Effects of lignocellulolytic enzymes on the fermentation profile, chemical composition, and in situ ruminal disappearance of whole-plant corn silage

Abstract

The objective of this study was to examine the enzyme activities of an enzymatic complex produced by Pleurotus ostreatus in different pH and the effects of adding increased application rates of this enzymatic complex on the fermentation profile, chemical composition, and in situ ruminal disappearance of whole-plant corn silage (WPCS) at the onset of fermentation and 30 d after ensiling. The lignocellulolytic enzymatic complex was obtained through in vitro cultivation of P. ostreatus. In the first experiment, the activities of laccase, lignin peroxidase (LiP), manganese peroxidase, endo- and exo-glucanase, xylanase, and mannanase were determined at pH 3, 4, 5, and 6. In the second experiment, five application rates of enzymatic complex were tested in a randomized complete block design (0, 9, 18, 27, and 36 mg of lignocellulosic enzymes/kg of fresh whole-plant corn [WPC], corresponding to 0, 0.587, 1.156, 1.734, and 2.312 g of enzymatic complex/kg of fresh WPC, respectively). There were four replicates per treatment (vacuum-sealed bags) per opening time. Bags were opened 1, 2, 3, and 7 d after ensiling (onset of fermentation period) and 30 d after ensiling to evaluate the fermentation profile, chemical composition, and in situ dry matter and neutral fiber detergent disappearance of WPCS. Laccase had the greatest activity at pH 5 (P < 0.01), whereas manganese peroxidase and LiP had the greatest activity at pH 4 (P < 0.01; P < 0.01). There was no effect of the rate of application of enzymatic complex, at the onset of fermentation, on the fermentation profile (P > 0.21), and chemical composition (P > 0.36). The concentration of water-soluble carbohydrate quadratically decreased (P < 0.01) over the ensiling time at the onset of fermentation, leading to a quadratic increase of lactic acid (P = 0.02) and a linear increase of acetic acid (P = 0.02) throughout fermentation. Consequently, pH quadratically decreased (P < 0.01). Lignin concentration linearly decreased (P = 0.04) with the enzymatic complex application rates at 30 d of storage; however, other nutrients and fermentation profiles did not change (P > 0.11) with the enzymatic complex application rates. Addition of lignocellulolytic enzymatic complex from P. ostreatus cultivation to WPC at ensiling decreased WPCS lignin concentration 30 d after ensiling; however, it was not sufficient to improve in situ disappearance of fiber and dry matter.

Introduction

Whole-plant corn silage (WPCS) is the main forage source in dairy diets (Ferraretto et al., 2018). However, only half of the neutral fiber detergent (NDF), approximately, in WPCS is digestible (Ferraretto and Shaver, 2015), primarily due to factors such as lignification (Adesogan et al., 2019). Lignin is a phenolic polymer that constitutes plant cell walls and negatively affects fiber degradability (Jung and Allen, 1995) due to the cross-linking of lignin to arabinoxylans (Hatfield et al., 2016). Hence, reducing lignin concentration by changing genotypes (Oba and Allen, 2000) or cleaving lignin linkages is a potential strategy to increase fiber degradability (Machado et al., 2020).

Some organisms, such as white-rot fungi, are known to produce lignocellulolytic enzymes as a mechanism to obtain energy and nutrients from fibrous substrates (Manavalan et al., 2015) and are considered the biological agents with the greatest potential to deconstruct lignocellulose (Adesogan et al., 2019). Pleurotus ostreatus, a white-rot fungus, also known as oyster mushroom, produces enzymes that degrade cellulose (endoglucanase and exoglucanase), hemicellulose (xylanase and mannanase), and lignin, such as laccase, lignin peroxidase (LiP), and manganese peroxidase (Leonowicz et al., 1999). Although these enzymes have the potential to break down lignin and possibly increase fiber degradability, few previous researches have focused on evaluating the effects of adding these enzymes to whole-plant corn (WPC) at ensiling.

Recently, we reported the potential of lignocellulolytic enzymes produced by P. ostreatus in reducing the concentration of lignin, cellulose, and hemicellulose and increasing in vitro degradability and antioxidant capacity of WPCS (Machado et al., 2020).

Although Machado et al. (2020) evaluated the effects of lignocellulolytic enzymatic complex application rates on WPCS composition, to our knowledge, no study has evaluated the effects of including lignocellulolytic enzymatic complex at ensiling on WPC composition at the onset of fermentation period or fermentation profile 30 d after ensiling.

Therefore, the objectives of the current study were to investigate the effects of increasing application rates of lignocellulolytic enzymatic complex from P. ostreatus at the time of ensiling on fermentation profile and composition of WPC during the first 7 d of ensiling and after 30 d of ensiling. We hypothesized that treating WPCS with the lignocellulolytic enzymatic complex from P. ostreatus would decrease the concentration of NDF and lignin in the material and consequently increase in situ dry matter disappearance.

Materials and Methods

Animal care and handling

The procedures for animal care and handling required were conducted under a protocol approved by the Institutional Animal Use and Care Committee of the University of Florida.

Production of the enzymatic complex and their activities

A sample of P. ostreatus (strain number 1833) was purchased from DSMZ (Leibniz Institute DSMZ, Braunschweig, Germany) and propagated in Petri dishes using potato dextrose agar (Sigma-Aldrich Co., St. Louis, MO) as a culture medium and incubated at room temperature. After 10 d of growth, 10 mm-diameter disks of P. ostreatus were incubated in 125 mL glass Erlenmeyer flasks with pre-autoclaved 25 mL of liquid culture medium (0.5% sugarcane diluted in distilled water) and 0.5 g of Coastal bermudagrass hay (Cynodon dactylon [L.] Pers) ground at 2 mm, as a carbon source. The flasks were placed in a platform shaker, incubated at 28 °C with constant agitation for 8 d. The liquid culture medium, proportion of carbon source, and incubation length were previously tested and chosen based on a pretrial on enzymatic activity. After each incubation, the material was frozen at −80 °C, freeze-dried (Labconco Freeze-dryer, Labconco Corporation, Kansas City, MO), and stored at 4 °C to prevent possible enzymatic denaturation or degradation.

The activities of lignocellulolytic enzymes were determined for laccase, manganese peroxidase, LiP, endoglucanase, exoglucanase, xylanase, and mannanase at pH 3, 4, 5, and 6. The liquid extracts of the enzymatic complex to determine the enzymatic activities were carried out using the respective buffers (McIlvaine buffer, sodium malonate buffer, sodium tartrate buffer, and citrate phosphate buffer in different pH, as described below for each assay), adding 1 g of the enzymatic complex to 19 mL of buffer in a 50-mL centrifuge tube. The extracts were vortexed for 1 min, filtered in two layers of cheesecloth, and centrifuged at 2,500 × g for 10 min at 4 °C, and the supernatants were used to determine the enzymatic activities.

Laccase (EC 1.10.3.2) activity was determined in a spectrophotometer (Spectra Max 340 PC, Molecular Devices Corporation, Sunnyvale, CA) at 420 nm, through the 2,2-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) (ABTS.⁺) oxidation, where 35 µL of ABTS^.+ solution (20 mM) as substrate, 35 µL of enzymes extract, and 280 µL of McIlvaine Buffer (corrected to each pH evaluated) were loaded in a 96-well microplate, incubated at 25 °C for 5 min. Laccase activity was expressed in units (U), and 1 U was defined as μmol of ABTS^.+ oxidized per minute (Li et al., 1999).

Manganese peroxidase (EC 1.11.1.13) activity was determined in a UV-Vis spectrophotometer (Jasco V-530, Jasco, Easton, MD) at 270 nm according to Wariishi et al. (1992), where 0.6 mL of sodium malonate buffer (50 mM, corrected to each pH evaluated), 1.2 mL of enzymes solution, 0.6 mL of MnSO₄ (4.5 mM) as substrate, and 0.3 mL of H₂O₂ (9 mM) reacted for 5 min at 25 °C. Manganese peroxidase activity was expressed in U, and 1 U was defined as 1 μmol MnSO₄ oxidized per minute.

Lignin peroxidase (EC 1.11.1.14) activity was determined in a UV-Vis spectrophotometer (Jasco V-530, Jasco, Easton, MD) at 310 nm, according to Tien and Kirk (1984) through veratryl alcohol oxidation to veratraldehyde (3,4 dimethoxybenzaldehyde). The analysis was carried out with 0.75 mL of sodium tartrate buffer (10 mM, corrected to each pH evaluated), 0.5 mL of enzyme solution, 0.25 mL of veratryl alcohol (3 mM) as substrate, and 0.10 mL of H₂O₂ (5 mM), with a reaction time of 5 min at 25 °C. LiP activity was expressed in U, and 1 U was defined as 1 μmol veratryl alcohol oxidized per minute.

Endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) activities were determined in a spectrophotometer (Spectra Max 340 PC, Molecular Devices Corporation, Sunnyvale, CA) at 540 nm, adapted from Wood and Bhat (1988). In the endoglucanase assay, 1 mL of carboxymethylcellulose (1%, w/v), as substrate, and 0.9 mL of citrate phosphate buffer (0.1 M, corrected to each pH evaluated) were added into a 19-mL borosilicate glass tube and incubated for 10 min at 39 °C. Posteriorly, 0.1 mL of enzyme extract was added and incubated at 39 °C for 5 min. Specifically, 3 mL of dinitrosalicylic acid was added into the tube and boiled for 5 min to mark the released reducing sugar, according to Miller (1959). Glucose was used as the standard. Endoglucanase activity was expressed in U, and 1 U was defined as 1 μmol of glucose released per min. The exoglucanase activity assay was similar, except the substrate was replaced with microcrystalline cellulose (1%, w/v).

Xylanase (EC 3.2.1.8) activity was determined in a spectrophotometer (Spectra Max 340 PC, Molecular Devices Corporation, Sunnyvale, CA) at 540 nm, adapted from Bailey et al. (1992). In the adapted xylanase assay, 1 mL of xylan (1%, w/v) as substrate and 0.9 mL of citrate phosphate buffer (0.1 M, corrected to each pH evaluated) were added to a 19-mL borosilicate glass tube and incubated for 10 min at 39 °C. Posteriorly, 0.1 mL of enzyme extract was added and incubated at 39 °C for 5 min. Released reducing sugar was marked as described previously, according to Miller (1959). Xylose was used as the standard. Xylanase activity was expressed in U, and 1 U was defined as 1 μmol of xylose released per minute.

Mannanase (EC 3.2.1.25) activity was determined in a spectrophotometer (Spectra Max 340 PC, Molecular Devices Corporation, Sunnyvale, CA) at 540 nm, adapted from Rättö and Poutanen (1988), where 1 mL of galactoglucomannan (0.5%, w/v), as the substrate, and 0.9 mL of citrate phosphate buffer (0.1 M, corrected to each pH evaluated) were added into a 19-mL borosilicate glass tube (Thermo Fisher Scientific Inc., Waltham, MA) and incubated for 10 min at 39 °C. Posteriorly, 0.1 mL of enzyme extract was added and incubated at 39 °C for 5 min. Released reducing sugar was marked as described previously, according to Miller (1959). Mannose was used as the standard. Mannanase activity was expressed in U, and 1 U was defined as 1 μmol of mannose released per minute.

Protein concentration was measured using the Bradford Protein Kit Assay (Sigma-Aldrich Co., St. Louis, MO) according to Bradford (1976). The bovine serum albumin was used as standard, and 1 mg of protein was defined as 1 mg of enzyme.

The enzymes produced under in vitro cultivation and freeze dried, as a powder, was named “enzymatic complex,” whereas the terminology “lignocellulolytic enzymes” was related to the protein concentration described earlier. The treatments were expressed as application rates of lignocellulolytic enzymes (mg/kg of fresh matter).

Silage preparation and experimental design

The WPC was harvested, and the ensiling process was carried out at the Plant Science Research and Education Unit (Citra, FL). The in situ incubation was carried out at the University of Florida Dairy Research Unit (Gainesville, FL).

The WPC hybrid Syngenta NK1694-3111 (Syngenta International AG, Basel, Switzerland) was manually harvested at 37.8% of dry matter (DM) from four different location areas at the research station. The average chemical composition is presented in Table 1. Each field area consisted of 30 × 60 m, and there was a distance between each area of 15 m to ensure the separation of each plot. Plants from the border of each area were avoided, and two lines from the internal part were sampled to ensure a representative sample from the field. In addition, each plot was individually seeded.

Table 1.

Chemical composition of unfermented whole-plant corn (n = 4; % DM, unless otherwise stated)

Item1	Mean	SD
pH	5.84	0.16
DM,%	37.8	2.77
NDF	35.7	2.44
ADF	18.5	1.21
Cellulose	16.6	1.39
Hemicellulose	17.2	1.32
Lignin	1.85	0.24
Starch	32.5	2.07
WSC	7.54	0.21
Ash	2.40	0.09

¹ADF, acid detergent fiber; DM, dry matter; NDF, neutral detergent fiber; WSC, water-soluble carbohydrate.

The silage trial was subdivided into two periods where the WPCS was evaluated. The first period consisted of the evaluation during the onset of fermentation stage (1, 2, 3, and 7 d after ensiling), and the second period was 30 d after ensiling. The evaluation during the onset of fermentation was carried out in a randomized complete block design using a 5 × 4 factorial split-plot arrangement of the treatments (five enzymatic complex application rates and four time periods of fermentation), totaling 20 treatments with 4 replicates per treatment. Each replicate originated from a different location area at the research station, named as a plot. The enzyme application rates were 0, 9, 18, 27, and 36 mg of lignocellulosic enzymes (determined by protein concentration in the enzymatic complex)/kg of WPC (fresh matter [FM] basis), corresponding to 0, 0.587, 1.156, 1.734, and 2.312 g of enzymatic complex/kg of FM, and the opening times were 1, 2, 3, and 7 d after ensiling (subplots). The same five enzyme application rates were used with four replicates for the material that was treated and ensiled for 30 d, consisted a randomized complete block design. The enzyme application rates (mg of enzyme/kg FM) were based on a previous study carried out with lignocellulolytic enzyme from P. ostreatus produced in our research group (Machado et al., 2020).

The WPC was chopped at 19 mm of theoretical length of cut with a single row silage chopper (model #707 SN: 245797; CNH Industrial America LLC, Burr Ridge, IL, USA). Approximately, 600 g of forage was weighed individually for each experimental unit. Each experimental unit was mixed individually with the respective application rates of lignocellulosic enzymes. Treated WPC was immediately placed into nylon–polyethylene vacuum bags (89 µm thickness, 25.4 × 35.6 cm; Doug Care Equipment Inc., Springville, CA) and heat-sealed using a vacuum machine (Bestvac; distributed by Doug Care Equipment Inc., Springville, CA). The silage bags were stored in a dark environment at room temperature (21 °C) until reaching their assigned fermentation length.

Sample collection and analyses

Bags were weighed to determine the DM losses and opened on the respective day of fermentation (n = 100/total study, as well as n = 20/opening time). The DM losses were determined by the difference between the weight of the plant ensiled and the weight of the material after the designated fermentation time, adjusted for DM content of the plant at ensiling and of the material at the opening. Two subsamples from each bag were dried in a forced-air oven at 55 °C for 72 h. After drying, the two subsamples were combined and ground in a Wiley mill (model No. 2; Arthur H. Thomas Co., Philadelphia, PA) to pass a 4-mm screen to determine the in situ DM and NDF disappearance and to pass a 1-mm screen to determine the chemical composition and in situ undigested NDF (uNDF).

An aqueous extract was prepared by mixing 20 g of silage (fresh sample) plus 200 mL of double-distilled water in a Stomacher (Lab-Blender 400, Tekmar Company, Cincinnati, OH) at high speed for 30 s and filtered through two layers of cheesecloth. The pH was determined using a digital pH meter (Accumet XL25, Thermo Fisher Scientific Inc., Waltham, USA). Aliquots of 40 mL of each extract were acidified with 0.4 mL of sulfuric acid in water (50/50, v/v), centrifuged at 7,000 × g for 15 min at 4 °C, and stored at −20 °C to determine organic acids and NH₃-N.

Organic acid concentrations were determined as described by Muck and Dickerson (1988) using high-performance liquid chromatography (Merck Hitachi Elite La-Chrome; Hitachi L2400, Tokyo, Japan). A Bio-Rad Aminex HPX-87H ion exclusion column (300 × 7.8-mm i.d.; Bio-Rad Laboratories, Hercules, CA) was used in an isocratic elution system containing 0.015 M sulfuric acid in the mobile phase with a UV detector at 210 nm, using a flow rate of 0.7 mL/min at 45 °C.

The NH₃-N concentration was determined according to Broderick and Kang (1980) and adapted to a plate reader, using 2 µL of the sample, 100 µL of phenol, and 80 µL of hypochlorite in each well of the microplate. The plate was incubated at 95 °C for 10 min and maintained at room temperature for 10 min for cooling. Absorbance readings were done utilizing a UV-Vis spectrophotometer at 620 nm (Spectra Max 340 197 PC, Molecular Devices Corporation, Sunnyvale, CA).

To determine the in situ disappearance of NDF, approximately 5 g of 4 mm ground material was weighed into filter bags (R1020, 10 × 20 cm, 50 ± 10-micron porosity; Ankom Technology, Macedon, NY) in duplicate for each replicate sample. Bags were incubated for 30 h in two rumen-cannulated lactating Holstein cows (1 bag per sample per cow). Cows were fed a diet consisting of 38% corn silage, 19% ground corn, 13% soybean meal, 11% cottonseed, 9% citrus pulp, 8.5% mineral premix, and 1.5% palmitic acid supplement, on a DM basis.

To determine uNDF, approximately 0.5 g of ground material (1 mm) was weighed into fiber filter bags (F57, 25-micron porosity; Ankom, Technology, Macedon, NY) in duplicate for each sample. Bags were incubated for 240 h in two rumen-cannulated lactating Holstein cows (one bag per sample per cow), and the same animals were used for the NDF in situ disappearance. After removal, bags were analyzed for NDF, and uNDF was expressed in percentage of DM.

All the bags from the incubations were dried in a forced-air oven at 60 °C for 48 h. The bags from 30 h of incubation were weighed to determine the DM disappearance, the replicates were composited and ground to pass a 1-mm sieve using a Cyclone sample mill (UDY Corporation, Fort Collins, CO), and samples were analyzed for NDF. The NDF disappearance was expressed in percentage of NDF.

The DM content was determined at 105 °C using an oven according to method no. 924.01 (AOAC, 1990). Ash was determined by combustion at 600 °C for 6 h in a furnace, according to method no. 924.05 (AOAC, 1990). The NDF was determined in an Ankom 200 Fiber Analyzer (Ankom Technologies, Macedon, USA) using thermostable α-amylase and sodium sulfite (Mertens et al., 2002). Acid detergent fiber (ADF) was determined sequentially according to method no. 973.18 (AOAC, 1990). The concentration of lignin was determined using the acid detergent lignin methodology, according to Van Soest and Wine (1968), by submitting the material to sulfuric acid (72/28, w/w in DI water) sequentially following ADF analysis in Daisy Incubator (Ankom Technology, Macedon, NY) with constant agitation. The cellulose concentration was obtained by the difference between the ADF and lignin, whereas hemicellulose concentration was obtained by the difference between NDF and ADF. Water-soluble carbohydrate (WSC) was determined using the anthrone reaction test (Weiss et al., 1990) and the starch concentration by colorimetric method, according to Hall et al. (2015).

Statistical analyses

All statistical analyses were carried out using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC). Differences were declared significant at P ≤ 0.05 and tendencies when 0.05 < P ≤ 0.10.

For the enzymatic activity, data were analyzed in a completely randomized design using the following model:

${\mathrm{Y}}_{\mathrm{i}\mathrm{j}}=\mathrm{\mu }+{\mathrm{H}}_{\mathrm{i}}+{\epsilon }_{\mathrm{i}\mathrm{j}}$,

where Y_ij = dependent variable, μ = overall mean, H_i = fixed effect of the pH (i = 3 to 6), and ${\epsilon }_{\mathrm{i}\mathrm{j}}$ = residual error. Enzymatic activity means were compared using the Bonferroni t-test option whenever differences were observed.

Data from the onset of fermentation stage were analyzed in a completely randomized block design using a 5 × 4 factorial split-plot arrangement (five application rates of enzymatic complex and four fermentation days from the onset of fermentation), using the following model:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{Y}}_{\mathrm{i}\mathrm{j}\mathrm{k}\mathrm{l}}=\mathrm{\mu }+{\mathrm{P}}_{\mathrm{i}}+{\mathrm{E}}_{\mathrm{j}}+{(\mathrm{P}\times \mathrm{E})}_{\mathrm{i}\mathrm{j}}+{\mathrm{D}}_{\mathrm{k}}+{(\mathrm{E}\times \mathrm{D})}_{\mathrm{j}\mathrm{k}}+{\epsilon }_{\mathrm{i}\mathrm{j}\mathrm{k}\mathrm{l}}}\end{array}}$$

where Y_ijkl = dependent variable, μ = overall mean, P_i = random effect of plot (i = 1 to 4), E_j = fixed effect of enzyme application rates (j = 0 to 36 mg of enzymes/kg of FM), (P × E)_ij = main-plot error, D_k = fixed effect of days after ensiling (k = 1 to 7), (E × F)_jk = fixed effect of interaction, and ɛ_ijkl = residual error. Linear and quadratic effects were tested for the enzyme application rates and days after ensiling using orthogonal polynomial contrasts. The coefficients for orthogonal polynomial contrasts were determined using the IML procedure of SAS to account for the unequal space among treatments.

Data from 30 d of fermentation were analyzed in a randomized complete block design, containing five enzyme application rates, using the following model:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{Y}}_{\mathrm{i}\mathrm{j}\mathrm{k}}=\mathrm{\mu }+{\mathrm{E}}_{\mathrm{i}}+{\mathrm{P}}_{\mathrm{j}}+{\epsilon }_{\mathrm{i}\mathrm{j}\mathrm{k}}}\end{array}}$$

where Y_ijk = dependent variable, μ = overall mean, E_i = fixed effect of enzyme application rates (i = 0 to 36 mg of enzymes/kg of FM), P_j = random effect of plot (j = 1 to 4), and ɛ_ijk = residual error. Linear and quadratic effects were tested for the enzyme application rates by using orthogonal polynomial contrasts.

Results

The characterization of the enzymatic complex produced by P. ostreatus is presented in Table 2. Laccase had the greatest activity (P < 0.01) at pH 5, which corresponded to 97.8 U/g of enzymatic complex/min. Laccase activity decreased at pH 4 and 6 approximately 23%, on average, compared with the activity at pH 5. The lowest laccase activity was observed at pH 3, and the reduction corresponded to 40.2% compared with the activity observed at pH 5.

Table 2.

Characterization of enzymatic complex produced by Pleurotus ostreatus

Enzymatic activity, U/g/min1	pH				SEM	P-value
	3	4	5	6
Laccase	58.5c	72.9b	97.8a	78.6b	2.01	<0.01
Mn peroxidase2	3.79b	22.2a	0.00b	0.00b	1.15	<0.01
LiP2	26.1b	81.8a	1.03c	0.0c	48.1	<0.01
Endoglucanase	16.3	14.3	19.3	14.1	0.17	0.61
Exoglucanase	23.4	27.9	35.4	28.8	1.82	0.64
Xylanase	26.6	28.2	28.6	27.1	0.07	0.42
Mannanase	17.0b	18.5a,b	19.0a	17.9a,b	0.03	0.04

¹The enzymatic activities were identified based on the final product obtained in the reaction. Enzymatic activity was expressed in U/g of enzymatic complex/min.

²LiP, lignin peroxidise.

^a–cMeans with different superscript letters differ by Bonferroni t-test (P ≤ 0.05).

Manganese peroxidase and LiP had the greatest activity at pH 4 (P < 0.01), whereas manganese peroxidase had no activity at pH 5 and 6 and low activity at pH 3. LiP activity decreased by approximately 68% at pH 3 and 99% at pH 5 compared with the greatest activity observed at pH 4. Mannanase had greater activity at pH 5 than at pH 3 (P < 0.04); however, it did not differ from the activities observed at pH 6 and 4. Activities of xylanase, endoglucanase, and exoglucanase were similar at pH 3 to 6.

The P-values for the effects of application rates of enzymatic complex, days from ensiling time, and their interactions at the onset of fermentation and the least-square means of treatments are presented in Table 3. No interaction effects were detected between application rates of enzymatic complex and days from the onset of fermentation (P > 0.11); also, the application rates of enzymatic complex did not affect fermentation profile (P > 0.21), chemical composition (P > 0.36), in situ DM and NDF disappearance (P > 0.57), and DM losses (P = 0.91) at the onset of fermentation. Differences observed for the main effect of days of fermentation are presented in Table 4. The pH quadratically decreased (P = 0.01), whereas the concentration of lactic acid quadratically increased (P = 0.01) as fermentation progressed. From 1 to 7 d, acetic acid (P = 0.02) and the total organic acids (P < 0.01) linearly increased. Concentrations of lignin, uNDF, and WSC quadratically decreased along with days of fermentation (P ≤ 0.02).

Table 3.

Least square means of treatments and P-values of the effect of enzymatic complex application rates, days of fermentation, and their interaction on profile, chemical composition, and DM and NDF disappearance at the onset of fermentation (1 to 7 d) in whole-plant corn silage treated with lignocellulolytic enzymes from Pleurotus ostreatus (% DM, unless otherwise stated)

Item1	Treatments (mg enzymes/kg FM)1					SEM	P-value
	0	9	18	27	36		Enzyme	Time	Enzyme × time
pH	4.07	4.03	4.06	4.08	4.07	0.045	0.62	<0.01	0.17
Total acids	5.31	5.46	4.54	4.58	5.19	0.431	0.26	<0.01	0.11
Lactic acid	3.01	3.16	2.67	2.80	3.12	0.271	0.43	<0.01	0.11
Acetic acid	1.43	1.45	1.17	1.11	1.32	0.127	0.21	<0.01	0.14
Propionic acid	0.83	0.81	0.70	0.68	0.75	0.758	0.57	0.02	0.46
NH3-N, % N	3.37	3.50	3.61	3.55	3.36	0.187	0.29	<0.01	0.15
DM, %	35.6	35.2	34.9	35.7	35.9	0.956	0.36	0.11	0.70
Ash	2.26	2.29	2.27	2.31	2.27	0.062	0.96	0.41	0.99
OM	97.7	97.7	97.7	97.7	97.7	0.062	0.96	0.41	0.99
NDF	37.1	35.9	37.0	36.3	36.0	0.742	0.57	0.48	0.82
ADF	20.2	19.7	20.3	19.5	19.5	0.457	0.52	0.19	0.71
Cellulose	18.5	18.0	18.5	17.7	17.8	0.422	0.43	0.22	0.66
Hemicellulose	16.7	16.2	16.8	16.8	16.7	0.338	0.58	0.13	0.57
Lignin	1.68	1.65	1.74	1.72	1.74	0.052	0.62	<0.01	0.3
WSC	3.13	3.28	2.88	3.27	2.96	0.230	0.37	<0.01	0.97
DMD	66.6	66.8	65.5	66.0	66.8	0.776	0.68	0.76	0.31
NDFD, % NDF	27.4	24.5	27.3	27.6	28.8	1.613	0.57	0.34	0.12
uNDF	13.0	12.7	12.6	12.4	12.7	0.315	0.65	0.01	0.38
DM loss	11.9	12.0	11.5	12.2	11.3	1.900	0.91	0.64	0.31

¹ADF, acid detergent fiber; DM, dry matter; DMD, in situ dry matter disappearance; NDF, neutral detergent; NDFD, in situ neutral detergent fiber disappearance; uNDF, in situ undigested neutral detergent fiber; WSC, water-soluble carbohydrate.

Table 4.

Fermentation profile and chemical composition of whole-plant corn silage treated at ensiling with lignocellulolytic enzymes from Pleurotus ostreatus, at days 1 to 7 after ensiling (% of DM, unless otherwise stated)

Item1	Day of ensiling				SEM	P-value
	1	2	3	7		Linear	Quadratic
Fermentation profile
pH	4.25	4.28	3.87	3.85	0.04	<0.01	<0.01
Lactic acid	1.82	2.18	3.37	4.43	0.25	<0.01	0.02
Acetic acid	1.19	1.06	1.50	1.43	0.11	0.02	0.16
Propionic acid	0.90	0.65	0.71	0.76	0.06	0.54	0.01
Total acids	3.91	3.89	5.65	6.62	0.41	<0.01	0.09
NH3-N, % N	2.90	3.34	3.58	4.09	0.18	<0.01	<0.01
Chemical composition
Lignin	1.85	1.63	1.66	1.69	0.04	0.10	<0.01
WSC	4.22	3.63	2.86	1.71	0.21	<0.01	<0.01
uNDF	13.4	12.2	12.6	12.5	0.28	0.16	0.02

¹DM, dry matter; uNDF, undigested neutral detergent fiber; WSC, water-soluble carbohydrate.

There was no effect of the enzymatic complex application rates at the time of ensiling on the fermentation profile observed 30 d later (Table 5). Ash concentration quadratically increased (P = 0.05), whereas organic matter quadratically decreased (P = 0.05) after 30 d of ensiling with the addition of enzymatic complex at ensiling. The lignin concentration in the WPCS, 30 d after ensiling, linearly decreased (P = 0.04) with the addition of the enzymatic complex at ensiling (Table 6). Other nutrients and in situ DM and NDF disappearance (P > 0.13) were not affected by enzyme application rates at ensiling 30 d later (P > 0.11).

Table 5.

Fermentation profile and dry matter loss after 30 d of ensiling of whole-plant corn silage treated with increasing application rates of lignocellulolytic enzymes from Pleurotus ostreatus at the time of ensiling (% DM, unless otherwise stated)

Item	Treatments (mg enzymes/kg FM)1					SEM	P-value
	0	9	18	27	36		Linear	Quadratic
pH	3.64	3.60	3.71	3.59	3.64	0.02	0.91	0.35
Lactic acid	5.82	5.33	5.08	6.17	5.35	0.56	0.92	0.65
Acetic acid	0.84	0.84	0.82	1.24	0.78	0.18	0.53	0.34
Propionic acid	0.52	0.73	0.55	0.99	0.34	0.18	0.81	0.12
Butyric acid	ND3	ND3	ND3	ND3	ND3	—	—	—
Total acids	7.18	6.91	6.44	8.36	6.46	0.71	0.99	0.67
NH3-N, % N	4.89	4.79	5.08	4.54	4.67	0.32	0.16	0.52
DM2 loss	13.3	14.4	13.1	12.5	12.7	1.95	0.33	0.74

¹Treatments corresponded to the following enzymatic activities: 0 (0 g of enzymatic complex/kg of fresh matter [FM]), 9 (0.587 g of enzymatic complex/kg of FM), 18 (1.156 g of enzymatic complex/kg of FM), 27 (1.734 g of enzymatic complex/kg of FM), 36 (2.312 g of enzymatic complex/kg of FM).

²Dry matter.

³ND, not detected.

Table 6.

Chemical composition and in situ disappearance after 30 d of ensiling of whole-plant corn silage treated with increasing application rates of lignocellulolytic enzymes from Pleurotus ostreatus at the time of ensiling (% DM, unless otherwise stated)

Item2	Treatments (mg enzymes/kg FM)1					SEM	P-value
	0	9	18	27	36		Linear	Quadratic
DM,%	35.4	34.9	35.4	35.8	35.7	1.28	0.28	0.63
Ash	2.37	2.66	2.70	2.50	2.56	0.09	0.44	0.05
Organic matter	97.6	97.3	97.3	97.5	97.4	0.09	0.44	0.05
NDF	36.4	39.1	39.2	37.1	35.8	2.06	0.57	0.15
ADF	19.0	20.9	21.1	20.5	19.6	1.15	0.78	0.12
Lignin	1.63	1.73	1.50	1.50	1.49	0.07	0.04	0.99
Cellulose	17.3	19.1	19.6	19.0	18.1	1.12	0.65	0.11
Hemicellulose	17.4	18.2	18.1	16.6	16.2	0.99	0.13	0.22
WSC	0.88	0.84	0.91	0.98	0.93	0.11	0.43	0.94
uNDF	11.6	12.6	12.9	12.3	12.1	0.53	0.73	0.12
DMD	66.1	66.7	66.3	67.9	69.2	1.42	0.15	0.55
NDFD, % NDF	23.6	28.6	28.7	28.8	26.8	2.73	0.39	0.13

¹Treatments corresponded to the following enzymatic activities: 0 (0 g of enzymatic complex/kg of fresh matter [FM]), 9 (0.587 g of enzymatic complex/kg of FM), 18 (1.156 g of enzymatic complex/kg of FM), 27 (1.734 g of enzymatic complex/kg of FM), and 36 (2.312 g of enzymatic complex/kg of FM).

²ADF, acid detergent fiber; DM, dry matter; DMD, in situ dry matter disappearance (30 h of incubation); NDF, neutral detergent fiber; NDFD, in situ neutral detergent fiber disappearance (30 h of incubation); uNDF, in situ undigested neutral detergent fiber (240 h of incubation); WSC, water-soluble carbohydrate.

Discussion

White-rot fungi, such as P. ostreatus, have been reported in the literature to produce enzymes that break down lignin and degrade cellulose and hemicellulose (Bánfi et al., 2015). In this study, we observed that among the lignocellulolytic enzymes evaluated, P. ostreatus mainly produced three enzymes that break down lignin (laccase, LiP, and manganese peroxidase), two types of cellulases (endoglucanase and exoglucanase), and two hemicellulases (xylanase and mannanase).

Enzyme activity is recognized to be affected by factors such as temperature, substrate concentration, and pH (Beauchemin et al., 2003). Therefore, the activity of enzymes from P. ostreatus cultivation was tested at different pH, simulating the range of the pH of the plant before ensiling and after an effective fermentation. As expected, the optimum pH varied among enzymes. The optimum pH observed for laccase was at 5 in our study, which was similarly described in previous studies (Manole et al., 2008; El-Batal et al., 2015).

Laccase activity was likely impaired during the onset of fermentation due to the pH drop, as silage pH decreased to almost 4 on the first day of fermentation, and also by anaerobiosis establishment in silages. As demonstrated, at pH 4, the activity of this enzyme was suppressed by approximately 23% compared with the optimum pH of 5. Anaerobiosis also may negatively affect laccase activity, as O₂ is an essential cofactor for its catalytic cycle (Shekher et al., 2011). In the silo, the residual oxygen is consumed by plant cell respiration and aerobic or facultative microorganisms shortly after silo sealing (McDonald et al., 1991), allowing a short time of optimal conditions for proper enzyme function.

The greater activities of manganese peroxidase and LiP at low pH may have allowed these enzymes to act during the whole fermentation period. Also, manganese peroxidase and LiP do not require aerobic conditions for the cycle activation (Wong, 2009). Therefore, the optimum pH, around 4, for LiP might be attributed to the pH required to complete part of the enzymatic cycle (Wong, 2009; Datta et al., 2017).

Manganese peroxidase does require Mn⁺² as a cofactor to cleave lignin (Hofrichter et al., 2010). WPC has approximately 914 mg of Mn/kg (DM basis; NRC, 2001), which might contribute to manganese peroxidase activity providing the mineral essential for the enzyme activity. Therefore, as the conditions did not favor laccase, the reduction in lignin concentration observed in the WPCS at 30 d after ensiling caused by the addition of enzymatic complex was mainly attributed to lignin cleavage by manganese peroxidase and LiP over the silage fermentation. We expected to observe a positive effect of enzymes on lignin concentration at the onset of fermentation, but this benefit was not observed before 30 d of ensiling. We attributed this lack of effect to insufficient accumulation of lignin cleavage until 7 d of fermentation.

Lignin acts as a barrier that hinders microorganisms from degrading fiber in the rumen (Hatfield et al., 2016). Thus, degradation of lignin would be a way to increase the access of the microorganisms to hemicellulose and cellulose and possibly increase fiber degradability (van Kuijk et al., 2015). However, despite reducing the lignin concentration, in situ DM and NDF disappearance did not increase by adding the enzymatic complex in this study. These results may indicate that reducing lignin concentration in a plant with low-lignin concentration would not be able to increase in situ disappearance.

The reduction of lignin concentration with the increase in enzymatic complex application rates at ensiling, observed 30 d later, was similar to that observed by Machado et al. (2020). However, in this previous study, the reduction in lignin increased in vitro digestibility. Differences between studies could be due to different lignin concentrations and the matrix organization in the cell wall. In the current study, lignin concentration in WPC was about four times lower than that observed by Machado et al. (2020; 1.63% vs. 6.64 % of DM). These factors might have influenced the magnitude of the effect (comparing the variation between control treatment and treatment with the highest application rate) observed in the lignin by enzyme treatment in the present study compared with Machado et al. (2020; 8.59% vs. 44.4%, respectively). We also speculated that there was a prevalence of cross-linking in the plants used by Machado et al. (2020) attributed to the greater lignin concentration than in the present study. Therefore, the combination of a greater reduction in lignin concentration with a prevalence of cross-linking may have favored an effect in the degradability in Machado et al. (2020).

Another important factor affecting lignin concentration and in situ disappearance was the range in lignocellulolytic enzyme activities. Machado et al. (2020) observed greater activity of laccase, cellulases, and hemicellulases than reported in the present study. Differences in enzymatic activities were attributed to fungal strain, and culture medium conditions as Machado et al. (2020) used KIRK medium (Kirk et al., 1986), coast cross grass hay (C. dactylon [L.] Pers), and the P. ostreatus strain was not determined. In the present study, the culture medium was prepared with bermudagrass (C. dactylon) hay, sugarcane, and water, chosen based on a pretrial to replace KIRK medium to decrease the production cost. Membrillo et al. (2008) also observed that two different strains of P. ostreatus produced different proportions of enzymes when the fungi were exposed to different medium conditions supporting our premise.

Although the enzymatic complex had activities of cellulases and hemicellulases, its addition onto WPC at ensiling did not affect the concentration of NDF and ADF at the onset of fermentation or 30 d after ensiling. In contrast, Colombatto et al. (2004) observed a reduction in the concentration of ADF and Lynch et al. (2015) in the concentration of NDF and ADF in WPCS treated with fibrolytic enzymes (a combination of cellulolytic and hemicellulolytic) at ensiling. Machado et al. (2020) also observed an effect in NDF and ADF concentrations in WPCS using a lignocellulolytic enzymatic complex. This divergence may be attributed to the enzymatic activities and the concentration and arrangement of NDF and ADF in the unfermented WPC. In those studies, the enzymatic activity per kilogram of WPC and the concentration of NDF and ADF were greater than in this study. In the study carried out by Lynch et al. (2015), the concentration of NDF and ADF in the WPC before ensiling were, respectively, 33% and 37% greater than in the present study.

Concentrations of lactic acid, acetic acid, propionic acid, and NH₃-N; pH; and the absence of butyric acid 30 d after ensiling were similar to the values described in the literature to an efficient fermentation, evidencing that the WPCS had a satisfactory fermentation (Kung et al., 2018).

Our suggestion for future studies is to test the enzymatic complex from the P. ostreatus cultivation in WPC or other forages containing high lignin and NDF concentration, usually observed in silage produced in tropical areas (Daniel et al., 2019), where the enzymes may express greater effects.

Conclusions

We observed that most enzyme activities were affected by pH, demonstrating the existence of an optimum pH at 5 for laccase, at 4 for manganese peroxidase and LiP, and at 5 and 3 for mannanase. Adding lignocellulolytic enzymatic complex from P. ostreatus cultivation at ensiling decreased WPCS lignin concentration 30 d after ensiling. We speculate that this reduction was possibly associated with the activities of LiP and manganese peroxidase, which are capable of degrading lignin under anaerobic and low pH conditions. However, there were no differences, at the onset of fermentation and at 30 d after ensiling, in the in situ disappearance of DM and NDF, fermentation profile, and other chemical entities in WPCS treated at ensiling with enzyme application rates.

Glossary

Abbreviations

ADF

acid detergent fiber

DM

dry matter

DMD

dry matter disappearance

FM

fresh matter

LiP

lignin peroxidase

NDF

neutral detergent fiber

NDFD

neutral detergent fiber disappearance

uNDF

undigested neutral detergent fiber

WPC

whole-plant corn;

WPCS

whole-plant corn silage

WSC

water-soluble carbohydrate

Conflict of interest statement

The authors declare no conflict of interest.