Sustainable Biomass Valorization by Solid-State Fermentation with the Mutant Strain Trichoderma viride M5-2 of Forage Legumes to Improve Their Nutritional Composition as Animal Feed

Abstract

The valorization of plant biomass is one of the main strategies for sustainable development. However, its use as energy, biofuels, fertilizers, value-added products, or even food is severely affected by the complexity of the plant cell wall. Therefore, the evaluation of fungi with high production of lignocellulolytic enzymes capable of efficiently degrading these substrates constitutes a viable, clean, and eco-friendly solution, allowing, for example, an increase in the digestibility and nutritional quality of alternative animal feed sources. For these reasons, the present study evaluated the ability of the mutant strain Trichodema viride M5-2 to improve the nutritional composition of the forage legumes Lablab purpureus and Mucuna pruriens through solid-state fermentation. Endo- and exoglucanase cellulolytic activity was assessed, as well as the effect of fermentation on the fiber’s physical properties and chemical composition. Molecular changes in the structure of plant fiber were analyzed using infrared spectroscopy. Increased production of the cellulolytic complex of the enzymes endoglucanase (3.29 IU/mL) and exoglucanase (0.64 IU/mL) was achieved in M. pruriens. The chemical composition showed an increase in true protein and a decrease in neutral fiber, hemicellulose, and cellulose, with a consequent improvement in nutritional quality. Fiber degradation was evident in the infrared spectrum with a significant decrease in the signals associated with cellulose and, to a lesser extent, with lignin. It can be concluded that the mutant strain T. viride M5-2 produced chemical, physical, and molecular changes in the fibrous and protein fractions of L. purpureus and M. pruriens through SSF, which improved their nutritional value as an alternative feed for animal nutrition. By promoting the use of this fungus, the nutritional quality of this source is increased through an effective and eco-friendly process, which contributes to mitigating the environmental impact of food production, in accordance with sustainability objectives and the need for more responsible agricultural practices.

1. Introduction

Global population growth will continue to generate greater demand for animal production. One of the solutions to the food shortage is improving the nutritional quality of agroindustrial waste and other alternative food sources. The lignocellulosic biomass produced from these wastes has low nitrogen content, high fiber content, and low nutrient density; its processing can increase its nutritional value [1]. Despite this, most of the processes used to revalue these residues are complex due to the structure of the plant cell wall.

In tropical and subtropical countries, the production of legume meals could be a very attractive alternative for feeding monogastric species due to their low cost and lack of competition with human food. Savón et al. [2] demonstrated the possibility of using forage meals from tropical legumes such as Lablab purpureus and Mucuna pruriens in poultry and pig rations. However, these feeds have limitations in terms of the biological availability of several nutrients [3], which could be ameliorated by the application of fungal solid-state fermentation (SSF) processes [4].

In recent years, solid-state fermentation has attracted the interest of scientists and industry due to its potential in the circular bioeconomy [5]. Microbial cultivation on waste-based substrates achieves the dual benefit of complete utilization of the waste and the production of value-added products, such as enzymes or secondary metabolites. They also generate biochemical and structural modifications in a large part of the fiber components, increasing their nutritional value [6] and the biological value of dietary protein [7,8,9], improving the structure of lignocellulosic compounds [10,11] of the resulting product, and producing high-value bioproducts, such as bioactive molecules for use as ingredients in biostimulants [12].

The literature reports the cellulolytic activity of many microorganisms, including several species of fungi such as Trichoderma [13], Aspergillus [14], and Penicilium [15], which account for more than 50% of the studies related to cellulases. Their use in a more productive way is conditioned by the utilization of highly efficient enzyme mixtures [16]. These microorganisms produce enzymes capable of improving the nutritional value and reducing the fiber content and antinutritional factors in Vigna unguiculata, L. purpureus, and M. pruriens [17]. Therefore, novel feeds obtained from processed legumes are undoubtedly useful for animal and human nutrition, due to their beneficial effects and use in disease prevention.

The mutant fungus Trichoderma viride M5-2 is a producer of cellulolytic enzymes (endo and exo β1-4 glucosidase, β glucosidase, and laccase) [18,19], and is resistant to catabolic repression with hydrolytic activity on sugarcane bagasse by a solid-state fermentation system. Its use was validated by increasing its added value in enzymes and microbial protein, with a significant reduction in cellulose content for animal feed purposes.

From these studies, it was recommended to evaluate this strain using the flours [20] and foliage [21] of tropical legumes Cannavalia ensiformis, Lablab purpureus, Vigna unguiculata, and Mucuna pruriens, and to determine the changes that occur in their nutrient content through solid-state fermentation. These legumes have several limitations, such as the presence of antinutritional factors, as well as a high content of the cell wall, which enables a decrease in their potential nutritional value and could have a greater or lesser effect on digestive physiology, depending on the monogastric species [22]. In addition, it was recommended to determine the quantification of its cellulase enzymes to achieve greater degradation of plant waste for nutrition.

Solid-state fermentation enabled improved nutritional value and reduced fiber content, reducing the content of antinutritional factors. In addition to being ideal substrates for obtaining high inoculant yields, without the use of other nitrogen or mineral sources, this ensures the production of inoculants for obtaining fibrolytic enzyme raw materials in the various substrates, as well as obtaining a new product from this substrate, different from the enzyme, as an unconventional feed alternative [17,18,20]. However, the T. viride M5-2 strain has not been evaluated for its effect in bioconversion processes of postharvest residues of forage legumes in which the entire plant material is mixed. In this study, we investigate its use and its potential to improve the nutritional quality of legume meals intended for animal feed. This approach not only enhances the value of plant biomass, but also contributes significantly to the search for alternatives to conventional protein sources. Therefore, the effect of the mutant strain T. viride M5-2 on improving the nutritional composition of whole forage flour meals of legumes through solid-state fermentation was evaluated.

2. Materials and Methods

2.1. Fungal Species

The mutant strain of lignocellulolytic fungus T. viride M5-2 was used. It was isolated from sugarcane bagasse, with a nucleotide sequence number registered in GenBank and accession number KY977981, and is part of the Institute of Animal Science (ICA) collection of microorganisms [23].

2.2. Whole Forage Flour Meals of Legumes Substrate Preparation

Whole forage flour meals of legumes from L. purpureus and M. pruriens forage were used for fermentation. The legumes were grown in the experimental area of the Institute of Animal Science (ICA). The flours were made with whole plants (leaves, stems, and seeds) cut at 5 cm above ground level, when 100% of the plants had pods and 50% of the pods were in a milky state. They were then spread on a plate and dried for 2–3 days until the humidity was reduced to 20–25% to allow grinding and prevent any fermentation process. The dried plants were reduced to a particle size of 1 mm ± 0.2 mm in a hammer mill.

2.3. Observation of the Cultural Characteristics of the Fungus T. viride M5-2 in Legume Flours

To determine the growth capacity of the fungus in the different flours, cultural characteristics (mycelium and sporulation) were observed at a temperature of 30 ± 1 °C and 70% humidity, without macro- and micronutrient additions to the flour. This trial only considered the growth or absence of the fungus in the different legume flours, depending on the experimental conditions of the subsequent solid-state fermentation (SSF) test.

2.4. Solid-State Fermentation Process

For the experiment, 500 mL Erlenmeyer flasks were used with 10 g of each of the whole forage flour meals, which were moistened with water up to 70%, according to a previous analysis of the dry matter of the whole flours. The moist whole forage flour meals were sterilized at 121 °C for 20 min and inoculated with 1 cm² of malt agar grown and completely covered with spores of the fungus, which represented 10% of the fungal inoculum. No urea or other nutrients were added during fermentation, only those from the plant material, which has a high nitrogen content. The mixture was homogenized, and the Erlenmeyer flasks were placed in an incubator at 30 °C for 96 h. The initial pH was 6.13 for L. purpureus and 6.03 for M. pruriens. Sampling was carried out every 24 h to perform the corresponding chemical, physical, and enzymatic analyses [20].

2.5. Cellulolytic Capacity of T. viride M5-2 in Solid-State Fermentation Process

To create the enzyme extract, 5 g of the fermented substrates were collected every 24 h during 120 h, 45 mL of sterile distilled water was added, and the suspensions were stirred for 30 min. The suspensions were filtered and then centrifuged at 4000× g for 20 min. The enzyme extract obtained (E.E) was immediately used for the cellulolytic assay and pH measurement; the unused residue was stored at −20 °C. Analyses were performed in triplicate [24].

2.6. Enzyme Activities

The enzymatic activities (EA) of endo β1-4 glucanase (CMCase) and exo β1-4 glucanase (PFase) were determined, calculated, and expressed in international units per milliliter (IU/mL), which refers to the micromoles of glucose released per minute of reaction under the activity assay conditions [25].

2.7. CMCase Enzyme Activity

An amount of 0.5 mL of (EA) was incubated in a 0.5 mL mixture of 1% carboxymethylcellulose (0.5 viscosity), 0.075 M sodium citrate buffer (pH = 4.8), and 3, 5-dinitrosalicylic acid (DNS) at 50 °C for 30 min. The reducing sugars released were determined after incubation at 100 °C for 5 min, and the reading was taken at λ = 540 nm [25].

2.8. PFase Enzyme Activity

An amount of 0.5 mL of the PFase enzyme (EE) was added to a mixture of 0.075 M sodium acetate buffer (pH = 4.8) and 3,5-dinitrosalicylic acid (DNS) on Whatman No. 1 filter paper (1 × 6 cm) at 50 °C for 60 min. The reducing sugars released were determined after incubation at 100 °C for 5 min, and the reading was taken at λ = 540 nm [25].

2.9. Chemical Analysis of Solid-State Fermentation Process

To determine the chemical composition of the fiber, 5 g of solid material were taken at 0, 24, 48, and 72 h, wherein the indicators studied were as follows: dry matter (DM) and crude protein (CP) according to the AOAC [26], true protein (PV) by the Berstein method cited by Scull [27], and neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose (Hem), cellular content (CC), lignin (Lig), and cellulose (Cel), according to Van Soest [28].

2.10. Physical Analysis of Solid-State Fermentation Process

Physical properties were determined according to the techniques compiled by Savón et al. [29]. To determine packing volume and solubility, the samples were passed through a 0.8 mm sieve and placed in an oven at 105 °C overnight.

2.11. Determination of Packing Volume

One gram of the sample was placed in a graduated centrifuge tube. Centrifugation was then carried out at 3000 rpm for 20 min. Finally, the volume (V%) occupied by the sample after centrifugation was determined [29].

2.12. Determination of Solubility

An amount of 60 mL of distilled water was added to two grams of the initially dried sample and left to stand for one hour. The samples were then filtered and dried in an oven at 60 °C for 12 h. The difference between the dry sample weight at the beginning and end of the analysis represented the percentage of the fraction that was solubilized in water [29].

2.13. The Water Adsorption Capacity (WAC)

The water adsorption capacity was determined by the gravimetric method using the following formula, WAC (g/g) = (wet sample weight − dry sample weight/dry sample weight), and the acid buffering capacity (ABC) and basic buffering capacity (BBC) were determined using the method described in [30].

2.14. Determination of T. viride M5-2 Structural Changes of the Whole Forage Flour Meals from the Legumes L. purpureus and M. pruriens, by Fourier Transform Infrared Spectroscopy (ATR-FT-IR) Analysis

The infrared (IR) spectrum was measured on an ATI Mattson Genesis Series FT-IR spectrometer with a special attachment for solid samples, set to attenuated total reflectance (ATR). The infrared spectrum was recorded in the range of 4000 to 600 cm⁻¹ at 72 h after the start of the fermentation process. Background interference from moisture and CO₂ was instantly eliminated during the scanning process. Each analysis was performed in quintuplicate, and the average value was taken as the final spectrum.

2.15. Statistical Analysis

An analysis of variance (ANOVA) was performed using a completely randomized design with a factorial arrangement (2 × 4). The mathematical model used for the analysis of variance was

$${\mathrm{Y}}_{\mathrm{i}\mathrm{j}\mathrm{k}}=\mathrm{\mu }+{\mathrm{\tau }}_{\mathrm{i}}+{\mathrm{\beta }}_{\mathrm{j}}+{({\mathrm{\tau }}_{\mathrm{i}}\ast {\mathrm{\beta }}_{\mathrm{j}})}_{\mathrm{k}}+{\mathrm{e}}_{\mathrm{i}\mathrm{j}\mathrm{k}}$$

where the following definitions apply:

Yijk: dependent variable;

μ: general mean common to all treatment observations;

τi: effect corresponding to the i-th level of the treatment factor τi; τi = 1 and 2;

βj= effect corresponding to the j-th level of the time factor βj; βj = 1, 2, 3……5;

(τi ∗ βj)k: interaction between the i-th level of factor τi and the j-th level of the time factor βj;

eijk = random error, normally and independently distributed with mean 0 and variance σ², N~(0, σ2).

The factors were the two whole forage meals, L. purpureus and M. pruriens, and four sampling times.

For enzyme activity, the values at hour zero were not considered since they correspond to the time of inoculation, when the fungus’s enzymatic activity on the substrate has not yet begun. Four time points were also used for this indicator, extending the fermentation to 96 h to determine whether a significant change in enzymatic activity occurred after 72 h, justifying further studies of the positive effect on nutritional quality of the degradation of these substrates. The remaining indicators related to chemical–physical characteristics were analyzed at 0, 24, 48, and 72 h.

The experiment was conducted in three replicates for each sampling schedule in different Erlenmeyer flasks, which were then removed. The data, selected in a simple and random manner, present an appropriate sample size, and comply with a normal distribution and homogeneity of variance, as describe in Supplementary Materials S1. For the analysis of variance, in a factorial arrangement, data were analyzed using the statistical package InfoStat (version 2017) [31]. Differences between means were established using the Duncan test [32]. The corresponding ANOVAs are presented as Supplementary Materials S2.

3. Results

3.1. Growth of the Lignocellulolytic Fungus T. viride M5-2 on Whole Forage Flour Meals of L. purpureus and M. pruriens

It is known that fungi can present different adaptations depending on nutritional conditions, which directly affect their development [33]. However, the cultural characteristics of the growth of T. viride M5-2 in whole forage flour meals of M. pruriens and L. purpureus (Figure 1) showed that, after 24 h, it developed a rapid growth (the mycelium was whitish at the beginning, and, after 72 h, a complete sporulation of deep green color was observed) in the two substrates independently of the nutritional conditions of these legumes. These results were achieved under optimal conditions of temperature and humidity. Late sporulation is an important characteristic for the greater effectiveness of the degradative potential of the fungus. For this reason, the present research justifies the study of this fungus to determine the specificity of the enzymatic action in the expression of the cellulase complex through SSF over time and to obtain greater action in the cell walls of the fibrous material.

3.2. Cellulolytic Capacity of T. viride M5-2 in the Degradation of Legumes in the Solid-State Fermentation Process (SSF)

The cellulolytic capacity of T. viride M5-2 to produce the main enzymes of the cellulases complex (endo-1,4-β-glucanase and exo-1,4-β-glucanase, respectively) during the fermentation of whole forage flour meals (L. purpureum and M. pruriens) is shown in

Table 1. Cellulolytic activity of endo β1-4 glucanase (CMCase) of the T. viride M5-2 strain in the whole forage flour meals of legumes.

Cellulolytic Activity	Legumes	Fermentation Time (h)				SE and p
		24	48	72	96
CMCase (IU/mL)	L. purpureus	2.09 c	1.18 b	1.22 b	1.00 a	±0.03 p < 0.0001
	M. pruriens	3.15 e	3.29 f	2.57 d	2.00 c

^a–f: Means with different letters in each row differ at p < 0.05 (Duncan, 1955) [32]. SE: standard error, p: level of significance.

and

Table 2. Cellulolytic activity of exo β1-4 glucanase (PFase) of the T. viride M5-2 strain in the whole forage flour meals of legumes.

Cellulolytic Activity	Legumes	Fermentation Time (h)				SE and p
		24	48	72	96
PFase (IU/mL)	M. pruriens	0.74 d	0.46 b	0.39 a	0.38 a	±0.02 p < 0.0001
	L. purpureus	0.49 b	0.60 c	0.64 c	0.63 c

^a–d: Means with different letters in each row differ at (p < 0.005) (Duncan, 1955) [32]. SE: standard error, p: level of significance.

. An interaction between the factors studied was detected for the activity of both enzymes (p < 0.0001). In the case of endo β1-4 glucanase activity (Table 1), the values are higher when the microorganism is degrading M. pruriens. In both cases, they maintain cellulolytic activity for up to 96 h. On the other hand, exo β1-4 glucanase activity (Table 2) showed a maximum potential for the cellulolytic production of this enzyme when the fungus was degrading M. pruriens, also. Differences in fiber degradation could be due to the fact that these cellulolytic enzymes are a complex that catalyzes the progressive conversion of cellulose and guarantees the efficiency of biotransformation in a synergistic way [34], also considering the differences in the physical–chemical characteristics of the flours used in the research [27].

As shown in the results in

Table 3. Determination of pH during fermentation of the whole forage flour meals of legumes with the T. viride M5-2 strain.

Indicator	Legumes	Fermentation Time (h)				SE and p
		0	24	48	72
pH	L. purpureus	6.13 b	6.83 d	7.46 g	7.37 f	±0.004 p < 0.0001
	M. pruriens	6.03 a	6.71 c	6.72 c	7.12 e

^a–g: Means with different letters in each row differ at (p < 0.05) (Duncan, 1955) [32]. SE: standard error, p: level of significance.

, during fermentation (SSF) of both whole forage flour meals, the initial pHs were 6.13 and 6.03 for L. purpureus and M. pruriens, respectively, and slightly increased over the course of fermentation (p < 0.0001). The lower pH values obtained in M. pruriens compared to L. purpureus are close to the optimum for cellulase activity, which could explain the higher CMCase and PFase activity in the degradation of M. pruriens forage meal. These differences show that there are changes in the levels of hydrolytic activity in the growth activity of this fungus in these legumes and that many factors may influence it, but greater stability of fermentation was observed in M. pruriens. However, the maximum values differed in time due to variations in enzyme production during the carbohydrate degradation of plant fiber with fungal growth. Under these conditions, it is evident that cellulolysis depends on the proportion of the enzymatic components, the nature of the fiber, the affinity for it, as well as the stereospecificity and concentration of the enzyme used [35]. Therefore, pH as a complementary variable in the bioconversion process allowed us to know that there is hydrolytic activity by the enzymes of T. viride M5-2 even at pH values above 7. At these values, the transformations in both flours continue and, as a consequence, the process can continue until it is stopped. An advantage of this indicator in SSF is that it does not need to be adjusted to any initial value for the SSF process to occur.

3.3. Chemical Analysis of Solid-State Fermentation Process by T. viride M5-2

The analysis of the solid-state fermentation of the whole forage flour meals of legumes L. purpureus and M. pruriens can be observed in

Table 4. Chemical composition of the fiber of the whole forage flour meals of the legumes L. purpureus and M. pruriens, and protein composition during fermentation with T. viride M5-2.

Indicators (% of DM)	Legumes	Fermentation Time (h)				SE and p
		0	24	48	72
DM *	L. purpureus	28.17 d	28.34 d	27.82 c	27.86 c	±0.10 p < 0.0001
	M. pruriens	28.76 e	26.38 a	27.14 b	27.14 b
CP	L. purpureus	13.63 a	17.15 c	15.36 b	17.03 c	±0.13 p < 0.0001
	M. pruriens	19.04 d	21.34 e	22.05 f	21.27 e
TP	L. purpureus	11.30 a	13.24 b	13.21 b	14.40 c	±0.13 p < 0.0001
	M. pruriens	17.58 d	19.53 e	21.74 g	20.61 f
NDF	L. purpureus	67.83 b	67.83 b	66.74 a	67.90 b	±0.29 p < 0.0001
	M. pruriens	73.06 d	68.48 b	69.56 c	69.83 c
ADF	L. purpureus	51.44 a	51.18 a	52.97 c	54.31 d	±0.18 p < 0.01
	M. pruriens	52.27 b	52.54 b,c	52.64 b,c	54.78 d
Cel	L. purpureus	42.07 f	39.28 b	41.67 e	43.88 g	±0.12 p < 0.0001
	M. pruriens	39.66 c	39.97 c,d	38.41 a	40.14 d
Lig	L. purpureus	9.17 a	10.87 c	10.93 c	10.20 b	±0.12 p < 0.0001
	M. pruriens	12.33 e	11.38 d	13.35 f	13.98 g
Hem	L. purpureus	16.39 c,d	16.65 c,d	13.77 a	13.59 a	±0.29 p < 0.0001
	M. pruriens	20.79 e	15.94 c	16.92 d	15.05 b

^a–g: Means with different letters in each row differ at p < 0.05 (Duncan, 1955) [32]. SE: standard error, p: level of significance. * dry matter [DM], crude protein [CP], true protein [TP], neutral detergent fiber [NDF], acid detergent fiber [ADF], cellulose [Cel], lignin [Lig], hemicellulose [Hem].

. The fermentation process of the whole forage flours with T. viride M5-2 achieved crude protein values of 20% in M. pruriens and approximately 18% in L. purpureus. True protein showed a similar behavior, with higher values for M. pruriens. The most relevant aspect is that protein increased between 3 and 4 percentage units for CP and TP, respectively (p < 0.0001), which coincided with the maximum cellulolytic activity values.

The variations (p < 0.0001), both in the CP and TP content, after fermentation represent an important effect from the nutritional point of view in the legumes L. purpureus and M. pruriens, since these are characterized by high protein content. Solid-state fermentation with the T. viride M5-2 strain generates biochemical and structural modifications that eliminate most of the antinutritional factors, with the consequent increase in the nutritional value of the resulting product. Through the sequential and synergistic enzymatic action of the cellulase enzymes that hydrolyze the β1-4 glycosidic bonds, it is possible to degrade the fiber and release part of the proteins bound to it, increasing the protein availability of the source. The present study managed to obtain increases of 3 and 4 percentage units, which were not achieved in other studies of the fermentation of the foliage of these legumes with other microorganisms [36,37]. Variations in dry matter during fermentation can also be observed. These variations could be the result of the volatilization of solid components, together with the production of water and CO₂ as a result of the metabolism of the fungus and the beginning of the degradation of the most complex components of the plant cell wall, such as cellulose and hemicellulose.

On the other hand, the results of the fiber fractionation analysis of the whole forage flour meals are shown. Interactions were found between fermentation time (h) and all indicators of the whole forage flour meals (L. purpureus and M. pruriens), which were expressed as percentages of dry matter. As can be seen in the table, the main component of the meals is insoluble fiber. The concentrations of NDF, ADF, and cellulose in the forages showed differences between species, with M. pruriens presenting the highest NDF value. The lowest NDF content was observed at 48 h in the L. purpureus forage meal and at 24 h in the M. pruriens forage meal. This indicator decreased by 4.18 percentage units in M. pruriens and presented a cellular content of 31.52%. However, in L. purpureus, it was only 1.09 percentage units lower than the initial value, and the cellular content of this fraction was 33.26%. With respect to hemicellulose, this fraction decreased due to the cellulolytic activity of the fungus on the plant fiber, but a higher percentage of enzymatic hydrolysis was observed in M. pruriens compared to the control.

The ADF did not vary in the first 48 h for the forage meal of L. purpureus and M. pruriens, respectively, then showed a relative increase (p < 0.01) in both complete forage meals. In the most notable decreases in the NDF and ADF fractions, it could be determined that the hemicellulose polysaccharide present in the cell wall of the plant fibers under study was on the order of 15.94% and 13.77% in 24 h of degradation for M. pruriens and 48 h for L. purpureus, respectively. Using the ADF, the cellulose and lignin contents were determined during the fermentation of flour from the legumes studied. The results showed a decrease in cellulose of 2.19 percentage units for L. purpureus after 24 h of degradation, while in M. pruriens, a decrease of only 1.29 percentage units was reached over a longer period (48 h). Differences were observed with the NDF, which did not coincide with the moments of maximum fungal activity. Regarding the values obtained for lignin, the results showed that it was concentrated during the fermentation of L. purpureus; however, for M. pruriens, it decreased 24 h after the start of the process by 1.7 percentage units.

The analysis of the physical properties showed differences between the two whole forage flour meals with respect to fermentation time in four of the five indicators analyzed (

Table 5. Physical composition of the fiber of the whole forage flour meals of the legumes L. purpureus and M. pruriens, during fermentation with T. viride M5-2.

Physical Indicators	Legumes	Fermentation Time (h)
		0	24	48	72	SE and p
V (g/mL)	L. purpureus	4.05 e	3.60 d	3.35 b	4.05 e	±0.04 p < 0.0001
	M. pruriens	3.48 c	3.22 a	3.32 a,b	3.25 a,b
WAC (g/g)	L. purpureus	3.71 b	3.76 b	2.85 a	4.36 c	±0.13 p = 0.0005
	M. pruriens	4.66 c,d	5.14 e	4.68 c,d	4.78 d,e
ABC (meq)	L. purpureus	0.51 c	0.42 a	0.59 g	0.47 b	±0.0018 p < 0.0001
	M. pruriens	0.63 h	0.58 f	0.53 d	0.57 e
BBC (meq)	L. purpureus	0.36 e	0.31 a	0.45 g	0.36 e	±0.0013 p < 0.0001
	M. pruriens	0.32 b	0.41 f	0.35 d	0.33 c

^a–h: Means with different letters in each row differ at p < 0.05 (Duncan, 1955) [32]. SE: standard error, p: level of significance. Physical composition of the fiber determined according to analysis: volume (V), water adsorption capacity (WAC), acid and base buffering capacity (ABC, BBC).

). Overall, they presented low physical composition values despite the high fiber and protein contents of these legumes. However, M. pruriens showed a greater water retention capacity in relation to its fiber volume. The results suggest greater acid buffering capacity than base buffering in both legumes, which favors the quality of the fiber for the introduction of unconventional feeds into animal diets.

Solubility, on the other hand, did not show interactions between the factors (flour × time), and the value of this indicator was found to be between 7.01 and 7.22 for L. purpureus and M. prurienns, respectively (p = 0.0613).

3.4. Determination of T. viride M5-2 Molecular Changes in Whole Forage Flour Meals from the Legumes L. purpureus and M. pruriens, by Fourier Transform Infrared Spectroscopy (ATR-FT-IR) Analysis

The ATR-FT-IR spectroscopic analysis (Figure 2 and Figure 3) shows the first spectra corresponding to whole forage flour meals from the legumes L. purpureus and M. pruriens. It can be observed that there is degradation of structures belonging to the carbon skeleton of the cellulose fibers, and lesser signals corresponding to the lignin in the fermented samples. The spectra of the forage meals generally present well-defined bands with good resolution. In the range of 1600–1400 cm⁻¹, the bands are more expanded in the biodegraded meals. However, between 1445 and 1390 cm⁻¹, the bands of bond formation in the OH plane of carbohydrates are of low intensity. The observed structural modification shows a characteristic band of the carbonyl groups present in amides at 1636–1449 cm⁻¹. This intense valence vibration band of the carbonyl group C=O γ C=O appears slightly split in two, suggesting the presence of amino acids or proteins in the samples. Furthermore, this band is so intense that it is unlikely to be associated with another bond, and its presence can be explained by the possible degradation of polysaccharides from the legume wall.

When comparing spectra of the whole forage flour meals with respect to the zero hour of fermentation of each one, a lower intensity of the vibration bands of benzene (C=CH) can be observed in the range 670 ± 20 cm⁻¹, and a new band also appears with intensity at 774.30 ± 20 cm⁻¹ where the aromatic rings that are mono-, di-, and tri-substituted by OH groups or methyl groups derived from benzene are found, which implies hydrolytic action of the fungus on the fiber of the legumes under study. In addition, an intense valence vibration band of the O-H γ OH bond can be observed in the 3291–3283 cm⁻¹ range. This band is a product of the polymeric association of the OH groups of carbohydrates. In the spectral area from 2852 to 2920 cm⁻¹, bands corresponding to CH₂ and CH₃ vibrations of carbonyl chains of organic compounds can be seen. The band is of medium intensity, according to the valence vibration of the C-H γ CH bond of alkyl groups in the same carbohydrate chain. Around 1730 cm⁻¹, bands characteristic of ester-type compounds are observed. Around 1028–1051 cm⁻¹, an intense band characteristic of C-O type vibrations is found. The bands corresponding to carbohydrates and aliphatic ethers are observed in all spectra; however, in the fermented samples, they are wider and more intense. This could be related to the hydrolytic action of the fungus on the carbohydrates present in the legumes, as cellulose decreases with fermentation time. These data, obtained from the ATR-FT-IR spectra, correspond to those obtained in the chemical composition analysis when observing the degradation of NDF in the range of 24–72 h compared to the control based on dry matter, suggesting a degradation of the fiber caused by the cellulolytic action in these whole forage flour meals of the legumes.

4. Discussion

To include any alternative raw material in production practices, it is necessary to understand the analytical characteristics of the product and its impact on the animal’s digestive physiology. Appropriate techniques must be used to evaluate, modify, and re-evaluate the product successively until optimal utilization is achieved. The evaluation of a high-fiber source includes determining its nutritional value and characterizing its fiber fraction. The nutritional value of a food depends on the consumption of the food in question and the degree to which the dry matter supplied by it provides the necessary amounts of energy, protein, minerals, and vitamins to the diet to meet the animal’s needs. Determining the physical and chemical characteristics (solubility, volume, buffering capacity, cation exchange capacity, and water adsorption capacity) is essential for estimating the nutritional value of fibrous foods for monogastric species. On the other hand, these response variables are a measure of the transformation that the substrates undergo due to the fermentation process with said strain.

Cellulolytic enzymes are a complex that catalyzes the progressive conversion of cellulose. The catalytic mechanisms of this enzymatic complex develop synergistically, ensuring the efficiency of bioconversion. Cellulolytic fungi such as T. viride M5-2 produce these enzymes under conditions of a deficit of alternative carbon sources with greater degradability and absorption, and of other nitrogen sources that are also rich in metabolizable energy and suppliers of carbon chains. This characteristic is due to the strict control by catabolic repression to which the cellulase genes are subject [34]. Under such conditions, these microorganisms use enzymes to release simple sugars from the solid substrate and use them as a carbon source [38].

As shown in the results, the T. viride M5-2 strain has the ability to grow on whole forage meals from the legumes L. purpureus and M. pruriens, without the addition of nutrients.

The quantitative evaluation of the activity of the main enzymes of the cellulase complex (endo-1,4-β-glucanase, and exo-1,4-β-glucanase), together with the degradation of plant fiber and its structural changes observed by infrared spectroscopy, as well as the increase in protein, demonstrated that this fungus, through a simple biotechnological process with SSF, was able to modify the nutritional composition of legume meals. According to this research, there are several factors that could have affected to some degree the degradation of whole legume flours, such as nitrogen concentration, enzyme inhibition by reaction products, adsorption of the enzyme to the substrate, and pH [39,40], the latter reaching values higher than 7 after 48 h of fermentation; even so, the cellulolytic activity was not affected in either of the two flours.

This relationship between pH and the activity of the cellulase complex depends on the acid–base behavior of the medium and the nature and concentration of the substrate [41,42,43]. It is known that the initial optimum pH for the hydrolytic action of cellulases is between 5 and 6 [44], depending on the substrate to be fermented and the growth temperature of each microorganism. The pH indicator is one of the variables that most influences the kinetics of the fermentation process and, therefore, the response of a microorganism in a given process. In the case of filamentous fungi such as the genus Trichoderma, it is more common for them to develop better in acidic environments, but this particular strain has demonstrated the ability to grow in a range that covers alkaline pH values [17]. In SSF, the pH cannot be easily controlled, and the formulation of the culture medium with salts allows it to vary within a known range depending on the combination of KH₂PO₄, urea, and (NH₄)₂SO₄ [21]. This combination allows the formulation of a medium with an initial value of 4.5 that varies up to 6. In this research, mineral salts and urea are not added, which offers advantages from the economic, environmental, and sustainability points of view in these fermentations. Thus, despite the baseline pH levels during the fermentation of both legume flours, hydrolytic action was maintained. The fermentation product exhibited positive physicochemical, microbiological, and functional properties under the pre-established conditions. These results also constitute an advantage for the use of this strain in the large-scale biotechnological production of alternative fibrous foods.

On the other hand, knowledge of the chemical composition of unconventional raw materials allows for estimating their nutritional quality [45] and performing specific evaluations for their use in animal feed [46]. That is why fungal fermentation results in the degradation of plant food carbohydrates into simpler sugars and ensures the biosynthesis of various metabolites with nutritional changes in fiber [36,47]. Therefore, the comprehensive characterization of the nutritional value of whole forage flour meals from these legumes fermented with T. viride M5-2 offers a novel aspect for research in the digestive physiology of monogastric animals.

According to Shubha et al. [48] and Sowdhanyaa et al. [49], the protein nitrogen content of legumes accounts for 75–85% of the total nitrogen in the plant. With this feature, it was possible to perform SSF without the need to add urea or other nitrogen sources, although the nitrogen concentration of whole forage flour meals from the legumes could influence the enzyme activity variably. Furthermore, fermentation processes of these legumes have been shown to produce a number of positive changes in nutrient content, such as increased essential amino acids, soluble proteins, and in vitro protein digestibility [2,50].

On the other hand, the fiber content of whole legume forage meals affects their nutritional quality by negatively influencing the digestibility of the nutrients that make up the feed [51]. Savón et al. [52] evaluated a group of legumes and other tree forage plants and suggested that the high fiber content of these legumes could be due to the complexes formed between carbohydrates and phenolic compounds.

In this experiment, the differences in the percentages of NDF and cellulose degradation by the fungus in both forages are related to the cellulolytic activity observed during SSF with T. viride M5-2. This fungus, in addition to utilizing easily degradable wall components, such as hemicellulose, was also able to utilize some of the nitrogen associated with the fiber as a nutrient, thanks to its rapid growth and short adaptation period before it began to degrade lignin. This demonstrated its valuable potential for the bioconversion of whole forage meals from the legumes used. However, Vázquez et al. [37] report that greater modifications in plant fiber can be obtained by combining cellulolytic and ligninolytic enzymes.

The observed lignin concentration was due to two possible causes: one, the physical barrier of this cell wall component, and the other, the chemical analysis of the fiber that could not be standardized in the SSF [53], mainly for whole forage flour meals from fungal delignified forage [17]. Lignin is very difficult to analyze accurately because it is insoluble and therefore cannot be determined directly with any specific procedure [54]. Furthermore, microscopic studies have shown that lignin in legumes is completely indigestible, while the lignin present in the cell walls of grasses is digested to some extent. If the composition of the cell wall is analyzed after the biodegradation of these types of fibrous sources, it is possible to find results that show an increase in lignin values, especially when used with indirect methods such as Van Soest fractionation, particularly in legumes [55].

The nature and chemical composition of the plant material to be fermented and the results that may arise should also be taken into account [19]. From the results obtained, an apparent concentration of ADF is observed, which could be due to the reduction in dry matter in both whole forage flours. However, comparing them with other results makes their interpretation difficult since the dry matter losses during this process are not reported. This is aggravated in the determination of lignin, since its value could be overestimated, mainly in M. pruriens, due to the presence of antinutritional factors such as tannins, which are of a chemical nature similar to lignin [56,57], and phytates and trypsin inhibitors that can have harmful effects on the animals that consume those [58].

For this reason, legume forage meals [59] and their seeds are consumed for their excellent nutritional properties [60], but their quality must also be taken into account, which is modified by their physical properties, such as fiber volume, solubility, water adsorption capacity, and acid–base buffering capacity, which also influence biological processes such as digestion and nutrient consumption [61].

The potential for greater water absorption favors fiber, due to the moisture content reached, which facilitates hydrolysis by cellulase enzymes in the matrix of polysaccharides, due to the greater hygroscopic power of hemicellulose compared to cellulose, and despite the hydrophobic nature of lignin. This explains the behavior of this indicator in whole forage flour meals. This process can occur not only during SSF with the fungus T. viride M5-2, but also due to the acidic buffering capacity of the legume flours studied for pH regulation in the gastrointestinal tract (GIT) of monogastric species. This is of great importance, since during digestion (GIT) in monogastric animals, a pH change occurs, from very acidic (pH 1) to almost neutral (pH 6.8–7.2) [62].

The formation of alcoholic, carboxylic, and carbonyl groups derived from the biodegradation of carbohydrates, as well as some groups formed by the oxidation of aromatic structures, confirms the molecular changes in the cell walls of legume flours by the fungus T. viride M5-2, which is capable of expressing specific enzymes responsible for degrading fiber components. Studies can be mentioned where the intensity of lignin and cellulose signals decreased with another ascomycete, Curvularia kusanoi L7 [36], corroborating previous approaches suggesting the expression of enzymes that modify lignin and cellulose and allow for more thorough degradation of the plant cell wall. This result provides useful information for fine-tuning solid-state fermentation processes of whole legume forage meals, enabling their efficient use in animal feed.

It was a condition of the bioconversion process that the fungal strain T. viride M5-2 managed to modify the cell wall structure of postharvest legume flour 72 h after the start of the process, at room temperature, demonstrating fermentation potential through the secretion of cellulolytic enzymes. In addition, it allowed the development of a biologically viable fermentation process compared to other fermentation processes with foliage of various legumes [21,43,63]. When comparing the whole forage flours of M. pruriens and L. purpureus fermented with the T. viride M5-2 strain, the final products have a different physical and chemical composition than other legume species, such as Mucuna deeringiana [64], Mucuna pruriens Georgina velvet, and Canavalia ensiformis [65], and other foliage flours, such as Lupinus mutabilis Sweet [66,67], Morus alba [62], and Trichantera gigantea [52]. Furthermore, the bioconversion of both meals achieved enzymatic activity of the cellulase complex and a high degradative capacity, improving the nutritional quality of alternative feeds. Its potential is evident in its higher DM content, crude and true protein content, and lower cell wall and lignin content. Regarding its physical composition, it has greater solubility and buffering capacity, and lower water absorption capacity as well as digesta transit speed, which allows for greater action of digestive enzymes and, therefore, better utilization of nutrients by the animal. These characteristics allow us to predict its effects on the gastrointestinal and metabolic functions of the animal’s organism. This contributes to achieving the most appropriate ration formulation and improved production performance.

5. Conclusions

It can be concluded that the mutant strain T. viride M5-2 produced chemical, physical, and molecular changes in the fibrous and protein fractions of L. purpureus and M. pruriens through SSF, which improved their nutritional value as an alternative feed source for animal nutrition. The valorization of this type of source significantly contributes to improving bioabsorption in animal nutrition, reducing competition with human food, and preserving the environment. Therefore, the conversion of fibrous biomass by cellulolytic fungi develops more efficient, sustainable, and environmentally friendly agricultural production.