Ligninolytic Enzyme Content of Mushroom Pleurotus ostreatus and Agaricus bisporus Extracts and In Vitro Rumen Fermentation of Fungal Extract‐Treated Wheat Straw

TL;DR

This study shows that mushroom byproducts can enhance the digestibility of wheat straw for livestock through their ligninolytic enzymes.

Contribution

The study evaluates the enzyme content and forage improvement potential of byproducts from two mushroom species.

Findings

Agaricus bisporus extracts had higher laccase activity than Pleurotus ostreatus extracts.

Pleurotus extracts increased gas production in wheat straw fermentation, while Agaricus reduced it.

Mushroom byproduct extracts improved wheat straw fermentability in vitro.

Abstract

Mushrooms get nutrients from the soil fibrous substrates by producing enzymes (mainly laccases and manganese peroxidases) via the radical apparatus to attack lignin and cellulose. In fungi cultivation, the spent mushroom substrate (SMS) is a mix of plant disintegrated lignocellulosic biomasses, residual fungal mycelium and other materials (e.g., straw, cereal grains, hays, livestock litter and manure, wood chip, sawdust, etc.) on which the mushrooms grow, whereas mushroom basal bodies (MBB) are parts discharged to remove the portion in contact with the SMS. The current lab‐scale project aims to evaluate the ligninolytic enzyme content of mushroom extracts from SMS and MBB of Pleurotus ostreatus (PO) and Agaricus bisporus (AB), using two extraction solvents (acetate buffer solution or only water) for 3 h. The extracts were used to treat wheat straw and to measure the rumen fiber degradation and gas production by in vitro fermentation assays. All extracts have been acidic (pH 5.2–6.0), the acidity being more pronounced for the acetate solvents (p < 0.01). AB extracts had higher laccase activity than PO extracts (155 vs. 29 U L−1, p < 0.01). On the contrary, manganese peroxidases showed a significant interaction (p < 0.01) between mushroom species and type of solvent, being higher (p < 0.01) in PO‐MBB extracts (35.2 U L−1) than in PO‐SMS, AB‐MBB and SMS (28.0, 20.1 and 26.1 U L−1, respectively). Wheat straw incubated in acetic solvent extracts showed significantly higher NDFD (p < 0.05) than untreated straw (CTRL) in both mushroom species and their byproducts. Wheat straw treated in acidic extracts from PO by products produced more gas (p < 0.05) than CTRL straw. On the contrary, extracts from AB reduced (p < 0.05) gas production compared to CTRL straw, except for the acidic AB‐MBB extract. In conclusion, extracts from mushroom by‐products, primarily those from SMS of Pleurotus, increase rumen in vitro fermentability of wheat straw.

Mushrooms produce enzymes via the radical apparatus to attack lignin and cellulose and we evaluated extracts from byproducts of Pleurotus ostreatus and Agaricus bisporus in terms of content of ligninolytic enzymes and as sustainable means of enhancing the degradability of forages. The extracts contained ligninolytic enzymes and were used to treat wheat straw. Extracts increased the rumen in vitro fermentability of extract‐treated wheat straw, hence fungal extracts are promising to improve the forage fiber digestibility for livestock.

Figures

NDFD (after 48 h of in vitro fermentation) of wheat straw incubated in extracts from Agaricus bisporus and Pleurotus ostreatus by products (spent mushroom substrate, SMS and mushroom basal body, MBB) obtained from buffer acetate (AC) or distilled water (H2O) and compared with CTRL (heights of bars and vertical lines are means and standard deviations of treatments, respectively; letters upper the bars indicate statistically difference compared to untreated straw at a p < 0.05, evaluated with Least Square Differences multiple comparison test, mean square error: 4.31).

Gas production (after 24 h of in vitro fermentation) of wheat straw incubated in extracts from Agaricus bisporus and Pleurotus ostreatus by products (spent mushroom substrate, SMS and mushroom basal body, MBB) obtained from buffer acetate (AC) or distilled water (H2O) and compared with CTRL (heights of bars and vertical lines are means and standard deviations of treatments, respectively; letters upper the bars indicate statistically difference compared to untreated straw at a p < 0.05, evaluated with Least Square Differences multiple comparison test, mean square error: 2.06).

Tables

TABLE 1. Proximate composition (means ± s.d.) of mushrooms by products used for the extract production and wheat straw used as substrate in the in vitro rumen fermentations.

	Agaricus bisporus (AB)		Pleurotus ostreatus (PO)		Wheat straw
	SMS	MBB	SMS	MBB
Dry matter, %	44.95	10.12	31.54	15.56	88.64
	±4.50	±0.25	±4.10	±1.95	±0.09
Crude protein, % DM	12.13	20.61	7.16	7.80	5.72
	±1.81	±0.66	±0.42	±0.39	±0.21
Crude ash, % DM	41.03	17.00	18.14	7.71	8.50
	±3.01	±1.00	±0.76	±0.31	±0.17
NDF, % DM	57.12	61.05	49.99	61.42	76.73
	±1.83	±2.12	±0.15	±1.40	±0.13
ADF, % DM	37.84	41.79	41.61	45.42	46.42
	±3.29	±2.18	±1.56	±2.24	±1.93
ADL, % DM	3.17	16.28	28.98	5.80	6.80
	±0.96	±0.63	±3.65	±2.20	±0.29
Hemicellulose, % DM	19.28	19.26	8.38	15.99	30.31
	±5.12	±0.06	±1.41	±0.84	±2.09
Cellulose, % DM	34.67	25.51	12.63	39.62	39.63
	±2.33	±1.55	±2.09	±4.44	±2.24

TABLE 2. Volume, pH and ligninolytic enzymes content of extracts and NDF recovery of wheat straw incubated in extracts from Agaricus bisporus and Pleurotus ostreatus by products (spent mushroom substrate, SMS and mushroom basal body, MBB) obtained from buffer acetate (AC) or distilled water (H2O).

	Agaricus bisporus (AB)				Pleurotus ostreatus (PO)				Significant effects 1						RSE 2
	SMS		MBB		SMS		MBB		M	B	S	M × B	M × S	B × S
	AC	H2O	AC	H2O	AC	H2O	AC	H2O
pH	5.4	5.9	5.2	6.0	5.2	5.2	5.5	6.3	ns	*	**	**	ns	ns	0.21
Extract volume 3 , mL	850	775	1125	1025	950	850	1075	950	ns	**	**	**	ns	ns	26.7
LAs activity, U L−1	139.8	146.8	145.6	188.2	61.3	7.5	34.7	11.1	**	ns	ns	ns	ns	ns	28.6
MnP activity, U L−1	32.6	19.5	22.4	17.8	31.9	24.1	42.0	28.3	**	ns	**	**	ns	ns	3.73
Wheat straw NDF content post‐extract treatment 4 , %	99	102	104	105	102	105	102	104	**	**	**	**	ns	*	0.5

Full text

Introduction

1

Ruminants have evolved to utilize plant fiber of forages (e.g., neutral detergent fiber, NDF) through the microbial fermentation in the rumen. Goeser (2008) in a total of 140 comparisons of diets containing different forages found NDF degradability ranged from 40% to 47% in corn silage and legume or grass‐based diets, respectively. A recent meta‐analysis (de Souza et al. 2018) based on 54 studies on dairy cows indicated that the in vivo average NDF degradability is roughly 42%. The incomplete utilization of dietary fiber is partly due to feeding regimes of high yielding ruminants (excessive consumption of starchy feeds, high feeding levels, fast rumen transit, etc.) and a significant constraint also arises from the lignification of the cell wall of forages, which hinders the action of cellulolytic rumen bacteria (Van Soest 1994).

To enhance the accessibility of cellulose and hemicellulose, various methods, including physical, physicochemical, and chemical treatments, have been explored (Adesogan et al. 2019; Ma et al. 2020). Physical treatments, including grinding, soaking, or pelleting, can effectively affect cellulose deconstruction, enhancing its vulnerability to rumen enzymatic degradation; however, these methods frequently necessitate specialized equipment or industrial processes, limiting their feasibility for on‐farm use (van Kuijk et al. 2015). While chemical treatments using acids (e.g., sulfuric or nitric acids) or alkalis (e.g., sodium hydroxide or potassium hydroxide) can efficiently reduce cellulose crystallinity and increase sugar yields, their high cost and potential environmental implications require careful evaluation (Adesogan et al. 2019).

A further promising area for improving fiber utilization is the pretreatment of forages with ligninolytic enzymes (Arriola et al. 2017). Mushrooms get nutrients from the soil fibrous substrates by producing enzymes via the radical apparatus to attack lignin and cellulose, mainly laccases and manganese peroxidases (Pérez‐Chávez et al. 2022). Fungal enzymes could be used to develop sustainable and cost‐effective ways for improving the fiber digestibility of forages, and Adesogan et al. (2019) in their recent review on strategies for enhancing fiber utilization assert that “…white rot fungi are perhaps the biological agents with the greatest potential for lignocellulosic deconstruction”. In commercial fungi farms, spent mushroom substrate (SMS) is a mix of plant disintegrated lignocellulosic biomasses, residual fungal mycelium and other materials (e.g., straw, cereal grains, hays, livestock litter and manure, wood chip, sawdust) used for mushroom growth, representing a significant byproduct biomass of the mushroom production (Leong et al. 2022). Typically, 3–5 kg of the substrate is required to produce at least 1 kg of mushrooms, and this abundant but underutilized resource is an interesting source of ligninolytic enzymes (Leong et al. 2022). A further byproduct is the basal bodies of the mushrooms (MBB), which are the tissue portion in contact with the growth substrate discharged immediately after harvesting (around 10% of commercial edible products), being a further promising source of ligninolytic enzymes. Currently, several studies have already tested the fungal treatments to improve forage utilization, such as the experiments based on direct inoculation of the ligninolytic substrate with the fungus (Martens et al. 2023; Sufyan et al. 2022, 2024). Other authors (Agustinho et al. 2021; Machado et al. 2020; Olagunju et al. 2023) used a complex of enzymes obtained from Pleurotus ostreatus (by direct cultivation or extracted from the spent substrate) and treated whole plant corn, sugarcane plant and corn stover and found a reduction in lignin concentration and an increase in rumen degradability of forages.

The current lab‐scale pilot project aims to evaluate the ligninolytic enzyme content of mushroom extracts (Pleurotus ostreatus and Agaricus bisporus) as well in vitro rumen fiber degradation and in vitro gas production of wheat straw treated with fungal‐extracts. As most of the available literature is focused on SMS from Pleurotus ostreatus or on its direct inoculation into forages (Agustinho et al. 2021; Martens et al. 2023; Sufyan et al. 2024; van Kuijk et al. 2015), the novelty of the study is to compare extracts obtained not only from SMS but also from MBB and to include the white champignon (Agaricus bisporus, AB), which is one of the main commercial edible mushrooms in the world (Mushroom Industry Team 2021). Moreover, aqueous extraction was examined as it is a more environmentally sustainable procedure compared to extraction in acidic conditions.

Materials and Methods

2

Sample Collection and Experiment Organization

2.1

The spent mushroom substrate (SMS) and the mushroom basal body (MBB) of Pleurotus ostreatus (PO) and champignon (Agaricus bisporus, AB) were provided by Consorzio Funghi and Fungamico s.r.l. (Treviso, Italy) in two separate amounts at the end of two cultivation cycles.

A 2 × 2 × 2 factorial experiment was performed to study the chemical composition and the liquid extract characteristics by considering two fungal species (PO and AB), two extraction solvents (water and acetate buffer), and two by‐products (SMS and MBB). All the determinations (chemical analysis, liquid extraction, and in vitro fermentations) were replicated separately for samples of each cultivation cycle (sampling session), which was considered as a block in the statistical analysis.

Chemical Analysis and Extract Preparation

2.2

Fresch samples were divided into two portions, which were used for the analysis of the chemical composition and to obtain a liquid extract, respectively. A first portion of samples was dried in an oven (48 h at 60°C) and then milled through a 0.1 cm screen (Pulverisette, Fritsch, Germany), as well as a sample of wheat straw. Samples were analyzed for residual DM, ash (CA), and crude protein (CP, methods 945.15, 942.05, 984.13, AOAC International, 2016). Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were sequentially determined using the Ankom^II^ Fiber Analyzer (Ankom Technology Corporation, Fairport, NY, USA), as described by Mertens (2002) for NDF, and as described by Van Soest (1973) for ADF and lignin. All fiber fractions were inclusive of residual ash.

A second portion of samples was used to obtain liquid extracts by using deionized water or 0.2 M acetate buffer (pH 5.4). A total of 500 g of fresh SMS or MBB in 1000 mL of acetic acid buffer or water was homogenized using A C‐Tronic 15 Plus homogenizer (Sirman, Italy) for 5 min at 2500 rpm at room temperature. Subsequently, we vacuum‐filtered the liquid homogenate portion through a 0.1 cm filter to separate it from the solid residue, and we kept it at −20°C until further analyses.

Enzymatic Assay of Liquid Extracts

2.3

For the determination of manganese peroxidase (MnP) in the extracts, one of the commonly used methods was used, based on phenol red substrate, following the protocol reported by Xu et al. (2023). For this scope, the extract aliquots were centrifuged for 10 min at 3000 × g. Subsequently, 1 mL of the supernatant of the experimental aliquots was mixed with the reaction mixture containing 0.5 mL of MnSO_4_ solution (10 mM), 1 mL of sodium malonate buffer (pH 4.5), and 0.5 mL of phenol red (0.25 mM). Afterward, 300 μL of this mixture was added in an ELISA microplate and the initial absorbance was measured at 624 nm using the microplate reader SUNRISE (Tecan, Germany); then, 1 μL of H_2_O_2_ solution (30% v/v) was added to the sample to start the reaction. The plate was incubated at 30°C for 5 min, and the reaction was terminated by adding 50 μL of NaOH solution (1 M). The absorbance was measured at 624 nm. The enzyme quantity, expressed as enzyme active unit (U), was calculated with the formula:

$$[eqn]$$

ΔOD represents the difference in absorbance between the initial and final readings. V1 denotes the total volume of the enzyme reaction mixture, while V2 is the volume of the enzyme aliquot. ε is the molar absorption coefficient of the oxidized phenol red product at 624 nm under the total volume of the blank reaction mixture. Δt represents the reaction time.

For the laccases (LAs) determination, the ABTS (2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid)) was used as the reaction substrate. For the analysis, 100 μL of the supernatant of the experimental extract aliquots was mixed with the reaction mixture containing 1 mL of buffer acetate (0.1 M, pH 4.5) and 250 μL of ABTS (1 nM). Subsequently, 300 μL of the reaction solution was added in an ELISA microplate and incubated at 25°C for 15 min as reported by Ghose et al. (2023). The absorbance was measured at 420 nm and the LAs activity was calculated with the above formula adjusting the ε to the wavelength applied.

Treatment of Wheat Straw With Extracts

2.4

To evaluate the ligninolytic activity of the SMS and MBB extracts, 4 g of milled straw was placed in a backer with a magnetic stirring bar and then submerged in 200 mL of each fungal extract treatment or just in H_2_O (CTRL). To activate the class of enzymes beyond the heme peroxidase, like the MnP, 10 μL of H_2_O_2_ was also added to all the backers, also in the CTRL. The straw–extract mixture was then incubated at 25°C for 48 h on a multiple magnetic stirrer (IKA RO10, IKA, Germany). After incubation, the straw was separated from the corresponding extract by centrifugation for 10 min at 14000 × g, and two additional washing steps with Milli‐Q water were performed in the same way to remove extract residues. After overnight drying at 60°C, 250 mg of milled straw was weighed into each of 6 (six) Ankom F57 bags (1500 mg in total for each treatment). Two bags were analyzed for NDF content, as previously described, while the other four bags were used to measure the rumen in vitro NDF degradability.

Measures of In Vitro Rumen Fermentation

2.5

To determine the rumen in vitro NDF degradability (NDFD), four Ankom F57 filter bags filled with wheat straw from each fungal extract treatment plus four bags for the untreated straw (CTRL) were incubated in buffered rumen fluid using the Ankom Daisy II Incubator (Ankom Technology Corporation, New York). Rumen fluid was collected at a slaughterhouse from culled dairy cows previously fed under controlled conditions (i.e., animals fed a production diet based on corn silage, slaughtered in good health, transported from farms located near the slaughterhouse). The rumen fluid was delivered to our laboratory within 30 min from slaughter in airtight glass‐bottles refluxed with carbon dioxide and maintained at 39°C. Bags were incubated for 48 h at 39°C in jars with 400 mL of filtered liquid rumen and two buffer solutions (266 mL of solution A: KH_2_PO_4_ 10 g L^−1^, MgSO_4_ 7H_2_O 0.5 g L^−1^, NaCl 0.5 g L^−1^, CaCl_2_ 2H_2_O 0.1 g L^−1^, Urea 0.5 g L^−1^; 1330 mL of solution B: Na_2_CO_3_ 15.0 g L^−1^, Na_2_S9H_2_O 1.0 g L^−1^), as previously described (Spanghero et al. 2010). At the end of incubation, the bags were removed from the jars, rinsed with deionized water, and then dried overnight at 105°C. Later, the bags were analyzed for their NDF content as previously reported.

To measure the gas production, 200 mg of DM of CTRL straw or fungal extract‐treated wheat straw was weighed into 100 mL glass syringes that were marked with a scale. Each syringe was then filled with 30 mL of diluted rumen fluid (Menke and Steingass 1988) and was placed vertically in a water bath at 39°C with three syringes without substrate as blanks. Collection and manipulation of rumen inoculum followed the same procedures previously described for the NDFD measure. Gas production was measured at 24 h, and syringes were gently shaken every 2 h until 8 h of incubation, and then again at 20 h of incubation.

Both in vitro tests (NDF degradability and gas production) were duplicated into two separate fermentation runs, and each fermentation contained eight combinations of experimental treatments plus the CTRL. Within each in vitro test, NDF degradation and gas measures were conducted in duplicate (analytical replications), and the average values of the replications were used as statistical units.

Statistical Analysis

2.6

Data regarding the characteristics of extracts were analyzed using the PROC MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA) with linear mixed models, according to the following factorial design:

$$[eqn]$$

where y_ijklm_ is the experimental data, μ is the overall mean, α i is the fixed effect of the mushroom specie (i = 1,2; PO and AB), β j is the fixed effect of the by product (j = 1,2; SMS and MBB), γ k is the fixed effect of the extraction solution (k = 1,2; water or acetate buffer), δ l is the random effect of the sampling session batch (block, l = 1, 2) and ε_ijklm_ is the random error.

The effect of incubation of wheat straw after incubation in different fungi extracts or in water (CTRL) on NDFD and gas production was analyzed with the following model:

$$[eqn]$$

where y_ijkl_ is the experimental data, μ is the overall mean, α i is the random effect of the sampling session (block i = 1, 2), β j is the random effect of rumen fermentation run (block j = 1, 2), γ k is the fixed effect of “straw treatments” having 9 levels (eight treated straws plus CTRL, k = 1, 9). Treated and CTRL wheat straws were compared using the Least Square Difference multiple comparison test, with a difference that was considered significant if the p value was below 0.05.

Results

3

The chemical composition of SMS and MBB by‐products and wheat straw is reported in Table 1.

SMS had higher DM than MBB by products (30%–45% vs. 10%–15%), while all the materials contained high levels of fiber (from 50% to 60% DM). The CP contents ranged between the highest values for MBB of AB (20.61% DM) to the lowest for SMS of PO (7.16% DM), and the ash content was particularly high for the AB‐SMS (41.03% DM) and ranged between 8 to 18% DM for the other products. The sum of CP, ash, and NDF percentages exceeds 100% in the AB SMS and approaches totality in the AB MBB; this is likely due to the specific substrate composition of AB, characterized by low starchy/sugar constituents alongside the inclusion of livestock litter and manure, peat, and compost which are rich in non‐protein nitrogen (such as urea, amides, ammonia, etc.).

Table 2 shows the extracts characteristics and the NDF recovery of wheat straw after treatment with extracts. The pH was higher for the water‐based extracts than the buffer acetate‐based extracts (5.9 vs. 5.3, p < 0.01). Furthermore, the interaction ‘mushroom × by‐product’ was significant because pH of PO‐MBB (5.9) was higher (p < 0.01) than all other treatments (from 5.2 to 5.7). The extract volumes of both fungi were similar for MBB (1075 and 1013 mL for AB and PO, respectively) while differed significantly (p < 0.01) for the SMS (813 vs. 900 mL for AB and PO, respectively). Laccase activity of AB extracts was higher than PO extracts (155 vs. 29 U L^−1^, p < 0.01). On the contrary, MnP showed a significant interaction (p < 0.01) “mushroom × by product”, being PO‐MBB extracts (35.0 U L^−1^) higher (p < 0.01) than PO‐SMS, AB‐MBB and SMS (28.0, 20.1 and 26.1 U L^−1^, respectively). Furthermore, the utilization of the acetic buffer solvent improved the MnP activity for all the fungal species and by product combinations (32 vs. 22 U L^−1^, p < 0.05). The percentage of NDF recovery of wheat straw treated with different extracts was significantly affected from all the considered factors excluding the “mushroom × solvent” and the three‐way interactions. However, the differences observed were, on average, ±2% between the treatments, with a maximum of up to 5%.

Results of NDFD and gas production from the in vitro trials are in Figures 1 and 2, respectively.

NDFD (after 48 h of in vitro fermentation) of wheat straw incubated in extracts from Agaricus bisporus and Pleurotus ostreatus by products (spent mushroom substrate, SMS and mushroom basal body, MBB) obtained from buffer acetate (AC) or distilled water (H2O) and compared with CTRL (heights of bars and vertical lines are means and standard deviations of treatments, respectively; letters upper the bars indicate statistically difference compared to untreated straw at a p < 0.05, evaluated with Least Square Differences multiple comparison test, mean square error: 4.31).

Gas production (after 24 h of in vitro fermentation) of wheat straw incubated in extracts from Agaricus bisporus and Pleurotus ostreatus by products (spent mushroom substrate, SMS and mushroom basal body, MBB) obtained from buffer acetate (AC) or distilled water (H2O) and compared with CTRL (heights of bars and vertical lines are means and standard deviations of treatments, respectively; letters upper the bars indicate statistically difference compared to untreated straw at a p < 0.05, evaluated with Least Square Differences multiple comparison test, mean square error: 2.06).

The straws treated with acidic extracts had a higher NDFD compared to those with water extracts for both by‐products (on average, 35.9% vs. 23.3% in AB and 38.5% vs. 30.0% in PO). All the straws treated with acidic extracts had NDFD higher (p < 0.05) than the CTRL straw. On the contrary, among the straws treated with water extracts, only that from SMS of PO had a higher NDFD (p < 0.05) than the CTRL straw.

In agreement to what found for fiber fraction degradation, the straws extracted with acidic solvent had a higher gas production than straws treated with water extracts (p < 0.05) for both PO substrates (25.8 vs. 21.5 mL for SMS and 24.3 vs. 20.0 mL for MBB) and for the MBB substrate from AB (21.7 vs. 17.3 mL). The acidic treated straws from SMS of PO had gas production higher (p < 0.05) than the CTRL straw. The gas production from straws treated with water and acidic extracts for the SMS from AB was similar and lower (p < 0.05) than the CTRL straw, as well as the gas production from the water extracted straw obtained from MBB.

Discussion

4

The differences in SMS chemical composition of the two fungal species considered reflected the mushrooms' ecological and nutritional requirements. According to Leong et al. (2022), Pleurotus species are classified as primary decomposers and grow on a high fiber substrate, composed of twigs, straw, wood chips, logs, or stumps. A. bisporus works together with many bacteria and other fungi in the soil and compost piles to mainly degrade organic waste that has already been partially degraded by primary decomposers and the substrate contains peat and is particularly rich in ash. The other by‐product of mushroom cultivation is the basal bodies, which showed similar DM and fiber fractions contents between the two species studied, but protein, ash, and lignin resulted in lower levels in Pleurotus.

The volume of extract recovered using water as a solvent was 10% lower than that using the buffer, whereas the SMS matrix produced 25% less extract than MBB in A. bisporus but only 10% less in Pleurotus. This reflects the difference in composition of the two spent substrates, with AB being formed of components (e.g., dust, manure, peat) that absorb solvents and determine a greater amount of retained material in the filtration step of the extracts production and consequently lower volumes of liquids.

However, the acidic conditions of all extracts (particularly pronounced in the acetic extracts) promoted enzymatic action, as low pH is known to provide optimum activity for ligninolytic enzymes (Yin et al. 2019).

We have chosen a length of treatment of the wheat straw in the extracts of 48 h: this mediates the long incubation adopted by Rodrigues et al. (2008) who maintained wheat straw in extracts from basidiomycetes of white‐rot type for 6 days, with more recent results showing a very quick action of the ligninolytic enzyme (less than 30 min, Machado et al. 2020). In addition, it was observed that longer incubations (72 h) in aerobic conditions promote the growth of mold on the surface which can interfere with the wheat straw extract treatment.

Ligninolytic Enzymes Extraction

4.1

The extraction methods employed in the study aimed to evaluate a suitable extraction environment for the enzymes, utilizing acetate buffer and a more environmentally friendly extraction, water‐based. Laccases activity was found to be higher in extracts from SMS and MBB of AB, with the solvent having minimal impact on its activity (Ghose et al. 2023; Zhang et al. 2018). Conversely, MnP activity was more pronounced in extracts from SMS and MBB of PO, reporting a significant solvent effect. This underlines that laccases are generally more stable and active over a wider pH range; MnP activity is significantly influenced by the extraction solvent, with higher activity observed in acetate buffer extracts compared to water extracts, suggesting an optimal pH range for MnP activity in the slightly acidic range.

It is important to note that fiber degradation often involves a cooperative process, with multiple enzymes working together. MnP is an extracellular heme‐containing enzyme that, utilizing hydrogen peroxide, catalyzes the oxidation of Mn^2+^ to Mn^3+^. The resulting Mn^3+^ acts as a diffusible redox mediator, crucial for the degradation of lignin's phenolic and non‐phenolic structures (Hofrichter 2002). Laccases, a subclass of multicopper oxidoreductases, catalyze the oxidation of a broad range of phenolic and non‐phenolic lignin subunits, utilizing molecular oxygen as the final electron acceptor, specifically oxidizing intermediate products generated by MnP activity, thereby enhancing the overall lignin degradation process (Bhardwaj et al. 2022; Higuchi 2004). While both laccases and MnP contribute significantly, their combined action can be more effective than using either enzyme alone.

To differentiate between true fiber degradation and mere structural modification derived from the extract treatment, the NDF recovery was assessed on the 4 g of straw residue following the 48‐h incubation period in the 200 mL extracts or in H_2_O. Across all extract treatments, the NDF recovery ranged from 99% to 105%, averaging 102%. Despite minor statistical differences, the recovery rate approached totality and indicates that no significant fiber degradation occurred under these conditions; the observed limited variations are attributed solely to analytical variability.

As a result, none of the evaluated extracts significantly degraded NDF throughout the incubation period, ensuring its availability for the next steps in the digestibility evaluation. Our findings are consistent with those of Rodrigues et al. (2008), who discovered no quantitative differences in fiber components of wheat straw following incubation with white‐rot fungus extracts. Conversely, Jafari et al. (2007) observed a reduction in NDF content in rice straw following 60 days of mushroom cultivation on this substrate. However, these findings are not directly comparable to the proposed treatment, as the latter utilized only an enzymatic extract rather than the whole fungal organism.

In Vitro Rumen Fermentation

4.2

Wheat straw is a commonly used substrate in the literature to examine the effectiveness of fungal degradation, and also other papers used the in vitro rumen degradation together with gas production to assess the improvements in fiber degradation (Martens et al. 2023; Rodrigues et al. 2008). In our trial, all extracts obtained using acetate buffer exhibited a higher potential to make fiber bioavailable compared to those extracted with water. This finding supports the notion that the enzymes of interest function optimally in a slightly acidic environment (Yin et al. 2019). Moreover, in both fungi types, the acidic extract determined NDFD to be higher than the CTRL, while the water solvents generally determined NDFD value no different from the CTRL straw (with the exception of SMS from PO).

Data from gas production confirms the higher fermentability of straw treated with acidic extracts from the PO substrates, which resulted in higher than CTRL straw. On the contrary, substrates from AB had a comparable fermentability (acidic solvent, MBB) or lower (water solvent and acid SMS) than the CTRL straw.

A contrasting result between NDFD and gas production was seen in AB spent mushroom substrates, which had high NDFD but low gas production when compared to the CTRL (compare Figures 1 and 2). We have no data to explain this different behavior, but a hypothesis could be the presence of anti‐fermentative compounds in the spent substrate which were extracted and adhered to the incubated straw. These substances could have had a negative effect in the closed system of the syringes used for gas production; the absence of such effect for the NDFD data could be due to the fact that fermentation jars contained straw treated with the different types of extracts and potential negative substances from SMS from AB were diluted and therefore not effective.

Other trials have provided evidence of the ligninolytic activity of Pleurotus on fiber, albeit the method of fungal treatment applied to the substrate differed significantly from what was used in the current experiment. Sufyan et al. (2022, 2024) tested the P. ostreatus pretreatment (solid state fermentation for 30 day) of wheat straw and discovered that the fungal treatment resulted in a 14%–16% higher in vitro gas production and a 20%–28% higher in vitro dry matter digestibility. Olagunju et al. (2023) tested the inoculation of corn stover with Pleurotus and measured an increase in in vitro fiber fractions degradation.

Furthermore, from our in vitro fermentation trials, there was a not clear difference between extracts from SMS compared to those from MBB. However, the SMS extract of both fungi produced high degradation of fiber, and this effect can be attributed to the physiology of the fungus. Mycelium, the vegetative part of the fungus, actively colonizes the substrate, producing enzymes locally for nutrient acquisition. As previously discussed for MnP, this localized production results in a higher enzyme concentration within the substrate compared to the aerial part (MBB), which primarily serves reproductive and structural functions.

Another important finding is the correlation observed between MnP and LAs with NDFD. While a high presence of LAs may be considered a contributing factor, we hypothesize that the addition of H_2_O_2_, a known catalyst of MnP activity, at the initiation of the straw treatment amplified the influence of MnP relative to LAs.

Limitations of the Study

4.3

This is a lab‐scale study that utilizes lab procedures to treat the wheat straw and to evaluate the ligninolytic action of the extracts and has limited practical on‐farm applications. Moreover, the in vitro rumen fermentation tests adopted are cheap and rapid procedures, but simplify the complex process of rumen metabolism and therefore results require verification with in vivo measures. Another potential issue could concern the adoption of a length of incubation of wheat straw in extracts from fungi for 48 h. This treatment of wheat straw has been suggested by some limited experimental experiences available in the literature (Machado et al. 2020; Rodrigues et al. 2008) but it is unlike this process that can be implemented in farms, being a lab‐scale approach. Lastly, we focused on wheat straw given its high lignification fiber, but it would be interesting to evaluate other, more common forages, such as wet crops to be ensiled.

Conclusions

5

The findings confirm that some extracts from commercial mushroom by‐products significantly improve in vitro rumen fermentability of treated wheat straw. Furthermore, buffer acetate improves the effectiveness of extracts over simple water and the extracts from Pleurotus ostreatus spent mushroom substrate are the most promising candidate to continue the research project in order to assess practical on‐farm applications.

Author Contributions

E. Daniso: data curation (equal), methodology (equal), writing – original draft (equal), writing – review and editing (equal). M. Spanghero: conceptualization (lead), data curation (equal), funding acquisition (lead), resources (lead), writing – original draft (equal), writing – review and editing (equal).

Funding

This work was supported by European Union Next‐GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR); MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.5—D.D. 1058 23/06/2022, ECS00000043).

Ethics Statement

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this experiment uses in vitro and not in vivo techniques. Moreover, the inoculums used for in vitro rumen fermentation were collected at the slaughterhouse from animals already slaughtered for productive purposes (as described in the manuscript).

Conflicts of Interest

The authors declare no conflicts of interest.