1. Introduction
Industrial production of bioethanol using cereal grains has increased significantly as a renewable fuel source [
1,
2]. Global ethanol production has registered a steady increase in the past few years, reaching 103 million liters in 2021 [
3]. This large-scale industrial operation generates a high amount of by-products, the main one being distiller’s dried grains with solubles (DDGS) [
4,
5,
6,
7]. Corn is the most used cereal in ethanol production [
8]. For each 3 kg of fermented corn, circa 1.4 L of ethanol and 1 kg of DDGS are produced [
9].
Aquaculture has a crucial role in global food security, being responsible for providing 87.5 million tons of aquatic animals, representing 49.2% of the total fishery production in 2020 [
10]. Until recently, aquaculture’s sustainability was threatened by its reliance on fisheries feedstuffs, mainly fishmeal and fish oil, used in aquafeeds. However, research efforts have led to a significant reduction in fishmeal and fish oil use with the increased utilization of feedstuffs [
11]. Many traditional agricultural feedstuffs used in aquafeeds, such as soybean, rapeseed, corn, and wheat, are internationally traded, used for direct human consumption, and therefore expensive, involving the use of arable land, water, and fertilizers. In order to foster the sustainability and cost-efficiency of aquafeeds, agro-industrial by-products should be explored as new and underutilized feedstuffs.
Corn DDGS is a granular by-product containing approximately 30–32% crude protein, 3–12% lipids, 8–11% fiber, and a higher energy value than corn, depending on the grain composition and the processing methods [
12,
13,
14,
15]. Corn DDGS color varies from light yellow to dark brown [
16]. The dark brown color may be associated with the increased CDS levels added to WDG, increasing energy, crude lipid, and mineral content. However, the dark color may also be associated with overheating during the drying process of DDGS, reducing lysine digestibility [
17]. Corn DDGS also holds spent yeast and yeast cell walls rich in beta-glucans and nucleotides, with potential functional properties in fish [
18].
Corn DDGS is already considered an efficient protein source for poultry, swine, and beef [
19,
20]. Dietary inclusion of up to 20% DDGS did not affect swine growth performance [
21] or milk production in dairy cattle. For beef cattle, DDGS may replace up to 40% of corn grain [
19]. The use of DDGS in aquafeeds also showed promising results, particularly for omnivore species. Corn DDGS may be included up to 30% and 40% in diets for hybrid tilapia,
Oreochromis spp. [
22] and Nile tilapia,
Oreochromis niloticus, respectively, without affecting growth or feed efficiency [
23]. The replacement of up to 30% of soybean and corn meals by DDGS, without lysine supplementation, did not decrease the growth and feed utilization of hybrid channel catfish juveniles,
Ictalurus punctatus × blue catfish
I. furcatus [
24], while the dietary supplementation with lysine increased the DDGS inclusion level up to 40% [
25]. The use of DDGS in diets for carnivorous fish is more limited, averaging 15% for rainbow trout,
Oncorhynchus mykiss [
26], although the inclusion level may increase when combined with amino acid supplementation [
23,
26]. In gilthead seabream,
Sparus aurata, voluntary feed intake, feed efficiency, or growth performance were not affected by the total dietary replacement of soybean meal with 35% of DDGS [
27]. However, for turbot,
Scophthalmus maximus, even low dietary inclusion of levels of DDGS, from 10 to 25% diet, reduced daily growth index and impaired nitrogen and energy metabolism [
28].
New approaches and technologies have been developed to potentiate DDGS use in aquafeeds, including high-protein DDGS [
29,
30] or dietary supplementation with commercial exoenzymes [
26,
29,
31]. DDGS is a suitable substrate for microbial fermentation [
20], so solid-state fermentation (SSF) may also be applied to increase its nutritional profile. SSF is a bioprocess with various applications, including animal nutrition [
32]. This type of fermentation is an ecological and economic bioprocess that uses low water volumes [
33] and cellulosic materials as substrates for microbial growth, such as agro-industrial by-products such as DDGS [
34]. The low free water and high moisture content during SSF make it more adequate for actinomyces and fungi growth [
35] since filamentous fungi, with hyphal growth and excellent tolerance to low water activity, are specially adapted to this process. During SSF, microorganisms produce hydrolytic enzymes, including lignocellulolytic enzymes [
36,
37]. The SSF of DDGS may promote hydrolysis is non-starch polysaccharides (NSP), releasing sugars and reducing the DDGS fiber content [
38], increasing its nutritional composition and potentiating DDGS utilization in aquafeeds [
39].
The present study aimed to improve DDGS nutritional value by applying SSF with three
Aspergillus sp. fungi, namely:
A. carbonarius, one of the most widely used fungi in biotechnological processes [
40];
A. ibericus, a recently discovered strain of the nigri section [
41]; and
A. uvarum. From these three fungi, the one resulting in a fermented DDGS with higher protein content and lignocellulolytic enzyme activity was selected. The fermentation process was then scaled up to include fermented DDGS in fish diets. The digestibility of diets containing both unfermented and fermented DDGS was determined in a digestibility trial with European seabass juveniles,
Dicentrarchus labrax, also assessing digestive enzyme activities.
2. Material and Methods
2.1. Solid-State Fermentation (SSF)
Three fungi from the Aspergillus section Nigri were obtained from Micoteca of the University of Minho (Braga, Portugal) and utilized in SSF: Aspergillus carbonarius 04.43, Aspergillus ibericus MUM 03.49, Aspergillus uvarum MUM 08.01. Fungi (preserved at −80 °C in glycerol) were revived in malt extract agar (MEA) plates (2% malt extract, 2% glucose, 0.1% peptone, and 2% agar), transferred to MEA slants, incubated for 7 days at 25 °C, and then stored at 4 °C until SSF.
Reduced-oil corn DDGS was used in the present trial. SSF of corn DDGS with each fungi species was performed in triplicate in 500 mL cotton-plugged Erlenmeyer flasks. SSF was carried out for 7 days at 30 °C using autoclaved corn DDGS (121 °C, 15 min), and humidity was adjusted to 75% (w/w wet basis). A sterile spores solution (0.1% peptone and 0.001% Tween-80) was added to the MEA slants. The spore concentration in each flask was adjusted to 106 spores/mL with a Neubauer counting chamber. The mixtures were incubated for 7 days at 25 °C. At the end of SSF, the fermented DDGS was frozen until the chemical composition and enzymatic and phenolic activity were analyzed. For enzymatic and phenolic activity analysis, an aqueous extraction was carried out in the fermented DDGS using a solution containing 1% NaCl and 0.5% Triton X-100 with a ratio of 1 g dry fermented solid to 5 mL of solution (1:5, w/v).
2.2. SSF Scale-Up
SSF of DDGS was scaled up to be included in the diets used in the digestibility trial with European seabass. The SSF scale-up was performed in tray-type bioreactors (trays measuring 43 × 33 × 7 cm) using the experimental conditions mentioned in
Section 2.1. Afterward, the fermented DDGS (SSF-DDGS) was dried at 40 °C for 24 h to reduce the moisture content to 10–15% to be included in the experimental diets.
2.3. Experimental Diets
The chemical composition and enzymatic activity of reference ingredients are described in
Table 1. A reference diet was formulated to include approximately 48% crude protein, 15% crude lipids, and an inert digestibility marker (chromium oxide). Two test diets were prepared by mixing 30% DDGS or SSF-DDGS with 70% of the reference diet (DDGS and SSF-DDGS diets, respectively). Dietary ingredients were grounded, mixed, and dry pelleted in a laboratory pellet mill (California Pellet Mill, Crawfordsville, IN, USA) through a 4 mm die. The resultant pellets were then dried at 40 °C for approximately 24 h and stored at −4 °C until further use. The formulation and composition of the reference and test diets are presented in
Table 2.
2.4. Digestibility Trial
The digestibility trial was carried out at CIIMAR’s Aquatic Organisms Bioterium (BOGA) under controlled conditions following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) Guidelines for Reporting Animal Research. BOGA is an aquatic animal facility that is accredited by the Direcção Geral de Alimentação e Veterinária (DGAV), the Portuguese National Authority for Animal Health, as a breeder and user establishment, according to National Decree (Decreto Lei n.° 113/2013).
This trial was subjected to an ethical review process by the CIIMAR animal welfare body (ORBEA-CIIMAR; reference ORBEA_CIIMAR_27_2019) and further approved by DGAV. All procedures were performed by certified scientists in compliance with the guidelines of the European Union (directive 2010/63/EU) and Portuguese law (Decreto Lei no. 113/2013, de 7 de Agosto) for the protection of animals used for scientific purposes.
European seabass (
Dicentrarchus labrax) juveniles were acquired at Maresa (Spain). After transport, fish were held in quarantine for 15 days. Fish were then transferred to the experimental system consisting of a thermo-regulated recirculation water system with nine 60 L fiberglass tanks, each equipped with fecal collectors (quadrangular tank; 62 × 42 cm; [
42]). Seawater flow was maintained at 4–5 L/min, 22 ± 1 °C, 34 ± 1‰ of salinity, and 90% oxygen saturation. Nitrogenous compounds were kept below 0.02 mg/L. Fish were subjected to 12 h light and 12 h dark.
At the beginning of the trial, nine homogenous groups of five European seabass were established (initial average weight of 171 g). Experimental diets were randomly assigned to triplicate groups, and fish were fed twice a day until apparent satiation, seven days a week. After an adaptation period of 7 days, feces were collected daily for 45 days. The fecal collectors were emptied each day before the morning meal. Collected fecal samples were centrifuged (3000 g, 10 min), separated from supernatant, pooled for each tank, and stored at −20 °C until the end of the trial. Thirty minutes after the last daily meal, uneaten feed and feces were removed by draining one-third of the water in the tanks.
Apparent digestibility coefficients (ADCs) of dry and organic matter, protein, lipids, starch, energy, and phosphorus of the experimental diets were determined using the following formula:
The apparent digestibility coefficients of the test ingredients, namely DDGS and SSF-DDGS, were determined according to Bureau et al. (1999) with the following formula:
where
Dref = % nutrient (or kJ/g) of reference diet (dry matter basis), and
Dingr. = % nutrient (or kJ/g) of experimental ingredients (dry matter basis).
At the end of the trial, intestines were sampled to measure digestive enzyme activity. Sampling was conducted 4 h after feeding to ensure full intestines, using 3 fish per tank. Fish were killed by anesthetic overdose (10 mL/L of ethylene glycol monophenyl ether) followed by decapitation. The gastrointestinal tract was then dissected on chilled trays, cleaned of adipose and connective tissue, and separated from the stomach. The intestine with pyloric caeca was divided into two portions, the anterior and distal intestine, and stored at −80 °C until enzymatic activity analysis.
2.5. Chemical Analyses
Chemical analyses of ingredients, including fishmeal, DDGS and SSF-DDGS, diets, and feces were performed following standard procedures [
43]: dry matter by drying samples at 105 °C until constant weight; ash by incineration in a muffle furnace at 450 °C for 16 h; crude protein (N × 6.25) using the Kjeldahl method with acid digestion, in a Kjeltec digestor and distillation units (Tecator Systems, Hõganäs, Sweden; model 1015 and 1026, respectively); lipids by petroleum ether extraction using a Soxtec system (Tecator Systems, Hõganäs, Sweden; extraction unit model 1043 and service unit model 1046); and gross energy by direct combustion of samples in an adiabatic bomb calorimeter (PARR Instruments, Moline, IL, USA; PARR model 1261). Total phosphorus content was determined using the colorimetric method described by Fiske [
44], and starch content following Beutler [
45]. Chromium oxide in diets and feces was determined by acidic digestion [
46]. Cellulose, hemicellulose, and Klason lignin content were determined with a two-stage acid treatment described by [
47], preceded by the removal of starch. Reducing sugars were measured using the 3,5-dinitrosalicylic acid method (DNS; [
48]), soluble protein by the Bradford method [
49], and total phenols using the Folin–Ciocalteau method (Commission Regulation (EEC) No. 2676/90), using caffeic acid for calibration.
2.6. Enzymes Activity Analyses
2.6.1. Lignocellulolytic Enzymes
The activity of lignocellulolytic enzymes in SSF-DDGS and experimental diets was determined by mixing SSF-DDGS or diet (1:5 w/v) with a solution containing 1% NaCl and 0.5% Triton X-100, stirred for 60 min at room temperature, filtered through a fine-mesh net (300 µm pore size), and centrifuged (7000 g, 10 min). The enzymatic activities were measured in the collected supernatants. Cellulase (endo-1,4-β-glucanase) activity was measured using an enzymatic kit “Azo-CM-Cellulose S-ACMC 04/07” (Megazyme International, Ireland). One unit of cellulase activity (U) was defined as the amount of enzyme required to release 1 µmol of glucose-reducing sugar equivalents from carboxymethylcellulose under the assay conditions (40 °C; pH 4.5). Xylanase (endo-1, 4-β-xylanase) activity was measured with an enzymatic kit “Azo Wheat arabinoxylan AWX 10/2002” (Megazyme International, Ireland). One unit of xylanase activity (U) was defined as the amount of enzyme that released 1 µmol of xylose-reducing sugar equivalents from the substrate under the assay conditions (40 °C; pH 4.5).
2.6.2. Digestive Enzymes Activity
Each portion of the intestines was homogenized in ice with ultrapure water buffer (pH 8.0), centrifuged (23,000×
g; 30 min; 4 °C), and the supernatants were stored at −80 °C until analysis. Alpha-amylase (EC 3.2.1.1), lipase (EC 3.1.1.3), and total proteases were measured according to Magalhaes et al. (2015). Trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) were determined following the methods described by [
50]. Soluble protein concentration was determined using the Bradford method [
49]. All digestive enzyme activity was expressed as mU/mg of soluble protein. One unit (U) of enzymatic activity was represented as µmol of product generated per minute under the specific assay conditions.
2.7. Statistical Analyses
All data were checked for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) and normalized if necessary. Data regarding DDGS composition after SSF and ADC of experimental diets were analyzed by one-way analysis of variance (ANOVA). ADC of DDGS and SSF-DDGS diets were analyzed using a t-test. Digestive enzyme activity was analyzed by a two-way ANOVA with the intestine section and diets as fixed factors; when interaction was significant, a one-way ANOVA was performed for each intestine section. Significant differences among groups (p < 0.05) were determined with Tukey’s multiple-range test. All data were analyzed in SPSS software package version 24.0 (IBM, Armonk, NY, USA).