The Effects of Solid-State Fermentation by Aspergillus spp. on the Nutritional Profile of Selected Agro-Industrial by-Products as Potential Feedstuffs for Weaner Rabbits

Abstract

This study evaluates the effects of solid-state fermentation inoculated with Aspergillus spp. on the nutritional profile of selected agro-industrial by-products (AIBPs: cowpea shell, groundnut shell, soybean hull, and maize shaft). These AIBPs were assessed as potential feedstuffs in weaner rabbit diets, which often exhibit digestive disorders when introduced to highly lignified feed ingredients. The AIBPs were milled to a particle size of 2 mm, sterilized, and subjected to fermentation with Aspergillus spp. under microaerophilic conditions at 28 ± 2 °C for 10 days. Samples (four replicates per treatment) were analyzed for chemical constituents (mineral and proximate composition, anti-nutritional factors, and fibre fractions) before and after fermentation. Digestible energy and digestibility coefficient of gross energy were calculated. Data were subjected to two-way analysis of variance (ANOVA). There was an increase (p < 0.05) in mineral profile, proximate composition, digestible energy, digestibility coefficient of gross energy, and dry matter, with a reduction (p < 0.05) in crude fibre, fibre fractions, and anti-nutritional factors. It was concluded that fermentation with Aspergillus spp. improved the nutritional value of the selected agro-industrial by-products. Therefore, fermented materials possess a better nutritional profile to be used in feeding programs for weaner rabbits. This will ensure sustainable animal production and add value to agricultural waste, which would otherwise constitute an environmental nuisance.

1. Introduction

Agro-industrial by-products are waste materials generated during the harvesting or processing of crops into marketable products. These by-products represent a substantial, yet underutilized, resource for animal feed [1]. Their fibrous nature and wide availability make them potential feedstuffs in rabbit nutrition, where dietary fibre is a major component due to the anatomical and physiological characteristics of the gastrointestinal tract. However, young rabbits often experience nutritional and digestive disorders when fed highly fibrous diets such as those containing agro-industrial by-products. This limitation is largely attributed to the immature digestive system of weaner rabbits and the high degree of lignification in plant structural carbohydrates, including cellulose and non-cellulosic polysaccharides such as hemicellulose, pectic substances, gums, mucilages, and lignin (a complex, non-carbohydrate phenolic polymer). These components hinder the efficient utilization of such by-products in weaner rabbit diets [2]. To overcome this challenge, biological treatments such as solid-state fermentation have been proposed to degrade lignocellulose fibre and enhance its nutritional value for growing rabbits.

Solid-state fermentation (SSF) is a fermentation process in which microorganisms (fungi, bacteria, or yeast) grow on solid substrates in the absence or near absence of free water, converting complex substrates into simpler, more digestible forms [3]. The use of SSF for degrading agro-industrial by-products offers several advantages over chemical treatments, as bioconversion products are typically less toxic, more cost-effective, and environmentally sustainable [4]. Solid-state fermentation systems have been applied to reduce undesired compounds and enhance the nutritional properties of various agricultural residues, including rapeseed meal [5], brewer’s dried grain [6], and millet straw [7], and for the bio-enrichment of Oryza sativa and Lablab purpureus [8]. Microbial hydrolysis of cellulosic materials is performed by cellulase-hydrolyzing enzyme complexes, which include three main enzymes: 1,4-β-D-endoglucanase (EG, EC 3.2.1.4), 1,4-β-D-cellobiohydrolase (CBH, EC 3.2.1.91), and β-glucosidase (BGL, EC 3.2.1.21). These enzymes work synergistically to convert complex cellulose into simple sugars [9].

Several fungal strains have been reported to produce multiple cellulolytic enzymes [10]. Aspergillus spp., a group of saprophytic fungi with ubiquitous distribution, are well known for colonizing cereal grains, legumes, and tree nuts. They have demonstrated the capacity to degrade the non-starch polysaccharides of agro-industrial by-products into simple sugars, resulting in improved energy and protein availability through solid-state fermentation [11]. Previous studies [6,12] have highlighted the effectiveness of fungal fermentation in improving the nutritional value of agro-industrial by-products such as brewers dried grains, distillers dried grains with solubles (DDGS), and corn cobs.

The purpose of this study was to evaluate the biodegradative effects of solid-state fermentation using Aspergillus spp. on the nutritive composition of selected agro-industrial by-products (cowpea shells, groundnut shells, soybean hulls, and maize shafts) as potential feedstuffs intended for weaner rabbits’ diets.

2. Materials and Methods

The experiment was conducted at the Animal Nutrition Laboratory, College of Animal Science and Livestock Production, located at latitude 7.23° N and longitude 3.44° E, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria.

2.1. Feedstuffs Used as Substrate for Solid-State Fermentation

The agro-industrial by-products were obtained from processing industries within the university environment. The materials used as substrate for solid-state fermentation were as follows: Maize shaft (MS), a by-product obtained from the fermentation of maize (Zea mays L.) grains used in the preparation of food; soybean hulls (SH), a by-product of the oil extraction process from soybean seeds (Glycine max L.); cowpea shells (CS), by-products of cowpea (Vigna unguiculata L.) processing plants after the seeds have been removed; and groundnut shells (GS), by-products of groundnut (Arachis hypogea L.) processing plants after seed removal. The materials were dry-milled using a hammermill to a 2 mm particle size prior to fermentation.

2.2. Source of Microorganism and Inoculum Preparation

Aspergillus spp. spores maintained in sterile soil at 4 °C were obtained from the Department of Microbiology, Federal University of Agriculture, Abeokuta. Under strict aseptic conditions, the spores were dislodged using a sterile inoculation loop into sterilized distilled water to prepare a spore suspension. The number of spores in the suspension was determined using a hemocytometer. Next, 1 mL of the prepared spore suspension, adjusted to a concentration of 5 × 10⁹ spores/mL, was inoculated onto sterile Petri dishes containing Sabouraud Dextrose Agar (SDA). The plates were incubated at 28 ± 2 °C for 72 h. Spores on plates containing pure cultures of fully grown lawn were sub-cultured bi-monthly and kept on SDA slants at 4 °C.

2.2.1. Molecular Characterization of Selected Isolates

Molecular characterization was performed following the protocol described by [13]. Genomic DNA was extracted from both parental and fusant Aspergillus isolates using the DNeasy Plant Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. DNA quality was assessed via agarose gel electrophoresis and Qubit Fluorometer (Invitrogen, Germany). The Internal Transcribed Spacer (ITS) region, recognized as a universal DNA barcode marker for fungi, was targeted for PCR amplification. The forward (ITS 1: 5′ TCC GTA GGT GAA CCT GCG G 3′) and reverse (ITS 4: 5′ TCC TCC GCT TAT TGA TAT GC 3′) primers, corresponding to the 5,8S ribosomal RNA gene region, were used for amplification. PCR conditions included initial denaturation at 94 °C for 5 min, followed by 35 cycles consisting of 30 s denaturation at 94 °C, 30 s primer annealing at 55 °C, 1.5 m extension at 72 °C, and a final extension step for 7 m at 72 °C. The nucleotide sequencing of the 5.8S-ITS region was carried out using a Gene Analyzer 3121 sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Forward and reverse sequences were checked, manually edited when necessary, and aligned to generate a consensus sequence using Molecular Evolutionary Genetics Analysis (MEGA) version 5.10 [14]. The resulting sequences were compared against the NCBI GeneBank database using the BLASTn algorithm to determine homology with known fungal sequences (http://www.ncbi.nlm.nih.gov/BLAST/). A phylogenetic tree was also constructed, and branch length variations were analyzed as described in the following section.

2.2.2. Molecular Identification and Phylogenetic Tree Representation of Sequences

The molecular characterization of parental and selected fusants is represented in Figure 1, which shows the phylogenetic tree representation of the parental and fusant isolates, respectively. The evolutionary history was inferred using the Neighbour-Joining method [15]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [14]. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates collapsed. The evolutionary distances were computed using the p-distance method [16] and are in the units of the number of base differences per site. The sum of branch lengths was calculated as 0.62235915. Parent and selected fusants were identified as Aspergillus flavus (parent TA and fusant T5) and Aspergillus tamarii (parent TC, fusant T13 and T14). The sequences were submitted to the GenBank MH836320 and identified as Aspergillus spp. strain TMOD 01.

Fungi 1-5: TA, TC, T5, T13, and T14, respectively. Note: The evolutionary history was inferred using the Neighbour-Joining method. The optimal tree with the sum of branch length = 0.62235915 is shown. The analysis involved 5 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Non-coding. All ambiguous positions were removed for each sequence pair. There were a total of 568 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

2.2.3. Screening for Cellulolytic Properties of Isolates

The cellulolytic ability of the fungal isolates to utilize cellulose as the sole carbon source was assessed using point-inoculating fungal spores onto Czapek Dox minimal medium (NaNO₃, 6; KH₂PO₄, 1.52; KCl, 0.52; MgSO₄·7H₂O, 0.52; FeSO₄·7H₂O, 0.01; and ZnSO₄.7H₂O, 0.01) supplemented with 3% (w/v) carboxymethyl cellulose (CMC), following a modified method described by [17]. The culture medium (pH 5.7) was sterilized at 121 °C for 15 min, allowed to cool (38 ± 2 °C), and poured aseptically into sterile Petri dishes until solidified. Spores from pure cultures of Aspergillus spp. were point-inoculated onto the surface of the solid medium. Inoculated plates were incubated at 28 ± 2 °C for 24–48 h. Plates with emerging mycelia were flooded with 1% Congo red solution and allowed to stand for 15 m at room temperature. Excess stains were removed using 1 M NaCl as a counterstain. The formation of a clear halo around the mycelium was considered indicative of cellulose activity. Isolates showing the widest zone of clearance were selected for use in the fermentation of the test agro-industrial by-products and were maintained on SDA slants at 4 °C.

2.3. Solid-State Fermentation of Agro-Industrial By-Products (AIBPs) by Selected Cellulolytic Fungi

Solid-state fermentation of AIBPs was carried out in a microaerophilic condition using a modified method described by the authors of [18]. Fifty grams (50 g) of each substrate (AIBP) was weighed into 100 mL Erlenmeyer flasks, moistened with 80 % (w/v) distilled water, and sterilized via autoclaving at 121 °C for 15 min. After cooling to room temperature, each flask was inoculated with a spore suspension (5 × 10⁹ spores/mL) of the selected cellulolytic fungal isolate (5 × 10⁹/ mL) and incubated at 28 ± 2 °C for 10 days under static conditions. Each treatment was prepared in quadruplicate.

The fermentation parameters adopted in this study were based on both literature precedent and practical suitability for fungal growth, substrate degradation, and potential inclusion in rabbit diets. A particle size of 2 mm was chosen to balance microbial colonization with physical characteristics compatible with rabbit feeding behaviour and digestive function and to allow optimum aeration needed for the growth of fungal spores within the fermentation medium. Moisture content was adjusted to 80% (w/v) to support fungal metabolism while avoiding anaerobiosis. The 10 d incubation period, supported by previous studies, ensured sufficient enzymatic activity and nutrient transformation, while also representing a practical duration for potential on-farm applications. Autoclaving was applied to sterilize the substrates and prevent microbial competition, allowing consistent colonization by Aspergillus spp. This process also served as a pre-treatment of the lignocellulosic material as well as its gelatinization, which rendered the AIBPs less recalcitrant to microbial colonization. Although effective under the present conditions, these parameters could be further optimized for large-scale or field use to enhance fermentation efficiency and nutritional outcomes.

2.4. Chemical Analyses

The chemical composition of the agro-industrial by-products samples was determined before and after microbial fermentation. All measurements were performed in quadruplicate.

2.4.1. Mineral Composition

Mineral contents: macro (calcium (Ca), magnesium (Mg), phosphorus (K), and sodium (Na)) and micro (iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn)) were determined according to the standard protocols of [19]. About 2.5 g of each sample was incinerated at high temperature (500–550 °C) to remove organic matter and leave behind the ash, which contains the mineral elements. The ash was then dissolved in an acidic solution to ensure complete dissolution of the ash. Atomic absorption spectrophotometry (AAS) was then used to quantify the concentrations of the mineral elements.

2.4.2. Anti-Nutritional Factors

Oxalates were determined according to the protocols outlined in [20]. About 5g of the samples was ground into powder and diluted with 60 mL of distilled water in a 250 mL volumetric flask, 11 mL (6 N HCl) was added, and boiled for 15 min and cooled. The volume was then made up to 100 mL with distilled water and left overnight, after which it was centrifuged. Thereafter, 25 mL of the supernatant was added to 5 mL of phosphotungstic acid. The solution was then left for 4 h and centrifuged. The supernatant was placed in a beaker, and ammonium hydroxide was added dropwise to maintain a pH of 4–4.5. Then, 5 mL of acetate buffer was added, and the solution was left overnight. The solution was centrifuged, and the supernatant was discarded. The precipitate was washed with 20 mL distilled water, further centrifuged, and the supernatant discarded. Next, 5 mL of sulphuric acid (1:9) was added to the precipitate, and the solutions were transferred into a conical flask with distilled water. The entire solution was boiled in a water bath and titrated hot with standardized KMnO₄ until the pink colour persisted for 30 s. The calculation was performed as follows: 1 mL of 0.002M KMnO₄ = 0.45 mg anhydrous oxalic acid.

Phytate was determined according to the modified methods described in [20]. First, 5 g of the ground sample was soaked in 100 mL of 2% HCl for 5 h and filtered. Next, 5 mL of 0.3% ammonium thiocyanate solution was added to 25 mL of the filtrate. The mixture was then titrated with iron (III) chloride solution until a brownish-yellow colour that persisted for 5 m was obtained. The phytate concentration of the sample was determined by measuring the absorbance of the sample in a Helios beta spectrophotometer at 500 nm and comparing it to a standard curve containing sodium phytate.

Tannin was quantitatively determined according to the methods of [21] using the colourimetric determination technique. Colourimetric estimation of tannins was based on the measurement of the blue colour formed by the reduction of phosphomolybdic acid by tannin-like compounds under alkaline conditions. The absorbance of the tannic acid standard solutions and the samples was measured at 760 nm using a spectrophotometer and compared to a standard curve containing tannic acid equivalent.

Trypsin inhibitor was determined using the protocols described by [22]. First, 1 g of ground samples was measured into a flask. Next, 50 mL of 0.5 M NaCl was added and stirred for 30 m. The mixture was then centrifuged at 1500 rpm for 5 m. The supernatant was discarded while 10 mL of the precipitate was pipetted into another flask. Thereafter, 2 mL of standard trypsin solution of known concentration was added to the precipitate and measured in a spectrophotometer at 410 nm absorbance while using 10 mL of the same substrate as a blank. Next, 1 mg, 2 mg, 4 mg, 6 mg, 8 mg, and 10 mg/L of standard trypsin inhibitor were also prepared and their absorbencies were measured at 410 nm. A standard curve of absorbance was compared against concentration.

2.4.3. Proximate Composition and Fibre Fractions

The samples were analyzed for dry matter and proximate composition (crude protein, crude fibre, ether extract, and ash) according to the protocols described by [23]. The fibre fractions of neutral detergent fibre (NDF), Acid Detergent Fibre (ADF) and Acid Detergent Lignin (ADL) were determined according to [24]. Cellulose and hemicellulose contents were calculated as the difference between ADF and ADL and NDF and ADF, respectively. Pectin contents were quantified using the protocol described by Sun et al. [25]. The digestibility coefficient of gross energy (GED) content of the samples was calculated using the equation described by [26]. The digestible energy (DE) values were calculated using the equation of [27]. The equations are presented below:

GED = 0.867 − 0.0012 × ADF_(g/kg),

(1)

DE_(MJ/kg) = 12.912 − 0.0236 × CF_(g/kg) + 0.010 × CP_(g/kg) + 0.020 × EE_(g/kg),

(2)

where CF, crude fibre; CP, crude protein; EE, ether extract.

2.5. Statistical Analyses

Data were checked for normality and outliers and then analyzed using two-way ANOVA of the GLM procedure in JMP^® Student Edition 18.2.0 (SAS Institute Inc., JMP Software, Cary, NC, USA) statistical package. The main effects of fibre source, fermentation, and their interaction were assessed. The means and pooled standard error of mean (SEM) were reported. Significant differences between the means were separated using Tukey’s HSD test at 5% probability.

3. Results

The macro and micro mineral composition of AIBPs presented in

Table 1. Macro and micro minerals of selected agro-industrial by-products.

		Macro Minerals (g/kg)					Micro Minerals (mg/kg)
		Ca	Mg	K	Na		Fe	Cu	Zn	Mn
Interaction
Fermentation:	Fibre source:
Unfermented	CS	3.731 c	3.889 c	2.110 e	3.956 f		95.700 f	8.10 c	28.40 d	84.50 c
Unfermented	GS	1.492 g	1.287 g	6.759 b	4.225 c		103.280 e	7.10 d	16.00 g	87.70 b
Unfermented	MS	0.810 h	0.963 h	1.309 h	3.842 g		11.430 h	2.30 g	12.90 h	60.00 f
Unfermented	SH	2.493 e	2.629 e	1.381 g	3.057 h		128.220 c	5.90 f	44.30 b	70.30 e
Fermented	CS	6.462 a	6.109 a	2.838 d	4.331 b		122.420 d	10.10 b	33.50 c	97.40 a
Fermented	GS	3.382 d	3.279 d	8.666 a	4.577 a		156.88 b	12.00 a	25.30 e	97.10 a
Fermented	MS	1.578 f	1.339 f	3.791 c	4.017 e		19.23 g	6.80 e	22.45 f	73.60 d
Fermented	SH	5.689 b	5.290 b	2.056 f	4.129 d		177.24 a	10.80 b	65.10 a	83.90 c
	SEM	0.0126	0.0597	0.1855	0.0845		0.7209	0.0647	0.1083	0.1698
Main effects
Fermentation:
Unfermented		2.132	2.192	2.890	3.769		84.658	5.85	25.40	75.62
Fermented		4.277	4.004	4.338	4.263		129.94	9.925	36.58	88.00
	SEM	0.5563	0.2989	0.0927	0.0423		0.3604	0.0323	0.0542	0.0849
Fibre source:
CS		5.097	4.999	2.474	4.144		109.06	9.10	30.95	90.95
GS		2.437	2.283	7.712	4.401		130.08	9.55	20.65	92.40
MS		1.194	1.151	2.551	3.928		37.33	4.55	17.67	66.80
SH		4.091	3.959	1.718	3.593		152.73	8.35	54.70	77.10
	SEM	0.7867	0.4227	1.3115	0.5976		0.5097	0.0457	0.0766	0.1200
p-Value
Interaction		<0.0001	<0.0001	<0.0001	<0.0001		<0.0001	<0.0001	<0.0001	<0.0001
Fermentation		<0.0001	<0.0001	<0.0001	<0.0001		<0.0001	0.0002	<0.0001	<0.0001
Fibre source		<0.0001	<0.0001	<0.0001	<0.0001		<0.0001	0.0013	<0.0001	<0.0001

^a–h Means with different superscripts across the same column are significantly (p < 0.05) different; Ca—calcium; Mg—magnesium; K—potassium; Na—sodium; Fe—iron; Cu—copper; Zn—zinc; Mn—manganese; CS—cowpea shell; GS—groundnut shell; MS—maize shaft; SH—soybean hull.

revealed that the minerals determined were significantly (p < 0.05) influenced by both fermentation, fibre source, and their interaction. All macro (Ca, Mg, K, and Fe) and micro (Fe, Cu, Zn, and Mn) minerals in all of the fibre sources (CS, GS, MS, and SH) in the study were significantly improved by fermentation. There was a significant (p < 0.05) increase in Ca (73% CS,126% GS,95% MS,128% SH), Mg (57%CS, 154%GS, 39%MS, 101%SH), Fe (27% CS, 51% GS, 68% MS, 38% SH), and Zn (18% CS, 58% GS, 74% MS, 47% SH) among others (Table 1) when the AIBPs were fermented.

Fermentation, fibre source, and an association between the two had a significant (p < 0.05) effect on all anti-nutritional factors examined (

Table 2. Anti-nutritional factors (g/kg) of selected agro-industrial by-products.

		Anti-Nutritional Factors
		Tannin	Phytate	TI	Oxalate
Interaction
Fermentation:	Fibre source:
Unfermented	CS	6.289 c	8.068 a	7.270 b	1.785 a
Unfermented	GS	8.031 b	7.017 c	8.708 a	1.572 b
Unfermented	MS	0.716 g	0.704 g	0.181 f	0.221 g
Unfermented	SH	8.938 a	7.163 b	8.730 a	1.536 c
Fermented	CS	2.270 f	3.512 d	3.243 e	0.430 d
Fermented	GS	2.863 e	2.488 e	3.490 c	0.255 f
Fermented	MS	0.423 h	0.247 h	0.082 g	0.110 h
Fermented	SH	3.019 d	2.310 f	3.408 d	0.287 e
	SEM	0.0073	0.0085	0.0106	0.0003
Main effects
Fermentation:
Unfermented		6.018	5.738	6.222	1.278
Fermented		2.143	2.139	2.556	0.270
	SEM	0.0036	0.0043	0.0053	0.0014
Fibre source:
CS		4.279	5.790	5.256	1.1076
GS		5.497	4.752	6.099	0.913
MS		0.569	0.475	0.132	0.166
SH		5.978	4.737	6.069	0.911
	SEM	0.0052	0.0060	0.0075	0.0020
p-Value
Interaction		<0.0001	<0.0001	<0.0001	<0.0001
Fermentation		<0.0001	<0.0001	<0.0001	<0.0001
Fibre source		<0.0001	<0.0001	<0.0001	<0.0001

^a–h Means with different superscripts across the same column are significantly (p < 0.05) different; TI—trypsin inhibitor; CS—cowpea shell; GS—groundnut shell; MS—maize shaft; SH—soybean hull.

). Fermenting the AIBPs significantly (p < 0.05) reduced tannin levels by 64% (from 6.018 to 2.143) g/kg; phytate levels by 63% (from 5.738 to 2.139) g/kg; the trypsin inhibitor by 59% (from 6.222 to 2.556) g/kg; and oxalate levels by 79% (from 1.278 to 0.270) g/kg across all fibre sources. The impact of fermentation was more pronounced (p < 0.05) in SH and GS compared to CS and MS (Table 2).

Agro-industrial by-products are rich sources of nutrients, as shown in

Table 3. Proximate composition and energy values of selected agro-industrial by-products.

		Proximate Composition (g/kg)							Energy
		DM	CF	CP	Ash	EE	NFE		GED 1	DE 2 (MJ/kg)
Interaction
Fermentation:	Fibre source:
Unfermented	CS	905.00	175.00 c	105.60 d	65.20 ab	62.38 f	496.83 e		0.55 e	11.09 e
Unfermented	GS	945.00	444.88 a	102.00 e	45.00 c	82.50 d	270.63 h		0.24 h	5.08 h
Unfermented	MS	880.00	65.10 e	44.13 g	10.00 d	2.45 h	758.33 b		0.67 c	11.87 d
Unfermented	SH	882.50	260.00 b	128.78 b	62.63 b	95.13 b	335.98 g		0.40 f	9.97 f
Fermented	CS	930.43	115.50 d	111.75 c	66.65 a	70.50 e	566.03 d		0.63 d	12.71 b
Fermented	GS	951.95	266.55 b	108.23 d	41.83 c	87.65 c	447.70 f		0.39 g	9.46 g
Fermented	MS	893.33	58.25 f	54.25 f	11.75 d	8.45 g	760.63 a		0.81 a	12.25 c
Fermented	SH	892.40	117.90 d	150.76 a	65.92 ab	103.80 a	454.02 c		0.72 b	13.71 a
	SEM	4.5390	1.8370	0.7120	0.8225	0.6836	4.3590		0.0017	0.0544
Main effects
Fermentation:
Unfermented		903.13 b	236.25	95.13	45.71	60.61	465.44		0.46	9.50
Fermented		917.03 a	139.55	103.25	46.54	67.60	557.09		0.64	12.03
	SEM	2.2700	0.9186	0.3560	0.4112	0.3418	2.1790		0.0009	0.0385
Fibre source:
CS		917.52 b	145.25	108.67	65.92	66.44	531.43		0.59	11.90
GS		948.5 a	355.71	105.11	43.41	85.08	359.16		0.31	7.27
MS		886.65 c	61.68	49.19	10.88	5.45	759.48		0.74	12.06
SH		887.45 c	188.95	139.77	64.28	99.46	395.00		0.56	11.84
	SEM	3.2100	1.2990	0.5034	0.5816	0.4834	3.0820		0.0012	0.0272
p-Value:
Interaction		0.2161	<0.0001	<0.0001	0.0041	0.0490	<0.0001		<0.0001	<0.0001
Fermentation		0.0002	<0.0001	<0.0001	0.1658	<0.0001	<0.0001		<0.0001	<0.0001
Fibre source		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001		<0.0001	<0.0001

^a–h Means with different superscripts across the same column are significantly (p < 0.05) different; ¹ Calculated according to De Blas et al. (1992) [26]; ² Calculated according to Wiseman et al. (1992) [27]; DM—dry matter; CF—crude fibre; CP—crude protein; EE—ether extract; NFE—nitrogen-free extract; GED—digestibility coefficient of gross energy; DE—digestible energy; CS—cowpea shell; GS—groundnut shell; MS—maize shaft; SH—soybean hull.

. The interaction between fermentation and fibre source did not significantly (p > 0.05) influence dry matter but had an impact (p < 0.05) on the proximate composition (CF, CP, Ash, EE, and NFE) and energy values (GED and DE) of AIBPs. The association between fermentation and fibre sources showed that fermentation led to a significant (p < 0.05) reduction in the CF (33% CS, 40%GS, 10%MS, 55% SH) but significantly (p < 0.05) increased the dry matter (1.49%) proximate composition (8.4% CP, 5.3% Ash, 9.6% EE and 27% NFE) and energy contents (39% GED; 27% DE) of the AIBPs.

Fermentation, fibre sources, and their interaction all had a significant (p < 0.05) impact on the fibre fractions of AIBPs (

Table 4. Fibre fractions (g/kg) of selected agro-industrial by-products.

		Fibre Fractions
		NDF	ADF	ADL	Cellulose	Hemicellulose	Pectin
Interaction
Fermentation:	Fibre source:
Unfermented	CS	485.00 d	263.25 d	120.10 c	143.20 c	221.8 b	5.14 b
Unfermented	GS	710.00 a	525.00 a	284.50 a	240.50 b	185.00 c	6.07 a
Unfermented	MS	420.00 e	168.25 f	47.50 f	120.80 d	251.80 a	2.78 g
Unfermented	SH	595.00 b	390.00 c	65.10 e	324.90 a	205.01 b	4.18 d
Fermented	CS	310.90 f	199.51 e	99.30 d	100.23 e	111.39 e	3.25 e
Fermented	GS	549.10 c	400.13 b	252.92 b	147.19 c	149.10 d	4.48 c
Fermented	MS	251.90 h	46.85 h	12.00 h	94.90 f	145.00 e	1.96 h
Fermented	SH	298.10 g	122.91 g	22.70 g	100.26 e	175.20 c	3.22 f
	SEM	3.0620	1.4460	0.8950	2.0460	3.6690	0.0065
Main effects
Fermentation:
Unfermented		552.50	336.63	129.30	207.35	215.86	4.54
Fermented		352.50	192.35	96.73	110.61	145.16	3.23
	SEM	1.5310	0.7229	0.4470	1.0230	1.8340	0.0032
Fibre source:
CS		397.95	231.38	109.70	121.70	166.55	4.19
GS		629.60	462.56	268.71	193.84	167.00	5.28
MS		335.95	107.55	29.75	107.85	198.35	2.37
SH		446.55	256.45	43.90	212.60	190.10	3.70
	SEM	2.1650	1.0220	0.6330	1.4470	2.5940	0.0046
p-Value
Interaction		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Fermentation		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Fibre source		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

^a–h Means with different superscripts across the same column are significantly (p < 0.05) different; NDF—neutral detergent fibre; ADF—acid detergent fibre; ADL—acid detergent lignin; CS—cowpea shell; GS—groundnut shell; MS—maize shaft; SH—soybean hull.

). The fibre fractions (NDF, ADF, ADL, cellulose, hemicellulose, and pectin) exhibited a significant reduction (p < 0.05) when the AIBPs were fermented. The interaction between fermentation and fibre sources thereby promoted a reduction in NDF (35% CS, 22% GS, 40% MS, 49% SH), ADF (24% CS, 24% GS, 37% MS 68% SH), cellulose (30% CS, 38% GS, 21% MS, 69% SH), and pectin (37% CS, 26% GS, 30% MS, 23% SH), among others, in the AIBPs.

4. Discussion

In animal nutrition, feed formulation involves not only meeting nutrient requirements but also developing cost-effective feeding strategies that consider the characteristics of feedstuffs and the animals’ physiological and digestive limitations. While the proximate and mineral compositions of feedstuffs are essential for formulating balanced diets, it is equally important to address fibre-related and anti-nutritional concerns, particularly those affecting nutrient utilization and energy availability. This becomes especially relevant when using non-conventional feed ingredients, such as agro-industrial by-products (AIBPs), which often contain high fibre levels and complex polysaccharide profiles. Even in herbivore species like rabbits, excessive or poorly digestible fibre can impair nutrient absorption, gut health, and overall performance, ultimately reducing feed efficiency and growth in vulnerable stages such as weaning.

The microbial fermentation process has been shown to induce biochemical modifications in primary food matrices through the action of microorganisms and their enzymes, enhancing the bio-accessibility and bioavailability of nutrients, improving organoleptic properties, and extending shelf life [28]. In the present study, solid-state microbial fermentation with Aspergillus spp. improved both the macro and micro mineral contents, reduced anti-nutritional factors, and increased concentrations of ash, ether extract, crude protein, and nitrogen-free extract, the digestibility coefficient of gross energy, and digestible energy by reducing fibre fractions. This increase in nutrient concentrations can be attributed to the metabolic capability of the fungal isolate to degrade the fibre, which serves as the primary substrate for fungal energy metabolism. As values are expressed on a percentage basis, the breakdown of fibrous components leads to a relative increase in the proportion of other nutrients. Overall, these improvements are associated with a reduction in fibre fractions and allow for enhanced digestible energy availability for animals.

Minerals are essential dietary nutrients that play critical roles in animal metabolism, enzyme activation, and physiological regulation. The metabolic capability of the fungal isolate to degrade carbohydrates, phytates, and oxalates that typically form insoluble complexes with minerals may also contribute to the enhanced mineral availability observed in the current study. Previous reports have shown that minerals from plant-based ingredients often exhibit very low bioavailability for monogastric animals due to their complexation with non-digestible components such as cell wall polysaccharides [29]. Therefore, the current findings suggest that fermentation with Aspergillus spp. may also enhance mineral release by disrupting these complexes, thereby increasing the mineral availability of the substrates. These results corroborate those reported by [30], who attributed the higher concentration of minerals following solid-state fermentation of oilseed cakes to the reduction in fibre fractions in the substrate. In addition, fungal biomineralization may enhance mineral solubility and bioavailability, contributing to improved mineral absorption and metabolic function.

Anti-nutrients are among the major limitations to the utilization of agro-industrial by-products in animal nutrition, particularly in monogastric animals. These compounds reduce the bioavailability of various nutrients, contributing to micronutrient and mineral-related deficiencies and malnutrition [31]. In the present study, a reduction in anti-nutrients was observed following SSF with Aspergillus spp., which aligns with findings from previous studies [32,33]. Tannin and trypsin inhibitors are commonly present in tree and shrub foliage, seeds, and agro-industrial by-products [34] and are known to impair feed intake, growth rate, feed efficiency, microbial enzyme activities, and protein digestibility in animals [35]. Trypsin inhibitors interfere with proteolytic activity in the digestive tract by inhibiting trypsin and chymotrypsin enzyme activities, ultimately impairing protein digestion [36]. Rabbits have been described as being sensitive to toxicity [37]. Although the tolerance levels of ANFs in rabbits are relatively scarce in the literature, the levels of tannin obtained after fermentation in this study fall below the 4% recommended threshold level [38]. The reduction in tannin concentration may be attributed to the presence or activity of tannase enzymes produced by the fermenting microbiota, which catalyze the hydrolysis of ester and depside bonds, releasing gallic acid and glucose [39]. Although the authors of study [30] reported a contrasting increase in tannin concentration after SSF of oilseed cake, the efficacy of solid-state fermentation in reducing tannin and trypsin inhibitor contents is nevertheless well documented [33,40]. Phytates are phosphorus-containing compounds that form complexes/chelate with minerals such as Ca and Mg, inhibiting absorption and digestive enzyme activity [41]. The reduction in phytate concentration observed in the current study suggests that phytase enzyme activity was present in the fermentative Aspergillus isolate community. Similar reductions in phytic acid following SSF have been reported [42]. Oxalates are anti-nutrients that negatively affect calcium and magnesium bioavailability by forming insoluble complexes with proteins and inhibit peptic digestion, causing hypocalcemia, gastrointestinal tract irritation, blockage of the renal tubules by calcium oxalate crystals, and development of urinary calculi and nephrotic lesions in the kidney [34]. The reduced concentrations of oxalates indicate the likely synthesis of oxalate-degrading enzymes (oxalate oxidase and oxalate decarboxylase) by Aspergillus spp., supporting earlier findings by [43], who reported similar effects following fungal SSF of Adansonia digitata seeds. It is worth noting that the lower initial concentration of ANFs observed in the maize shaft (MS) may be attributed to partial natural fermentation that this by-product had undergone prior to the controlled SSF process.

The report in this study indicates that fermentation with Aspergillus spp. improved the proximate composition of agro-industrial by-products. The improved proximate composition observed might be attributed to the amylolytic and proteolytic enzymatic activities of the fermenting fungi, which are capable of breaking down complex organic macromolecules into simpler ones such as glucose and amino acids [43]. The findings are consistent with those of previous studies [44,45], wherein increased ash, ether extract, and crude protein concentrations following solid-state fermentation of agricultural residues were documented. The increased ether extract and crude protein concentrations may also be linked to the extended fermentation period (10 days), which enhanced the ability of Aspergillus spp. to synthesize long-chain fatty acids from acetyl-CoA and other complex unsaturated lipids and protein through the bioconversion of carbon and nitrogen sources into microbial protein [46]. The increased digestible energy and digestibility coefficients of gross energy seen in this study reflect the ability of SSF to improve the energy feeding values of agro-industrial waste, as these components indicate the amount of potential energy in a feed stuff that is actually available for the rabbits or any other suitable animals’ use after the losses due to undigested feedstuffs in their faeces have been accounted for.

Agro-industrial by-products typically contain high levels of cellulose and other structural fibre fractions, which limit their digestibility in animal feed. The increase in nitrogen-free extract observed in the current study might be attributed to the hydrolysis of insoluble fibre and the degradation of structural polysaccharides into fermentable energy substrates by Aspergillus spp., during fermentation. The marked reduction in the concentration of crude fibre and fibre fractions (NDF, ADF, ADL, cellulose, and hemicellulose) observed in the current study confirms the biodegradative capability of Aspergillus spp., on plant cell wall components [47] and its ability to secrete hemicellulase, cellulase, pectinase and polygalacturonase that cleave/breakdown lignocellulolytic bonds. These findings are consistent with those of [44], the authors of which reported a reduction in fibre fractions following solid-state fermentation of olive leaves. However, contradictory reports by [48,49] documented reductions in total carbohydrates of agricultural waste after SSF with Aspergillus niger, attributing this to the sustained consumption of carbohydrates by the fungal biomass during fermentation. On the other hand, the authors of [30] declared ADL as an undigestible fibre fraction and recalcitrant to degradation in a study in which SSF was proposed to enhance the nutritional value of oilseed cakes. These divergent findings underscore the influence of substrate composition, fungal strain, and fermentation conditions (temperature, pH, humidity, duration, etc.) on fibre degradation outcomes.

Solid-state fermentation (SSF) is a low-cost and operationally simple strategy to enhance the nutritional quality of fibrous agro-industrial by-products, with potential applicability in practical on-farm or small-scale feed production systems, and also allows for easier downstream processing of crude enzymes. In this study, SSF with Aspergillus spp. led to consistent improvements in nutrient composition and reduction in anti-nutritional factors, reinforcing its value as a viable approach for feed enhancement. Beyond these compositional changes, fermented feedstuffs are also known to exert functional effects—such as improving gut health, modulating the microbiota, and enhancing nutrient digestibility—which are particularly relevant for weaner rabbits. Although these functional outcomes were not directly assessed herein, the results provide a strong foundation for future studies. In vitro digestion models and in vivo feeding trials are recommended to confirm the bioavailability and physiological impacts of nutrients from fermented substrates and to guide their evidence-based inclusion in rabbit feeding programs.

5. Conclusions

Agro-industrial by-products are potential sources of nutrients for animals. Solid-state fermentation by Aspergillus spp. improved the feeding value of agro-industrial by-products by reducing the concentration of fibrous components, which led to increased mineral levels and bioavailability of other nutrients, besides the reduction in anti-nutrients. The reported better nutritional profile is expected to enhance the digestibility and nutrient utilization of agro-industrial by-products in young rabbits whose digestive systems are yet to be fully developed to tolerate high-fibrous feedstuffs. For sustainable animal production, waste valorization, and improved animal feed, the findings of this study provide further ways to utilize agro-industrial by-products in the livestock sector.