposed process through proposal of scenarios for process integration and future research studies.

Brewer’s Spent Grain as a Substrate for Fungal Cultivation

The main components of BSG were polysaccharides (64.9 ± 4.34%), proteins (22.65 ± 0.10%), and lignin (15.68 ± 0.20%). The main polysaccharides were glucan, accounting to 17.52 ± 1.43%, starch, accounting to 20.88 ± 0.10%, and xylan, accounting to16.30 ± 0.17% (Table 2).

The batch cultivation of BSG without the addition of nutrients resulted in recovery of solids ranging from 15.62 to 20.61 g/L. The concentrations of recovered solids and respective protein contents following cultivation of A. oryzae and R. delemar were not significantly different, while cultivation with N. intermedia led to the lowest concentration of recovered solids and respective protein content (p-value = 0.003 and 0.008, respectively) (Table 3). The fungal cultivation with each of the different filamentous fungal strains resulted in an increase in protein content in comparison with the initial protein content of BSG (Fig. 2). The cultivation with A. oryzae resulted in the highest protein content increment (19.43 ± 0.16%; p-value = 0.008) in comparison with the other two fungal strains.

Table 2. Chemical Composition (% w/w) of BSG and of the Recovered Solids from Cultivations with Three Fungal Strains and Addition of Supplementary Nutrients

AIL – acid-insoluble lignin; ASL – acid-soluble lignin