Chitosan improves the chemical composition, microbiological quality, and aerobic stability of sugarcane silage

Abstract

The purpose of the current study was to evaluate the effects of inoculants on chemical composition, dry matter (DM) and neutral detergent fiber (aNDF) in vitro degradation, fermentative and effluent losses, microbiology, fermentative profile, and aerobic stability of sugarcane mini-silos. Treatments were randomly distributed to the mini-silos, in which: (1) Control (CON); (2) Lactobacillus buchneri (Lb), addition of Lb at 2.6 × 1010 cfu/g; (3) Lactobacillus buchneri and Bacillus subtilis (Lb + Bs), addition of Lb at 2.6 × 1010 cfu/g and Bs at 1 × 109 cfu/g; and (4) Chitosan (CHI), addition of 1% of CHI on wet basis of sugarcane ensiled. Treatments 2 and 3 were incorporated to the silage at 2 g/t of natural matter ensiled. Lb and Lb + Bs did not alter the in vitro degradation of DM and NDF. Chitosan incorporation increased the DM content (P = 0.013, 18.7 g/kg DM) and improved (P = 0.029, 45.6 g/kg DM) the NDF in vitro degradation of sugarcane silage. In addition, CHI incorporation showed higher (P = 0.002) DM content in silage than Lb and Lb + Bs. Microbial inoculants (Lb and Lb + Bs) reduced the total losses (P = 0.009) of sugarcane silage. Moreover, CHI incorporation showed lower (P = 0.001, 84.9 g/kg DM) total losses and higher (P = 0.031, 84.8 g/kg DM) dry matter recovery than Lb and Lb + Bs. Lactic acid bacteria concentration was increased (P = 0.001) with additives, and CHI incorporation showed higher (P = 0.001) lactic acid bacteria concentration than silages treated Lb and Lb + Bs. All additives decreased the ethanol concentration in sugarcane silage, but CHI showed lower (P = 0.002) ethanol concentration compared to Lb and Lb + Bs. Inoculants improved the aerobic stability of sugarcane silage. In general, the incorporation of CHI to sugarcane silage showed better results of NDF in vitro degradation and gas and effluent losses than Lb and Lb + Bs. Moreover, CHI incorporation showed higher concentration of lactic acid bacteria and lower concentration of ethanol compared to silages treated Lb and Lb + Bs. Chitosan may be an alternative additive to microbial inoculants used in sugarcane ensiling.

Full text

Animal
Feed
Science
and
Technology
214
(2016)
44–52
Contents
lists
available
at
ScienceDirect
Animal
Feed
Science
and
Technology
journal
homepage:
www.elsevier.com/locate/anifeedsci
Chitosan
improves
the
chemical
composition,
microbiological
quality,
and
aerobic
stability
of
sugarcane
silage
J.R.
Gandraa,∗,
E.R.
Oliveiraa,
C.S.
Takiyac,
R.H.T.B.
Goesa,
P.G.
Paivad,
K.M.P.
Oliveirab,
E.R.S.
Gandraa,
N.D.
Orbacha,
H.M.C.
Harakia
aDepartment
of
Animal
Science,
Universidade
Federal
da
Grande
Dourados,
Rodovia
Dourados-Itahum,
km
12,
Zip
Code:
79804-970,
Dourados,
MS,
Brazil
bDepartment
of
Biology,
Universidade
Federal
da
Grande
Dourados,
Rodovia
Dourados-Itahum,
km
12,
Zip
Code:
79804-970,
Dourados,
MS,
Brazil
cDepartment
of
Animal
Nutrition
and
Production,
School
of
Veterinary
Medicine
and
Animal
Science,
University
of
São
Paulo
(USP),
Av.
Duque
de
Caxias
Norte,
225-Campus
da
USP,
Zip
Code:
13635-900,
Pirassununga,
SP,
Brazil
dDepartment
of
Animal
Sciences,
UNESP
−
Universidade
Estadual
Paulista
“Júlio
de
Mesquita
Filho”/Campus
Jaboticabal,
Rod.
Prof.
Paulo
Donato
Castellane
km
5,
Rural,
Zip
Code:
14884-900,
Jaboticabal,
SP,
Brazil
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
12
August
2015
Received
in
revised
form
23
February
2016
Accepted
26
February
2016
Keywords:
Aerobic
stability
Bacillus
subtilis
Chitin
Fermentation
Inoculant
Lactobacillus
buchneri
a
b
s
t
r
a
c
t
The
purpose
of
the
current
study
was
to
evaluate
the
effects
of
inoculants
on
chemical
composition,
dry
matter
(DM)
and
neutral
detergent
fiber
(aNDF)
in
vitro
degradation,
fer-
mentative
and
effluent
losses,
microbiology,
fermentative
profile,
and
aerobic
stability
of
sugarcane
mini-silos.
Treatments
were
randomly
distributed
to
the
mini-silos,
in
which:
(1)
Control
(CON);
(2)
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
(3)
Lac-
tobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
(4)
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
Treatments
2
and
3
were
incorporated
to
the
silage
at
2
g/t
of
natural
matter
ensiled.
Lb
and
Lb
+
Bs
did
not
alter
the
in
vitro
degradation
of
DM
and
NDF.
Chitosan
incorporation
increased
the
DM
content
(P
=
0.013,
18.7
g/kg
DM)
and
improved
(P
=
0.029,
45.6
g/kg
DM)
the
NDF
in
vitro
degradation
of
sugarcane
silage.
In
addition,
CHI
incor-
poration
showed
higher
(P
=
0.002)
DM
content
in
silage
than
Lb
and
Lb
+
Bs.
Microbial
inoculants
(Lb
and
Lb
+
Bs)
reduced
the
total
losses
(P
=
0.009)
of
sugarcane
silage.
More-
over,
CHI
incorporation
showed
lower
(P
=
0.001,
84.9
g/kg
DM)
total
losses
and
higher
(P
=
0.031,
84.8
g/kg
DM)
dry
matter
recovery
than
Lb
and
Lb
+
Bs.
Lactic
acid
bacteria
con-
centration
was
increased
(P
=
0.001)
with
additives,
and
CHI
incorporation
showed
higher
(P
=
0.001)
lactic
acid
bacteria
concentration
than
silages
treated
Lb
and
Lb
+
Bs.
All
additives
decreased
the
ethanol
concentration
in
sugarcane
silage,
but
CHI
showed
lower
(P
=
0.002)
ethanol
concentration
compared
to
Lb
and
Lb
+
Bs.
Inoculants
improved
the
aerobic
stability
of
sugarcane
silage.
In
general,
the
incorporation
of
CHI
to
sugarcane
silage
showed
better
results
of
NDF
in
vitro
degradation
and
gas
and
effluent
losses
than
Lb
and
Lb
+
Bs.
Moreover,
Abbreviations:
aADF,
acid
detergent
fiber;
aNDF,
neutral
detergent
fiber;
Bs,
Bacillus
subtilis;
CHI,
chitosan;
CON,
control;
CP,
crude
protein;
DM,
dry
matter;
DMR,
dry
matter
recovery;
Lb,
Lactobacillus
buchneri;
TDN,
total
digestible
nutrient.
∗Corresponding
author.
E-mail
addresses:
jeffersongandra@ufgd.edu.br
(J.R.
Gandra),
caio.takiya@usp.br
(C.S.
Takiya),
bocazoo@hotmail.com
(P.G.
Paiva).
http://dx.doi.org/10.1016/j.anifeedsci.2016.02.020
0377-8401/Published
by
Elsevier
B.V.

J.R.
Gandra
et
al.
/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
45
CHI
incorporation
showed
higher
concentration
of
lactic
acid
bacteria
and
lower
concentra-
tion
of
ethanol
compared
to
silages
treated
Lb
and
Lb
+
Bs.
Chitosan
may
be
an
alternative
additive
to
microbial
inoculants
used
in
sugarcane
ensiling.
Published
by
Elsevier
B.V.
1.
Introduction
Sugarcane
(Saccharum
officinarum)
is
often
used
in
Brazil
as
a
forage
source
for
dairy
and
beef
cattle,
since
the
harvesting
phase
coincides
with
the
winter
which
is
a
period
of
shortage
of
feed.
Ensiling
sugarcane
may
be
a
strategy
to
decrease
daily
manpower
and
theoretically
maintain
similar
nutrient
composition
from
the
beginning
until
the
end
of
the
silo.
Sugarcane
crop
has
a
high
DM
production
per
hectare
(25–40
t;
Ávila
et
al.,
2009),
high
water-soluble
carbohydrates
content,
and
a
low
buffering
capacity
that
enables
rapid
decrease
of
pH
(Freitas
et
al.,
2006).
However,
its
fermentation
can
produce
high
amounts
of
ethanol,
increasing
DM
losses
(Kung
and
Stanley,
1982),
and
accumulating
fiber
components
causing
a
decrease
of
DM
digestibility
(Santos
et
al.,
2009);
thus,
the
advantages
of
ensiling
sugarcane
may
be
limited
by
these
factors.
Microbial
inoculants
have
been
used
to
shift
alcoholic
fermentation
and
improve
sugarcane
silage
digestibility.
Several
studies
have
evaluated
Lactobacillus
buchneri
as
a
silage
additive
during
the
last
decade
(Santos
et
al.,
2015;
Carvalho
et
al.,
2012;
Pedroso
et
al.,
2010).
This
heterolactic
bacteria
has
been
shown
to
improve
silage
fermentation
due
to
a
reduction
of
ethanol
production
and
pH
values
(Pedroso
et
al.,
2008).
Kleinschmidt
and
Kung
(2006)
evaluated
forty-three
experiments
and
reported
the
effectiveness
of
L.
buchneri
to
reduce
the
pH
and
yeast
population,
and
its
effectiveness
to
increase
the
acetic
acid
concentration
and
aerobic
stability
of
silages
from
several
plants
species
(corn,
sorghum,
wheat,
barley,
and
grass
forages).
However,
studies
related
to
Bacillus
subtilis
treatment
during
the
ensiling
process
are
scarce
in
literature.
Todovora
and
Kozhuharova
(2010)
reported
that
B.
subtilis
produces
metabolites
with
antifungal
and
antibacterial
activity.
Phillip
and
Fellner
(1992)
evaluated
the
addition
of
B.
subtilis
in
corn
silage
and
reported
improvements
of
the
aerobic
stability.
Chitosan
is
a
biopolymer
obtained
by
the
partially
deacetylation
of
chitin,
the
second
most
abundant
biopolymer
in
nature,
and
the
major
component
of
crustaceans
and
insects
exoskeleton
(Senel
and
McClure,
2004).
The
antimicrobial
activity
of
CHI
is
well
known
against
bacteria
and
fungi
(Senel
and
McClure,
2004),
and
have
been
used
as
rumen
modulator.
Chitosan
was
able
to
completely
inhibit
the
growth
of
dimorphic
fungus
(Olicón-Hernández
et
al.,
2015).
Araújo
et
al.
(2015)
reported
that
CHI
quadratically
affected
the
ruminal
ammonia
nitrogen
concentration
and
the
molar
proportions
of
propionate
in
beef
steers.
In
addition,
the
same
authors
found
that
CHI
increased
the
digestibility
of
DM,
NDF
and
crude
protein
(CP;
Araújo
et
al.,
2015).
Our
hypothesis
was
that
inoculants
would
positively
affect
the
fermentation
pattern
and
aerobic
stability,
decreasing
the
DM
losses
of
sugarcane
silage.
Furthermore,
CHI
would
alter
microbiology
and
reduce
fungi
amounts
in
the
silage.
The
objective
of
the
current
study
was
to
evaluate
the
effects
of
three
inoculants
on
chemical
composition,
DM
and
NDF
in
vitro
degradation,
fermentative
and
effluent
losses,
microbiology,
fermentative
profile,
and
aerobic
stability
of
sugarcane
mini-silos.
2.
Material
and
methods
The
experiment
was
conducted
between
May
and
September
of
2013
at
the
Department
of
Animal
Science,
School
of
Agrarian
Sciences,
Federal
University
of
Grande
Dourados,
Dourados,
Brazil;
22◦14S
latitude,
54◦49W
longitude
and
450
m
altitude.
2.1.
Treatments
and
ensiling
Sugarcane
variety
RB
84-5257
was
manually
harvested
from
10
batches
within
one
0.35-ha
plot
after
10
months
of
regrowth
(second
cut).
Approximately
50.0
kg
of
sugarcane
tillers
from
each
location
was
separately
chopped
in
a
stationary
cutter
to
a
theoretical
cut
length
of
10
mm.
A
randomized
experimental
design
was
used,
and
contained
4
treatments
distributed
into
40
mini-silos.
Mini-silos
were
produced
in
plastic
bucket
(30
cm
of
height
and
30
cm
of
diameter)
containing
Bunsen
valves
to
avoid
the
gas
scape.
Two
kilograms
of
sand
were
placed
at
the
bottom
of
mini-silos,
separated
from
the
forage
by
a
nylon
screen
to
determine
the
effluent
production.
Silos
were
packed
(650
kg/m3,
on
wet
basis),
sealed,
weighed,
and
stored
at
room
temperature
(28.5
±
2.3 ◦C)
for
60
days.
Mini-silos
were
weighed
immediately
after
the
opening
to
record
DM
and
gas
losses.
Treatments
were
randomly
distributed
to
the
mini-silos,
in
which:
(1)
Control
(CON);
(2)
L.
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
(3)
L.
and
B.
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
(4)
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
Chitosan
used
during
all
experiment
had
the
technical
specifications:
apparent
density
of
0.64
g/mL,
20.0
g/kg
of
ash,
7.0–9.0
of
pH,
viscosity
<200
cPs
and
deacetylation
level
of
95%
(Polymar
Industria,
Ceara,
Brazil).
In
addition,
the
CHI
had
873
g/kg
of
DM
and
316
g/kg
of
CP.
The
treatments
2
(Lb)
and
3
(Lb
+
Bs)
were
added
at
2
g/t
of
natural
matter
ensiled.
Microbial
inoculants
were
diluted
in
water
(2
g/L)
and
sprayed
onto
the
forage,

46
J.R.
Gandra
et
al.
/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
Table
1
Chemical
composition
of
sugarcane
before
the
ensiling
(g/kg
DM,
otherwise
stated).
Dry
matter
(g/kg)
275
Organic
matter
963
Protein
28.4
Neutral
detergent
fiber
502
Acid
detergent
fiber 272
Lignin
(sa)
55.9
Ash
36.6
and
CHI
was
top
dressed
and
mixed
into
the
fresh
forage.
The
same
amount
of
water
was
added
to
the
CON
and
CHI.
All
inoculants
were
added
separately
in
each
mino-silo.
2.2.
Chemical
composition
and
in
vitro
degradation
Prior
to
the
ensiling,
sugarcane
was
sampled
and
stored
at
−20 ◦C
until
chemical
analyses.
Dry
matter
(#950.15),
CP
(#984.13),
and
ash
(#942.05)
were
determined
according
to
the
procedures
of
AOAC
(2002).
Crude
protein
was
calculated
as
Kjeldal
N
×
6.25.
Neutral
detergent
fiber,
acid
detergent
fiber
(aADF)
and
lignin
(sa)
were
determined
according
to
Van
Soest
et
al.
(1991).
Total
digestible
nutrient
(TDN)
was
estimated
following
the
equations
of
NRC
(2000).
The
soluble
solid
content
in
the
stalk
juice
was
18.4◦Brix.
The
chemical
composition
of
sugarcane
is
shown
in
Table
1.
At
the
silos
opening,
5
samples
(0.2
kg)
of
each
mini-silo
were
collected
to
form
a
composite
sample,
and
then
were
analyzed
to
determine
DM,
CP,
NDF,
ADF,
lignin
(sa),
ash
and
TDN
content
as
previously
described.
The
in
vitro
degradation
of
DM
and
NDF
was
performed
according
to
Tilley
and
Terry
(1963).
2.3.
Fermentative
and
effluent
losses
Mini-silos
were
weighed
on
days
15,
30,
45
and
60
after
the
ensiling.
On
day
60
of
ensiling,
mini-silo
were
opened
to
determine
gas
losses.
The
silage,
silo
assembly,
sand
layer
and
nylon
screen
were
weighed
to
quantify
the
effluent
production.
Gas
losses
were
calculated
as
follows:
GL
=
(SWE
−
WSO)/DME
×
100
in
which
GL
=
gas
losses
(%DM),
SWE
=
silo
weight
prior
to
the
ensiling
(kg),
WSO
=
silo
weight
after
the
mini-silos
opening
(kg)
and
DME
=
dry
matter
ensiled
(kg
of
forage
×
%
DM).
Effluent
production
was
calculated
according
to
the
equation:
EP
=
(WSAO
−
WSAE)/DME
×
100
in
which
EP
=
effluent
production
(kg
of
effluent/t
of
natural
matter
ensiled),
WSAO
=
weight
of
the
silo
assembly
after
the
mini-silos
opening
(kg),
WSAE
=
silo
weight
before
ensiling
and
DME
=
dry
matter
ensiled
(kg
of
forage
×
%
DM).
Dry
matter
recovery
(DMR)
was
calculated
as:
DMR
=
(FDM/IDM)
×
100
in
which
FDM
=
dry
matter
after
the
mini-silos
opening
(kg)
and
IDM
=
dry
matter
before
the
ensiling.
Changes
of
DM
content
were
calculated
as
the
difference
in
module
of
DM
percentage
at
the
ensiling
moment
and
the
DM
percentage
at
the
mini-silos
opening.
2.4.
Microbiology
Samples
(0.2
kg)
were
collected
on
day
60
after
the
ensiling
from
five
different
sites
of
all
mini-silos
and
homogenized
to
form
a
composite
sample.
Then,
subsamples
of
10
g
of
each
treatment
were
diluted
in
90
mL
of
sterilized
sodium
chloride
solution
(0.9%)
and
a
serial
dilution
was
performed
from
10−1until
10−6in
test
tubes.
The
microorganism
counting
was
performed
in
triplicate
from
each
dilution
using
culture
medium
of
MRS
agar
(De
Man,
Rogosa
and
Sharpe)
to
lactic-acid
bacteria,
nutrient
agar
to
aerobic
and
anaerobic
bacteria
(48
h
of
incubation
at
37 ◦C)
and
agar
PDA
(potato
dextrose
agar,
120
h
of
incubation
at
26 ◦C)
for
mold
and
yeast.
2.5.
Fermentative
profile
After
the
opening
of
mini-silos
(on
day
60),
sugarcane
silage
was
homogenized
and
one
sample
(500
g)
of
each
bucket
was
collected,
and
then
the
juice
from
samples
was
extracted
by
a
hydraulic
press.
Silage
juice
aliquots
(50
mL)
were
collected
to
determine
pH
using
a
digital
potentiometer.
Aliquots
of
2
mL
of
silage
juice
were
transferred
to
test
tubes
containing
1
mL
of
sulfuric
acid
(1N)
and
stored
at
−20 ◦C.
Ammonia
nitrogen
analysis
was
performed
by
colorimetric
method
described
by
Kulasek
(1972)
and
adapted
by
Foldager
(1977).

J.R.
Gandra
et
al.
/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
47
The
analyses
of
short-chain
fatty
acids,
ethanol
and
acid
lactic
concentration
were
carried
out
at
the
Department
of
Animal
Nutrition
and
Production,
School
of
Veterinary
Medicine
and
Animal
Science
−
University
of
São
Paulo,
Pirassununga,
Brazil,
according
to
the
methods
described
by
Rodrigues
et
al.
(2012).
Aliquots
of
1
mL
of
silage
juice
were
mixed
with
0.2
mL
formic
acid
in
amber
glass
bottles
and
stored
at
−18 ◦C
until
analysis.
Short-chain
fatty
acids
and
ethanol
concentrations
were
determined
by
a
gas
chromatograph
(Focus
GC,
Thermo
Fisher
Scientific
Inc.,
Waltham,
MA,
USA)
equipped
with
an
automatic
sample
injector
(model
AS-3000,
Thermo
Electron
Corporation®,
MA,
USA),
a
glass
packed
column
(2.0
m
×
1/5,
80/120Carbopack®B-DA/4%
Carbowax®20
M
phase)
and
a
flame
ionization
detector
set
at
270 ◦C.
The
chromatograph
oven
and
injector
temperatures
were
set
to
190 ◦C
and
220 ◦C,
respectively.
Hydrogen
was
used
as
the
carrier
gas
flowing
30
mL/min.
The
acid
lactic
concentration
was
measured
by
high
performance
liquid
chromatography
(LC-10ADVP
Shimadzu
HPLC
system,
Shimadzu
Inc.,
Kyoto,
Japan)
according
to
Ding
et
al.
(1995).
2.6.
Aerobic
stability
Silo
temperatures
were
obtained
using
an
infrared
digital
thermometer
every
8
h
during
7
days
after
the
silos
opening.
The
aerobic
stability
was
defined
as
the
period
(h)
in
which
silage
remained
stable
before
rising
more
than
1◦C
above
the
room
temperature
(Driehuis
et
al.,
2001).
In
addition,
during
the
aerobic
stability
assessment,
one
mini-silo
per
treatment
was
randomly
assigned
to
sample
collections
every
24
h,
to
determine
DM
(AOAC
2002,
#950.15)
and
pH
(Kung
et
al.,
1984).
2.7.
Statistical
analyses
Data
related
to
the
silage
chemical
composition,
in
vitro
degradation,
total
losses,
microbiology,
fermentative
profile,
and
aerobic
stability
period
were
analyzed
by
the
MIXED
procedure
of
SAS
(9.1
version,
SAS
Institute
Cary,
NC,
2004)
after
the
normality
of
residues
and
homogeneity
of
variances
tested
by
the
UNIVARIATE
procedure,
using
the
model
below:
Yi=
␮
+
Ai+
ei
in
which
Yi=
dependent
variable,
␮
=
overall
mean,
Ai=
fixed
effect
of
additive,
and
ei=
residual.
Satterthwaite
method
(ddfm
=
satterth)
was
applied
to
calculate
degrees
of
freedom.
Microbiological
data
were
log
transformed.
Data
of
DM
losses
and
pH
over
the
aerobic
stability
were
analyzed
as
repeated
measures
using
the
MIXED
procedure
(SAS
9.1,
SAS
Institute),
and
normality
of
residues
and
homogeneity
of
variances
were
also
checked
as
previously
described.
The
model
used
was:
Yij =
␮
+
Ai+
Tj+Ai×
Tj+
eij
in
which
Yij =
dependent
variable,
␮
=
overall
mean,
Ai=
fixed
effect
of
additive,
Tj=
random
effect
of
time
(hours),
Ai×
Tj=
interaction
effect
of
additive
by
time,
and
eij =
residual.
Differences
among
treatments
were
determined
using
orthogonal
contrasts:
C1
=
control
versus
Lb
and
Lb
+
Bs,
C2
=
control
versus
chitosan
and
C3
=
chitosan
versus
Lb
+
Bs.
Significance
level
was
set
at
0.05.
3.
Results
3.1.
Chemical
composition
and
in
vitro
degradation
Microbial
inoculants
increased
(P
=
0.001)
CP
content
of
sugarcane
silage
and
did
not
affect
in
vitro
degradation
of
DM
and
NDF
compared
to
CON
(Table
2).
Nevertheless,
CHI
increased
(P
≤
0.043)
DM,
OM,
CP
and
TDN
silage
content,
and
decreased
(P
=
0.033)
ash
content
compared
to
CON.
Chitosan
incorporation
improved
(P
=
0.029)
NDF
in
vitro
degradation.
In
addition,
incorporation
of
CHI
increased
(P
≤
0.042)
the
DM,
OM,
CP
and
TDN
content
and
decreased
(P
=
0.022)
the
ash
content
of
sugarcane
silage
compared
to
silages
treated
Lb
and
Lb
+
Bs.
3.2.
Fermentative
and
effluent
losses
Microbial
inoculants
decreased
(P
≤
0.004)
gas
(%)
and
effluent
losses
(kg/t
and
g/kg
DM),
and
consequently
decreased
(P
=
0.009)
total
losses
(Table
3).
However,
Lb
and
Lb
+
Bs
did
not
alter
the
DMR
(P
=
0.089).
Chitosan
incorporation
ameliorate
(P
≤
0.043)
the
gas,
effluent
and
total
losses
compared
to
CON,
increasing
(P
=
0.031)
the
DMR.
Chitosan
incorporation
showed
lower
(P
≤
0.035)
gas
and
total
losses,
but
increased
(P
=
0.001)
the
losses
by
effluent
compared
to
Lb
and
Lb
+
Bs.
Furthermore,
CHI
incorporation
showed
higher
(P
=
0.022)
DMR
than
microbial
inoculants.
3.3.
Microbiology
The
three
inoculants
increased
(P
≤
0.002)
the
number
of
lactic-acid
and
anaerobic
bacteria
and
decreased
(P
≤
0.009)
aerobic
bacteria
and
fungi
in
relation
to
CON
(Table
4).
Likewise,
CHI
incorporation
increased
(P
≤
0.003)
lactic-acid
and

48
J.R.
Gandra
et
al.
/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
Table
2
Effects
of
three
inoculants
on
chemical
composition
and
in
vitro
degradation
of
sugarcane
silage
(g/kg
DM,
otherwise
stated).
Item TreatmentaSEM Pb
CON
Lb
Lb
+
Bs
CHI
C1
C2
C3
Chemical
Dry
matter
(g/kg)
230
227
221
249
0.29
0.542
0.013
0.002
Organic
matter
941
944
945
953
0.09
0.714
0.003
0.042
Crude
protein
21.8
24.1
23.2
26.5
0.04
0.001
0.001
0.001
Neutral
detergent
fiber
636
642.4
659
621
0.79
0.803
0.504
0.120
Acid
detergent
fiber 343
349
340
338
0.39
0.349
0.561
0.651
Lignin
(sa) 72.1 61.0
61.2
61.1
0.55
0.128
0.197
0.998
Ash
59.3
55.4
55.5
47.0
0.09
0.574
0.033
0.022
Total
digestible
nutrientc529
548
553
571
0.22
0.774
0.043
0.032
In
vitro
degradation
Dry
matter
607
589
592
622
0.48
0.291
0.728
0.369
Neutral
detergent
fiber
(g/kg
NDF)
623
635
629
669
0.37
0.350
0.029
0.496
aControl
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010
cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
bOrthogonal
contrasts:
C1:
control
versus
Lb
and
Lb
+
Bs,
C2:
control
versus
chitosan,
and
C3:
Lb
and
Lb
+
Bs
versus
chitosan.
cAccording
to
NRC
(2000)
model.
Table
3
Effects
of
three
inoculants
on
fermentative
and
effluent
losses
of
sugarcane
silage
(g/kg
DM,
otherwise
stated).
Item TreatmentaSEM Pb
CON
Lb
Lb
+
Bs
CHI
C1
C2
C3
Losses
Gas
losses
(%
natural
matter)
2.99
2.26
2.72
2.24
0.09
0.001
0.001
0.981
Gas
losses
264
207
201
119
0.88
0.074
0.001
0.035
Effluent
losses
(kg/t
natural
matter)
38.9
29.9
30.5
30.9
0.99
0.003
0.043
0.760
Effluent
losses
31.8
24.3
23.0
27.8
0.08
0.004
0.001
0.001
Total
losses
287
226
239
147
0.87
0.009
0.001
0.001
Dry
matter
recovery
736
799
793
880
0.88
0.089
0.031
0.022
aControl
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
bOrthogonal
contrasts:
C1:
control
versus
Lb
and
Lb
+
Bs,
C2:
control
versus
chitosan,
and
C3:
Lb
and
Lb
+
Bs
versus
chitosan.
Table
4
Effects
of
three
inoculants
on
microbiology
of
sugarcane
silage.
Item TreatmentaSEM Pb
CON
Lb
Lb
+
Bs
CHI
C1
C2
C3
Bacteria
(log10 cfu/g
fresh
silage)
Lactic-acid
4.29
5.40
5.38
6.19
0.10
0.001
0.001
0.001
Aerobic
5.38
4.56
4.30
4.53
0.07
0.004
0.031
0.435
Anaerobic
4.25
4.92
4.97
5.77
0.11
0.002
0.003
0.760
Total
5.74
5.08
5.50
5.97
0.09
0.344
0.671
0.781
Fungi
(log10/g
fresh
silage)
6.75
5.09
4.72
5.02
0.12
0.009
0.031
0.122
aControl
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
bOrthogonal
contrasts:
C1:
control
versus
Lb
and
Lb
+
Bs,
C2:
control
versus
chitosan,
and
C3:
Lb
and
Lb
+
Bs
versus
chitosan.
anaerobic
bacteria,
and
decreased
(P
=
0.031)
aerobic
bacteria
and
fungi
compared
to
CON.
Furthermore,
CHI
incorporation
showed
higher
(P
=
0.001)
number
of
lactic-acid
bacteria
than
Lb
and
Lb
+
Bs.
3.4.
Fermentative
profile
All
inoculants
decreased
(P
=
0.031)
pH
and
increased
(P
=
0.002)
ammonia
nitrogen
compared
to
CON
(Table
5).
Sugarcane
silages
treated
with
inoculants
showed
higher
(P
≤
0.034)
concentrations
of
acetate,
butyrate
and
lactic
acid
than
CON.
In
addition,
inoculants
decreased
(P
≤
0.004)
ethanol
concentrations
compared
to
CON.
Although
CHI
incorporation
showed
lower
(P
≤
0.034)
pH
and
ethanol
concentration,
and
higher
(P
=
0.001)
acetate,
butyrate
and
lactic
acid
concentrations
than
CON,
CHI
strongly
increased
(P
=
0.001,
294.9%)
the
NH3-N
concentration
in
mini-silos.
Chitosan
incorporation
also
showed
higher
(P
≤
0.003)
NH3-N
and
butyrate
concentrations
compared
to
the
silages
treated
with
Lb
and
Lb
+
Bs.
Furthermore,
CHI
incorporation
demonstrated
lower
(P
=
0.002)
ethanol
concentration
in
silage
than
Lb
and
Lb
+
Bs.

J.R.
Gandra
et
al.
/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
49
Table
5
Effects
of
three
inoculants
on
fermentative
profile
of
sugarcane
silage.
Item TreatmentaSEM Pb
CON
Lb
Lb
+
Bs
CHI
C1
C2
C3
pH
4.22
3.33
3.34
3.32
0.01
0.031
0.034
0.981
NH3-N
(mg/dL)
5.47
5.68
7.15
16.13
0.79
0.002
0.001
0.002
Acetate
(g/kg
DM)
5.24
5.07
6.73
8.02
0.01
0.004
0.001
0.345
Propionate
(g/kg
DM)
1.2
0.9
3.1
1.2
0.03
0.872
0.563
0.785
Butyrate
(g/kg
DM) 4.2 7.5 3.1 6.3 0.03 0.034 0.001 0.003
Ethanol
(g/kg
DM) 33.9 23.7 20.8 11.3
0.14
0.004
0.001
0.002
Lactic
acid
(g/kg
DM) 33.4
50.3
56.5
67.8
0.23
0.003
0.001
0.289
aControl
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
bOrthogonal
contrasts:
C1:
control
versus
Lb
and
Lb
+
Bs,
C2:
control
versus
chitosan,
and
C3:
Lb
and
Lb
+
Bs
versus
chitosan.
Table
6
Effects
of
three
inoculants
on
7-day
aerobic
stability
of
sugarcane
silage.
Item TreatmentaSEM Pb
CON
Lb
Lb
+
Bs
CHI
C1
C2
C3
Aerobic
stability
(h) 32.00 43.20 41.60
49.6
2.48
0.026
0.013
0.224
pH
5.86
5.74
5.53
6.08
0.11
0.233
0.132
0.045
Dry
matter
losses
(g/kg)
311
293
254
299
0.38
0.123
0.654
0.034
aControl
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
bOrthogonal
contrasts:
C1:
control
versus
Lb
and
Lb
+
Bs,
C2:
control
versus
chitosan,
and
C3:
Lb
and
Lb
+
Bs
versus
chitosan.
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
24
48
72
96 12
0 14
4 168
pH
Aerobic stability
(h)
CON Lb
Bs+Lb
CHI
Treatment = 0.034
Time = 0.022
Treat
ment*ti
me = 0.
050
SEM = 0.11
Fig.
1.
pH
values
of
sugarcane
silage
treated
with
three
inoculants
during
aerobic
stability
assessment.
Control
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
3.5.
Aerobic
stability
Inoculants
decreased
(P
=
0.010)
silo
stability
temperatures
(Table
6).
Moreover,
the
period
of
aerobic
stability
was
higher
(P
=
0.026)
in
silages
treated
with
inoculants
compared
to
CON.
Chitosan
incorporation
decreased
(P
=
0.004)
the
temperature
of
stability
in
mini-silos
and
prolonged
(P
=
0.013)
the
period
of
aerobic
stability
of
sugarcane
silage.
Chitosan
incorporation
showed
higher
(P
≤
0.045)
pH
and
DM
losses
than
silages
treated
Lb
and
Lb
+
Bs.
The
average
pH
values
after
24
h
of
silos
opening
was
3–3.5
and
progressively
increased
until
6.5–7.2
(Fig.
1).
Dry
matter
content
of
sugarcane
silage
showed
effects
of
time,
treatment
and
treatment
by
time
interaction
(Fig.
2).
4.
Discussion
Despite
the
potential
of
sugarcane
as
a
forage
source
along
drought
periods,
sugarcane
silages
are
characterized
by
high
DM
losses,
ethanol
production
and
yeast
activity.
The
current
experiment
showed
that
CHI
ameliorated
DM
losses,
and
consequently
increased
the
DM
content
of
sugarcane
silage.
However,
the
increased
DM
content
of
sugarcane
silage
may
be
related
to
the
DM
content
of
CHI
added
to
the
sugarcane,
which
may
also
influenced
the
effluent
and
fermentation
losses.
The
absence
of
L.
bucheneri
effects
on
DM
sugarcane
silage
content
have
been
reported
in
several
studies
(Ávila
et
al.,
2009;
Santos
et
al.,
2009).
In
addition,
B.
subtilis
did
not
affect
DM
content
of
sugarcane
silage
(Basso
et
al.,
2012).

50
J.R.
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Science
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44–52
180
230
280
330
380
24
48
72
96 12
0 14
4 168
Dry Matter (g/kg)
Aerobic stability
(h)
CON Lb Bs+Lb CHI
Treatment = 0.022
Time
= 0.043
Treat
ment*ti
me = 0
.034
SEM = 0.38
Fig.
2.
Dry
matter
content
of
sugarcane
silage
treated
with
three
inoculants
during
the
aerobic
stability
assessment.
Control
(CON);
Lactobacillus
buchneri
(Lb),
addition
of
Lb
at
2.6
×
1010 cfu/g;
Lactobacillus
buchneri
and
Bacillus
subtilis
(Lb
+
Bs),
addition
of
Lb
at
2.6
×
1010 cfu/g
and
Bs
at
1
×
109cfu/g;
and
Chitosan
(CHI),
addition
of
1%
of
CHI
on
wet
basis
of
sugarcane
ensiled.
Incorporation
of
CHI
increased
the
DM
content
of
sugarcane
silage
due
to
the
lower
gas
and
effluent
losses,
and
higher
DMR
than
CON.
The
effluent
release
during
the
ensiling
process
represents
a
loss
of
DM
and
a
reduction
of
the
nutritional
value
of
silage
(Gabrehanna
et
al.,
2014).
Despite
all
additives
diminished
the
effluent
losses,
the
chemical
composition
of
sugarcane
silage
was
not
extensively
altered
by
microbial
inoculants.
The
DM
losses
of
sugarcane
silage
is
mainly
from
CO2production
by
the
fermentation
pathway
which
yields
ethanol,
that
may
represents
up
to
54%
of
total
DM
losses
of
sugarcane
silage
(Pedroso
et
al.,
2005).
To
our
knowledge,
there
is
no
study
evaluating
CHI
as
additive
in
silages.
Interestingly,
CHI
decreased
the
silage
ethanol
concentrations
and
fungi
activity.
The
ethanol
production
in
sugarcane
silage
is
raised
from
fermentation
of
water-soluble
sugars
catalyzed
by
yeasts
(Kung
et
al.,
2003).
The
antifungal
effect
of
CHI
is
related
to
the
capacity
of
suppressing
sporulation
and
spore
germination
(Hernandez-Lauzardo
et
al.,
2008),
and
may
be
even
greater
in
sugarcane
silages
compared
to
other
crops,
because
CHI
antifungal
activity
is
increased
at
lower
pH
values
(Roller
and
Covill,
1992).
Finally,
we
highlight
that
the
association
of
Lb
+
Bs
showed
the
lowest
value
of
fungi
concentration
in
silage.
Chitarra
et
al.
(2003)
reported
that
strains
of
B.
subtilis
are
able
to
produce
antifungal
compounds
which
block
the
spore
germination;
and
Basso
et
al.
(2012)
reported
a
linear
decrease
of
spoilage
microorganisms
concentration
in
maize
silage
treated
B.
subtilis,
due
to
its
antifungal
activity
(Todovora
and
Kozhuharova,
2009).
The
current
study
showed
an
increase
of
NDF
degradation
when
adding
CHI
to
sugarcane
silage,
but
the
reason
is
not
clear.
Several
studies
reported
stronger
antibacterial
activity
against
Gram-negative
bacteria
than
Gram-positive
bacteria
(Chung
et
al.,
2004;
No
et
al.,
2002).
Lactic
acid
bacteria
are
gram-positive
and
their
metabolic
activity
is
important
to
silage
quality
(Duniere
et
al.,
2013).
In
general,
lactic
acid
is
the
goal
of
end
product
of
fermentation
in
the
silo,
due
to
lactic
acid
be
a
stronger
acid
(pKa3.86)
than
acetic
(pKa4.76;
Muck,
2010).
High
concentrations
of
lactic
acid
can
rapidly
drop
the
silage
pH,
and
reduce
the
activity
of
spoilage
microorganisms
and
production
of
butyric
acid.
Weinberg
et
al.
(2007)
ensiled
corn
and
wheat
forages
with
lactic
acid
bacteria
and
reported
improve
of
the
in
vitro
NDF
degradation
of
forages.
In
addition,
Nsereko
et
al.
(2008)
demonstrated
that
several
lactic
acid
bacteria
produce
ferulic
acid
esterase
in
the
silo,
which
has
the
potential
to
improve
fiber
degradation.
All
additives
decreased
the
pH
of
sugarcane
silage,
because
they
increased
the
acetate,
propionate
and
lactic
organic
acids
in
mini-silos.
Kleinschmidt
and
Kung
(2006)
evaluated
43
studies
that
inoculated
L.
buchneri
in
different
forages
to
ensilage
(corn,
sorghum,
barley
and
grasses)
and
reported
that
microbial
inoculant
decreased
pH
and
improved
the
aerobic
stability
of
silages.
Data
of
pH
values
of
CHI
and
Bs
as
additives
for
silages
are
scarce
in
literature.
Meanwhile
inoculants
decreased
the
silage
pH,
they
increased
the
silage
ammonia
nitrogen
concentration.
Increased
concentrations
of
ammonia,
as
well
as,
the
increased
butyrate
production,
are
not
expected
once
ammonia
is
related
to
excessive
protein
breakdown
caused
by
a
slow
drop
in
pH,
and
the
butyrate
production
is
related
to
yeast
activity.
The
highest
values
of
ammonia
concentrations
when
CHI
was
incorporated
to
the
silages
may
be
related
to
the
CP
of
chitin
(precursor
of
CHI)
that
may
reach
10.8%
(Manni
et
al.,
2010).
In
addition,
CHI
is
a
weak
base
soluble
in
aqueous
acid
solution
below
its
pKa(6.3),
in
which
glucosamine
units
(−NH2)
are
converted
into
soluble
protonated
form
(−NH+3;
Goy
et
al.,
2009).
Heinl
et
al.
(2012)
reported
that
Lb
metabolism
can
turn
lactic
acid
into
acetic
acid
under
an
anaerobic
environment.
Despite
L.
buchneri
have
been
reported
to
strongly
decrease
the
pH
and
increase
the
acetate
concentrations
in
crimped
wheat
grains
(Adesogan
et
al.,
2003),
the
acetate
production
values
were
increased
when
Lb
+
Bs
were
added
or
when
CHI
was
incorporated
into
the
sugarcane.
In
contrast,
Basso
et
al.
(2012)
did
not
observe
differences
in
production
of
acetic
and
lactic
acid
when
corn
silage
was
inoculated
with
B.
subtilis.
The
acetate
is
the
major
organic
acid
to
prevent
the
growth
of
spoilage
microorganisms
(Danner
et
al.,
2003;
Kleinschmidt
and
Kung,
2006).
Aerobic
deterioration
occurs
when
fermentation
products
of
the
silo
(i.e.
lactic
acid)
became
substrate
to
microbial
growth
(Pahlow
et
al.,
2003).
Microbial
organisms
oxidize
acids
and
water-soluble
carbohydrates
to
CO2and
water,
resulting
in
an
increase
of
silo
temperature
that
rises
above
the
ambient
temperature
(Ranjit
and
Kung,
2000).
The
current
experiment
showed
that
both
microbial
inoculants
and
CHI
were
effective
to
decrease
the
temperature
in
which
silo
reached
the
stability

J.R.
Gandra
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/
Animal
Feed
Science
and
Technology
214
(2016)
44–52
51
and
increased
the
length
of
aerobic
stability
period.
Chitosan
treated
silages
had
higher
pH
and
DM
losses
than
microbial
inoculants.
However,
the
higher
DM
losses
of
CHI
may
be
related
to
its
chemical
composition,
since
the
incorporation
of
CHI
increased
the
DM
and
TDN
content
of
sugarcane
silage.
If
more
substrate
is
available
to
microbial
growth,
higher
DM
loss
occurs.
In
a
meta-analysis,
Kleinschmidt
and
Kung
(2006)
reported
that
the
length
period
of
aerobic
stability
was
increased
when
corn
and
small-grain
silages
were
treated
with
L.
buchneri.
Besides
the
alterations
in
organic
acids
production,
L.
buchneri
may
produce
antimicrobial
substances
that
are
responsible
to
enhance
aerobic
stability,
including
bacteriocins
(Yildirim,
2001).
Finally,
CHI
is
the
second
most
abundant
biopolymer
in
nature,
is
a
byproduct
of
marine
bioprocessing
plants,
and
has
proved
environmentally
attractive
and
economically
feasible
(Avanitoyannis
and
Kassaveti,
2008).
Furthermore,
CHI
have
shown
positive
effects
on
nutrient
total
tract
digestion,
ruminal
fermentation
and
milk
yield
in
cattle
(Araújo
et
al.,
2015;
Vendramini
et
al.,
2016;
Paiva
et
al.,
2016).
Thus,
adding
CHI
to
silages
may
have
also
positive
effects
besides
those
found
during
the
ensiling
process.
5.
Conclusion
Both
CHI
and
microbial
inoculants
improved
the
chemical
composition
of
sugarcane
silage.
However,
CHI
incorporation
to
the
sugarcane
showed
higher
DM
and
TDN
concentrations
compared
to
inoculants
treatment.
In
addition,
CHI
increased
in
vitro
degradation
of
NDF,
which
was
not
observed
in
silages
treated
with
microbial
inoculants.
Moreover,
CHI
showed
higher
concentrations
of
lactic
acid
bacteria
and
lower
ethanol
concentration
than
silages
treated
with
microbial
inoculants.
Furthermore,
CHI
improved
the
aerobic
stability
compared
to
CON.
Chitosan
may
be
an
alternative
to
microbial
inoculants
used
in
sugarcane
ensiling.
Conflicts
of
interest
The
authors
declare
no
conflicts
of
interest
related
to
this
publication
and
there
has
been
no
financial
support
to
the
present
study
that
could
have
influenced
its
outcome.
Acknowledgment
Authors
acknowledge
BIOMART
(Biocampo,
Nutricao
Animal
Imp.
e
Exp.
LTDA,
Presidente
Prudente,
Brazil)
for
microbial
inoculants
donation.
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