Conversion of brewers’ spent grain into proteinaceous animal feed using solid state fermentation

Brewers’ spent grain (BSG) represents the 85% of the total residue produced during the beer brewing process, with a global annual production volume exceeding 30 Mtons. The current study concerns the application of solid state fermentation (SSF) as a bioprocess where the nutritional value of BSG is improved for further use as animal feed with increased value. The investigated SSF procedure was initiated by the edible fungi Pleurotus ostreatus, which constitutes a natural source of proteins, β-glucans, and various metabolites (vitamins, nutrients, etc.). Herein, the SSF of BSG resulted in a significant increase of protein content by 49.49%, a 10-fold increase of 1,3-1,6 β-glucans, and a respective reduction of cellulose by 11.42%. The application of this method is expected to provide some useful information on the utilization of BSG as substrate for fungi-initiated SSF, a bioprocess allowing the significant reduction of the environmental impact caused by the beer brewing industry and simultaneously producing animal feed with higher protein content and improved nutritional characteristics. Such studies contribute to confront the unavailability of proteinaceous animal feed observed in the last decade.


Introduction
The adoption of the European Strategy and Action Plan for the Circular Economy in 2015 initiated a vigorous search towards the exploitation of more efficient utilization means for natural resources and anthropogenic products in terms of an environmentally oriented economy (https://ec.europa.eu/info/ research-and-innovation/research-area/environment/ bioeconomy_en). In this respect, a recent global trend towards the reduction of environmental pollution caused by the remaining and/or by-products of industrial activities is being arisen. Among the broad variety of diverse industrial residues, those of the agro-industrial activities that characterized by lignocellulosic materials present an intriguing case. They are produced in large quantities and are recyclable or reusable through the application of appropriate techniques and procedures of circular economy. In addition, their nutritional composition justifies their characterization as raw materials and not as wastes or by-products, since the lignocellulosic residues are mainly composed of cellulose, hemicellulose, and lignin. These constituents are also indicative of their suitability for serving as substrates of solid state fermentation (SSF), a procedure that can be initiated by various fungi (Mussatto et al. 2006;Ritota and Manzi 2019), such as the white rot fungi Pleurotus ostreatus. The latter is well known for the degradation of various lignocellulosic materials and their transformation into useful substrates. In this context, P. ostreatus is an edible basidiomycete, displaying the capability of colonizing and degrading lignocellulosic materials by secreting lignocellulolytic enzymes, which are classified as hydrolytic (cellobiohydrolases, endoglucanases, β-glucosidases) and oxidative enzymes (laccases, lignin peroxidases, manganese peroxidases) (Akinfemi et al. 2010;Han et al. 2020). Their ability of initiating bioconversion via a SSF process has been previously utilized for the production of high added value products, such as antibiotics, vitamins amino acids, enzymes, biofuels, organic acids, and biopolymers (Cooray and Chenc 2018;Liguori et al. 2015).
Beer is considered as the fifth most consumed beverage worldwide, following tea, coffee, soda carbonates, and milk. For 2018 global beer's annual production reached almost 182 Mtons, whereas Europe's production was almost 52 Mtons (FAOSTAT 2019). The brewing procedure produces large amounts of wastes, with prevailing by-product the brewers' spent grain (BSG), which represents almost the 85% of the total brewing industry residues. BSG is derived from malted grain, the main raw material of brewing industry (Khidzir et al. 2010). It must be noted that the annual production of BSG exceeds 30 Mtons, since every hectoliter of beer results in the production of 15-20 kg of BSG (Radosavljevic et al. 2017). The annual European production of BSG is estimated to be 3.4 Mtons, from which 2 Mtons are produced in Germany and 288,000 in Italy (Bianco et al. 2020).
BSG's disposal is a high-cost procedure and may constitute a potential environmental hazard due to its chemical composition. In particular, the high moisture (approximately 70%) and nutritional content, makes BSG an ideal and available substrate for microbial contamination, initiated mainly by filamentous fungi almost immediately after its production. This situation poses environmental risks, regarding the presence of large populations of bacteria migrating and growing in the environment. Additionally, every ton of BSG which is disposed to the environment contributes to the emission of 513 kg CO 2 equivalent to greenhouse gases (Kavalopoulos et al. 2021;Mathias et al. 2014).
After the mashing process, all the exploitable ingredients are extracted. Nevertheless, the remaining barley is still consisted of digestible fibers and protein justifying the utilization of spent grains in agricultural applications (Westendorf and Wohlt 2002). On the other hand, the BSG content of exploitable compounds indicates its potential use for the production of high added value commodities, such as novel proteinaceous animal feed, enriched with bioactive compounds (Cooray and Chenc 2018).
To date, 70% of BSG is used as animal feed, 10% for the production of biogas, and 20% is disposed as landfills (Bianco et al. 2020). Its precise chemical composition depends on the variety of barley, the harvest period, and the applied malting and mashing processes (Liguori et al. 2015). Furthermore, BSG is rich in fibers (cellulose, hemicellulose, and lignin), proteins, essential amino acids, minerals and antioxidants (polyphenols, flavonoids), vitamins, and lipids (Bianco et al. 2020;Cooray and Chenc 2018). Currently, brewery waste is mainly used as a low nutritional value animal feed of low economic cost, since BSG constitutes a source of lower protein quality content compared to other proteinaceous supplements such as soybean meal, fishmeal, and milk. On the other hand, its unique nutritional profile consisted of Ca, Na, and K justifies its utilization as predominant material that should be combined with other cereal grains, forages, and protein supplements for the creation of feedstuff with high impact on animals' growth (Westendorf and Wohlt 2002). Thus, there is an emerge for the upgrade of its nutritional content, in order to obtain higher commercial value and simultaneously provide animals with nutrients and bioactive compounds. In this respect, the increased protein content, improved amino acid profile and enhanced β-glucans content contribute in animals' good health and welfare. These constituents also contribute on the improvement of the quality value of produced meat. As it was already mentioned, the lignocellulosic nature of BSG highlights its suitability as substrate for SSF and its colonization by fungi, bacteria, or microorganisms (Ritota and Manzi 2019;Tan et al. 2019). This constitutes an environmentally friendly process that is capable of upgrading raw materials to overcome the problem of low protein content and high lignin concentration. So far in the literature, there is limited information regarding the upgrading of BSG by SSF focusing mainly on the fungus Rhizopus. The aim of the present study is to investigate an exploitation pathway of BSG by its conversion into proteinaceous animal feed by SSF with the fungus P. ostreatus. The study herein highlights the proteins' increase, as well as the presence of β-glucans, indicating BSG potential as a novel animal feed with high nutritional content and value.

Materials and microorganisms
BSG substrate for the growth of P. ostreatus was kindly provided by the Athenian Brewery, one of the biggest beer industries of Greece. To achieve the optimum moisture content (approximately 70%) for an unhindered SSF process, tap water was added and renewed every day. All samples were weighed to a final weight of 200g (fresh matter). The substrate was placed into test vessels of 750 ml volume and sterilized at 121°C for 15min. P. ostreatus White 2000 P67 LOTTO 1551 MN 01827 strain was used for the inoculation of substrates. Throughout all experiments, the fungi stock cultures were stored at 4°C.

Inoculation and solid state fermentation
Inoculation was performed under aseptic conditions. Inoculum of P. ostreatus strain (5% w/w) was added at substrate's surface and transferred into a bioclimatic chamber with stable temperatures of 25°C and 60% humidity. The total incubation time was 12 days, while samples of day 0, day 2, day 4, day 6, day 9, and day 12 were taken for analyses purposes. Every sample was prepared and studied in triplicate.

Analytical methods
Moisture, protein content, and ashes were determined according to AOAC methods (AOAC 1995). Total soluble sugars were determined according to method developed by Dubois et al. (1951), and reducing sugars were determined according to Miller (1959). The determinations of sugars were performed on the aqueous extracts of the respective samples. Crude fiber substances content was determined in accordance with AOAC method (AOAC 1995). Cellulose and lignin presence were evaluated according to the acid-detergent fiber (ADF) method (AOAC 1995). Finally, β-glucans were assessed using the Megazyme enzymatic assay kits (β-Glucan Assay Kit (Mixed Linkage), Megazyme Product code: K-BGLU and β-Glucan Assay Kit Yeast & Mushroom, Megazyme Product code: K-YBGL). The results of the aforementioned analyzes are expressed as g/100g of dry weight.

Statistical analysis
All analyses were performed in triplicates, and the results were expressed in means ± standard deviation (± S.D.). Data normality was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk test. The differences between the groups were analyzed by paired t-test (p ≤ 0.05 was considered significant). To all proportions of both substrates, a statistical analysis was performed between day 0 and day 12. Figure 1 illustrates the gradual mycelium growth during days 2 to 12, while Table 1 presents the physicochemical parameters (moisture content, total and reducing soluble sugars concentration, and ash content) of the examined substrates, which were analyzed throughout all stages of mycelium growth, from the beginning (day 0) until the end (day 12) of the fermentation process. As shown in Table 1, the moisture content, although fluctuated during incubation, remained practically unaffected since only a slight increase (no statistically significant, p ≥ 0.05) was observed between days 0 and 12. The concentration of the total soluble sugars endured a statistically significant decrease (p ≤0.05), reduced from 10.34 to 7.66% from day 0 to day 12. A similar pattern was observed for the reducing soluble sugars content, since a statistically significant decrease (p ≤ 0.05) by 41.91% was observed between days 0 and 12. Finally, the ash content displayed a 2fold significant increase (p ≤ 0.05), as the ash content ranged from 1.13 to 2.27% between days 0 and 12. Statistical analysis was performed between day 0 and day 12 Figure 1. Representative images of stages of mycelial growth during days 2 to 12 of fermentation As shown in Figure 2, protein content (%DW) increased gradually during the incubation period. More specifically, a statistically significant increase (p ≤ 0.05) by 49.49% was observed, since the protein content reached 25.01% from 16.73 to between days 0 and 12. According to Figure 3, the crude fiber substances content was practically unaffected (no statistical significance; p ≤0.05) by the process. In particular, crude fiber substances content had a slight increase of 9.24%, i.e., from 13.63 to 14.89% between days 0 and 12. Figure 2. Assessment of protein content. Differences between same symbols represent significance >5%. Statistical analysis was performed between day 0 and day 12 Figure 3. Assessment of crude fiber substances content. Differences between same symbols represent significance >5%. Statistical analysis was performed between day 0 and day 12

Results
The cellulose content displayed a fluctuation during the process. In particular, cellulose content was found to be slightly reduced by 11.42% without however having statistical significant (p ≤0.05) differences, reaching 21.10% (day 12) from 18.69% (day 0). According to Figure 5, lignin concentration resulted to a slight increase by 27.59% at the end of fermentation without statistical significance (p ≤ 0.05). Lignin content increased slightly from 5.58 to 7.12% between days 0 and 12 by presenting the highest value. Figure 4. Evaluation of cellulose content. Differences between same symbols represent significance >5%. Statistical analysis was performed between day 0 and day 12 Figure 5. Evaluation of lignin content. Differences between same symbols represent significance >5%. Statistical analysis was performed between day 0 and day 12 As presented in Table 2, 1,3-1,4 β-glucans content was reduced between days 0 and 12, whereas the presence of 1,3-1,6 β-glucans was increased. In particular, the 1,3-1,4 βglucans content ranged from 3.24 (w/w) to 0.78% (w/w) between days 0 and 12 presenting a statistically significant reduction (p ≤ 0.05) of 75.92%, while on the contrary 1,3-1,6 βglucans content resulted to a statistically significant 10-fold increase (p ≤0.05) on the respective days increasing from 0.1 (w/w) to 11.68% (w/w).

Discussion
Due to the significant concern regarding the use of meat and soy flour derived from genetically modified varieties, the search for proteinaceous supplements is essential, especially when simultaneously contributes to agro-industrial waste management. It is crucial to find reliable components as alternatives of animal feed, which should be economically viable but also environmentally friendly. Candidate ingredients should have specific nutritional characteristics, such as high protein content, favorable amino acid profile, high digestibility, good taste, but also low levels of indigestible fibrous substances, starch, and anti-nutrients. Study herein aimed the utilization of BSG as substrate for the solid state fermentation process initiated by P. ostreatus. The final goal of the study was the evaluation of fermentation products as proteinaceous animal feed. In this respect, BSG's moisture content constitutes a crucial factor for fungi growth, since the increased moisture content inhibits the oxygen transfer, generating concurrently a suitable environment for contamination. On the contrary, low moisture levels prevent the fungi/microbial growth and enzyme production and confines the nutrition availability (He et al. 2019). In our experiments, the moisture content always maintained around 76%, a value that is in line with previous literature reports (Khidzir et al. 2010;Mussatto et al. 2006;Wang et al. 2001;Xiros and Christakopoulos 2012). On the other hand, the most crucial variable for BSG upgrade is the protein content of the fermentation outcome. Previous studies have demonstrated the ability of fungi-initiated SSF procedure to increase the protein content. In this respect, Xiros and Christakopoulos (2012) have reported that the initial protein content of unfermented BSG, which varied between 10 and 30%, was significantly increased upon SSF initiated by P. ostreatus, Trichoderma pseudokoningii, and Rhizopus sp., respectively (Wang et al. 2001;Bayitse et al. 2015;Ibarruri et al. 2019). These findings are in accordance with our results indicating that the protein content was gradually increased during incubation to conclusively result in a statistically significant increase at the end of fermentation (day 12). In similar studies, according to Akinfemi et al. (2010), proteins' content was increased probably because of the excretion of certain extra cellular enzymes which are proteinaceous in nature into the waste during their breakdown and its subsequent metabolism. Additionally, proteins' increase could probably be explained by the intake of nitrogen excess via aerobic fermentation. Darwish et al. (2012) and Terrasan and Carmona (2015) have reported that proteins' content increase is probably due the fungal biomass accumulation.
BSG is considered that constitutes a source of lower quality protein content as compared to other proteinaceous supplements such as soybean meal, fishmeal, and milk (Westendorf and Wohlt 2002). This upgrade through SSF could probably improve its amino acid profile by upgrading the protein content, thus recommending the treated BSG as suitable and enriched with proteins substrate for livestock. On the other hand, the content of crude fiber substances varied between days 0 and 12, recording a slight increase at the end of fermentation (day 12), possibly as a result of a parallel lignin increase. Darwish et al. (2012) and Ritota and Manzi (2019) noted that Pleurotus spp. excrete hydrolytic enzymes (lignin peroxidase and manganese peroxidase) displaying the potential of degrading lignocellulosic raw materials, resulting to their improved digestibility (Akinfemi et al. 2010). Herein, the cellulose content was diminished without significance, whereas the lignin content showed a slight increase at the end of fermentation. It needs to be noted that fibers' concentration affects the feed conversion rate. According to Lao et al. (2020), growing/ finishing pigs that were fed with BSG up to 23% did not result to a significant gain reduction or a decline in quality's carcass.
The augmented BSG's ratio, over more than 6%, initially increased fiber content but concurrently caused a decreased conversion rate of feed in pigs. Thus, a decreased performance was observed, with the authors concluding that, in order to have an essential management, BSG could be absorbed at a rate up to 50%, fulfilling the additional protein needs without a decrease in performance (Westendorf and Wohlt 2002). Thus, the present study highlights the importance of BSG's biotransformation by SSF where the entire BSG's proportion can be used since a reduction of cellulose is observed.
Both total and reducing soluble sugar concentrations varied until day 6, and then they were gradually diminished until the end of fermentation. This may be rationalized considering that P. ostreatus consumed fermentable sugars as an energy source for its growth.
Barley and mushrooms are composed by many bioactive compounds such as β-glucans, which constitute polysaccharides composed by D-glucose monomers linked through β-glycosidic bonds. β-glucans from barley consist of 1,3-1,4 bonds, while β-glucans originated from mushrooms consist of 1,3-1,6 Table 2. Assessment of β-glucans content Β-glucans content (g/100g) Day 0 Day 12 1,3-1,4 β-glucans 3.24±0.07 a 0.78±0.09 b 1,3-1,6 β-glucans 0.1±0.01 a 11.68±0.84 b Differences between same symbols represent significance >5% Statistical analysis was performed between day 0 and day 12 linkages (Jin et al. 2004;Zhu et al. 2015). Previous studies have displayed the β-glucans beneficial impacts on human and animal health, which vary due to the differences between their linkage bonds. Barley's β-glucans have been displayed on the list of the European Food Safety Authority (EFSA) by claiming that they can exhibit positive health effects under certain circumstances. Furthermore, the consumption of (1,3-1,4) β-glucans is associated with a decrease of blood cholesterol (Steiner et al. 2015;Zhu et al. 2015). Additionally, β-glucans derived from mushrooms are known for their antitumor and immunestimulating properties that are responsible for health promotion and welfare. β-glucans derived from barley are the predominant constituent (70%) of barley endosperm cell walls. According to previous literature regarding brewing, barley's β-glucans content ranged from 2 to 6% (w/w) (Jin et al. 2004), which is also in line with the results of the present work. Additionally, the content of 1,3-1,4 β-glucans derived herein from BSG was metabolized and therefore resulted in a significant reduction between days 0 and 12, while the content of 1,3-1,6 β-glucans was significantly increased. This notable increase of 1,3-1,6 β-glucans content was due to the fungal growth. Finally, the fungal growth was achieved by breaking the cell walls, utilizing enzymes such as cellulases and glucosidases which resulted to reducing sugars release. P. ostreatus consumed these fermentable sugars as an energy source as well as structural components. Overall, according to Ibarruri et al. 2019, BSG proteins increased by 54% ranging from 20.5 to 31.7% via SSF with Rhizopus. Nevertheless, BSG used in this study had lower initial protein concentration probably due to its variety or cultivation practices; a similar pattern was observed, via SSF initiated by P. ostreatus demonstrated a notable increase of 50% ranging from 16.73 to 25.01%. The commercial price of a supplement as animal feed is inextricably linked on its protein content, so any bioprocess that contributes to proteins' increment impacts on its augmented commercial price. BSG is not considered as an ideal option used in animal feeding, fact that justifies its low commercial price. It needs to be mentioned that BSG's indicative commercial price as feedstock in the US reaches $40.23/ ton which is lower compared to the other cellulosic materials by their commercial value fluctuating between $50 and 150/ton. This difference is due to BSG's high moisture content which poses several management issues. This can be exemplified by assuming that if other lignocellulosic materials' moisture content is 10%, their economic cost would range from $57.2 to $171 in dry tons. Concerning BSG's moisture content which is approximately 70%, the economic value in dry tons reaches $134.10. Hence, the beer manufacturers are willing to sell it without any processing "as-is" (Buffington 2014). SSF is a process which upgrades its quality characteristics such as proteins, by consequently increasing the final commercial price and making BSG an attractive possible animal feed. BSG's high moisture content is the most important issue. By drying BSG which is considered as the most efficient conservation method, moisture content is reduced resulting in a more convenient management regarding storage and/or transportation since BSG's weight and volume are not increased. Drying cost has to be included on BSG's commercial price. According to the latter, breweries facilities constitute the most suitable places for this activity from the aspect that BSG is placed, and there is no transportation needed (Mathias et al. 2014). Also, BSG's bioconversion through SSF initiated by filamentous fungi such as P. ostreatus may form the basis for a new profitable activity in the feed sector. Finally, BSG is consisted of 1,3-1,4 β-glucans which contribute to the cholesterol and blood glucose regulation. SSF initiated by P. ostreatus enriched BSG with 1,3-1,6 β-glucans, which impact to the immunostimulation and the animals' good health and welfare. This procedure generates a novel fermentation outcome as a proteinaceous animal feed consisted of 1,3-1,4 and 1,3-1,6 β-glucans contributing to the economic and environmental impact in the context of the circular economy.

Conclusion
In conclusion, the current study aimed to the valorization of BSG bioconversion to a novel proteinaceous animal feed. According to the results, BSG bioconversion into a high added value product through SSF with P. ostreatus enhanced its nutritional value, displaying, thus, its potential use for animal nutrition. SSF initiated by P. ostreatus, using exclusively BSG as substrate, increased protein content by 49.49%, reduced cellulose concentration of 11.42%, and enriched BSG with a significant amount of 1,3-1,6 β-glucans of 11.68g, indicating its potential as animal feed, since so far it was used for different commercial purposes. Furthermore, from an economic point of view, the current study highlighted BSG's potential for augmented commercial price which is in the line/related with the protein's content increase achieved by SSF bioprocess. Overall, BSG is strongly recommended for exploitation in the field of animal nutrition for its physicochemical properties as well as for reducing its environmental impact in the context of the circular economy.
Availability of data and materials All data generated or analyzed during this study are included in this published article.
Author contribution CE conducted the experiments, analyzed the data, discussed the results, and was a major contributor in writing the manuscript. DA had the idea for the article, designed the experiments, discussed the results, and was a major contributor in writing the manuscript. NC conducted the literature survey, analyzed the data, discussed the results, and was a major contributor in writing the manuscript. GM conducted the literature survey, analyzed the data, discussed the results, and was a major contributor in writing the manuscript. SAH had the general supervision and revised the manuscript. All authors read and approved the final manuscript.
Funding This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T1EDK-04331).

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable.

Competing interests
The authors declare no competing interests.