Chitosan decreases fermentation losses and improves aerobic stability of rehydrated corn silage

Quitosana diminui as perdas fermentativas e melhora a estabilidade aeróbica da silagem de milho reidratado

ABSTRACT:

Ensilage of rehydrated corn kernels (RC) has been used to improve nutritional value and facilitate on-farm storage. This study evaluated the effects of chitosan and lactic acid microbial inoculants on rehydrated corn silage microbiology, fermentation profile and losses, chemical composition, in vitro degradation, and aerobic stability. Forty experimental silos were used in a completely random design to evaluate the following treatments: 1) Control (CON): RC silage without additives; 2) Chitosan (CHI): RC silage with 6 g/kg dry matter (DM) of chitosan; 3) Lactobacillus buchneri (LB): RC ensiled with 5 × 10⁵ colony forming units (CFU) of L. buchneri per gram fresh weight; and 4) Lactobacillus plantarum and Pediococcus acidilactici (LPPA): RC ensiled 1.6 × 10⁵ of L. plantarum and 1.6 × 10⁵ P. acidilactici per gram fresh weight. Additives increased lactic acid bacteria and concentration of lactic and propionic acid, decreased mold and yeast count and gas and fermentative losses, and improved DM recovery. The CHI-silos had lower silage pH, Ammonia-N concentration, fermentative losses, and higher acetic acid concentration compared to microbial inoculated-silos. In addition, CHI and LB decreased silage pH and temperature after aerobic exposure. Although, treatments showed slight effects on the nutritional value of RC, CHI improved aerobic stability and decreaseds fermentation losses.

Key words: fermentation profile; kernel silage; lactic acid bacteria; L. buchneri

RESUMO:

A ensilagem de grãos de milho reidratados (RC) tem sido usada para melhorar o valor nutricional e facilitar o armazenamento na fazenda. Este estudo teve como objetivo avaliar os efeitos de inoculantes microbianos de quitosana e ácido lático na microbiologia de silagem de milho reidratada, perfil e perdas de fermentação, composição química, degradação in vitro e estabilidade aeróbia. Quarenta silos experimentais foram utilizados em delineamento inteiramente casualizado para avaliar os seguintes tratamentos: 1) Controle (CON): silagem RC sem aditivos; 2) Quitosana (CHI): silagem de RC com 6 g/kg de matéria seca (MS) de quitosana; 3) Lactobacillus buchneri (LB): RC ensilados com 5 × 10⁵ unidades formadoras de colônias (UFC) de L. buchneri por grama na alimentação; e 4) Lactobacillus plantarum e Pediococcus acidilactici (LPPA): RC ensilado 1,6 × 10⁵ de L. plantarum e 1,6 × 10⁵ P. acidilactici por grama na alimentação. Os aditivos aumentaram as bactérias láticas e a concentração de ácido lático e propiônico, diminuíram a contagem de fungos e leveduras e as perdas gasosas e fermentativas e melhoraram a recuperação da MS. Os silos CHI apresentaram menores pH da silagem, concentração de N amoniacal, perdas fermentativas e maior concentração de ácido acético em comparação com silos inoculados microbianos. Além disso, CHI e LB reduziram pH e temperatura da silagem após a exposição ao ar. Embora os tratamentos mostrem pequenos efeitos sobre o valor nutricional do RC, a CHI melhoroua a estabilidade aeróbia e diminui as perdas por fermentação.

Palavras-chave: bactérias láticas; L. buchneri; perfil de fermentação; silagem de grãos

INTRODUCTION

Rehydrated corn grain silage (RC) is an economical alternative to store corn grain and improve the nutritional quality of the grain in ruminant feed (FERRARETTO et al., 2018; MOMBACH et al., 2019). Mature corn kernels (between 16 and 13% humidity) contain varying concentrations of soluble carbohydrates and low counts of lactic acid bacteria responsible for the rapid acidification of ensiled material (CARAVALHO-ESTRADA et al., 2020). Therefore, fermentation occurs at a lower intensity and slower than whole-plant corn silage (CARVALHO et al., 2016), favouring the growth of yeasts and resulting in inefficient fermentation (MUCK, 2010). Thus, additives can improve the fermentation of RC.

Homofermentative lactic acid bacteria (LAB) are the oldest and most common silage microbial inoculants (MUCK et al., 2018). These inoculants can increase lactic acid production and reduce silage aerobic stability. Conversely, Lactobacillus buchneri is a heterofermentative bacteria that has been used to increase the aerobic stability of silage due to its ability to increase acetic acid levels (ARRIOLA et al., 2021). Furthermore, L. buchneri can affect the microbial profile of the silage and increase the proteolytic activity and starch degradability (JUNGES et al., 2017). Chitosan is a natural biopolymer derived from the deacetylation of chitin that can establish interactions with anionic sites in the cell walls of microorganisms, causing structural damage and membrane permeability (GOY et al., 2009). Therefore, its antifungal activity is well documented (MENDES et al., 2016). DEL VALLE et al. (2018) observed that chitosan addition during sugarcane ensiling increases dry matter (DM) recovery compared to microbial inoculants containing Pediococcus acidilactici and Propionibacterium acidicipropionici. Studies from our research group evaluated chitosan as an additive to sugarcane (DEL VALLE et al., 2018; DEL VALLE et al., 2020) and whole-plant soybean silage (GANDRA et al., 2018; MORAIS et al., 2021). The addition of chitosan improved fermentative, increasing lactic acid production, reducing gas losses, and improving aerobic stability of silage.

However, material fermentability can greatly affect additives effects on silage. In addition, to the best of our knowledge, there are no studies that make a direct comparison between chitosan and microbial inoculants in RC ensiling. Therefore, we hypothesized that chitosan decreases yeast and mold count and fermentation losses, improving nutritional value and aerobic stability of RC compared to microbial inoculants. The present study evaluated chitosan and microbial inoculation (facultative and obligatory homofermentative LAB) effects on rehydrated corn silage microbial count, fermentation profile, fermentation losses, DM recovery, chemical composition, in vitro degradation, and aerobic stability.

MATERIALS AND METHODS

The present trial was conducted from July to October 2019, on the School of Agrarian Sciences of Universidade Federal da Grande Dourados, (Dourados, Brazil; 22° 14′ S latitude, 54° 49′ W longitude, and 450 m of altitude) and analysis were performed on Agricultural Sciences Center of Universidade Federal de São Carlos (UFSCar, Araras, Brazil).

Experimental design, treatments, and management

Forty experimental silos (PVC tubes with 30 cm height and 30 cm diameter) were used in a completely randomized design to evaluate the following treatments: 1) Control (CON): rehydrated corn silage without additives; 2) Chitosan (CHI): rehydrated corn silage with 6 g/kg DM of chitosan (Polymar Indústria, Fortaleza, Brazil); 3) Lactobacillus buchneri (LB): rehydrated corn ensiled with 5 × 10⁵ colony forming units (CFU) of L. buchneri (NCIM 40788, Lasil Cana^®, Lallemand, Montreal, Canadá) per gram fresh weight; and 4) Lactobacillus plantarum and Pediococcus acidilactici (LPPA): rehydrated corn ensiled 1.6 × 10⁵ of L. plantarum and 1.6 × 10⁵ P. acidilactici (Kera SIL^®, Kera Nutrição Animal, Bento Gonçalves, Brazil) per gram fresh weight.

The corn kernels (cultivar DKB 330 PRO3, Dekalb, St. Louis, MO) were harvested with 840 g/kg of DM content and processed in a hammer mill through a 4-mm sieve. Ground corn of each silo was weighed, received 0.33 L/kg of distilled water, and four composite samples were obtained to evaluate chemical composition, as described below. Treatments were provided during the corn rehydration. High moisture corn before the ensiling had 620 g/kg DM, 983 g/kg organic matter (OM), 670 g/kg starch, 90.2 g/kg neutral detergent fiber (NDF), 23.2 g/kg acid detergent fiber (ADF), 90.2 g/kg crude protein (CP), and 25.2 g/kg ether extract (EE). Empty silos containing dried sand and a screen to obtain effluent, were weighed and rehydrated corn was manually compacted to achieve 1000 kg/m³ of specific density. Then, silos were stored at room temperature for 100 days.

Sampling and microbiology analysis

After storage period, whole silos were weighed and opened. We discarded top and bottom layers (5 cm), homogenized, and sampled silage of the center of silos. Silage samples were used for microbial analysis (~ 100 g), fermentation profile (~ 400 g), chemical analysis (~ 500 g), and aerobic stability assay (3 kg). After silage removal, empty-silos (sand, effluent, and screen) were weighed to calculate effluent losses. Silage juice was extracted using a hydraulic press (PHE-45^®, Engehidro, Piracicaba, Brazil) and pH was immediately recorded using a bench pHmeter (LUCA-210, Lucadema, São José do Rio Preto, Brazil). Then, silage juice was frozen for ammonia-N (NH₃-N) and organic acids analysis. Aerobic stability samples were housed in plastic buckets without compaction and storage at 25.0 ^oC for 144 h. The temperature was evaluated every 8 h using spit thermometers (K29-5030^®, Kasvi Produtos Laboratoriais, Pinhais, Brazil) positioned in the center of the silage mass. The pH was recorded every 24 h, as previously described.

Silage samples for microbial analyses (10 g) were diluted in a 9 g/L sodium chloride solution (90 mL). Subsequent dilution was performed and plated in MRS agar for LAB evaluation (BRICEÑO & MARTINEZ, 1995) and nutrient agar under aerobic and anaerobic conditions. Incubations were performed for 48 h at 30 ^oC. In addition, diluted samples were plated in potato-dextrose agar for 120 h at 26 ^oC to evaluate yeast and mold (RABIE et al., 1997). Counts were performed manually, and results were converted in log₁₀ of CFU before the analysis.

Chemical analysis and in vitro assay

Ammonia nitrogen (NH₃-N) in silage juice was analyzed using colorimetric method. Organic acids (except lactic) were analyzed using a gas chromatograph (Focus GC, Thermo Fisher Scientific Inc., Waltham, MA), as described by DEL VALLE et al. (2020). Briefly, flame ionization detector temperature was 270 ^oC, whereas temperature of oven and injector were 190 and 220 ^oC, respectively. Hydrogen was used as carrier gas. The lactic acid concentration was analyzed using the spectrophotometric method (PRYCE, 1969).

Chemical composition and in vitro degradation was analyzed after oven dried (60 ^oC for 72 h) and ground using a Wiley mill (MA580, Marconi, Brazil) equipped with 1 and 2-mm sieves, respectively. Samples were analyzed for DM (method 950.15), ash (method 942.05), CP (Kjeldahl method 984.13), and ether extract (EE, method 920.39) according to AOAC (2000). Futhermore, starch was analyzed using the spectrophotometric method (BACH KNUDSEN, 1997), and neutral and acid detergent fiber were assessed using VAN SOEST et al. (1991) detergents and alpha-amylase without sodium sulfite. In vitro degradation of DM and NDF was evaluated after 48 h of incubation using a daisy incubator (TE150, Tecnal, Piracicaba, Brazil), according to HOLDEN (1999). Briefly, samples (500 mg, processed at 2-mm sieve) were placed in 5 × 5 non-woven fabric (100 g/m²) in triplicates. Ruminal fluid was sampled at 8:00, from a Holstein heifer with almost 450 kg of body weight, maintained in Megathyrsus maximus (Mombaça grass) pasture, with no supplementation. Ruminal fluid sampling was performed using an oral-ruminal probe. After sampling, ruminal fluid was placed in thermal bottles and diluted (1 ruminal fluid and 4 buffer, v:v) in MCDOUGALL (1948) buffer to produce ruminal inoculum.

Calculations and statistical analysis

Fermentation losses were calculated according to JOBIM et al. (2007), using the following equations: $\mathit{Effluent\; production}\left(\mathit{EP},\frac{g}{\mathit{kg}}\right)=({\mathit{WOS}}_{\mathit{op}}-{\mathit{WOS}}_{\mathit{en}})\times \frac{1000}{\mathit{DME}}$ where: WOS_op is silo weight without silage at opening; WOS_en is silo weight without silage at ensiling, and DME is ensiled DM. $\mathit{Gas\; losses}\left(\mathit{GL},\frac{g}{\mathit{kg}}\right)={(\mathit{WIS}}_{\mathit{en}}-{\mathit{WIS}}_{\mathit{op}})\times \frac{1000}{\mathit{DME}}$ where: WIS _en is whole silo weight (within silage) at ensiling and WIS _op is whole silo weight (within silage) at silos opening. Total fermentation losses (TFL, g/kg) was calculated as effluent production and gas losses sum. Dry matter recovery (DMR, g/kg) was obtained by opening DM (g) to ensiled DM (kg) ratio.

Data analysis was performed using PROC MIXED of SAS (version 9.4, SAS Institute Inc., Cary, NC) and the following model:

$Y_{\mathit{ij}}=\mu +T_i+e_{\mathit{ij}}$

with e _ij ≈ N (0, σ_e ²), where Y _ij is the dependent variable; µ is the overall mean; T _i is the fixed effect of treatments (i = 1 to 4); e _ij is the random effect of error (j = 1 to 10); N stands for Gaussian distribution; and σ_e ² is residual variance.

Silage pH and temperature after aerobic exposure were evaluated considering the model:

$Y_{\mathit{ijk}}=\mu +T_i+{\omega }_{\mathit{ij}}+H_k+\mathit{T\; x}{H}_{\mathit{ik}}+e_{\mathit{ijk}}$

with ω_ij ≈ N (0, σ_ω ²) and e _ijk ≈ MVN (0, R), where: Y _ijk is the observed value of variable; µ, T _i , and N were previously defined; is the error associated with experimental unit (silo; ij = 40); H _k is the fixed effect of time/hour after aerobic exposure; T x H _ij is treatment and time interaction effect; e _ijk is the random residual effect; σ_ω ² is variance of experimental unit random effect; MVN stands multivariance with Gaussian distribution; R is the matrix of variance and covariance due to repeated measures. Matrixes were evaluated considering Bayesian method. Treatment effect was decomposed into orthogonal contrasts: 1. Additives effect (C1): CON vs. LB + LPPA + CHI; 2. Chemical (CHI) vs. microbial inoculants (LB + LPPA); and 3. Microbial inoculant evaluation (LB vs. LPPA). Significance was declared at P ≤ 0.05.

RESULTS

Microbial analysis

In general, additives increased (P < 0.01) total and lactic acid bacteria and reduced (P < 0.01) mold and yeast count of silage (Table 1). Chitosan-treated silos showed lower (P ≤ 0.04) mold and yeast count, than inoculated silos. Chitosan, LB, and LPPA inoculants showed similar (P ≥ 0.23) silage count of total and lactic acid bacteria. Silage inoculation of LPPA decreased (P < 0.01) mold count compared to LB inoculation.

Table 1
Microbial and fermentation profile, losses, and dry matter recovery of rehydrated corn ensiled with microbial inoculant and chitosan.

Item	-----------------Treatments1--------------------				SEM	--------------------P2-------------------
	CON	LB	LPPA	CHI		C1	C2	C3
---------------------------------------------------------------Microbial count, log10/g fresh matter---------------------------------------------------------
Lactic acid bacteria	4.32	6.64	7.16	6.44	0.161	<0.01	0.56	0.47
Total bacteria	5.39	8.59	7.45	8.48	0.214	<0.01	0.65	0.23
Mold	6.67	4.81	3.22	1.54	0.476	<0.01	<0.01	<0.01
Yeast	7.40	5.93	5.53	3.45	0.186	<0.01	0.04	0.76
--------------------------------------------------------Fermentative profile, g/kg DM, unless stated------------------------------------------------------
pH	3.94	4.02	3.87	4.12	0.041	0.26	0.02	0.04
NH3-N, mg/dL	10.4	8.35	8.09	10.6	0.632	0.13	0.02	0.55
Lactic acid	41.2	50.2	67.8	52.1	2.41	0.03	0.11	0.02
Acetic acid	9.87	11.6	8.78	7.52	0.732	0.48	0.02	0.01
Branched-chain FA	2.12	2.14	2.19	2.17	0.123	0.46	0.45	0.50
Butyric acid	1.04	1.04	1.05	0.948	0.104	0.54	0.48	0.49
Propionic acid	0.566	0.667	1.07	0.866	0.054	<0.01	0.14	<0.01
-------------------------------------------------------------------Fermentative losses, g/kg DM-------------------------------------------------------------
Effluent	25.4	28.8	18.4	10.9	1.17	0.12	0.02	<0.01
Gas	126	115	70.0	61.5	3.09	0.02	<0.01	<0.01
Total	151	144	88.4	72.4	3.33	0.04	<0.01	<0.01
DM recovery	849	856	912	928	3.33	0.04	<0.01	<0.01

• ¹
  Control (CONT): rehydrated corn silage without additive; Chitosan (CHI): rehydrated corn silage with 6 g/kg DM of chitosan (Polymar Indústria, Fortaleza, Brazil); Lactobacillus buchneri (LB): rehydrated corn ensiled with 5 × 10⁵ colony forming units (CFU) of L. buchneri (NCIM 40788, Lasil Cana^®, Lallemand, Montreal, Canadá) per g of fresh matter; and Lactobacillus plantarum and Pediococcus acidilactici (LPPA): rehydrated corn ensiled 1.6 × 10⁵ of L. plantarum and 1.6 × 10⁵ P. acidilactici (Kera SIL^®, Kera Nutrição Animal, Bento Gonçalves, Brazil). SEM (standard error of mean). ²Probabilities:C1: Control vs. (LB+LPPA+CHI); C2: CHI vs. (LB+LPPA); C3: LB vs. LPPA.

Fermentative profile and losses

In general, additives increased (P ≤ 0.03) lactic and propionic acid, reduced (P ≤ 0.04) gas and total fermentative losses, and improved (P = 0.04) silage DM recovery when compared to CON (Table 1). Relative CHI, inoculated silos had lower (P = 0.02) pH value and NH₃-N, and higher (P = 0.02) acetic acid concentration. In addition, CHI reduced (P < 0.01) effluent, gas, and total losses, whereas increased (P < 0.01) DM recovery in relation to microbial inoculated silos. Comparing microbial inoculants, LPPA increased (P ≤ 0.02) lactic and propionic acid and reduced (P ≤ 0.04) silage pH and acetic acid concentration. Besides that, LB increased (P < 0.01) effluent, gas, and total losses and decreased (P < 0.01) DM recovery when compared to LPPA.

Chemical composition and in vitro degradation

Additives did not affect (P ≥ 0.13) silage DM, starch, CP, and EE content (Table 2). Chitosan increased rehydrated corn silage DM content, relative to inoculated silos. Besides, LB tended to reduce (P = 0.07) silage DM concerning LPPA.

Table 2
Chemical composition and in vitro degradation of rehydrated corn ensiled with microbial inoculant and chitosan.

Item	------------------------Treatments1----------------------				SEM	---------------P2--------------
	CON	LB	LPPA	CHI		C1	C2	C3
-------------------------------------------------------Chemical composition, g/kg DM unless stated-----------------------------------------------------
Dry matter, g/kg fresh weight	585	560	572	587	3.4	0.13	0.01	0.07
Organic matter	984	984	984	984	2.8	0.32	0.55	0.65
Starch	637	643	635	635	6.3	0.43	0.33	0.76
NDF	85.2	87.9	101	85.8	2.93	0.34	0.23	0.12
ADF	21.9	22.1	25.2	22.6	0.90	0.50	0.65	0.24
Crude protein	85.6	83.8	85.9	87.8	1.99	0.13	0.32	0.44
Ether extract	23.8	22.0	22.9	23.8	0.76	0.43	0.39	0.29
------------------------------------------------------------------In vitro degradation, g/kg--------------------------------------------------------------------
Dry matter	898	908	908	926	7.2	0.33	0.32	0.99
NDF	522	542	517	520	38.0	0.96	0.92	0.82

• ¹
  Control (CONT): rehydrated corn silage without additive; Chitosan (CHI): rehydrated corn silage with 6 g/kg DM of chitosan (Polymar Indústria, Fortaleza, Brazil); Lactobacillus buchneri (LB): rehydrated corn ensiled with 5 × 10⁵ colony forming units (CFU) of L. buchneri (NCIM 40788, Lasil Cana^®, Lallemand, Montreal, Canadá) per g of fresh matter; and Lactobacillus plantarum and Pediococcus acidilactici (LPPA): rehydrated corn ensiled 1.6 × 10⁵ of L. plantarum and 1.6 × 10⁵ P. acidilactici (Kera SIL^®, Kera Nutrição Animal, Bento Gonçalves, Brazil). SEM (standard error of mean). ²Probabilities: C1: Control vs. (LB+LPPA+CHI); C2: CHI vs. (LB+LPPA); C3: LB vs. LPPA.

Aerobic stability

There was a treatment and time interaction effect (P < 0.01) on silage pH and temperature after aerobic exposure (Figures 1 and 2). In general, additives decreased (P ≤ 0.04) pH and temperature of silage. Chitosan decreased (P = 0.01) silage temperature compared to microbial inoculation. Microbial inoculation of LP had lower (P < 0.01) silage pH and temperature after aerobic exposure when compared to LPPA inoculation.

Figure 1
Silage pH after aerobic exposure of rehydrated corn ensiled with microbial inoculant and chitosan.

Figure 2
Silage temperature after aerobic exposure of rehydrated corn ensiled with microbial inoculant and chitosan.

DISCUSSION

In the present study we hypothesized that chitosan decreases yeast and mold count and fermentation losses, whereas increases DM degradation and aerobic stability of RC compared to microbial inoculants. Chitosan decreased silage yeast and mold count and, fermentation losses. However, treatments showed no effects on chemical composition and DM degradation of RC. On the other hand, CHI and LB reduced silage pH and temperature after aerobic exposure.

In general, additives increased LAB and reduced mold and yeast count of silage. Microbial inoculation is used to stimulate LAB establishment and lactic acid production (FILYA, 2003), reducing the activity of other microorganisms. Furthermore, additives increased propionic acid, which inhibits the growth of yeasts (SELWET, 2008). The silos treated with CHI had lower mold and yeast counts compared to microbial-inoculated ones. Similar results have been reported by GANDRA et al. (2016) and DEL VALLE et al. (2020) when studying chitosan addition in sugarcane ensiling. According to TANTALA et al. (2019), chitosan acts through an electrostatic interaction with the anionic elements of the bacterial cell wall membrane. Chitosan-treated silos showed similar LAB count when compared to microbial-inoculated silos. It reinforces that CHI shows no detrimental effect on LAB metabolism (DEL VALLE et al., 2018) and a negative effect on other microorganism growth favors LAB establishment.

In the present study, the additives added to RC increased lactic and propionic acids, however, LB had lower lactic acid content compared to LPPA. Increased lactic acid concentration is a direct consequence of increased LAB count. Conversely, the increase in propionic acid concentration is due to the presence of L. buchneri which can convert lactic acid and glucose into acetic acid and propionic acid under anaerobic conditions (PAHLOW et al., 2003). The positive effect of LB on aerobic stability is well documented. Acetic acid, acummuled during the silage fermentation, prevents spoilage microorganisms (FILYA, 2003; MUCK, 2010).

Microbial inoculation decreased NH₃-N content and silage pH compared to chitosan. Ammonia-N is produced mainly by protein degradation. In the present study, CHI did not affect CP content and increased NH₃-N because chitosan has 70.0 g/kg of N (438 g/kg CP) and NH₃-N delivery occurs after acids solubilization. In addition, ammonia has an alkalization effect, increasing the silage pH of CHI-treated-silos. Furthermore, CHI-treatment showed lower acetic acid content and numerical lower (P = 0.11) lactic acid content compared to microbial inoculants treatment. Increased acids content and lower alkalization compounds concentrations results in decreased silage pH when microbial inoculation was provided. In the present study, the negative effect of CHI on RC acetic acid concentration occurred when compared to LB and LPPA because LAB inoculation increases lactic acid production in a short period and later, acetic acid concentration.

According to MUCK (2010), acetic acid production results in additional CO₂ production, increased gas losses, and reduced DM recovery. Therefore, CHI decreased acid production and fermentation (gas and effluent) losses and improved DM recovery compared to LB and LPPA in the present study. In addition, during the silage process, the release of effluents (GABREHANNA et al., 2014), as well as the activity of undesirable microorganisms (MUCK, 2010) represents a reduction in the nutritional value and DM losses of the silage. Considering the positive effects of CHI on fermentation profile, losses, and DM recovery, it was expected and positive effect on silage nutritional value.

Chitosan increased silage DM content compared to inoculants. The same was reported by GANDRA et al. (2016) and DEL VALLE et al. (2018). According to MINSON (1990), silage intake by cattle increases with increasing DM content. The addition of CHI reduced the effluent losses, which increased DM content. During the silage fermentation process, water production occurs (MUCK, 2010) by microorganisms’ fermentation. Although, we did not evaluate silage fermentation through time, the positive impact of CHI on effluent production suggests a low intense fermentation process in CHI compared to microbial inoculated silos. Lower water production and effluent losses are directly connected with increasing the DM content of the silage. However, treatments showed no effects on OM, starch, fiber, CP, and ether extract content of the silage. Increased fiber content has been associated with higher fermentation losses once fiber losses during the fermentation process are almost null. However, treatments showed no effects on chemical composition and in vitro degradation. It is essential to consider the degradation assay limitation in the current study: 1 assay was performed for 48 h instead of 7-h, which is more common nowadays; and 2. starch degradation analysis was not performed.

After aerobic exposure, the yeasts consume lactic acid and increase silage pH and temperature (MUCK, 2010). Aerobic stability is more relevant in high fermentable materials. Other studies of our research group observed positive effects of CHI on sugarcane silage aerobic stability. In the present study, CHI decreased silage pH and temperature after aerobic exposure compared to inoculants. These results are prone to improving aerobic stability. On the other hand, MORAES et al. (2021), evaluating chitosan and microbial inoculant in whole-plant soybean silage (low fermentable) observed no effects on aerobic stability. As previously mentioned, chitosan inhibited yeast and mold growth after aerobic exposure. In addition, LB reduced silage pH and temperature compared to LPPA, which is associated with well-documented organic acids protection against deterioration by aerobic microorganisms (MOON, 1983).

CONCLUSION

Although the treatments did not show any effects on the chemical composition, RC, CHI and LB improved aerobic stability, and CHI reduced fermentation losses, being a better additive in RC ensilage.

ACKNOWLEDGEMENTS

The authors appreciate the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) for the trial financial support (2017/15457-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (NCPq) for TADV and FBF grants.

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• CR-2023-0164.R2

BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL

• All procedures were previously approved by the Ethics Committee of UFSCar Ethics Welfare Committee of UFSCar previously approved the procedures (approval number: 1395120219).

Edited by

• Editors: Leandro Souza da Silva (0000-0002-1636-6643) Márcia Helena Machado da Rocha Fernandes (0000-0001-8787-9668)

Publication Dates

• Publication in this collection
  02 Dec 2024
• Date of issue
  2025

History

• Received
  17 Mar 2023
• Accepted
  22 May 2024
• Reviewed
  15 Oct 2024