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Article

Bioconversion in Ryegrass-Fescue Hay by Pleurotus ostreatus to Increase Their Nutritional Value for Ruminant

by
Rita Astudillo-Neira
1,
Evelyn Muñoz-Nuñez
2,
Soledad Quiroz-Carreno
2,
Jorge Avila-Stagno
1 and
Julio Alarcon-Enos
2,*
1
Laboratorio de Nutrición Animal y Sistemas Ganaderos, Departamento de Ciencia Animal, Universidad de Concepción, Chillán PC3780000, Chile
2
Laboratorio de Síntesis y Biotransformación de Productos Naturales, Departamento Ciencias Básicas, Universidad del Bío-Bío, Chillán PC3780000, Chile
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(4), 534; https://doi.org/10.3390/agriculture12040534
Submission received: 18 February 2022 / Revised: 30 March 2022 / Accepted: 7 April 2022 / Published: 9 April 2022
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Abstract

:
Carbohydrates from lignocellulosic feed can be released by basidiomycete fungi for ruminal fermentation. This study aimed to evaluate the bioconversion of hay of ryegrass-fescue (Lolium perenne—Festuca arundinácea) by solid state fermentation with Pleurotus ostreatus, to obtain superior quality hay. After only 14 days of fermentation, crude protein (CP) (4.73 to 5.16%), and non-fibrous carbohydrates (NFC) (20.84 to 25.04%) increased, while neutral detergent fiber (NDF) (68.72 to 64.87%) and acid detergent lignin (5.88 to 1.98%) decreased. The enzymatic biodegradation carried out by P. ostreatus was verified, through measurements of enzymatic activity. Lignin peroxidase (LiP) and laccase (Lac) reached the higher activity on day 14 (19.51 U/L and 34.17 U/L, respectively), whereas manganese peroxidase (MnP) displayed stability up to 21 days of fermentation (between 6.54 and 7.75 U/L). In conclusion, results indicate that lignocellulosic feed bioconversion by P. ostreatus is promising to improve the ruminal fermentation of fibrous feedstocks and 14 days were considered to be optimal for hay fermentation.

1. Introduction

The major limitation of ruminant production based on forages is the fluctuation of their production and quality [1]. Traditionally, ruminant diets are characterized by forages high in lignocellulose and low in available carbohydrates, which are fermented to short-chain fatty acids, mainly acetic, propionic and butyric. Acetate and butyrate are the main energy sources, propionate is used in gluconeogenesis and acetate itself is a lip- genic precursor. The production of these acids is related to the diet in terms of its content of cellulose, hemicellulose, lignin, starch, and sugars, among other nutrients. In general, the higher the fibrous carbohydrate content, the higher the acetate production, and the higher the NFC content, along with higher propionate production, which also translates into lower energy losses by methane production, in most cases [2]. Due to the seasonality in forage growth, the conservation of them for use in periods of scarcity ensures the feeding of the livestock. Of the various options available, haymaking is usually performed at the beginning of the maturation stage or once maturation has already begun, which implies a decrease in available nutrients and an increase in fiber (and, therefore, lignin) content [1].
The cell wall contains cellulose, hemicellulose, pectin and the phenolic polymer lignin [3]. Lignin provides strength and rigidity to cell walls and tissues of all vascular plants [4]. It is indigestible for ruminants and their rumen microflora and its presence makes cell wall carbohydrates unavailable as it limits microbial and enzymatic attack in the rumen [5,6] by linking to both hemicellulose and cellulose, forming an impenetrable seal [7,8]. Therefore, to optimize and improve its nutritive value the use of agricultural products, forage (hay) and by-products, as feed for ruminants, it is desirable to increase the lignin degradation of the lignocellulosic complex [6,9,10].
Xylophagous fungi (such as Ganodermas spp., Phlebia radiata, Lentinula edodes or Pleurotus spp.) are the main lignin degraders and the only group of microorganisms able to mineralize lignin; among them, only white rot fungi completely degrades lignin to CO2 and H2O [11,12]. Fungi have two types of extracellular enzymatic systems: the hydrolytic system, which produces hydrolases that are responsible for polysaccharide degradation and a unique oxidative and extracellular ligninolytic system, which degrades lignin and opens phenyl rings [13]. The lignin-degrading ability of these species is due to the strong oxidative activity and low specificity of their ligninolytic enzymes (laccases, (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and multifunctional peroxidase (versatile)) [12]. Laccase corresponds to a phenol oxidase, thus catalyzing the degradation of polyphenols; this oxidizes one-electron of phenolic compounds with an associated reduction of oxygen to water as a secondary product and produces phenoxy radicals than can be converted to quinones (Figure 1). Within the group of oxidoreductases, LiP uses H2O2 to oxidize cationic aryl radicals as the initial substrate, achieving lignin depolymerization (Figure 2) and MnP acts on phenolic and aromatic compounds as shown in Figure 3. Finally, multifunctional peroxidase is a combination of LiP and MnP activities [10].
A complex system has been proposed, where laccase, LiP and MnP are secreted by fungal hyphae close to the hyphae environment, where they co-operate with each other as well as with mediating factors [14]. This lignin degradation allows the entry of simple products into their hyphae and their incorporation into their catabolic pathways [13].
In this way, the degradation of lignin breaks down the lignocellulose complex, leaving these carbohydrates susceptible to rumen microflora and enzymatic attack [15], thus improving ruminant digestibility [9,16] (Figure 4).
It has been established that the solid state fermentation (SSF) system with a previously submerged fermentation is suitable to study the morphological and metabolic differences in the growth of fungi with ligninolytic activity, as well as for the study of enzymes [17]. SSF is defined as the growth of microorganisms on solid materials in the absence or near absence of free water (minimum 12% [18]) and is considered an accessible, low-cost and environmentally friendly system that can be used with various materials high in lignocellulose, such as agricultural by-products, industrial wastes, among others, delivering value-added products or compounds [19,20,21]. SSF is a polyfactorial event, in which the fungus, its enzymes, the physical structure of the substrate, physiological factors of fermentation and cultivation, and nutritional conditions have an important role in controlling lignin degradation and digestibility of the fermented substrate [6]. On the other side, submerged fermentation involves the nurturing of microorganisms in high oxygen concentrated liquid nutrient medium. The viscosity of the broth is the major problem associated with the fungal submerged fermentations [22] combined with a high cost for enzyme production [23]. It has been established that white rot fungi produces ligninolytic isoenzymes depending on whether their growth occurs in liquid medium or lignocellulosic substrate, either encoded by different genes or with allelic variations. These differences are responsible for the differences in the proportions of enzymes produced by the fungus between media [24].
The Pleurotus genus (Family Pleurotaceae, Basidiomycetes) is one of the most studied edible lignolyitic fungi due to its nutritional properties, anti-inflammatory, antioxidant, antidiabetic, antitumor and immunostimulatory effects. Globally distributed, the genus is estimated to include more than 200 species. [25]. Pleurotus spp. can colonize a wide variety of lignocellulosic substrates [26], without compromising cellulose and hemicellulose (unlike other fungi [27]), thus performing a bioconversion that improves the nutritional contribution of these materials when included in ruminant diets, making them the preferred fungi for improving feed quality [6]. In this sense, the bioconversion of lignocellulosic residues using Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm, has generated positive results in by-products destined for animal feed [11,26]. In contrast to the direct application of lignolytic enzymes, which have a high price, discarding their massive use as a strategy for lignin degradation in animal feed products [12].
Most reports [6,11,26,28] extend the cultivation of P. ostreatus until harvest, which implies the depletion of the substrate in terms of nutrients, to then dedicate them to animal feed, which is detrimental to their nutrition. However, those studies that have performed a short fermentation (without the formation of a fruiting body of the fungus), have managed to improve the quality of the fermentation substrates. For example, a 21-day fermentation of wheat straw with different strains of P. ostreatus, reported significant decreases NDF and ADF, increased crude protein and in vitro digestibility [29].
The research aimed to achieve bioconversion of ruminant feeds, specifically using a P. ostreatus native to Chile, to achieve the release of carbohydrates for ruminal fermentation considering the processing time and enzymatic activity.

2. Materials and Methods

2.1. Biological Material

A P. ostreatus strain 136UBB, belonging to the Macromycete collection of the University of Bío-Bío (Chillán, Chile) was selected due to its high stability. The origins of these strains are fruiting bodies collected in 2001, of the genus Nothofagus sp., located in forests of the regions of Araucanía, Bío Bío, Ñuble and Maule (south and central part of Chile). The strain was kept under refrigeration (4 °C), in a sowing system in an inclined tube with potato dextrose agar (Merck KGaA, 64271 Darmstadt, Germany).

2.2. Growth Activation of P. ostreatus

The representative strain tubes were incubated for 24 h at 30 °C in the dark (Heating incubator DHP-9052), to promote the activation of the mycelium [30].

2.3. Cultivation in Plates

A mycelium sample (1 cm2) was taken and placed in the center of Petri dishes with a PDA culture medium (under aseptic conditions, under a laminar airflow hood), and thereafter incubated at 30 °C in the dark, until complete growth of the mycelium inside the dish [30,31].

2.4. Submerged Fermentation

Mycelium samples in active growth were extracted from Petri dishes, to be seeded in 250 mL Erlenmeyer flasks with 150 mL of Hagen culture medium, which contains (g/L): CaCl2 0.044, KH2PO4 0.025, (NH4)2HPO4 0.25, MgSO4 × 7H2O 0.15, FeCl2 × 6 H2O 0.022, malt extract 3.0 and glucose 10.0 [31]. Fermentation was carried out under agitation (150 rpm) and at room temperature, for 25 days [8], until the complete colonization of the flasks by the fungal biomass [32].

2.5. Substrate for Solid State Fermentation

Hay of ryegrass (Lolium perenne) with fescue (Festuca arundinacea) harvested in the 2016 season, was milled with a 2 mm screen (Grain Mill, Breuer, Temuco, Chile). Then, ground hay was autoclaved (Vertical type steam sterilizer, model HL-340) at a temperature of 121 °C, 120 W/KA pressure, in a 15-min cycle [33]. Afterward, the hay was dried at 50 °C for 48 h.

2.6. Solid State Fermentation (SSF)

This step was conducted in 48 Erlenmeyer flasks (250 mL) with 50 mL of Hagen medium, vegetal substrate (5%), and the piece of mycelium (pellets) (1.15 ± 0.20 g dry weight, contained in 100 mL of Hagen medium from the submerged fermentation). Flasks were immediately incubated at 30 °C in the dark for 21 days, under stationary conditions [34,35]. It has been established that under these conditions P. ostreatus reaches full colonization of the substrate [36] (Figure 5).

2.7. Crude Enzymatic (CE) Obtainment from Submerged Fermentation

Samples of the biomass content in the flasks from the submerged fermentation were analyzed according to the following methodology. Briefly, 1 g of the biomass mixture (mycelium and Hagen medium) was ground with 10 mL of acetate buffer (pH 4.5) in sand, filtered (filter paper Whatman N° 1), and centrifuged at 5000 rpm at 4 °C for 20 min. The supernatant was removed and stored at −80 °C until the determination of enzymatic activity (between 3–5 days) [19,34,36].

2.8. Crude Enzymatic (CE) Obtainment from Solid State Fermentation (SSF)

Randomly selected samples (three units, corresponding to Erlenmeyer flasks) were taken at 1, 7, 14 and 21 days to obtain different fermentation times. One gram of the biomass mixture (hay, mycelium and Hagen medium) was ground with 10 mL of acetate buffer (pH 4.5) in sand, stirred at 150 rpm for 2 h at room temperature, filtered and centrifuged as previously described. The supernatants were stored at −80 °C [20].

2.9. Laccase (Lac EC 1.10.3.2) Enzymatic Activity

Laccase activity was measured by monitoring the change in absorbance at 436 nm due to the oxidation of 2,2′-Azino-bis [3-ethylbenzothiazolin-6-sulfonic] acid (ABTS) using a spectrophotometer (Epoch Microplate Spectrophotometer, Biotek Instruments, Highland Park, IL, USA). The reaction mixture included 0.5 mM ABTS in acetate buffer (0.1 M, pH 5.0) (0.712 mL) and enzymatic crude (0.0375 mL). The enzymatic crude substituted by acetate buffer was considered as a blank sample. The control was considered as the mixture without reaction [37].

2.10. Manganese Peroxidase (MnP EC 1.11.1.13) Enzymatic Activity

Manganese peroxidase activity was determined according to the following method modified using a reaction mixture of phenol red (0.01%) (100 µL), sodium phosphate (250 nM) (100 µL), serum albumin (0.5%) (200 µL), manganese sulfate (2 mM) (50 µL), hydrogen peroxide (2 mM) in acetate buffer (0.1 M, pH 5.0) (50 µL), and enzymatic crude (500 µL). After 5-min incubations at 30 °C absorbance readings were taken at 610 nm in a spectrophotometer (Epoch Microplate Spectrophotometer, Biotek Instruments, Highland Park, IL, USA). The mixture of the control contained all the reagents, but enzymatic crude and buffer acetate; whereas the control blank contained all the reagents, but serum albumin, enzymatic crude and hydrogen peroxide that was substituted by buffer phosphate. The blank sample was the enzymatic crude and acetate buffer [19].

2.11. Lignin Peroxidase (LiP EC 1.11.1.14) Enzymatic Activity

The lignin peroxidase activity was determined using the reaction mixture of acetate buffer (0.1 M, pH 3.0) (0.427 mL), ABTS (10 mM) (0.107 mL), hydrogen peroxide (0.1M) (0.107 mL) and crude enzyme (0.107 mL). Changes in absorbance were determined at 420 nm, after 1-h of incubation at 30 °C by spectrophotometry. The control contained all the reagents but the enzymatic crude. The control blank contained all except the CE and hydrogen peroxide [38].
The three enzymes here measured were expressed as one enzyme unit (U), defined as the amount of enzyme that causes the increase of 1 unit of absorbance per minute. The specific activity was reported as a unit per gram of total extracellular protein (U/g TP) [39,40]. The total protein content in the CE was determined by employing Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific®, Rockford, IL, USA).

2.12. Chemical Composition of Hay before and after Solid State Fermentation (SSF)

A sample composed of hay (consisting of three subsamples) was analyzed according to the methodology described as follows: dry matter (DM) (AOAC method #934.01) [41], total ash (TA) (AOAC method #942.05) [41], crude protein (CP) (AOAC method #954.01) [41], ether extract (EE) (AOAC #920.39) [41], theoretical metabolizable energy for ruminants (EM) [41], acid detergent fiber (ADF) (AOAC method #973.18) [41] and acid detergent lignin, neutral detergent fiber (NDF) [42]. Similarly, once the SSF of each flask reached 14 days, considered the minimum time of colonization of the substrate, a sample was taken to form a composite sample which was analyzed with the same methodology used to analyze the hay.

2.13. Structural Analysis of Ryegrass Hay with Fescue

Hay samples before and after 14 days of fermentation were taken and dried in a drying oven (model UNB 500, memmet GmbH+Co.KG, Schwabach, Germany) (50 °C) until constant weight, to observe microscopic structural changes. Samples were observed through an electron microscope (Hitachi SU 3500, Tokyo, Japan).

2.14. Statistical Analysis

In the submerged fermentation experiment, 54 Erlenmeyer flasks were considered as experimental units, while the sampling units were three flasks of 21 days of incubation; they were compared with descriptive statistics of means and standard deviations. On the other hand, in the solid state fermentation experiment, the experimental unit was 48 flasks and the sampling units were 12 Erlenmeyer flasks. An ANOVA was used for the analysis of the results in a completely randomized model design, of the model Yij = µ + αi + εij, where Yij is each of the observations of the sampling unit (enzymatic activity and specific enzymatic activity of each enzyme), µ is the general mean, αi is the fermentation time and εij is the experimental error. When indicated by the ANOVA, a Tukey mean comparison test (significance level 0.05) was performed. The statistical software Infostat [43] was used.

3. Results

The use of P. ostreatus in submerged fermentation and later in solid, with ryegrass-fescue as substrate, was carried out as expected, with the sporadic occurrence of environmental contamination. The solid state fermentation generated the biodegradation of the plant substrate used, due to the enzymatic activity of the fungus and as a consequence its bioconversion for its use in ruminant feeding.

3.1. Chemical Composition of Ryegrass-Fescue Hay before and after 14 Day Solid State Fermentation by Action of P. ostreatus

Table 1 shows the comparative results of the nutritional contributions of ryegrass-fescue hay before and after SSF with P. ostreatus. The bromatological analysis of the fermentation residue indicated 5.16% CP, 1.10% EE, 1.95 Mcal/kg DM of ME, 64.87% NDF, 42.05% ADF and acid detergent lignin 2.82% (Table 1). The SSF of the hay caused an increase in the content of CP and NFC, and decreases in NDF, ADF and acid detergent lignin, without altering the theoretical ME contributions for ruminants.

3.2. Structural Analysis

The changes in the vegetal material before and after the SSF with P. ostreatus are shown in Figure 6. As shown in Figure 6a, the surface structure of the hay in its natural state shows a low degree of damage, possibly due to the effect of cutting and grinding the material prior to its use. While after SSF and mechanical milling of the resulting material, disorganization, fragmentation and damage to the integrity of the structure are observed (Figure 6b), and a closer approach (Figure 6c) reveals that the damage is greater.

3.3. Enzymatic Activity of P. ostreatus in Submerged Fermentation

The total protein evaluation in submerged fermentation had a value of 0.02 ± 0.005 g/L. The enzymatic activities expressed in U/L were 4.38 ± 0.35, 9.39 ± 5.14, and 10.60 ± 5.71 for MnP, LiP and Lac, respectively, while expressed as specific enzymatic activity (U/g PT), for the same enzymes it was 175.48 ± 19.94, 380.05 ± 140.49 and 477.98 ± 334.24, respectively.

3.4. Enzymatic Activity of P. ostreatus in SSF

The enzymatic activity (U/L) (Figure 7) varied according to the enzyme type and time. MnP was stable between (6.54 and 7.75 U/L) days 1, 7, 14 and 21, indicating high stability up to 21 days of fermentation. The activity of LiP on day 1 was 14.61, 10.82 on day 7, 19.51 on day 14 and 13.43 U/L on day 21, being statistically the highest on day 14. The enzymatic activity of Lac was highest (p ˂ 0.05) on day 14, corresponding to 34.17, decreasing later to 30.55 on day 21. Total protein concentration did not differ on days 1, 7 and 14 (varying between 1.1 and 1.2 g/L), and then decreased to 0.760 g/L on day 21 of SSF.
Specific enzymatic activity (U/g PT) considers the total protein present in the enzymatic crude, for MnP, the highest value was reached on day 21 (10.99 U/g PT), doubling the activity measured on days 1, 7 and 14 (varying between 5.83 and 6.55). Similarly, LiP and Lac reached the highest values on 21 days of SSF (18.28 and 40.60, respectively), with fluctuations along the evaluated days (Figure 8).

4. Discussion

4.1. Bioconversion of Hay by the Action of P. ostreatus and Structural Analysis

Taking into account the characteristics of hay as feed for ruminants, used in this study (Table 1), this is classified as forage, although the CP value (4.73%) is slightly over half the value indicated for ryegrass hay (8.21%) and closer to pasture hay (7.25%) [42]. Theoretical ME for ruminants of the hay is low (1.94 Mcal/kg DM), as compared to ryegrass hay (2.15 Mcal/kg DM) and pasture hay (2.17 Mcal/kg DM) [45]. Considering CP and ME as the main nutrients, the hay used in this study is of inferior quality for its use in ruminant feeding. This situation could be due to a late cutting moment, in relation to the state of maturation of the pasture.
The SSF of ryegrass hay with fescue for 14 days using P. ostreatus modified its nutritional composition. These positive changes can be attributed to the contribution of the mycelium itself, the Hagen medium and the enzymatic action of the mycelium on the forage, as a consequence of its growth [46]. The availability of nutrients in ruminant feeds can be estimated by their chemical composition, linked to available components, such as protein, lipids, minerals, organic acids, and alpha-bound carbohydrates (glucose, fructose, sucrose, fructans, and starch), and less available ones (cellulose, hemicellulose) to factors that may limit the availability of those components [47]. In the case of forages, it is represented by lignin [6]. Thus, the nutritional value is conditioned by the relative proportion of cell content, cell wall, and the degree of lignification.
In this study, after 14 days of fermentation (Table 1), the CP value increased from 4.73 to 5.15%, the NDF representing the cell wall, decreased from 68.72 to 64.87% and also ADF. This probably indicates the rupture of the union of the lignocellulose complex (cellulose-hemicellulose-lignin). Acid detergent lignin had a considerable decrease of 66.33%. The non-fibrous carbohydrate content (NFC), represented mainly by starch, free sugars, pectins among others, increased from 20.84 to 25.04%. This final NFC value is above the content of average alfalfa hay (22.0%) and well above average mixed hay (16%) [44]. Although the optimal concentration of NFC in the diets of lactating dairy cows is not well defined, a maximum of 30–40% of the dry base diet avoids acidosis and other metabolic problems [44]. The correct formulation of rations should seek a balance between the maximum energy intake (reducing the intake of NDF and increasing non-fiber carbohydrates) and the maintenance of ruminal functions, which should be considered when using this hay biodegraded by the activity of P. ostreatus. These characteristics of hay after 14 days of SSF show a feed that at the ruminal level will present greater microbial degradability, with a faster adhesion of microorganisms to the plant wall due to the lower degree of lignification of the same [48], and consequent increased production of short chain fatty acids. Ruminal fermentation of feeds or diets high in structural carbohydrates generates a greater amount of methane per unit of feed ingested, compared to the fermentation of feeds or diets rich in non-structural carbohydrates [49]. Therefore, the bioconversion and its alteration of the ratio of structural versus nonstructural carbohydrates positively alter methane production in ruminal fermentation, in other words, by decreasing the production by intake feed. In addition, energy concentration is not modified by the effect of biodegradation [50].
SSF employing P. ostreatus on three types of solid olive milling residues mixed with wheat straw, showed the NDF fraction decreased for all three types of residues and the ADF fraction remained unchanged or decreased concluding that bioconversion with P. ostreatus causes selective degradation of the fiber fraction and improves the nutritional potential of lignocellulosic residues for ruminant feeding due to increased digestibility [47]. Similar effects have been obtained with wheat straw, after 15 days of SSF [6], and rice straw after 21 days [35].
The increased CP after 14 days of SSF in this study is consistent with the results of fermentation in olive milling residues [47], wheat straw [51], sorghum stubble [50,52], and rice straw [35]. The increase in the CP is attributed by the mentioned authors to mycelial growth and [35] suggested that the increase of fungal biomass is proportional to the CP enhancement. Another reason for this increase in crude protein could be the breakdown of the carbohydrate-protein bond [46]. Although agricultural residues are abundant, their use in animal feed must take into account their low nutritional value, due to their high fiber content and low levels of crude protein, vitamins and minerals. Within the range of physical, biological and chemical methods to improve quality, biological pretreatment with white rot fungi would be the best alternative for developing countries [35,46,52].
A compilation study of bromatological analyzes of 5 years carried out on samples of forages conserved in the Animal Nutrition Laboratory of INIA Quilamapu, of samples from the Maule and Bío Bío Region, Chile [53] indicate that Lolium perenne hay has an average CP concentration of 8.5%, with a maximum of 13.2% and a minimum of 3.7%, also noting that over 36% of the samples are below the average, which implies that these are in the lower minimum values to those required for medium to high milk production. The minimum values are usually associated with advanced phenological stages of the plants, which also affect the ruminal degradability of the remaining nutrients. In this situation, carrying out a pretreatment of forages with fair to poor characteristics, such as the bioconversion carried out by P. ostreatus, which improves nutritional quality, degradability and also in a short period of time, should have a positive impact on both milk and meat production costs, in ruminal health, as in the use of more rustic forage species, less competitive with human food or crop by-products. Studies of lignin biodegradation in wheat straw, and its subsequent in vitro digestibility, established that lignin degradation and in vitro digestibility have a correlation coefficient of 0.915, which implies a strong positive correlation [9,28].
The changes in the structure of the vegetal material were evidenced by electron microscopy analysis. SSF with P. ostreatus disorganizes the structure, causes structure breakdown, and small cavities appear where subsequent microbial attacks could take place. These results agree with studied the effect of P. ostreatus on corn stover [54].

4.2. Enzymatic Activity of P. ostreatus in Submerged Fermentation

The total protein had a value of 0.02 ± 0.005 g/L. This value is much lower than the value of the enzymatic activity of P. ostreatus in submerged fermentation for 25 days [55]. The author reported a peak total protein concentration of 0.17 g/L at 18 days. Similarly, the evaluation of Oxyporus latamerginatus reported an increase in soluble protein up to 22.72 g/L, on day 14 and then a reduction on day 17 [32]. Others reported enzymatic activity of P. ostreatus evaluated after day 3 of submerged fermentation were Lac (300 U/L) and MnP (0.41 U/L), however, LiP activity was not detected [56]. These values contrast with the results herein reported, probably due to differences in the fermentation times and growth medium employed in this study. Extracellular laccases can be consistently produced in small quantities; however, their production can be increased by different substrates, namely aromatic or phenolic compounds related to lignin [40]. The evaluation of enzymatic activity in submerged fermentation in peat-added medium, indicated greatest enzymatic value of Lac (67 U/L) was at day 15 while MnP was less than 2 U/L and LiP activity was not detected in significant quantities [8]. These authors stated that P. ostreatus LiP production is very low and difficult to detect, the reason why it is usually not studied. In contrast, in this work LiP activity in submerged fermentation was 9.39 U/L. This situation was also described in P. ostreatus culture in submerged fermentation [57,58] and solid fermentation using wheat straw [56].

4.3. Enzymatic Activity of P. ostreatus in SSF

The transition from the mycelial biomass enzymatic activity to SSF with hay implied an increase in three enzymes values. Increases in MnP (153%), LiP (176%) and Lac (207%) were detected after the first 24 h of fermentation, as compared to enzymatic activities of submerged fermentation. This change in enzymatic activity can be attributed to the solid substrate used, which contributed to the fungal development [8,39]. Beforehand, the solid fermentation of P. ostreatus on coffee pulp [40], presented Lac activity values of 7.10, 11.70 and 12.38 U/L on days 7, 14 and 21 of fermentation, respectively. These authors reported lower or similar values for sorghum and corn, attributable to the polyphenol contents in coffee pulp and sorghum. Similarly, the potato dry skin residues in SSF were evaulated, with P. ostreatus for up to 23 days [20] showing enzymatic activity values for Lac, MnP and LiP of 6708.30, 2503.60 and 231.20 U/L, respectively on day 17 of fermentation. These values are much higher than those reported in this study. Probably the substrate plays the most important role, as potato peels are highly hydrophobic and the surface load plus the capability of the fungus to metabolize sugars consequently improves P. ostreatus enzymatic activity, which may have contributed to the higher values. The variation in enzyme activity at 21 days of fermentation, specifically in LiP and Lac, can be attributed to the fact that the fungus preferentially degrades lignin and only once this substrate is exhausted, does it start producing enzymes for cellulose and hemicellulose degradation.

4.4. Producer-Level Applications

The Agricultural Research Institute (INIA) of Chile presented a bulletin on the production of oyster mushroom (Pleurotus ostreatus) for human consumption [59]. This is aimed at small and medium-sized agricultural producers, since in INIA’s opinion; this is a low-investment crop and is easier to grow than other mushrooms. The authors propose as a means of field pasteurization the immersion of vegetable material in boiling water for a few minutes and incubation in bags. This methodology could be suitable for the objectives of our research, with 14-day incubations, in the dark, to improve lignolytic substrates for animal consumption, but for small volumes of initial vegetable material.
An alternative for scaling up the production of plant material fermented by P. ostreatus would be the use of cereal grains inoculated with the fungus, as an additive to pasture hay (or other product intended for animal consumption high in lignocellulose), for storage in silage. Under these conditions, the lignolytic activity of the mycelium would act on the ensiled material, as long as oxygen remains available in the silage, improving the nutritional quality of the final product to be consumed by ruminants. This possible form of production opens new lines of research to study the interactions between the mycelium of the fungus and the bacteria of the ensiled material, the duration of the ensiled material, animal consumption tests and animal performance tests.

5. Conclusions

Pleurotus ostreatus actively carried out the solid state fermentation of hay ryegrass fescue in 14 days due to the high enzymatic activity and nutrient availability. This bioconversion resulted in a decrease in lignocellulosic fractions and an increase in the protein and non-fibrous carbohydrates, which implies a positive bioconversion, with a possible increased supply of nutrients and energy for ruminal microorganisms. Consequently, the use of native P. ostreatus in low quality forages improved feed quality by biodegradation of lignocellulosic material, in a period of only 14 days.

Author Contributions

Conceptualization, J.A.-E. and R.A.-N.; methodology, J.A.-E., E.M.-N. and S.Q.-C.; formal analysis, R.A.-N.; investigation, R.A.-N., E.M.-N., S.Q.-C. and J.A.-E.; resources, J.A.-E., R.A.-N.; writing—original draft preparation, R.A.-N., E.M.-N., S.Q.-C., J.A.-S. and J.A.-E.; writing—review and editing, J.A.-E. and J.A.-S.; funding acquisition, J.A.-E. and R.A.-N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Advanced human capital training program, of the National commission for scientific and technological research (CONICYT), of the Chilean Government, grant number Scholarship sheet N° 21191877.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors want to thank to internal grant from Research Direction, University of Bio Bio, Chillán, CHILE: DIUBB # 132209 GI/C.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Callejo, A. Conservación de Forrajes (II): Fundamentos de la henificación. Frisona Española 2017, 220, 104–109. [Google Scholar]
  2. Van Soest, P.J. Nutritional Ecology of the Ruminant, 2nd ed.; Cornell University Press: Ithaca, NY, USA, 1994. [Google Scholar]
  3. Ortiz, M. Aproximaciones a la comprensión de la degradación de la lignina. Orinoquia 2009, 13, 137–144. [Google Scholar]
  4. Hofrichter, M. Review: Lignin conversion by manganese peroxidase (MnP). Enzym. Microb. Technol. 2002, 30, 454–466. [Google Scholar] [CrossRef]
  5. Arora, D.S.; Sharma, R.K. Effect of different supplements on bioprocessing of wheat straw by Phlebia brevispora: Changes in its chemical composition, in vitro digestibility and nutritional properties. Bioresour. Technol. 2011, 102, 8085–8091. [Google Scholar] [CrossRef]
  6. Shrivastava, B.; Thakur, S.; Khasa, Y.P.; Gupte, A.; Puniya, A.K.; Kuhad, R.C. White-rot fungal conversion of wheat straw to energy rich cattle feed. Biodegradation 2011, 22, 823–831. [Google Scholar] [CrossRef]
  7. Howard, R.L.; Abotsi, E.; van Rensburg, E.J.; Howard, S. Lignocellulose biotechnology: Issues of bioconversion and enzyme production. Afr. J. Biotechnol. 2003, 2, 602–619. [Google Scholar] [CrossRef]
  8. Hong, Y.; Dashtban, M.; Chen, S.; Song, R.; Qin, W. Enzyme production and lignin degradation by four basidiomycetous fungi in submerged fermentation of peat containing medium. Int. J. Biol. 2012, 4, 172–180. [Google Scholar] [CrossRef]
  9. Arora, D.S.; Sharma, R.K. Comparative ligninolytic potential of Phlebia species and their role in improvement of in vitro digestibility of wheat straw. J. Anim. Feed Sci. 2009, 18, 151–161. [Google Scholar] [CrossRef]
  10. Kumla, J.; Suwannarach, N.; Penkhrue, W.; Kakumyan, P.; Jatuwong, K.; Vadthanarat, S.; Lumyong, S. Cultivation of mushrooms and their lignocellulolytic enzyme production though the utilization of agro-industrial waste. Molecules 2020, 25, 2811. [Google Scholar] [CrossRef]
  11. Have, R.T.; Teunissen, P.J. Oxidation mechanisms involved in lignin degradation by white rot fungi. Chem. Rev. 2001, 101, 3397–3413. [Google Scholar] [CrossRef]
  12. Janusz, G.; Pawlik, A.; Sulej, J.; Swiderska-Burek, U.; Jarosz-Wilkołazka, A.; Paszczyski, A. Lignin degradation: Microorganisms, enzymes involved genomes analysis and evolution. FEMS Microbiol. Rev. 2017, 41, 941–962. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Sánchez, C. Lignocellulosic residues: Biodegradation and bioconversion by fungi. Biotechnol. Adv. 2009, 27, 185–194. [Google Scholar] [CrossRef] [PubMed]
  14. Leonowicz, A.; Cho, N.S.; Luterek, J.; Wilkolazka, A.; Wojtas-Wasilewska, M.; Matuszewska, A.; Hofrichter, M.; Wesenberg, D.; Rogalski, J. Fungal laccase: Properties and activity on lignin. J. Basic Microbiol. 2001, 41, 185–227. [Google Scholar] [CrossRef]
  15. Dashtban, M.; Schraft, H.; Syed, T.A.; Qin, W. Fungal biodegradation and enzymatic modification of lignin. Int. J. Biochem. Mol. Biol. 2010, 1, 36–50. [Google Scholar]
  16. Shrivastava, B.; Nandal, P.; Sharma, A.; Jain, K.K.; Khasa, Y.P.; Das, T.K.; Mani, V.; Kewalramani, N.J.; Kundu, S.S.; Kuhad, R.C. Solid state bioconversion of wheat straw into digestible and nutritive ruminant feed by Ganoderma sp. rckk02. Bioresour. Technol. 2012, 107, 347–351. [Google Scholar] [CrossRef]
  17. Díaz-Godínez, G.; Téllez-Téllez, M.; Sánchez, C.; Díaz, R. Characterization of the solid-state and liquid fermentation for the production of laccases of Pleurotus ostreatus. In Fermentation Processes; Jozala, A., Ed.; IntechOpen: London, UK, 2017; Chapter 4; pp. 57–74. [Google Scholar]
  18. Cueva, C.M. Aprovechamiento De Residuos De Plátano, Cacao Y Maíz Como Sustratos Para La Producción Del Hongo “Pleurotus ostreatus”, En La Comunidad La Magdalena De Francisco De Orellana; Presentado Para Optar El Grado Académico De: Ingeniera En Biotecnología Ambiental. Facultad De Ciencias, Escuela Superior Politécnica de Chimborazo: Orellana, Ecuador, 2018. Available online: http://dspace.espoch.edu.ec/handle/123456789/10172 (accessed on 11 April 2020).
  19. Chan-Cupul, W.; Heredia-Abarca, G.P.; Rodríguez-Vázquez, R. Aislamiento y evaluación de la actividad enzimática ligninolítica de acromicetos del estado de Veracruz, México. Rev. Int. Contam. Ambient. 2016, 32, 339–351. [Google Scholar] [CrossRef] [Green Version]
  20. Ergun, S.O.; Urek, R.O. Production of ligninolytic enzymes by solid state fermentation using Pleurotus ostreatus. Ann. Agrar. Sci. 2017, 15, 273–277. [Google Scholar] [CrossRef]
  21. Cano y Postigo, L.O.; Jacobo-Velázquez, D.A.; Guajardo-Flores, D.; Garcia-Amezquita, L.E.; García-Cayuela, T. Solid-state fermentation for enhancing the nutraceutical content of agrifood by-products: Recent advances and its industrial feasibility. Food Biosci. 2021, 41, 1–10. [Google Scholar] [CrossRef]
  22. Shraddha, R.S.; Sehgal, S.; Kamthania, M.; Kumar, A. Laccase: Microbial sources, production, purification, and potential biotechnological applications. Enzym. Res. 2011, 2011, 1–11. [Google Scholar] [CrossRef] [Green Version]
  23. Iandolo, D.; Piscitelli, A.; Sannia, G.; Faraco, V. Enzyme Production by Solid Substrate Fermentation of Pleurotus ostreatus and Trametes versicolor on Tomato Pomace. Appl. Biochem. Biotechnol. 2011, 163, 40–51. [Google Scholar] [CrossRef] [Green Version]
  24. García, A. Estudio De La Degradación De Residuos Lignocelulósicos Derivados Del Procesamiento Industrial Del Cranberry (Vaccinium macrocarpon Ait.). Ph.D. Thesis, Universidad Austral de Chile, Valdivia, Chile, 2006. [Google Scholar]
  25. Rodriguez-Barbosa, J.; dos Santos-Freitas, M.M.; da Silva-Martins, L.H.; de Carvalho-Junior, R.N. Polysaccharides of mushroom Pleurotus spp.: New extraction techniques, biological activities and development of new technologies. Carbohydr. Polym. 2020, 229, 115550. [Google Scholar] [CrossRef] [PubMed]
  26. Martínez, D.A.; Buglione, M.B.; Filippi, M.V.; Reynoso, L.C.; Rodríguez, G.E.; Agüero, M.S. Evaluación del crecimiento mycelial de Pleurotus ostreatus y Agrocybe aegerita sobre orujos de pera. An. Biol. 2015, 37, 1–10. [Google Scholar] [CrossRef]
  27. Tuyen, D.V.; Phuong, H.N.; Cone, J.W.; Baars, J.J.P.; Sonnenberg, A.S.M.; Hendriks, W.H. Effect of fungal treatments of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and methane production. Bioresour. Technol. 2013, 129, 256–263. [Google Scholar] [CrossRef] [PubMed]
  28. Arora, D.S.; Sharma, R.K.; Chandra, P. Biodelignification of wheat straw and its effect on in vitro digestibility and antioxidant properties. Int. Biodeterior. Biodegrad. 2011, 65, 352–358. [Google Scholar] [CrossRef]
  29. Fazaeli, H.; Mahmodzadeh, H.; Azizi, A.; Jelan, Z.A.; Liang, J.B.; Rouzbehan, Y.; Osman, A. Nutritive value of wheat straw treated with Pleurotus fungi. Asian-Australas. J. Anim. Sci. 2004, 17, 1681–1688. [Google Scholar]
  30. Hoa, H.T.; Wang, C.L. The effects of temperature and nutritional conditions on mycelium growth of two oyster mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus). Mycobiology 2015, 43, 14–23. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  31. Alarcón, J.; Águila, S.; Arancibia-Ávila, P.; Fuentes, O.; Zamorano-Ponce, E.; Hernández, M. Production and purification of statins from Pleurotus ostreatus (Basidiomycetes) strains. Z. Nat. C 2003, 58, 62–64. [Google Scholar] [CrossRef]
  32. González-Caloch, I.; Nava-Galicia, S.; Díaz-Godínez, R.; Garrido-Bazán, V.; Tlecuitl-Beristain, S.; Bibbins-Martínez, M.D. Determinación de la actividad de oxidasas producidas por Oxyporus latemarginatus crecido en fermentación sumergida en presencia y ausencia de colorante amarillo azo. Rev. Latinoam. El Ambiente Y Las Cienc. 2015, 6, 72–88. [Google Scholar]
  33. Rahman, M.M.; Lourenco, M.; Hassim, H.A.; Baars, J.J.P.; Sonnenberg, A.S.M.; Cone, J.W.; De Boever, J.; Fievez, V. Improving ruminal degradability of oil palm fronds using white rot fungi. Anim. Feed Sci. Technol. 2011, 169, 157–166. [Google Scholar] [CrossRef]
  34. Akpinar, M.; Urek, R.O. Production of ligninolytic enzymes by solid-state fermentation using Pleurotus eryngii. Prep. Biochem. Biotechnol. 2012, 42, 582–597. [Google Scholar] [CrossRef]
  35. Akinfemi, A.; Ogunwole, O.A. Chemical composition and in vitro digestibility of rice straw treated with Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus tuber-regium. Slovak J. Anim. Sci. 2012, 45, 14–20. [Google Scholar]
  36. Ardón, C. La Producción De Hongos Comestibles. Master’s Thesis, Universidad de San Carlos de Guatemala, Ciudad de Guatemala, Guatemala, 2007. [Google Scholar]
  37. Páez, M. Determinación De La Actividad Enzimática De Lacasa Y Lignina Peroxidasa De Hongos Degradadores De Colorantes Seleccionados Para El Tratamiento De Aguas Residuales De La Industria Textil; Memora de título para obtención de título de Ingeniería en Biotecnología; Escuela Politécnica del Ejército: Sangolqui, Ecuador, 2012. [Google Scholar]
  38. Usnayo, P. Optimización De Medios De Cultivo, Para La Producción De Enzimas Ligninolíticas, Por Cepas Fúngicas Aisladas Del Altiplano Boliviano. Trabajo de grado para optar al título de Lic. En Bioquímica. Bachelor’s Thesis, Universidad Mayor de San Andrés, La Paz, Bolivia, 2007. [Google Scholar]
  39. Téllez-Téllez, M.; Fernández, F.J.; Montiel-González, A.M.; Sánchez, C.; Días-Godínez, G. Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation. Appl. Microbiol. Biotechnol. 2008, 81, 675–679. [Google Scholar] [CrossRef] [PubMed]
  40. García-Oduardo, N.; Bermúdez-Savon, R.; Téllez-Suarez, I.; Chávez-Toledano, M.; Perraund-Gaime, I. Enzimas lacasa en inóculos de Pleurotus spp. RTQ 2017, 27, 39–45. [Google Scholar]
  41. AOAC (Association of Official Analytical Chemists Official). Methods of Analysis of AOAC International, 16th ed.; Association of Official Analytical Chemists: Arlington, VA, USA, 1997. [Google Scholar]
  42. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  43. InfoStat Versión 2017. Grupo InfoStat, FCA. Universidad Nacional de Córdoba: Córdoba, Argentina, 2017. Available online: http://www.infostat.com.ar (accessed on 19 March 2020).
  44. NRC. Nutrient Requirement of Dairy Cattle, 7th ed.; National Academic Press: Washington, DC, USA, 2001. [Google Scholar]
  45. Anrique, R. Composición De Alimentos Para El Ganado Bovino; Cuarta Ed. Consorcio Lechero; Imprenta América Ltd.: Región Metropolitana, Chile, 2014. [Google Scholar]
  46. Jonathan, S.G.; Okorie, A.N.; Garuba, E.O.; Babayemi, O.J. Bioconversion of sorghum stalk and rice straw into value added ruminant feed using Pleurotus pulmonarius. Nat. Sci. 2012, 10, 10–16. [Google Scholar]
  47. Shabtay, A.; Hadar, Y.; Eitam, H.; Brosh, A.; Orlov, A.; Tadmor, Y.; Izhaki, I.; Jerem, Z. The potential of Pleurotus-treated olive mill solid waste as cattle feed. Bioresour. Technol. 2009, 100, 6457–6464. [Google Scholar] [CrossRef]
  48. Calsamiglia, S. Nuevas Bases Para La Utilización De La Fibra En Dieta De Rumiantes. Avances En Nutrición Y Alimentación Animal: Xiii Curso De Especialización Fedna/Coord; Rebollar, P.G., Beorlegui, C.d., Mateos, G.G., Eds.; Universidad Autónoma de Barcelona: Barcelona, Spain, 1997; pp. 3–19. [Google Scholar]
  49. Carro-Travieso, M.D.; de Evan, T.; González-Cano, J. Emisiones de metano en los animales rumiantes: Influencia de la dieta. Rev. Albéitar 2018, 220, 32–35. [Google Scholar]
  50. Bonilla-Cárdenas, J.A.; Lemus-Flores, C. Emisión de metano entérico por rumiantes y su contribución al calentamiento global y al cambio climático. Revisión. Rev. Mex. Cienc. Pecu. 2012, 3, 215–246. [Google Scholar]
  51. Fazaeli, H.; Azizi, A.; Amile, M. Nutritive value index of treated wheat straw with Pleurotus Fungi fed to sheep. Pak. J. Biol. Sci. 2006, 9, 2444–2449. [Google Scholar] [CrossRef] [Green Version]
  52. Akinfemi, A.; Adu, O.A.; Doherty, F. Conversion of sorghum stover into animal feed with white-rot fungi: Pleurotus ostreatus and Pleurotus pulmonarius. Afr. J. Biotechnol. 2010, 9, 1706–1712. [Google Scholar] [CrossRef] [Green Version]
  53. Cofré, P.; Velasco, R. Forrajeras: Calidad Y Costos De Producción [En Línea]. Informativo Agropecuario Bioleche INIA Quilamapu. 2007. Available online: https://biblioteca.inia.cl/handle/123456789/34598 (accessed on 25 March 2020).
  54. Zuo, S.; Niu, D.; Zheng, M.; Jiang, D.; Tian, P.; Li, R.; Xu, C. Effect of Irpex lacteus, Pleurotus ostreatus and Pleurotus cystidiosus pretreatment of corn stover on its improvement of the in vitro rumen fermentation. J. Sci. Food Agric. 2018, 98, 4287–4295. [Google Scholar] [CrossRef] [PubMed]
  55. Díaz, R. Efecto Del Ph Inicial De Desarrollo De Pleurotus Ostreatus En Fermentación Sumergida Sobre Su Actividad De Lacasas. Master’s Thesis, Instituto politécnico nacional Tlaxcala, Tlaxcala, Mexico, 2009. [Google Scholar]
  56. Guillén-Navarro, G.K.; Márquez-Rocha, F.J.; Sanchez-Vásquez, J.E. Producción de biomasa y enzimas ligninolíticas por Pleurotus ostreatus en cultivo sumergido. Rev. Iberoam. Micol. 1998, 15, 302–306. [Google Scholar] [PubMed]
  57. Vyas, B.R.M.; Molitois, H.P. Involvement of an Extracellular H2O2-Dependent Ligninolytic Activity of the White Rot Fungus Pleurotus ostreatus in the Decolorization of Remazol Brilliant Blue R. Appl. Environ. Microbiol. 1995, 61, 3919–3927. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Muñoz-Duarte, L.D. Evaluación De Enzimas Degradadoras De Lignina Producidas Por Aislamientos Fúngicos De Cultivos De Arroz; Trabajo de Grado Presentado Para Optar al Titulo de Microbiología Industrial; Pontificia Universidad Javeriana: Bogota, Colombia, 2012. [Google Scholar]
  59. France, A.; Cañumir, J.A.; Cortez, M. Produccion De Hongos Ostras (Pleurotus ostreatus); No. 23; Boletin INIA—Instituto de Investigaciones Agropecuarias: Primera Edición; Rodríguez, H., Ed.; Imprenta Trama: Chillán, Chile, 2000. [Google Scholar]
Agriculture 12 00534 g001 550
Figure 1. Reaction of laccase on phenols oxidation [10].
Figure 1. Reaction of laccase on phenols oxidation [10].
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Figure 2. Reaction catalyzed by LiP of cleavage C-C [10].
Figure 2. Reaction catalyzed by LiP of cleavage C-C [10].
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Figure 3. Lignin depolymerization with MnP [10].
Figure 3. Lignin depolymerization with MnP [10].
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Figure 4. Schematic diagram of lignin degradation by basidiomycetes white-rot fungi [15].
Figure 4. Schematic diagram of lignin degradation by basidiomycetes white-rot fungi [15].
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Figure 5. Flow chart for the production of biodegraded hay with P. ostreatus.
Figure 5. Flow chart for the production of biodegraded hay with P. ostreatus.
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Figure 6. Electron microscopic structural analysis of ryegrass fescue hay before and after SSF with P. ostreatus for 14 days, (a) vegetal material before SSF; (b,c) vegetal material after SSF, ×65 and ×250.
Figure 6. Electron microscopic structural analysis of ryegrass fescue hay before and after SSF with P. ostreatus for 14 days, (a) vegetal material before SSF; (b,c) vegetal material after SSF, ×65 and ×250.
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Figure 7. Enzymatic activity (U/L) of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac) and total protein concentration (g/L), in SSF with P. ostreatus for 21 days. Differences were assessed by ANOVA with Tukey mean comparison test. Graph bars with different superscripts (a, b) indicate significant differences between fermentation days (p < 0.05). Capital letters on the secondary axis of the total protein indicate significant differences between the days of fermentation (p < 0.05). Above the superscripts of the bars and the capital letters of the secondary axis, the standard error of the mean is indicated.
Figure 7. Enzymatic activity (U/L) of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac) and total protein concentration (g/L), in SSF with P. ostreatus for 21 days. Differences were assessed by ANOVA with Tukey mean comparison test. Graph bars with different superscripts (a, b) indicate significant differences between fermentation days (p < 0.05). Capital letters on the secondary axis of the total protein indicate significant differences between the days of fermentation (p < 0.05). Above the superscripts of the bars and the capital letters of the secondary axis, the standard error of the mean is indicated.
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Figure 8. Specific enzyme activity (U/g PT) of manganese peroxidase (MnP), lignin peroxidase (LiP) and lacase (Lac) and total protein concentration (g/L), in SSF with P. ostreatus for 21 days. Differences were assessed by ANOVA with Tukey mean comparison test. Graph bars with different superscripts (a, b) indicate significant differences between fermentation days (p < 0.05). Capital letters on the secondary axis of the total protein indicate significant differences between the days of fermentation (p < 0.05). Above the superscripts of the bars and the capital letters of the secondary axis, the standard error of the mean is indicated.
Figure 8. Specific enzyme activity (U/g PT) of manganese peroxidase (MnP), lignin peroxidase (LiP) and lacase (Lac) and total protein concentration (g/L), in SSF with P. ostreatus for 21 days. Differences were assessed by ANOVA with Tukey mean comparison test. Graph bars with different superscripts (a, b) indicate significant differences between fermentation days (p < 0.05). Capital letters on the secondary axis of the total protein indicate significant differences between the days of fermentation (p < 0.05). Above the superscripts of the bars and the capital letters of the secondary axis, the standard error of the mean is indicated.
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Table
Table 1. Chemical composition of ryegrass fescue hay before and after 14 day SSF.
Table 1. Chemical composition of ryegrass fescue hay before and after 14 day SSF.
Determinations *UnitsRyegrass-Fescue HayFermentation Residue
Proximal analysis
Dry matter (DM)g/100g91.2914.52
Total ashes (TA)g/100g4.413.83
Crude protein (CP) ag/100g4.735.16
Etheric extract (EE)g/100g1.301.10
Non-fibrous carbohydrates (NFC) bg/100g20.8425.04
Metabolizable energy (ME) cMcal/kg DM1.941.95
Van Soest analysis
Neutral detergent fiber (aNDFom)g/100g68.7264.87
Acid detergent fiber (aADFom)g/100g42.4542.05
Acid detergent lignin dg/100g5.881.98
* Except for the dry matter value, the nutrient values are reported on a dry matter basis. a CP: Crude protein applying factor 6.25, b NFC: 100—(aNDFom + CP + EE + TA) [44]. c ME: Theoretical metabolizable energy for ruminants. d Acid detergent lignin expressed base as offered.
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Astudillo-Neira, R.; Muñoz-Nuñez, E.; Quiroz-Carreno, S.; Avila-Stagno, J.; Alarcon-Enos, J. Bioconversion in Ryegrass-Fescue Hay by Pleurotus ostreatus to Increase Their Nutritional Value for Ruminant. Agriculture 2022, 12, 534. https://doi.org/10.3390/agriculture12040534

AMA Style

Astudillo-Neira R, Muñoz-Nuñez E, Quiroz-Carreno S, Avila-Stagno J, Alarcon-Enos J. Bioconversion in Ryegrass-Fescue Hay by Pleurotus ostreatus to Increase Their Nutritional Value for Ruminant. Agriculture. 2022; 12(4):534. https://doi.org/10.3390/agriculture12040534

Chicago/Turabian Style

Astudillo-Neira, Rita, Evelyn Muñoz-Nuñez, Soledad Quiroz-Carreno, Jorge Avila-Stagno, and Julio Alarcon-Enos. 2022. "Bioconversion in Ryegrass-Fescue Hay by Pleurotus ostreatus to Increase Their Nutritional Value for Ruminant" Agriculture 12, no. 4: 534. https://doi.org/10.3390/agriculture12040534

APA Style

Astudillo-Neira, R., Muñoz-Nuñez, E., Quiroz-Carreno, S., Avila-Stagno, J., & Alarcon-Enos, J. (2022). Bioconversion in Ryegrass-Fescue Hay by Pleurotus ostreatus to Increase Their Nutritional Value for Ruminant. Agriculture, 12(4), 534. https://doi.org/10.3390/agriculture12040534

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