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Effect of chitosan on the preservation quality of sugarcane silage

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DOI: 10.1111/gfs.12356

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Received: 8 July 2017 | Revised: 4 February 2018

DOI: 10.1111/gfs.12356

ORIGINAL ARTICLE

Effect of chitosan on the preservation quality of sugarcane

silage

T. A. Del Valle1,2 | T. F. Zenatti1 | G. Antonio1 | M. Campana1 | J. R. Gandra3 |

E. M. C. Zilio2 | L. F. A. de Mattos1 | J. G. P. de Morais1

1
Department of Biotechnology and Vegetal
and Animal Production, Center of Abstract
Agricultural Sciences, Federal University of We aimed to evaluate the effects of chitosan and microbial inoculant addition to
S~ao Carlos, Araras, Brazil
2 sugarcane silage fermentation, gas and effluent losses, chemical composition, in situ
Department of Animal Nutrition and
Production, University of S~ao Paulo, dry matter (DM), neutral detergent fibre (NDF) degradation and aerobic stability. A
Pirassununga, Brazil
completely randomized design with four treatments (n = 40) was performed. It was
3
Department of Animal Science, Federal
University of Grande Dourados, Dourados, arranged in a 2 9 2 factorial scheme with chitosan [0 and 6 g/kg of sugarcane DM
Brazil —1.66 g/kg of natural matter (NM)] and microbial inoculant (0 and 8 mg/kg on
Correspondence NM). Each g of inoculant contained 3.9 9 1010 UFC/g of Pediococcus acidilactici
T. A. Del Valle, Department of and 3.75 9 1010 UFC/g of Propionibacterium acidicipropionici. The addition of micro-
Biotechnology and Vegetal and Animal
Production, Center of Agricultural Sciences, bial inoculant increased lactic acid concentration in silos treated with chitosan. Fur-
Federal University of S~ao Carlos, Araras, thermore, chitosan increased pH and tended to increase acetic acid of silage. In
Brazil.
Email: tiagodelvalle@usp.br contrast, the inoculant decreased pH and acetic acid, besides increasing ethanol
concentration. As chitosan addition increased DM recovery, inoculant addition
decreased it. Chitosan decreased NDF and acid detergent fibre (ADF) level and
increased DM degradation, while inoculant decreased DM content, DM and NDF
degradation. In addition, chitosan improved the aerobic stability only in non-inocu-
lated silos. Thus, chitosan has a positive effect on silage fermentation, reducing fer-
mentative losses, and improving silage chemical composition and degradation.
Conversely, the addition of microbial inoculant negatively affected silage DM recov-
ery, chemical composition, and its association with chitosan decreased the aerobic
stability when compared to the exclusive use of chitosan.

KEYWORDS
fibre degradation, lactic acid, silage additives, silage storage losses, silo

1 | INTRODUCTION of dry matter (DM) and poor quality of forages (Pedroso et al.,
2005). Despite the high fermentative losses, a favourable point of
In subtropical conditions, sugarcane can be used as cattle feed dur- sugarcane would be its high production per area unit if compared to
ing periods of low pasture availability (Santos et al., 2015). However, corn or other crops.
this food source has to be harvested and chopped before being fed Microbial inoculants can be used in sugarcane silage to regulate
to animals, which demands labour-intensive procedures in large-scale yeast growth and alcoholic fermentation, reducing total losses and
systems (Fortaleza et al., 2012). Sugarcane silage can reduce farm improving DM digestibility (Freitas et al., 2006). The use of inocu-
labour but its intense alcoholic fermentation by yeasts entails losses lants containing homofermentative bacteria during ensiling leads to

Grass Forage Sci. 2018;1–9. wileyonlinelibrary.com/journal/gfs © 2018 John Wiley & Sons Ltd | 1
2 | DEL VALLE ET AL.

higher lactic acid production and lower pH (Freitas et al., 2006) as T A B L E 1 Chemical composition of ensilated sugarcane (n = 4)a
suitable factors. Nevertheless, some studies with homofermentative Item Mean SD
bacteria have shown little efficiency to control alcoholic fermenta-
Composition, g/kg DM
tion (Freitas et al., 2006; Pedroso et al., 2008; Santos et al., 2015).
DM, g/kg 277 8.5
According to Fitzsimons et al. (1992), Pediococcus acidilactici exhibits
Organic matter 970 1.3
a short lag phase on glucose and fructose, which allows rapid estab-
Non-fibre carbohydrate 436 6.9
lishment of a low silage pH when the conditions are pH almost 7.0
NDF 507 6.1
and 20°C. In addition, Propionibacterium spp can produce propionic
Acid detergent fibre 289 3.4
acid using lactic acid as substrate (McDonald, Henderson, & Heron,
1991); however, it is well known that propionic acid has negative Ether extract 14.0 1.74

effects on yeast metabolism (Moon, 1983). Crude protein 13.6 0.43

Recently, Gandra et al. (2016) proposed chitosan as an additive NEL3xb, MJ/kg 6.28 0.134
to sugarcane silage. This polymer is obtained from chitin, which com- In situ degradation, g/kg
poses the exoskeleton of crustaceans and insects, being the second DM 602 24.3
most abundant biopolymer in nature after cellulose (Senel & McClure, NDF 376 10.1
2004). This polysaccharide is insoluble at neutral pH and has antimi-
DM, Dry matter; NDF, Neutral detergent fibre; SD, Standard deviation.
crobial activity against fungi and bacteria (Senel & McClure, 2004). a
First cut of RB025799 cultivar sugarcane, which showed BRIX 21, at
Gandra et al. (2016) found increased DM content, lower total losses 11.5 months after plantation.
b
and improved in vitro neutral detergent fibre (NDF) degradation of Net energy for lactation at three times maintenance level: calculated
according to Weiss et al. (1992) and NRC (2001).
sugarcane silage with chitosan use. However, there is still limited
information on the use of chitosan as an antimicrobial in forage
silage. It is unclear whether chitosan can influence LAB growth or Separator (Maulfair, Fustini, & Heinrichs, 2011). The results showed
whether inoculant acidification effect can affect chitosan efficiency in 149 g/kg for particles larger than 19 mm, 290 g/kg between 19 and
silage. Therefore, we hypothesized that the chitosan use improves 8 mm, 275 g/kg between 8 and 4 mm, and 285 g/kg lower than
the response of sugarcane silage to inoculant treatment. In this study, 4 mm.
we evaluated the effect of associating chitosan with microbial inocu- The plots comprised forty experimental silos (plastic buckets with
lant on sugarcane silage parameters such as fermentation, degrada- 28 9 25 cm, diameter 9 height) equipped with Bunsen valves, to
tion, total losses and nutritional composition. avoid gas escape. Four treatments were arranged in completely ran-
domized design, in 2 9 2 factorial scheme. The evaluated factors were
chitosan use [0 (control—CONT) or 6 g/kg DM—1.66 g/kg of NM]
2 | MATERIAL AND METHODS and microbial inoculant addition (0 and 8 mg/kg NM of a commercial
inoculant). Each kg of inoculant contained 3.9 9 1010 UFC/g of Pedio-
The experiment was conducted between August 2015 and February coccus acidilactici and 3.75 9 1010 UFC/g of Propionibacterium
2017, by the Research Group of Agriculture Study and Labor acidicipropionici. The doses of microbial inoculant were established
(GETAP), in the Agrarian Sciences Center (CCA) of the Federal according to manufacturer’s recommendation. Chitosan was obtained
University of S~ao Carlos (UFSCar), in Araras—SP, Brazil. The area is from Fagron (S~ao Paulo, Brazil) and showed 0.697 of density, pH
0 00 0 00 10.15 and concentration of heavy metals lower than 1 mg/kg, besides
located at 22°18 32 S latitude, 47°22 52 W longitude and 665 m
altitude. The local climate is classified as subtropical humid. During the absence of countable fungi, yeasts and bacteria. Chitosan level
the experiment, the daily averages of temperature and relative was established in a previous study performed in our laboratory,
humidity were 22.3  3.16°C (mean  SD) and 85.3%  11.3%. where the doses 0, 1, 2, 4 and 8 g of chitosan per kg of DM were eval-
Throughout the cultivation period, from August 2015 to August uated. In that study, we observed that chitosan decreased silage losses
2
2016, rainfall was of 1,633 mm/m . Yet, during the 60 days of ensil- and fungal counts: after a regression, the optimal level of 6 g of chi-
ing, temperature and relative humidity averaged 19.5  2.99°C and tosan per kg of DM was chosen (Del Valle et al., unpublished).
78.1%  10.9%.

2.2 | Ensiling and sampling

2.1 | Treatments and experimental design
About 5-kg sand was placed underneath silage, separated by a nylon
As shown in Table 1, plants of RB02-5799 sugarcane variety were screen to determine effluent production. Ensiled material was com-
used in the experiment, after 11.5 months of growth, and with 21% pacted (around 600 kg/m3), sealed, weighed and stored at room
of Brix degrees (°Brix). Almost 800 kg of sugarcane from five differ- temperature for 60 days. Before opening the silos, they were
ent batches in the same area was manually harvested and chopped weighed to calculate gas loss. After opening, the topmost content
in a forage harvester (90 z-10, JF, Itapira, Brazil). The processed sug- was discarded, and one sample (300 g) was collected from the cen-
arcane was analysed for particle size using the Penn State Particle tre of the silos. One subsample was frozen for chemical composition
DEL VALLE ET AL. | 3

and in situ evaluations. Another subsample (15 g) was diluted with The injector and detector temperatures were 250 and 300°C, respec-
150 ml distilled water and processed in a blender for 30 s (Cherney tively, and the initial temperature of the column was 40°C. The tem-
& Cherney, 2003). This sample was then filtered through a cheese- perature ramp of the column started with a gradient from 40 to
cloth, and pH was immediately measured (LUCA-210, Lucadema, S~ao 120°C at a 40°C/min rate, followed by ranges of 120 to 180°C at
 do Rio Preto, Brazil). The filtered samples were frozen for sub-
Jose 10°C/min rate and from 180 to 240°C at a 120°C/min, keeping it at
sequent evaluation of organic acids, ethanol and NH3-N. 240°C for 3 min at the end. For quantification, a calibration of the
After removing the silage, silos were weighed for effluent loss method was made using diluted solutions of the WSFA-2 standard
determination. Aerobic stability was determined using about 3 kg (Ref. 47056, Supelco©) and of HPLC grade ethanol (Ref. 459828,
silage, placed in plastic buckets (28 9 25 cm, diameter 9 height) Sigma-Aldrich©) analysed under the above-described conditions. Peak
and maintained in a controlled temperature room (21.5  1.66°C, detection and integration were made using the GCsolution v. 2.42.00
mean  SD) for 5 days. Every 8 hr, the temperature of the centre of software (Shimadzu©). Lactic acid concentration was measured using
silage mass was registered, using an infrared digital thermometer a spectrophotometric method (Pryce, 1969).
(DT-8380, Tianjin Cheerman Technology Co. Ltd, Tianjin, China).
Then, the aerobic stability was defined as the number of hours the
temperature difference between silage and the environment 2.4 | Calculations and statistical analysis
remained within 2°C (Ranjit & Kung, 2000). Silage pH was evaluated Gas and effluent losses were calculated according to the following
every 24 hr, using the previously described method, up to 144 hr.
equations:

SWE ðgÞ  SWO ðgÞ

2.3 | Chemical analysis GL ðg=kgÞ ¼
FME ðkgÞ

Samples were pre-dried in a forced-air oven at 60°C for 72 hr and where GL is the gas loss; SWE and SWO are the silo weight at ensil-
ground to pass through a 2-mm screen (SL-31, Solab Cientıfica, ing and opening, respectively, and FME is the ensilaged fresh matter.
Piracicaba, Brazil). Then, we evaluated DM (method 950.15), ash
ESWO ðgÞ  ESWE ðgÞ
(method 942.05), crude protein (CP, N 9 6.25; method 984.13) and EL ðg=kgÞ ¼
FME ðkgÞ
ether extract (EE, method 920.39) according to AOAC (2000). Neu-
tral detergent fibre (with heat stable amylase, without sodium and where EL is the effluent loss; ESWO and ESWE are the empty silo
expressed inclusive of residual ash) and acid detergent fibre (ADF, weight at opening and ensiling respectively.
with heat stable amylase, without sodium sulphite and expressed
ODM (kg)
inclusive of residual ash) were determined according to Van Soest, DMR ¼
EDM (kg)
Robertson, and Lewis (1991). Net energy concentration of sugarcane
was estimated according to Weiss, Conrrad and St-Pierre (1992) and where DMR is dry-matter recovery; ODM is dry matter in the silos

considering the method described in the NRC (2001). Two cannu- after silos opening, and EDM is the ensiled DM.

lated dairy cows, previously adapted to a diet with forage to concen- Data were analysed using PROC MIXED of SAS 9.3. (SAS Inst.

trate ratio of 60:40, were used for an in situ degradation assay. Inc., Cary, NC), according to the statistical model:

Ruminal incubation was performed for 96 hr (Ruiz, Van Soest, Van Yijk ¼ l þ Ci þ Ij þ C  Iij þ eijk
Amburgh, Fox, & Robertson, 2001) using 5 9 5 cm non-woven tis-
sue (100 g/m2) (Casali et al., 2008) and almost 500 mg sample in in which eijk  Nð0; r2e Þ; where, Yijk is the value of dependent vari-

each bag. After removal, bags were washed in running water and able; l is the overall mean; Ci is the fixed effect of chitosan (i = 1,

evaluated for NDF content as previously described. 2); Ij is the fixed effect of microbial inoculant (j = 1, 2); C 9 Iij is an

Frozen samples of silage juice were thawed to room temperature interaction term; eijk is the residual error; N stands for the Gaussian

and centrifuged (500 g for 15 min). The supernatant was used for distribution. The degrees of freedom were corrected using the

determination of NH3-N, organic acid and ethanol concentrations. method of Kenward and Roger (1997). When interaction term was

The ammonia nitrogen was evaluated by the colorimetric phenol- significant, comparisons between treatments were made using Fish-

hypochlorite method (Broderick & Kang, 1980). The concentrations er’s protected LSD. The significance level was set at .05.

of ethanol and acetic, propionic and butyric acids and the supernatant Silage pH and temperature after aerobic exposure were analysed

of the samples after centrifugation (500 g for 15 min) were deter- as repeated measures, according to the following statistical model:

mined by gas chromatography (GC-2010 Plus chromatograph, Shi- Y ijkl ¼l þ Ci þ Ij þ C  Iij þ xk:ij þ Tl þ T  Cli þ T
madzu, Barueri, Brazil), equipped with a AOC-20i auto-sampler,  Ilj þ T  C  Ilij þ eijkl
Stabilwax-DATM capillary column (30 m, 0.25 mm ID, 0.25 lm df;
Restek©), and a flame ionization detector. The samples were acidified in which xijk  Nð0; r2x Þ and; eijkl  MVN ð0; RÞ where, Yijkl is the

using formic acid at a 1:4 ratio. A 1-ll aliquot of each sample was value of dependent variable; l is the overall mean; Ci is the fixed

injected with a split ratio of 40:1, using helium as the carrier gas with effect of chitosan (i = 1, 2); Ij is the fixed effect of microbial inocu-

a linear velocity of 42 cm/s, in a chromatographic run of 11.5 min. lant (j = 1, 2); Tl is time fixed effect (l = 1 to 5 for pH and l = 1 to
4 | DEL VALLE ET AL.

15 for temperature); xk:ij is the random effect of silo k, within ith inoculant-treated ones (p ≤ .05). Regardless of inoculant addition, chi-
level of chitosan and jth level of inoculant (k = 1 to 40); C9Iij, T9Cli, tosan decreased effluent losses (p < .001), which resulted in lower
T9Ilj and T9C9Ilij are interaction term; eijkl is the residual error; N total losses (p ≤ .05). In contrast, inoculant addition increased total
stands for the Gaussian distribution; r2x is the variance associated losses (p ≤ .05) in silos not treated with chitosan and had no effect on
with silos; MVN stands for the multivariate normal; and R is the vari- the treated ones (p > .05). Thus, while the addition of chitosan
ance-covariance matrix of residuals due to the repeated measure- increased DM recovery, the microbial inoculant decreased it
ments. Compound symmetry (CS), heterogeneous compound (p ≤ .005).
symmetry (CSH), autoregressive (AR), heterogeneous autoregressive
(ARH), Toeplitz (TOEP), heterogeneous Toeplitz (TOEPH), factor ana- 3.3 | Chemical composition
lytic (FA), Huynh-Felt (HF), unstructured (UN) and variance compo-
nents (VC). Variance and covariance matrixes were evaluated using Chitosan decreased organic matter (OM), NDF and ADF contents
Bayesian method. The covariance matrices chosen were CSH and (p < .001; Table 4), besides increasing non-fibre carbohydrate (NFC)
ARH for silage pH and temperature respectively. concentration (p < .001) and improving silage DM degradation
(p = .001), but had no effect on NDF degradation (p = .133). Silos
treated with inoculant showed lower DM (p < .001) and increased
3 | RESULTS
CP, EE, NDF and ADF contents (p ≤ .017) than did those not trea-
ted. Furthermore, the inoculant use decreased NFC concentration,
3.1 | Fermentative profile
and DM and NDF degradation (p ≤ .040) of sugarcane silage.
There was an interaction effect of chitosan and inoculant use on lac-
tic acid concentration (p < .001; Table 2). Regardless of chitosan
3.4 | Aerobic stability
addition, microbial inoculant increased the lactic acid content in sug-
arcane silage (p ≤ .05), while chitosan decreased it only in silos non- There was an interaction effect of chitosan and microbial inoculant
treated with inoculant (p ≤ .05). Moreover, chitosan increased silage on aerobic stability (p = .021; Table 5). Chitosan increased the aero-
pH (p < .001) and tended to increase acetic acid (p = .058). On the bic stability (p ≤ .05) in silos with no inoculant (p ≤ .05), but had no
other hand, microbial inoculant addition decreased silage pH, NH3-N effect on those receiving inoculant (p > .05). There was an interac-
concentration (g/kg of CP) and acetic acid concentration (p ≤ .033), tion effect of treatment and time on silage pH and temperature after
but increased silage ethanol concentration (p = .017). Nonetheless, aerobic exposure (p ≤ .013; Figures 1 and 2). In general, the treat-
the evaluated factors showed no effect on propionic and butyric acid ments with chitosan showed a rapid increase in pH after aerobic
concentrations (p ≥ .119). exposure, especially on those with inoculant addition. On the other
hand, the effects of inoculant use on silage pH depended on the
addition of chitosan. The inoculant was able to increase (p ≤ .05)
3.2 | Fermentative and effluent losses
silage pH in silos containing chitosan at 96 and 120 hr after aerobic
There was an interaction effect of chitosan and microbial inoculant on exposure. Besides, this effect was reversed in silos without chitosan
gas and total losses (p ≤ .035; Table 3). Despite the lack of effect in (p ≤ .05), where silage pH was decreased at 96, 120 and 144 hr
silos without inoculant (p > .05), chitosan affected gas loss in the after aerobic exposure.

T A B L E 2 Effects of chitosan and microbial inoculant on fermentative profile of sugarcane silage

Without inoculant With inoculant† p
‡ § ‡ §
Item CONT CHI CONT CHI SEM CHI INO INT
pH 3.47 3.55 3.40 3.54 0.001 <.001 .033 .116
NH3-N, g/kg N 40.9 40.7 28.0 32.4 1.01 .308 <.001 .266
Ethanol, g/kg DM 6.45 6.40 7.22 6.81 0.117 .328 .017 .462
Organic acids, g/kg DM
Lactic acid 28.8a 21.4b 26.0a 28.1a 1.74 .015 .072 <.001
Acetic acid 19.3 22.5 17.8 18.7 0.53 .058 .018 .275
Propionic acid 1.23 1.50 0.94 0.79 0.138 .822 .119 .491
Butyric acid 0.865 0.836 0.852 0.903 0.0133 .683 .336 .164

CHI, chitosan effect; DM, dry matter; INO, inoculant effect; INT, chitosan and inoculant interaction effect; SEM, Standard error of mean.
†
Inoculant: 8 mg/kg NM of inoculant, which each g contained 3.9 9 1010 UFC/g of Pediococcus acidilactici and 3.75 9 1010 UFC/g of Propionibacterium
acidicipropionici.
‡
CONT: Without chitosan addition.
§
CHI: With 6 g of chitosan per kg of silage DM.
a-b
LSD means test.
DEL VALLE ET AL. | 5

T A B L E 3 Effects of chitosan and microbial inoculant on fermentative and effluent losses of sugarcane silage
Without inoculant With inoculant† p
‡ § ‡ §
Item CONT CHI CONT CHI SEM CHI INO INT
Losses, g/kg
Gases 24.0b 20.9b 31.6a 23.2b 0.52 <.001 <.001 .016
Effluent 47.2 29.2 56.9 22.8 2.32 <.001 .724 .090
Total 71.2 b
50.1 c
88.5 a
46.0 c
2.45 <.001 .185 .035
DM recovery, g/kg DM 784 819 757 780 4.9 .005 .002 .563

CHI, chitosan effect; DM, dry matter; INO, inoculant effect; INT, chitosan and inoculant interaction effect; SEM, Standard error of mean.
†
Inoculant: 8 mg/kg NM of inoculant, which each g contained 3.9 9 1010 UFC/g of Pediococcus acidilactici and 3.75 9 1010 UFC/g of Propionibacterium
acidicipropionici.
‡
CONT: Without chitosan addition.
§
CHI: With 6 g of chitosan per kg of silage DM.
a-b
LSD means test.

T A B L E 4 Effects of chitosan and microbial inoculant on chemical composition of sugarcane silage

Without inoculant With inoculant† p

Item CONT‡ CHI§ CONT‡ CHI§ SEM CHI INO INT

Chemical composition, g/kg DM
DM, g/kg 234 239 230 227 1.1 .720 .001 .070
Organic matter 961 957 960 955 0.5 <.001 .090 .483
NDF 745 674 761 708 5.0 <.001 .017 .403
ADF 455 411 472 432 3.6 <.001 .013 .801
NFC 187 253 157 210 5.3 <.001 .001 .537
Crude protein 15.4 18.3 23.2 22.0 0.6 .465 <.001 .073
Ether extract 14.1 12.4 16.8 15.7 0.53 .205 .007 .769
In situ digestibility, g/kg
DM 439 480 425 454 4.7 .001 .040 .535
NDF 339 325 315 307 3.5 .133 .007 .677

ADF, acid detergent fibre; CHI, chitosan effect; DM, dry matter; INO, inoculant effect; INT, chitosan and inoculant interaction effect; NDF, neutral
detergent fibre; NFC, non-fibre carbohydrate; SEM, Standard error of mean.
†
Inoculant: 8 mg/kg NM of inoculant, which each g contained 3.9 9 1010 UFC/g of Pediococcus acidilactici and 3.75 9 1010 UFC/g of Propionibacterium
acidicipropionici.
‡
CONT: Without chitosan addition.
§
CHI: With 6 g of chitosan per kg of silage DM.
a-b
LSD means test.

If compared to control treatment, silos treated only with chitosan range suggested by McDonald et al. (1991) (from 3.8 to 4.2) as ideal for
showed higher aerobic stability, which results in lower (p ≤ .05) silage fermentation. According to these authors, lactic acid is the main
silage temperatures between 52 and 72 hr of aerobic stability and fermentation product end product of traditional silages (e.g., corn, grass,
higher temperatures at 120 hr. Although silos treated only with inoc- and alfalfa silages). Besides having an elevated concentration of NFC,
ulant showed no effects on silage stability (in hr), they had lower sugarcane silages are also rich in water-soluble carbohydrates (WSC),
(p ≤ .05) temperature than control treatment, both at 56 and 64 hr which are mainly metabolized by lactic acid bacteria into organic acids
after aerobic exposure. (McDonald et al., 1991). Therefore, in the present study, the use of
microbial inoculant showed a positive effect on lactic acid concentra-
tion, decreasing silage pH. One of the microorganisms in the evaluated
4 | DISCUSSION inoculant (Pediococcus acidilactici) is classified as a homofermentative
microorganism (Driehuis, Oude Elferink, & Van Wikselaar, 2001), once it
In the present study, in natura sugarcane had Brix degree and NFC con- exhibits a short lag phase, what provides a fast acid production rate and
tent of 21.0 and 436 g/kg respectively. These conditions provide a hence a high sugar-to-lactate conversion efficiency (Fitzsimons et al.,
great substrate for lactic acid bacteria growth (Santos et al., 2015), 1992). The effects of homofermentative LAB inoculant on the lactic
resulting in a silage with an average pH ≤ 3.55, which is lower than the 
acid concentration and pH of silages have been well documented (Avila
6 | DEL VALLE ET AL.

T A B L E 5 Effects of chitosan and microbial inoculant on silage aerobic stability

Without inoculant With inoculant† p
‡ § ‡ §
Item CONT CHI CONT CHI SEM CHI INO INT
Aerobic stability, hr 47.6b 65.2a 56.2ab 49.3b 2.56 .304 .478 .021
Silage pH 4.18 4.36 3.91 4.66 0.505 <.001 .925 .008

CHI, chitosan effect; INO, inoculant effect; INT, chitosan and inoculant interaction effect; SEM, Standard error of mean.
†
Inoculant: 8 mg/kg NM of inoculant, which each g contained 3.9 9 1010 UFC/g of Pediococcus acidilactici and 3.75 9 1010 UFC/g of Propionibacterium
acidicipropionici.
‡
CONT: Without chitosan addition.
§
CHI: With 6 g of chitosan per kg of silage dry matter.
a-b
LSD means test.

6.80 Probabilities (P): Elferink et al. (2001), acetic acid production by heterofermentative LAB
CHI < 0.001
INO: 0.925
fermentation depends on the presence of an electron acceptor. Further-
6.30 a
CHI×INO: 0.008 more, a metal linked to one or more chitosan chains could act as an
Time < 0.001
5.80 a electron acceptor (Goy et al., 2009), which is associated with its antimi-
Time×CHI: 0.002 a
Time×INO: 0.179
Silage pH

5.30 Time×CHI×INO: 0.013 crobial activity. Thus, despite the lactic acid reduction, chitosan was able
a b
b to increase acetic acid. This positive effect of chitosan on silage acetate
4.80
b c concentration has already been observed in sugarcane silage (Gandra
a b
4.30 et al., 2016).
b
b b c Sugarcane silage losses have been associated with alcohol fermen-
3.80 c
b 
tation by yeasts (Avila, Pinto, Figueiredo, & Schwan, 2009; Carvalho,
3.30
24 48 72 96 120 144 Avila, Pinto, Neri, & Schwan, 2014; Kung & Stanley, 1982). The positive
Hours after aerobiosis
effects of the heterofermentative LAB on silage losses are associated
F I G U R E 1 pH after aerobic exposure of sugarcane silage treated with higher concentration of acetate and lower of lactic acid (Carvalho
with chitosan and microbial inoculant et al., 2014). In the present study, chitosan decreased gas and effluent
losses, with a positive effect on DM recovery. However, chitosan
et al., 2010; Muck, 2010; Santos et al., 2015). According to Ranjit and showed no effect on silage ethanol though there was a lower numerical
Kung (2000), lactic acid shows a lower antifungal effect than acetic acid concentration in silos containing (0.41 g/kg DM) or not (0.05 g/kg DM)
and, therefore, yeast is more prone to act in accord with this condition, microbial inoculant. Rodrigues et al. (2012) have already reported the
providing ethanol production and DM losses (Kung Jr. Schmidt, Ebling, lack of effect on ethanol concentration in silos with lower acetic acid
& Hu, 2007). concentrations. These authors associated this outcome to the magni-
Chitosan solubilization in an acid solution converts glucosamine tude of responses, as observed in the present study. Besides environ-
units (-NH2) into its soluble protonated form (-NH+3; Goy, de Brito, & mental conditions, the potential antifungal effect of chitosan has been
Assis, 2009). The level of NH3-N averaged 35.5 g/kg of total nitrogen, already proven (Hernandez-Lauzardo et al., 2008; Munhuweyia, Calebb,
which is in accordance with Ohshima and McDonald (1978), who sug- Lennoxc, Van Reenend, & Oparaa, 2017) and could provide a positive
gested values below 10%, with a high level of lactate. However, chi- effect on DM recovery of sugarcane silage (Gandra et al., 2016).
tosan showed no effect on the level of NH3-N (g/kg of N). Besides an According to Santos et al. (2009), additives with positive effects on sug-
increased protonation, chitosan inhibits effluent production, which had arcane silage pH improve DM recovery, probably due to a decrease in
no effect on the silage NH3-N level (g/kg of N) in the silage. Devlie- fermentation extension. On the other hand, microbial inoculant
ghere, Vermeulen, and Debevere (2004) have already demonstrated a decreased acetic acid concentration and increased those of lactic acid
higher chitosan antimicrobial activity at lower environmental pH values and ethanol, which are prone to increase yeast metabolism (Pedroso
(around 4.0). Therefore, this antimicrobial activity of chitosan decreased et al., 2005). Chitosan addition affected the effect of microbial inocu-
the production of acids, mainly lactic acid. Even though this chitosan lant on gas and total silage losses. We agree that this might be related
effect on lactic acid concentration occurred only in silos without micro- to the antifungal action of chitosan as the inoculant effects on silage pH
bial inoculant. Casquete, Castro, and Teixeira (2016), found an associa- and ethanol concentration were lower in silos treated with chitosan
tive effect between chitosan and LAB (Pediococcus spp.) on food than in those without it.
conservation, in which chitosan inhibits LAB growth after 7 days of Although microbial inoculant showed no effect on effluent losses,
incubation. We agree that chitosan was not able to inhibit both according to Pedroso et al. (2005), ethanol production occurs with
microorganisms used in the inoculation process (Pediococcus acidilactici H2O synthesis. Therefore, inoculant increased silage moisture con-
and Propionibacterium acidicipropionici), which resulted in no effects on tent. Organic acid production occurs with WSC (NFC) consumption,
lactic acid concentration in inoculated silos. Additionally, chitosan which results in increased fibre, CP and EE contents in the silage as
tended to increase acetic acid concentration. According to Oude compared to the ensiled material (Pedroso et al., 2005). Increased
DEL VALLE ET AL. | 7

12
Probabilities (P):
CHI: 0.673
INO: 0.711 a
10 a
CHI×INO: 0.072
(oC under environmental temperature)

Time <0.001 a
8 Time×CHI: 0.574 ab ab
a b
Time×INO: 0.440
Time×CHI×INO: 0.006 b
a ab
Temperature

6 b b
a
ab
a
4 b
b
2 b b
b
0
8 16 24 32 40 48 56 64 72 80 88 96 104 112 120
F I G U R E 2 Temperature of silage after
–2
Hours after aerobiosis aerobic exposure

concentrations of CP and EE in silos treated with inoculant were of (lower fibre and higher CP), which increases the challenge and ren-
small importance (5.75 g/kg CP and 3.0 g/kg EE) for silage nutritional ders chitosan inefficient when associated. Finally, silos treated only
value and could also be associated with an increased microbial with inoculant had intermediate aerobic stability with no difference
growth, which increases their contribution to silage composition. of any other conditions. This result is associated with the negative
Values of CP were lower than the expected concentration for sugar- effect of inoculants on silage composition.
cane, which could be associated with advanced maturity degree of
sugarcane. In addition, microbial inoculant decreased NFC and
increased fibre concentration, which reduces digestibility (NRC, 2001) 5 | CONCLUSION
and degradation of DM and NDF. Differently, On the other hand, chi-
tosan decreased fibre concentration of silages by reverse mechanism, Chitosan reduced fermentative losses, besides improving silage
with a positive effect on NFC concentration and DM degradation. chemical composition and DM degradation. However, the microbial
In general, chitosan had a positive effect on silage pH in silos inoculant, containing Pediococcus acidilactici and Propionibacterium
treated with the inoculant, after aerobic exposure. Gandra et al. acidicipropionici, increased fermentative losses and showed a worse
(2016) also found a positive effect of chitosan on silage pH after silage chemical composition, in addition to reducing DM and NDF
aerobic exposure. These authors associated this effect with the degradations. In addition, the association of chitosan and inoculant
lower fibre content of the tested silage, which enhanced the micro- promoted a lower aerobic stability than when using chitosan singly.
bial action. Furthermore, microbial inoculant effect on silage pH after Therefore, the use of chitosan without microbial inoculant is recom-
aerobic exposure was dependent on chitosan addition. These results mended as a good sugarcane silage additive.
are associated with lactic acid concentration in the silages. The pH
level is an indicator of aerobic deterioration of silages as yeasts con-
CONFLICT OF INTERESTS
sume the lactic acid during the aerobic exposure (Basso et al., 2012).
Although Propionibacterium acidicipropionici is classified as propionic The authors declare that is no conflict of interests in this study.
acid bacteria, which affects silage aerobic stability (Filya, Sucu, &
Karabulut, 2004), the inoculant showed no effect on propionic acid
ACKNOWLEDGMENTS
silage concentration. According to Danner, Holzer, Mayrhuber, and
Braun (2003), acetic acid concentration is another important acid for The authors wish to thank Professor Dr. Reinaldo Gaspar Bastos,
providing silage aerobic stability. In the opposite sense, the microbial from UFSCar, for providing the physical infrastructure and staff the
degradation of lactic acid increases silage pH after aerobic exposure. evaluations of for organic acids.
The interaction between inoculant and chitosan had an effect on
silage temperatures after aerobic exposure, especially during aerobic ORCID
stability (when silage temperatures are up to 2°C above the environ-
mental one). Chitosan by itself and its effects on fermentation (e.g., T. A. Del Valle http://orcid.org/0000-0001-8093-7132

higher acetic acid concentration) inhibited yeasts upon air exposure

after silage feed out (Basso et al., 2012; Danner et al., 2003). In con-
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