Solid-state fermented brewer's spent grain enzymatic extract increases in vitro and in vivo feed digestibility in European seabass

Brewer’s spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0—control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0—control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds.


Materials and methods
This study was approved by the CIIMAR Animal Welfare Committee (ORBEA) and by the Portuguese National Authority for Animal Health (DGAV); reference ORBEACIIMAR_27_2019) and was carried out according to ARRIVE guidelines 29  Solid-state fermentation of brewer's spent grain. Brewer's spent grain (BSG) was provided by UNICER S.A. (Porto, Portugal). BSG proximal composition is presented in Table 1. SSF of BSG was performed with Aspergillus ibericus (MUM 03.49), maintained at 4 °C in Potato Dextrose Agar medium until utilization. This fungus was selected as it has GRAS (Generally Regarded As Safe) status and a high ability to produce carbohydrases, namely xylanase and cellulase. The SSF was carried out using 4 trays (43 × 33 × 7 cm) as bioreactors. In each tray, 400 g of dried BSG (bed height 2.5 cm) was inoculated with a spore's suspension of A. ibericus with 10 6 spores' concentration. BSG humidity was then adjusted to 75%, followed by incubation at 25 °C for 7 days. At the end of SSF, water-soluble compounds of fermented BSG were recovered. For that, the fermented BSG was mixed with distilled water (1:5 w/v) under constant agitation for 30 min 31 , sieved (0.45 µm pore-size filter), centrifuged (7000g for 5 min), and the supernatant (crude extract) was recovered and lyophilized for 48 h. The xylanase and cellulase activities of the lyophilized fermented BSG extract (SSF-BSG extract) are presented in Table 1 www.nature.com/scientificreports/ Diets formulation. Six diets were formulated to be isoproteic (48% crude protein) and isolipidic (16% crude lipids), containing 15% of fishery products as protein source (fish meal and fish protein concentrate) and fish oil as the main lipid source. Chromium oxide was used as a digestibility marker. SSF-BSG extract was included in the diets at 0, 0.1, 0.2, and 0.4% of dry matter (control, BSG0.1, BSG0.2, and BSG0.4 diets, respectively), corresponding to a cellulase supplementation of 0, 1000, 2000, and 4000 U g −1 of diet (DM basis). Another BSG0.4 diet was formulated, including a commercial catalyzer (Silica+; CERESCO NUTRITION; inclusion level 0.02% of the diet) and a plant-derived detoxifier (Ena-Detox; EUROPEAN NATURAL ADDITIVES, inclusion level 0.07% of the diet) (diet BSG0.4+). For comparison purposes, a diet supplemented with a commercial carbohydrases-complex (endo-1,4-β-xylanase, and endo-1,4-β-glucanase complex; Natugrain TS, BASF, Germany) was formulated with a cellulase activity of 4000 U g −1 of diet (DM basis; NAT diet). The diets were produced as pellets according to Magalhães et al. 5 . The proximate composition of the control diet is presented in Table 2.
In vitro and in vivo trials performed are schematically indicated in Fig. 1 and described below.
In vitro digestibility assays. Three different in vitro assays were carried out to determine the: Assay 1 > effect of SSF-BSG extract on phosphorus (P) availability (total and phytate P) of control and BSG0.4 diets, measured by total P and phytate P release. The fish enzyme extracts used in all the in vitro digestibility assays were obtained from full-fed European seabass juveniles as described by Morales and Moyano 27 . Briefly, fish were starved for 6 h prior to sampling and sacrificed by immersion in ice-cold water. Fish extracts were obtained after individual homogenization of the stomach and proximal intestine with pyloric caeca with distilled water (1:10 w/v), followed by centrifugation (12,000 g, 3 °C, 15 min). Fish extracts were lyophilized and resuspended when needed. The fish enzymes extract:diet ratio used was 13,000 U g −1 and 6800 U g −1 for acid and alkaline digestion. This ratio was defined considering the average total acid and basic proteases produced by European seabass (with circa 50 g body weight) fed a 45% crude protein diet at a ration level of 3% of body weight.
All in vitro digestibility assays were performed using bioreactors modified from those described by Morales and Moyano 27 . The mixture of fish enzyme extracts and the desired diet is placed in the upper chamber, and the released products of hydrolysis (amino acids, pentoses, P, and others) pass through the membrane into the lower chamber, being recovered at different time intervals during the reaction time. Acid and alkaline stages of the hydrolysis were carried out at pH 5 and 8.5, respectively, according to the average pH measured in the stomach and intestine of fed European seabass (50 g average weight) 32 .
All in vitro digestibility assays were run in triplicate for each diet. At the beginning of each run, the diet, fish acid enzymatic extract, and acid buffer (0.2 M citrate buffer with NaCl 50 mM, pH 5) were placed in the upper chamber and maintained under continuous agitation (270 rpm) for 2 h. During this time, three samples were taken from the upper and lower chambers at 30 min, 1 h, and 2 h. Samples taken from the upper chamber were centrifuged (14,000 g, 4 °C, 15 min), and the supernatant was stored at − 20 °C for determination of residual enzyme activities. After acid digestion, the pH of the upper chamber was adjusted to 8.5 with NaOH 6 N, and the crude alkaline enzymatic extract was added. Subsequently, an alkaline buffer (0.01 M Borax-Boric buffer www.nature.com/scientificreports/ with 20 mM CaCl 2 and 45 µM taurocholate, pH 8.5) was added in the lower chamber to begin the alkaline digestion. Alkaline hydrolysis was maintained for 4 h, and a sample was taken every hour from the lower chamber. Moreover, at the end of the digestion, a sample was also collected from the upper chamber. The entire digestion process (6 h) was carried out at 25 °C, being stopped by adding trichloroacetic acid (20%) before the last sample collection. Specific assays aimed to evaluate the effect of SSF-BSG extract or commercial product (NAT) in the absence of active fish enzymes were carried out after inactivating fish enzyme extracts by heating at 100 °C for 30 min.
In vivo digestibility assay. Based on the in vitro assays results and to reduce the number of fish used in in vivo assays, the control, BSG0.1, and BSG0.4 diets were selected to be tested in an in vivo digestibility assay with European seabass juveniles. The assay was performed in a recirculating aquaculture system (RAS) equipped with 9 fiberglass tanks (60 l water capacity each) provided with a feces settling column connected to the outlet, designed according to Cho et al. 33 . During the assay, a constant water flow of 5 l min −1 was kept, the water temperature was regulated to 22 ± 1 °C, salinity averaged 32 ± 1 ‰, dissolved oxygen averaged 92% of saturation, ammonia and nitrites levels were kept below 0.02 mg ml −1 , and photoperiod was adjusted to 12 L: 12 D.
Nine groups of 15 fish (initial body weight of 22 ± 1 g) were randomly distributed to each tank, and each experimental diet was randomly assigned to triplicate groups. Fish were fed by hand, until apparent satiety, twice a day, 7 days a week, for 64 days. During the last 30 days of feeding, feces accumulated in each settling column were collected daily before the first meal. Feces were then centrifuged at 3000 g for 15 min, pooled for each tank, and stored at − 20 °C until further analysis. After the last daily meal, the tanks, pipes, and settlings columns were cleaned to remove remaining feces and uneaten feed. Apparent digestibility coefficients (ADC) of dry matter, protein, gross energy, starch, cellulose, hemicellulose, glucans, xylan, arabinan, and lignin of the experimental diets were calculated as follows: Physical-chemical analysis. Enzymatic activity analysis. Acid protease activity on fish stomach extracts used in the in vitro assays was measured 34 using 0.5% w/v hemoglobin in a glycine-HCl buffer (100 mM, pH 2.0) as substrate. Alkaline protease activity in intestinal extracts was measured 35 using 1% w/v casein dissolved on Tris-HCl buffer (100 mM + 10 mM CaCl 2 , pH 8) as substrate. For both assays, one unit of activity was defined as 1 µg of tyrosine released per minute. Xylanase and cellulase activities were measured in SSF-BSG extract and experimental diets, while β-glucosidase, phytase, proteases, and lipase activities were measured only in the experimental diets. Xylanase (endo-1,4-β-xylanase), cellulase (endo-1,4-β-glucanase), and β-glucosidase were measured according to Fernandes et al. 36 . www.nature.com/scientificreports/ Phytase activity was assessed 37 using sodium phytate in acetate buffer (0.2 M, pH 4.5) as substrate, and the activity was defined as the enzyme needed to release 1 µM of inorganic phosphate.
Protease and lipase activities were measured according to Charney and Tomarelli 38 and Gomes et al. 39 , respectively.
Analysis of products released during in vitro assays. In the in vitro digestibility assays, variations in phytate P, pentoses, and amino acids were measured as indicators of the hydrolysis of phytic acid, non-starch polysaccharides, and protein, respectively.
Phytate P was determined according to Haug and Lantzsch 40 . Pentoses were measured according to Douglas 41 . Total amino acids were quantified according to Church et al. 42 .

Chemical analyses of ingredients, experimental diets, and feces.
Chemical analyses were performed according to the Association of Official Analytical Chemists methods (AOAC79). Cellulose, hemicellulose, and lignin contents were determined according to Fernandes et al. 36 .
Statistical analysis. Before analysis, normality and homogeneity of variances were verified using the Shapiro-Wilk test. When necessary, data were normalized using log-transformation or arc-sin square-root transformation for percentage values.
In vitro assays data of total and phytate P release were evaluated using two-way ANOVA, with digestion stage (acid, alkaline, or total) and diets as factors. In vitro assay data of pentoses and amino acids release for each digestion stage (acid, alkaline, or total) were analyzed by two-way ANOVA, with diets and fish digestive enzymes as factors, followed by Bonferroni's multiple range test to detect significant differences among means (p < 0.05).
Data from in vivo digestibility assay was analyzed by one-way ANOVA, followed by Bonferroni's multiple range test to detect significant differences among means (p < 0.05). All statistical analyses were conducted using IBM SPSS Statistics software version 26 (IBM, NY, USA; https:// www. ibm. com/ produ cts/ spss-stati stics).

Results
Enzyme production by SSF of BSG. SSF of BSG with Aspergillus ibericus allowed the production of 50 g of lyophilized SSF-BSG extract per kg of BSG. The lyophilized SSF-BSG extract had 15,885 and 1343 U g −1 of xylanase and cellulase activities, respectively. The enzyme activity of the experimental diets reflected the amount of lyophilized SSF-BSG extract added to the diets. The NAT supplemented diet had no protease activity, and cellulase and xylanase activities of this diet were within the range values of the BSG0.2 diet, while β-glucosidase and phytase activities were within the range values of the BSG0.4 diet ( Table 1).
In vitro assays. In Assay 1, aimed to evaluate differences in bioavailability of total and phytate P for control and BSG0.4 diets, a steady release of total and phytate P was observed with time for both diets. However, the inclusion of 0.4% of SSF-BSG extract in the diet significantly increased the release of total P (Fig. 2, Table 3).
In Assay 2, designed to assess the interaction effect between endogenous (fish) and exogenous (SSF-BSG extract) enzymes on BSG0.4 diet carbohydrates hydrolysis, it was observed (i) a basal release of pentoses from the diet in the absence of any active enzymes; (ii) no significant effect of fish enzymes on such release; (iii) a fourfold increase in pentoses release with active relative to inactive BSG0.4 diet and; (iv) a 14% reduction on pentoses release when BSG0.4 diet was mixed with active fish enzymes compared to the amount released when fish enzymes were inactivated ( Table 4). The dynamic of pentoses release during the in vitro hydrolysis is shown in Fig. 3.
In Assay 3, designed to evaluate the effect of dietary inclusion of SSF-BSG extract or Natugrain on the hydrolysis of carbohydrates and protein in all experimental diets, it was observed (i) a positive effect of the       In vivo digestibility assay. The digestibility of dry matter, starch, cellulose, and glucans was significantly higher with the BSG0.4 diet than with the BSG0.1 or control diets. Energy digestibility was higher with the BSG0.4 diet than the control diet, but it was not significantly different from the BSG0.1 diet. The digestibility of hemicellulose, xylan, and arabinan tended to be higher with the BSG0.4 diet than in the other dietary treatments, though differences were not statistically significant (Table 7).

Discussion
Enzyme production by SSF of BSG. BSG is an underutilized agro-industrial by-product with the potential to replace high carbon footprint conventional feedstocks, given its high production rate, acquisition price, and supply chain 42 . However, for the moment, no "established market function" for BSG exist, and its high moisture content and low sugars content hinder the development of economic models for its valorisation 43 . Considering costs associated with BSG disposal, new and promising utilizations for this by-product must be envisioned to meet circular economy guidelines and ensure sustainability and food security. New and alternative applications for BSG are increasing, as construction materials, alternative fuels, textile applications, human food, and animal feeds 44 . www.nature.com/scientificreports/ The reutilization of BSG towards the obtention of value-added compounds following eco-friendly and circular economy approaches, such as SSF, is of utmost importance. BSG is a lignocellulosic by-product with 24% of hemicellulose and 21% of cellulose, with a high potential to produce carbohydrases by SSF, an under-explored process. In the present study, the biotechnological treatment of BSG through SSF with A. ibericus resulted in high xylanase and cellulase production (15,885 and 1343 U g −1 , respectively). Previously, SSF of BSG with Penicillium janczewskii produced 371 U xylanase g −1 BSG 23 , a lower value than obtained in the present study. These results confirm the potential of agro-industrial by-products to produce added-value compounds, such as enzymes, through SSF processes [45][46][47] .
The production of enzymes through SSF processes presents several advantages compared to SmF, including higher enzymatic productivity and stability within a broader range of conditions, and lower susceptibility to inactivation by the substrate 15 . The present study confirmed that the enzymes of SSF-BSG extract remained functional throughout the acid and alkaline digestion. Indeed, even though endogenous fish enzymes may negatively interact with the SSF-BSG extract activity, the in vitro carbohydrates bioavailability increased fourfold when comparing an active versus inactive SSF-BSG extract supplemented diet (BSG0.4 diet). Accordingly, Vasconcellos et al. compared the production of endoglucanase and β-glucosidase through SSF or SmF and concluded that SSF  In vitro and in vivo assays. Experimental approaches based on in vitro models simulating biological functions are increasingly encouraged to reduce the number of animals used for experimental purposes 27,49 and are in line with the eco-friendly approach of the present study. In vitro assays simulating the European seabass gut biochemical conditions and previously optimized for this species 27,28 , allowed a detailed assessment of the effect of SSF-BSG extract and Natugrain on nutrients bioavailability, the interactive effects of SSF-BSG extract and Natugrain with fish enzymes during digestion, and the fine-tune selection of the most potential experimental diets to be further evaluated during an in vivo digestibility trial. Phytate P is present in a wide range of plant feedstuffs but is unavailable for most fish species as they lack intestinal phytases, impairing mineral utilization, growth, and feed utilization. Phytate P can bind with minerals, such as calcium, zinc, sodium, and others, decreasing its solubility and, concomitantly, minerals bioavailability 50 . Dietary supplementation with phytase has been used as a nutritional strategy to increase phosphorus bioavailability for fish and other animals 50 . Even though A. ibericus used in the present study was selected based on its high capacity to produce carbohydrases, phytases were also produced during SSF of BSG. In vitro assay 1 confirms that supplementation of a plant-based diet with 0.4% of SSF-BSG extract significantly increased total P release but not that of phytate P. This result may be interpreted as an increase in the potential bioavailability of this element.
In vitro digestibility assay 2 confirms the potential of SSF-BSG extract to hydrolyze dietary carbohydrates. Dietary inclusion of 0.4% SSF-BSG extract (BSG0.4 diet) increased more than fourfold the pentoses release during in vitro digestion compared to the same diet in which the enzymatic activity was previously inactivated by heat. Moreover, combining active or inactive BSG0.4 diet with inactive fish enzymes during in vitro digestion did not affect pentoses release, corroborating the main role of SSF-BSG extract on carbohydrates hydrolysis. However, combining active BSG0.4 diet with active fish enzymes during in vitro acid or alkaline digestion decreased pentoses release by about 10% to 16%, respectively, suggesting a negative interaction between endogenous fish enzymes and SSF-BSG extract carbohydrases. The efficacy of feed enzymes is affected by different factors, including pH specificity and gastrointestinal inhibitors, limiting the potential benefits of dietary supplementation with exogenous enzymes 4,50 . For example, Morales et al. observed that both activity and stability of a fungal phytase (from Peniophora lycii) were significantly compromised when evaluating the in vitro acid digestion of rainbow trout (Oncorhynchus mykiss) 51 .
Moreover, in assay 2, the in vitro digestion of inactive BSG0.4 diet combined with inactive endogenous fish enzymes also resulted in pentoses release. Pentoses release without the presence of active carbohydrases indicates that some non-starch glucosidases may be present in plant feedstuffs and retained some activity after pelleting and digestion processes. In fact, cellulases have been found in various plants, being involved in the defense mechanisms, lignification process, cell-wall development, and symbiotic activities of the plant 52,53 .
The in vitro digestibility assay 3 confirms a dose-response effect of SSF-BSG extract on carbohydrates hydrolysis, as pentoses release linearly increased with dietary SSF-BSG extract inclusion level (R = 0.75; P < 0.05, including total digestion data with active or inactive fish enzymes). Moreover, dietary supplementation with 0.4% SSF-BSG extract combined with the commercial catalyzer, Silica+, and a plant-based detoxifier, Ena-Detox, promoted similar carbohydrates or protein hydrolysis than to the diet supplemented with 0.4% SSF-BSG extract alone. Contrarily to the present results, silicon dioxide has been utilized in poultry and pigs diets with promising results on growth performance and feed utilization 54,55 .
Additionally, the in vitro digestibility assay 3 confirmed that SSF-BSG extract enzymes exhibited carbohydrates and protein hydrolytic activity at both acid and alkaline pH (5 and 8.5, respectively), being more efficient during the alkaline than the acid stage. Although optimum enzyme activity occurs under certain pH and temperature conditions 8 , the higher carbohydrates hydrolysis observed during the alkaline digestion is not aligned to the optimum pH for maximum activity of carbohydrases produced in SSF by filamentous fungi. In general, cellulases produced by filamentous fungi have maximum activities at acidic pH (3.6-5) 56 . For example, xylanases www.nature.com/scientificreports/ produced by A. fumigatus showed higher activity at pH between 3 and 6 57 , and xylanases and β-glucosidases produced through the SSF of Nopalea cochenillifera with A. niger and Rhizopus sp. presented optimum activities at pH between 4 and 7 58 . Besides the pH and temperature effect, enzymes activity may also be affected by enzymes accessibility to the substrate. In the present study, the higher pentoses and amino acids release observed in the alkaline stage may have been triggered by the previous stomach digestion, which facilitated protein and carbohydrate hydrolysis during intestinal digestion by increasing substrate accessibility to the enzymes. Similarly, other authors also observed that the acid pre-digestion of fishmeal and plant feedstuffs increased intestinal protein hydrolysis 27,59 , reflecting the significant contribution of alkaline digestion in the total protein hydrolysis 60 .
In vitro trials are well suited to evaluate differences in the bioaccessibility of nutrients and the effect of diverse factors that may influence their potential bioavailability upon intestinal absorption. For this reason, in vitro trials provide a pre-absorption estimation of nutrient bioavailability, while in vivo digestibility measures are considered a post-absorption estimation of nutrients bioavailability, being affected by other factors such as microbial biomass or products of their metabolism present in fecal mass 28 . This could explain why not always both types of determinations present a good correlation. Nevertheless, the results of the in vivo digestibility trial of the present study corroborated to a great extent the results obtained in vitro. In both trials, dietary SSF-BSG extract supplementation did not significantly affect protein hydrolysis, whereas bioavailability of carbohydrates increased with 0.4% SSF-BSG extract incorporation. Dietary supplementation with 0.4% of SSF-BSG extract increased the pentoses release by circa 45% in the in vitro digestibility trial. Accordingly, in the in vivo digestibility trial, 0.4% SSF-BSG extract supplementation increased cellulose and hemicellulose digestibility by circa 52% and 16%, respectively, while xylan, glucan, and arabinan digestibility increased 20%, 16%, and 11%, respectively. For carnivorous fish, as European seabass, the digestibility of NSPs was never evaluated. Nevertheless, the digestion of some NSPs fractions has been reported for some non-carnivorous fish species. For example, in Nile tilapia (Oreochromis niloticus), depending on dietary fiber composition, NSPs digestibility averaged 24% 61 , ranging between 23 and 73% 62,63 , with soluble NSPs digestibility being 60% and insoluble NSPs undigested 64 . The beneficial effect of dietary supplementation with SSF-BSG extract on carbohydrates bioavailability of European seabass is probably not only related to NSPs digestion. The effect of SSF-BSG extract carbohydrases on the digesta physical properties, decreasing its viscosity, releasing entrapped nutrients by facilitating the fish enzymes action 4,7 , and promoting microbiota fermentation processes 3,61,65 may also have contributed to the present results. Fish microbiota plays a significant role in digestion and metabolism 66 and is modulated by dietary supplementation with exoenzymes, supporting the development of a more favorable microbiota community for nutrient hydrolysis 6,67 .
Present results confirmed that dietary incorporation of SSF-BSG extract is a promising nutritional tool to increase nutrient digestibility by hydrolyzing NSPs. The use of carbohydrases in fish feeds was revised by Castillo and Gatlin and Zheng et al. with promising results 4,8 . For example, in European seabass, dietary supplementation with a commercial carbohydrases complex increased dry matter, protein, lipids, energy, and phosphorus digestibilities, decreased chyme pH along the intestine but did not affect digestive enzymes activity 68 . In white seabream (Diplodus sargus), the same carbohydrases complex increased feed, nitrogen, and energy utilization efficiency as well as intestinal amylase and lipase activities 5 . This carbohydrases complex also increased dry matter and some amino acids digestibility, intestinal lipase, and protease activity, and modulated intestinal microbiota in turbot (Scophthalmus maximus) 6 . Dietary supplementation with cellulase in a Chlorella-based diet increased dry matter, energy, and protein digestibility, digestive enzymes activity, and growth performance of crucian carp (Carassius auratus) 69 . In juvenile yellow croaker (Larimichthys crocea), dietary supplementation with xylanase improved zootechnical performance, intestinal morphology structure, and microbiota constitution 70 . An enzymatic-rich extract produced from SSF of agro-industrial by-products with Aspergillus niger and A. oryzae increased protein, mineral, energy, and lipids digestibility of Nile tilapia 71 .

Conclusions
SSF of BSG with Aspergillus ibericus allowed the production of highly active enzymatic enriched extract. The in vitro assays confirmed that the inclusion of 0.4% SSF-BSG extract in a plant-based diet for European seabass increases phosphorus and carbohydrates hydrolysis, being more effective during the alkaline than the acid stage of digestion. Fish endogenous enzymes reduce the efficacy of SSF-BSG extract by about 7% to 16%. The in vivo digestibility assay results corroborated the data obtained in in vitro assays. Dietary supplementation with 0.4% SSF-BSG extract increased digestibility of dry matter, energy, starch, cellulose, and glucans. This study also highlighted the partial digestibility of NSPs in plant-based diets for carnivorous fish, requiring further research.
Overall, the results showed the potential of SSF of BSG to produce an added-value enzymatic supplement that increases the bioavailability of plant ingredients. Additionally, the use of BSG, an underutilized agro-industrial by-product, as substrate for filamentous fungi enzymes production, represents a synergy between economic and environmental efforts under the circular economy guidelines. Further research is needed to develop technological strategies to incorporate the SSF-BSG extract into aquafeeds, including coating, to reduce the negative interaction with fish enzymes, and assess the economic and environmental impact of SSF-BSG extract utilization.