Effect of Amylolytic and Cellulolytic Enzymes on Whole Plant Corn Silage: Characteristics of Silage and Animal Digestion

JEFFERSON RODRIGUES GANDRA (  jeffersongandra@unifesspa.edu.br ) Universidade Federal do Sul e Sudeste do Para https://orcid.org/0000-0002-4134-5115 Alanne T. Nunes USP FMVZ: Universidade de Sao Paulo Faculdade de Medicina Veterinaria e Zootecnia Euclides R. Oliveira UFGD: Universidade Federal da Grande Dourados Mávio S. J. Silva UFGD: Universidade Federal da Grande Dourados Cibeli A. Pedrini UFGD: Universidade Federal da Grande Dourados Fabio S. Machado UFGD: Universidade Federal da Grande Dourados Geleice K. R. Silva UFGD: Universidade Federal da Grande Dourados Erika R. S. Gandra UFGD: Universidade Federal da Grande Dourados Paulo V. C. Mendes UNIFESSPA: Universidade Federal do Sul e Sudeste do Para Alzira G. S. Pause UNIFESSPA: Universidade Federal do Sul e Sudeste do Para


INTRODUCTION 29
A variety of enzyme additives have been added to forage at ensiling to improve fermentation and the nutritive value of silage. Inclusion 30 of enzyme additives to forage aims to break down plant cell walls at ensiling, which can improve silage fermentation once provide 31 sugars for homofermentative lactic acid bacteria. Besides that, enzymes may also increase the digestibility of cell walls, enhancing the 32 nutritive value of silage (Muck et al., 2018). 33 Cellulase is an enzyme that breaks down cellulose into beta-glucose and short-chain polysaccharides. Cellulase is made up of a complex 34 of several different enzymes, including exoglucanases (also called cellobiohydrolases), endoglucanases, and beta-glucosidases. 35 Fibrolytic enzymes added to silages can increase silage digestibility and decrease aerobic stability, as released sugars are rapidly used 36 by spoilage yeasts and molds (Kung and Muck, 2015). Cellulolytic enzymes may act on the more-digestible components of NDF, leaving 37 indigestible components intact what reduces the overall digestibility of consumed NDF (Nadeau et al., 2000;Dehghani et al., 2012;Jin 38 et al., 2015). 39 Glucoamylases are amylolytic enzymes considered exoamylases, which cleave 1,4-α-glycosidic bonds from the nonreducing end of the 40 glycosidic chains releasing d-glucose. Thus, these enzymes can increase the content of fermentable carbohydrates and reduce the 41

Fermentative Losses 74
After 70 days of fermentation, mini silos were weighed to calculated gas losses. Effluent losses were calculated based on the 75 difference between weight of silo assembly (plastic bucket, nylon screen, and sand layer) before the storage and weight of silo assembly 76 (plastic bucket, nylon screen, and sand layer containing silage effluent) after 60 d. 77 The gas losses, effluent losses and dry matter recovery were calculated according to Jobim et al. (2007), as follows: 78 in which: SWE is the silo weight at the ensiling, SWO is silo weight at the opening, and DME is total DM ensiled. 80 where: WSAO is the weight of silo assembly after the opening (g) and WSAE is the weight of silo before the ensiling (g). 82 in which: DMO is total DM after the opening of silo (kg) and DME is total DM before the ensiling (kg). 84 85

Silage Aerobic Stability 86
Aerobic stability was considered as the period (h) in which corn silage temperature remained less than 1°C above the room 87 temperature (Driehuis et al., 2001). During the 5 days period of aerobic stability evaluation, silos were maintained at room temperature 88 (28.55 ± 4.27, mean ± SD), and temperature of silage was measured every 12 h after oxygen exposure using an infrared thermometer 89 (MS6530, Wiltronics Research Pty. Ltd., Victoria, Australia). In addition, samples (100 g) from silos of each treatment were collected 90 every 24 h to determine pH (Kung et al., 1984). 91 92

Chemical Composition and In Vitro Degradation 93
Forage samples (500 g) from each experimental silo were collected to assess DM, OM, NFC, CP, EE, NDF, ADF, lignin, ash, 94 NEL and macro minerals as previously described. Dry matter and NDF in vitro digestibility were determined using filter bags and 95 artificial rumen incubator (TE-150, Tecnal, Piracicaba, Brazil) according to Tilley and Terry (1963) and adapted by Holden (1999). 96 8 Briefly, filter bags with samples were incubated for 48 h at 39°C in a buffer-inoculum solution (1,600 mL of buffer solution and 400 97 mL of rumen inoculum). Jars containing the buffer-inoculum solution were purged with CO2 and lids had gas relief valves. After the 98 incubation period, the buffer-inoculum was drained from the jars and the filter bags were gently squeezed against the sides of jar to 99 remove the gas trapped in inflated bags. Afterward, bags were rinsed in jars with 3 changes of warm tap water. 100 101

Fermentative Profile 102
Silage liquid was extracted from forage samples using a hydraulic press and pH was measured using a digital potentiometer 103 (MB-10, Marte, Santa Rita do Sapucai, Brazil). Silage liquid aliquots (2 mL) were mixed with 1 mL of sulfuric acid (1 N) for 104 determination of ammonia nitrogen concentration through the colorimetric method described by Foldager (1977). and injector temperatures were set to 190°C and 220°C, respectively. Hydrogen was used as the carrier gas flowing at 30 mL/min. The 112 lactic acid concentration was measured by HPLC (LC-10ADVP Shimadzu HPLC system, Shimadzu Inc., Kyoto, Japan) according to 113 Ding et al. (1995). 114 115

Microbiological Quality and Enzymatic Activity 116
Samples (200 g) from the middle layer within each mini silo were collected at the opening for microbiological population counts. 117 Ten grams from samples were diluted in sterilized sodium chloride solution (0.9%, 90 mL) and a serial dilution was performed. 118 Microorganism counts were carried out in triplicate through decimal dilution series in plates with De Man, Rogosa, Sharpe agar for 119 LAB (Briceño and Martinez, 1995), nutrient agar for aerobic and anaerobic bacteria (48 h of incubation at 30°C), and potato dextrose 120 agar (120 h of incubation at 26°C) for mold and yeast as described by Rabie et al. (1997). The absolute values were obtained as colony-121 forming units and then log-transformed. 122 For enzymatic activity evaluation, samples (5 g) were constantly shaked at 100 rpm for 1 h with distilled water (40 mL). Then, 123 it was filtered through nylon cloth and centrifuged (3000×g for 5 min at 5 °C). The enzymatic activity was determined by adding 0.1 124 mL of enzymatic suspension (supernatant) to 0.9 mL of sodium acetate buffer (0.1M and pH 5. Cellulases increased (P ≤ 0.038) gas losses and effluents production (Table 3). Interaction effect (P ≤ 0.039) was observed on 163 losses by gases (DM) and total (DM), which was greater for silages treated with cellulases and glucoamylases compared with CON but 164 not differ from GLU+CEL. At the same way, recovery DM was smaller for CEL and GLU compared with CON but not differ from 165 GLU+CEL (P = 0.039). 166 After aerobic exposure, no differences were observed between silages to measure temperature of all treatments (Table 3) GLU silage showed lower DM and NFC content than CON, but not differ from GLU + CEL silages. Unlike CON silages presented 176 lower NDF content compared with GLU and CEL silages, not differing from GLU + CEL. Additionally, silages treated with cellulases 177 demonstrated higher levels of NEL compared to CON, but not differ to GLU and GLU + CEL. 178 Corn silages treated with cellulases presented lower (P = 0.012) ethanol content and GLU silages showed higher (P = 0.012) 179 lactate concentration (Table 5) and lower (P ≤ 0.002) counts of anaerobic, aerobic, total bacteria, and fungi (Table 6). However, CEL 180 silage presented higher anaerobic bacteria counts (P = 0.02). An interaction effect (P = 0.003) was observed for lactic acid bacteria. 181 GLU+CEL silage showed greater counts than GLU silage, but not differ from CON and CEL. 14 In the intake and digestion trial, an interaction effect (P ≤ 0.043) was observed for feed intake. Lambs fed CEL silage showed 184 greater intake of DM, OM, CP and NDF than those in the GLU + CEL group, but not differ from animals fed CON and GLU silages. 185 For nutrient digestibility, lambs fed CEL silages presented higher (P ≤ 0.012) digestibility coefficients for DM, OM, CP and NDF. Enzymes incorporation increased gas and total losses (DM) resulting in 6,31% drop in DM recovery. CEL increased gas and 194 effluents losses, probably due to enhances on anaerobic bacteria count, as a greater microbial activity in silages treated with enzymes is 195 likely related to increases on the fermentative losses observed in this study. In contrast, despite of greater total losses in GLU treatment, 196 corn silage with GLU showed lower counts of anaerobic, aerobic, total bacteria and fungi. 197 Enzymes can decrease aerobic stability because of excessive release of WCS, increasing available sugars that can be quickly 198 used by undesirable microorganisms, such as spoilage yeasts and molds (Kung and Muck, 2015). According to Higginbotham et al. 199 (1998) yeasts usually initiate aerobic deterioration, and molds continue the deterioration process, because yeasts grow faster but tolerate 200 less heat than molds. In this study, fungi counts were reduced in GLU and no altered in CEL treatment, consequently no effects on 201 aerobic stability were observed. 202 Cellulolytic enzyme added to corn silage increased starch and crude protein content and reduced ADF. The last can be related to 203 the increase in the degradation of fiber fractions, which is also confirmed for improvements on in vitro degradation of DM and NDF by 204 CEL. Amylolytic enzyme increased ADF and starch content, with no effects on in vitro degradation. Dry matter content was greater in 205 CON, compared to GLU and CEL, but not differ from GLU + CEL. CEL probably showed a lower dry matter content because of greater 206 effluents losses, but the same was not observed in GLU. This is also observed by Lynch et al. (2015) when adding cellulase and xylanase 207 to corn forage before ensiling alone, causing a decrease on DM recovery in the enzyme-treated silage. 208 Exogenous enzymes hydrolyze complex carbohydrates into different products (malto-, cello-, and xylo-oligosaccharides), 209 supporting growth of fibrolytic microorganisms, which was called cross-feeding mechanism and could cause a synergistic effect between 210 fibrolytic and amylolytic enzymes (Zilio et al., 2019). However, in the present study the combination treatments resulted in no further 211 beneficial effects, which agrees with the low cellulase activity observed on GLU treatment. The overwhelming majority of studies with enzymes have applied cellulases and hemicellulases for improve the release of plant 218 cell wall carbohydrates, increasing its availability for LAB to ferment to lactic acid (Muck et al., 2018). However, different than 219 expected, in the present study CEL did not affect LAB count, but increased anaerobic bacteria count and reduced ethanol concentration. 220 Eun et al. (2017) demonstrated that fibrolytic enzymes products could greatly improve forage utilization, but the optimum doses and the 221 activities supplied are critical for achieving this response. 222 Exogenous fibrolytic enzyme products can greatly improve forage utilization (Muck et al., 2018). In fact, lambs fed silages 223 containing CEL had greater total tract digestibility. CEL positive effects on DM and NDF digestibility were somewhat expected, as 224 demonstrated by the in vitro assay. Despite of increases on NDF degradability, which could allow greater voluntary intake by reducing 225 physical fill in the rumen (Dado and Allen, 1995), feed intake was not influenced by adding none of the enzymes. On the other hand, 226 increased NDF degradability could also enhance the energy density of diets and stimulates microbial N production (Oba and Allen, 227 2000) being economically viable. Thus, the increases in NDF degradation observed in our study have the potential to substantially 228 improve the performance of animals fed diets containing corn silage. 229 Despite of amylolytic enzymes have potential to increase nutrients digestibility by acting on starch-protein matrix, which could 230 enhance microbial attachment and enzymatic digestion of starch granules (Giuberti et al., 2014), no beneficial responses were observed 231 on nutrient intake and digestibility.

Funding (information that explains whether and by whom the research was supported) 241
The study was supported by Federal University da Grande Dourados 242

Conflicts of interest/Competing interests (include appropriate disclosures) 243
The authors declare no competing interests. 244

Ethics approval (include appropriate approvals or waivers) 245
All the procedures in the present study involving animals were in accordance with the Animal Ethics Committee of the Federal 246 University of Grande Dourados, Brazil, number 0285/2017. 247

Consent to participate (include appropriate statements) 248
Not applicable 249

Consent for publication (include appropriate statements) 250
All the authors give consent for publication 251

Availability of data and material (data transparency) 252
All data generated and analyzed during this study are included in this published article 253 Code availability (software application or custom code) 254