Can Fermentative and Nutritional Quality of Panicum maximum Silage be Improved with
the Use of Corn Silage Juice as a Bioinoculant?
Keuven dos Santos Nascimento1*†, Ricardo Loiola Edvan2†, Santos Jeovanny Aguilera Vallecillo2†, Romilda Rodrigues do Nascimento2†, Dhiéssica Morgana Alves Barros2†, Mirella Almeida da Silva3†, Marcos Jácome de Araújo2† and Leilson Rocha Bezerra3†
1São Paulo State
University (Unesp), São Paulo, Brazil
2Federal University
of Piauí, Piauí, Brazil
3Federal University
of Campina Grande, Paraíba, Brazil
*For correspondence: keuvensantos03@gmail.com
†Contributed equally to this work and are co-first
authors
Received 11 May 2021; Accepted 18 June 2021; Published 18
September 2021
Abstract
The aim of this study was to
evaluate the quality of Paredão grass silage (Panicum
maximum Jacq.) with fermented juice produced from corn silage in different
fermentation periods, as bioinoculant, through the characteristics: gas and
effluent losses, dry matter recovery, chemical composition, microbiological
profile and aerobic stability. The treatments consisted of fermented corn
silage juices obtained in different fermentation periods, which were: 0, 5, 10,
15 and 30 days of fermentation, and were used as a bioinoculant additive in Paredão grass silage. The lowest effluent losses were
observed in silages containing corn silage juice fermented for 10 days, while
the highest dry matter recovery values were observed in silages containing corn
silage juice fermented for 30 days. The highest values of crude protein were
observed in silages containing corn silage juice fermented for 10 days. The
highest content of lactic acid (53 ± 2.2 g/kg DM) was observed in the Paredão grass silage containing corn silage juice fermented
for 10 days. The inclusion of corn silage juice fermented for 10 days as
bioinoculant improves the quality of Paredão grass
silage, as it presented greater recovery of the silage dry matter, higher crude
protein content and greater amount of beneficial acids. ©
2021 Friends Science Publishers
Keywords: Additive; Fermentation; Fermented juices; Microorganisms; Paredão grass
Introduction
The Panicum maximum Jacq. grass presents easy cultivation, in addition
to high production and good nutritional value for animals (Borjas‐Ventura
et al. 2019). Thus, the silage
production with this tropical grass allows the storage of a large volume of
forage mass which increases food security for the herd.
The production of silage using
tropical grasses is subjected to high losses, because during the harvest
despite having good nutritional value, they have a high moisture content, high
buffering capacity and low concentrations of soluble carbohydrates, which
results in a low-quality silage (Bureenok et al. 2011). In addition, plants
ensiled with high humidity favor undesirable fermentations, reducing the
nutritional value of the silage. The production of quality silage requires the
presence of lactic acid epiphytic bacteria (LAB) and water-soluble carbohydrate
(WSC) to produce sufficient lactic acid for rapid pH reduction (Kung et al.
2018).
The inoculants used in the
silages are mainly composed of homofermentative lactic bacteria and aim to
increase the initial population available in the forage to accelerate the
process of organic acids production, and a consequent drop in the pH of the
silage (Ludovico et al. 2014; Moraes et al.
2017; Cardoso et al. 2019; Costa et al. 2021). This also improves the
aerobic stability of the silage after opening the silo and beneficial
microflora (Silva et al. 2020).
Currently the use of natural additives has been preferred to improve the
fermentative quality of grass silages; however, the use of this technique is
still in vogue.
The fermented juice of epiphytic
lactic acid bacteria has been recommended as additive for tropical grass silage
(Bureenok et al.
2005a, b). However, this additive can be ineffective because of the low LAB and
low WSC content, especially when coming from plants that already have these
characteristics in their natural composition (Bureenok
et al. 2011). The corn silage stands
out in the production of LAB, mainly due to the chemical composition of the
plant that is beneficial for the action of LAB. When used as additive, the
bioinoculant of this silage can improve the fermentation of the ensiled mass,
reducing losses and preserving the nutritional value (Silva et al. 2020). Thus, using fermented corn
silage juice in grass silages may allow a greater conservation potential of
silages inoculated with this product.
The fermented corn silage juice
can be easily produced in the farm (Hang et
al. 2003; Silva et al. 2020), but
there is no information on its production made in silages of different storage
periods, since these silages present different types of microorganisms at
different fermentation stages in terms of days. It is expected that the corn
silage, after fermentative stabilization may produce a fermented juice of
better quality to be used as bioinoculant in grass silages. Therefore, it was
hypothesized that a longer storage period of corn (Zea mays L.) silage for the production of fermented juice as
bioinoculant, will provide better quality of Paredão
grass (P. maximum) silage. Thus, the
objective in this study was to evaluate the quality of P. maximum silage containing different fermented corn silage juices
as bioinoculants through the analyses of gas and effluent losses, dry matter
recovery, chemical composition, microbiological profile and aerobic stability
of the silage.
Materials and Methods
All the methods used for the
cultivation and collection of the vegetable material cultivated were carried
out in compliance with relevant institutional, national and international
guidelines and legislation.
The research was performed in
the city of Bom Jesus, Piauí, Brazil. The city is
located in the south of the state of Piauí, in the
microregion of Alto-Médio Gurguéia,
(09°04′S, 44°21′W, 277 m a.s.l.), with
climatic classification BSh, with rainy summer and
dry winter rains according to the Köppen
classification of 1936, described by Alvares et al. (2013). The region presents an
average minimum temperature of 18ºC, average maximum temperature of 36ºC and
average annual rainfall of 900 mm. A completely randomized design was adopted,
with five replications. The treatments consisted of five production periods of
fermented corn silage juice to be used as bioinoculant, which were 0, 5, 10, 15
and 30 days of silage storage.
In the experimental field of
corn crop (BAS hybrid) and Paredão grass, the soil
was representatively sampled at a depth of 0˗20 cm. After sampling, the
samples were sent to the Soil Analysis Center Laboratory of the Professora Cinobelina Elvas Campus (CPCE) of the Federal University of Piauí (UFPI).
The soil was
classified as Dystrophic Yellow Latosol, with a clay texture (clay, 257 g/kg;
mud, 34 g/kg, sand, 709 g/kg) and had the following characteristics: 5.3 pH in
water;, 15.7 mg/dm3 of phosphorus; 116.9 mg/dm3 of
potassium; 1.7 cmol c/dm3 of calcium; 0.4 cmol c/dm3 of magnesium; 0.0 cmol
c/dm3 of aluminum; 0.1 cmol c/dm3
of hydrogen + aluminum; 4.3 cmol c/ dm3 of
sum of bases; 7.5 cmol c/dm3 of cation
exchange capacity at pH 7.0; 660 g/kg of base saturation; and 0.0 g/kg of
aluminum saturation.
The Paredão grass
plants were sown in January 2016, under irrigated system. The cultivation of
corn was performed in March 2017 in the same experimental field. In both crops,
40 kg/ha of phosphorus (simple superphosphate, 180 g/kg of P2O5,
110 g/kg of S and 180 g/kg of Ca) were applied and 30 days after planting
(corn) and right after uniform cutting of Paredão
grass, 120 kg/ha of nitrogen (urea, 450 g/kg of N) were applied. It was not
necessary to correct the soil or fertilize with potassium, because according to
the soil analysis, the chemical values were sufficient for both species (Martha Júnior et al. 2007).
A stationary silage machine regulated to cut
the material into particles of 2 to 3 cm was used to chop the material for the
production of the Paredão grass silage. After
chopping, the bioinoculant was added in the material through spraying, applying
the equivalent of 500 mL/ton of fresh forage.
Two types of experimental silos were
produced, silos in plastic buckets with a storage capacity of 3.0 kg of silage
and experimental PVC silos (100 mm in diameter) with a length of 50 cm and a
capacity of 2.5 kg of silage. Both were filled with silage from the same
processing, and at the same time and place of filling of the silos, to allow
homogeneity of the ensiled material. The aerobic stability was analyzed in the
silages from the experimental PVC silos, while the analyses of chemical
composition, losses through effluents and gas, organic acids content (lactic
acid, acetic acid, propionic acid and butyric acid) and microbial population
count were performed in the silage from the 3 kg experimental silos. The Paredão grass silages already inoculated were stored with
an average density of 500 kg m˗3. In order to characterize the
silage, both the fresh forage and the product of the silage were analyzed 120
days after ensiling. The analyses were performed at the Microbiology and Animal
Nutrition Laboratories of CPCE/UFPI, in the city of Bom Jesus, Piauí, Brazil.
Production
of fermented corn silage juice
Fig 1: Procedures to produce and use the
bioinoculant
For the production of fermented
corn silage juice, the plants of corn crop were cut and chopped when they
reached R4 stage (dough). The silage was produced following all procedures to
obtain quality silage. Samples for the production of fermented corn silage
juice were obtained according to periods of 0, 5, 10, 15 and 30 days of
fermentation, always removing from the same silo. All ensiling procedures were
also performed in the silage stored for 0 days (Fig. 1).
For the production of fermented
corn silage juice, at the end of each fermentative period (0, 5, 10, 15 and 30
days) 500 g of silage were collected and processed in a blender, then the
collected liquid was strained through a 1.0-mm sieve, and added 500 g of corn
glucose and distilled water until filling to 2 L of mixture (Silva et al. 2020). The resulting mixture was
stored in plastic bottles for 30 days.
Determination of losses through effluents, dry matter and gas
To obtain the values of losses
through gas and effluents, 1.0 kg of sand was deposited at the bottom of each
experimental silo of 3 kg of capacity, separated from the forage by a layer of
cotton fabric, making it possible to measure the amount of effluent retained.
The losses through gas, effluents and the dry matter recovery were obtained
according to equations described by Zanine et al.
(2010). Based on the weight difference of the dry forage mass, the gas losses
were obtained using the following equation: G = (iFW-fFW)
/ (iFM × iDM) × 100, where:
G: gas losses (% DM); iFW: weight of the full silo
after closing (kg); fFW: weight of the full silo
after opening (kg); iFM: forage mass after closing
(kg); iDM: forage dry matter content after closing.
The effluent losses were calculated through the following equation,
based on the difference in weight of the sand and related to the fresh forage
mass after closing. E = [(fEW-St)-(iEW-St)]/iFM×100, where: E: effluent production (kg/ton of
silage); iEW: weight of empty bucket + weight of sand
after closing (kg); fEW: weight of empty silo +
weight of sand after opening (kg); St: silo tare; iFM:
forage mass after closing (kg). The following equation was used to estimate the
dry matter recovery: DMR (g kg˗1 of DM) = [(foGM×foDM)/(iSM×siDM)]×100, where: DMR (g kg˗1 of DM):
DM recovery as percentage; foGM: forage green mass
(kg) during ensiling; foDM: forage DM (%) during
ensiling; iSM: silage mass (kg) before opening the
silos; siDM: Silage DM (%) after opening of the
silos.
Fresh samples of Paredão grass and corn (Table 1) and silages were properly
stored and frozen in a freezer (-20°C) for further analysis. The samples were
stored in paper bags, identified, weighed and pre-dried in a forced air
circulation oven at 55ºC for 72 h and later, ground in a Willey knife mill with
a 1.0-mm sieve.
To determine the chemical
composition the analyses were performed in triplicate, following the AOAC
recommendations (1990) regarding the contents of dry matter (method 967.03),
crude protein (method 981.10), ether extract (method 920.29) and ash (method
942.05). The determination of neutral detergent fiber (NDF) and acid detergent
fiber (FDA) was carried out
Fig 2: Microorganisms count values in fermented corn
silage juice of different fermentation periods
LAB: lactic acid bacteria
¥FM: fresh material
according to the methods described by Van Soest et al.
(1991) with the modification proposed by Senger et al. Table 1: Chemical
composition of Paredão grass and corn before ensiling
Chemical composition |
Ingredients |
|
Paredão grass |
Corn |
|
Dry matter (g kg-1 of feed) |
262 |
332 |
Crude protein (g kg˗1 of DM) |
879 |
794 |
Neutral detergent fiber (g kg˗1 of
DM) |
621 |
562 |
Acid detergent fiber (g kg˗1 of DM) |
- |
278 |
Ashes (g kg˗1 of DM) |
61.0 |
45.0 |
pH |
6.10 |
5.20 |
Ether extract (g kg˗1 of DM) |
- |
316 |
Nin-fiber carbohydrates (g kg˗1 of
DM) |
- |
322 |
(2008). The temperature of the autoclave was adjusted to 110°C for 40
min. Determination of non-fibrous carbohydrates (NFC) was done according to
Weiss (1999): NFC (g/kg)=1000-(NDF+PB+EE+ash).
To determine volatile fatty acids (lactic, acetic, propionic and
butyric), 10g of each silage was weighed in triplicate and added to 90 mL of
distilled water, this material was later homogenized and filtered through a fine
mesh sieve covered with gauze. From the solution, a 10 mL sample of the
filtrate was taken and placed in tubes for centrifugation, with 2.0 mL of
metaphosphoric acid (3M) being added, and then the solution was centrifuged for
15 min at 13,000 × g. After this process, the supernatant was transferred to
Eppendorf tubes sealed, identified and frozen for analysis of organic acid by
high performance liquid chromatography (HPLC) (Vrátný
and Mudřík 1985).
For the enumeration of microbial
groups of lactic acid bacteria (LAB), enterobacteria, molds and yeasts,
homogenization of replicates of each treatment was performed with 90 mL of
distilled water added to the samples, in order to obtain a dilution of 10˗1.
Dilutions were performed in order to obtain variations from 10˗1
to 10˗9.
Plating was
performed in sterile Petri dishes (duplicate). For the quantification of
microbial populations selective culture media were used for each microbial
group, as follows: Agar Rogosa (Difco),
for enumeration of LABs after 48 h of incubation at 37°C; Bright Green Agar
(Disc), for enumeration of enterobacteria (ENT) after 24 h of incubation at
35°C; and Potato Dextrose Agar, which was added with 1 kg of 0.01 g/kg tartaric
acid, after sterilization, to count molds and yeasts (M and Y) after incubation
for 3–7 days at room temperature. The incubations were performed in a B.O.D.
oven. The plates petri dish considered susceptible to counting were those in
which there were values between 30 and 300 CFU (colony forming units). The
differentiation between yeasts and molds was given by the physical structure of
the colonies, which was visually perceptible, since yeasts are unicellular and
molds multicellular (González and Rodríguez 2003). The enumeration of the microbial
groups present in the fermented juices of corn silage in each fermentative
period was carried out as described above (Fig. 2).
Table 2: Losses of Paredão grass silage with the
inclusion of fermented corn silage juice of different fermentation periods
Variables |
Fermented
corn silage juice (days) |
Mean |
SEM£ |
P-value |
||||
0 |
5 |
10 |
15 |
30 |
||||
E† (kg t˗1 AF) |
141b |
67a |
11b |
59a |
66a |
437 |
54 |
<
001* |
G‡ (% DM) |
130 |
162 |
096 |
111 |
066 |
113 |
023 |
014ns |
DMR§
(% DM) |
721ab |
604b |
594b |
599b |
85a |
674 |
346 |
<
001* |
Means followed by different letters in the row are statistically
different by the Tukey’s test with P < 0.05; *significant at P
< 0.05; nsnon significant at P > 0.05; †E:
Loss through effluents kg/t on as fed basis (AF); ‡G: Loss through gas,
percentage (%) on dry matter basis (DM); §DMR: Dry Matter Recovery
in percentage (%); £SEM: standard error of the mean
Aerobic stability and pH
To determine aerobic stability,
the silages were taken to a closed room with temperature control, where a
suspended digital thermometer measured the temperature constantly. The aerobic
stability of the silage surface layer was determined after opening and
unpacking the top layer of the silo. The material was exposed to air for a
period of 0 to 48 h.
After opening the silo in the determined time, the surface temperature
and the mass of the silage were checked every hour, over a period of 12 h (0,
12, 24, 36 and 48 h) (Silva et al.
2020). The surface temperature of the silage was measured by a digital infrared
thermometer and the internal temperature of the forage mass by a digital
immersion thermometer, inserted in the center of the silo (at a depth of 10
cm). Aerobic stability was determined according to Taylor and Kung Jr. (2002),
to considers the time necessary for the silage, after exposure to air, an
increase of 2°C above room temperature, thus considering the break in stability
aerobic use of the material.
The determination of pH in distilled water was carried out in duplicate.
Being determined from the addition of 100 mL of water in samples of 25 g of
ensiled material for each treatment. After 1 h, a reading was taken with a
bench microprocessor pH meter (Bolsen et al. 1992); Marconi – Piracicaba, São
Paulo, Brasil). The data were collected during the
periods of exposure of the silage to air at 0, 12, 24, 36 and 48 h.
The data were subjected to an
analysis of variance for determine the level of P < 0.05. The data related
to the losses through effluents, gases and dry matter, chemical composition and
volatile fatty acids of the Paredão grass with
fermented corn silage juices of different fermentation periods, were analyzed
by the Tukey’s Test, through the iterative process of the SAS® PROC NLIN
implementation of the Marquardt algorithm. The statistical model used was: Zij=μ+Fi+εi, where Z represents the observed value,
and Fi the fixed effect of the fermentation period i (i = 0, 5, 10, 15, 30
days).
The aerobic stability data of Paredão grass silage was performed for the analysis of
interaction between the fermented juices from corn silage of different
fermentation periods (0, 5, 10, 15 and 30 days) × air exposure times of the Paredão grass silage (0, 12, 24, 36 and 48 h). The
statistical model Zij=μ+Fi+Ej+(F×E)ij+εij was used, where Z
represents the observed value, Fi the fixed effect of the fermentation period i
(i = 0, 5, 10, 15, 30 days), Ej the fixed effect of
the air exposure times j (j = 0, 12, 24, 36, 48 h), and (F × E)ij the interaction between fermentation periods and the air
exposure times. A regression analysis (linear and quadratic) was performed with
the data of air exposure time of the Paredão grass
silage using the MIXED and REG procedures implemented in the statistical
software SAS® (version 9.1. Cary, NC, USA).
Results
Losses through effluents, dry matter and gas
There was a significant effect
of the treatments (P < 0.01) on
effluent losses and dry matter recovery (DMR). The highest losses through
effluents were observed in silages containing corn silage juice fermented for
5, 15 and 30 days (Table 2) showing mean values of 67, 59 and 66 kg/t AF,
respectively. However, the silage containing the bioinoculant fermented for 10
days presented the lowest effluent loss values (11±5.4 kg/t AF). No effect (P = 0.14) was observed on losses through
gas of Paredão grass silage, averaging 1.13±0.23 g/kg
DM. The highest DMR value was observed in the silages containing corn
silage juice fermented for 30 days, presenting 85±3.46 g/kg DM, followed by the
silages treated with corn silage juice fermented for 0 days, which presented
72±3.46 g/kg DM. The silages inoculated with corn silage juice fermented for 5,
10 and 15 days presented DMR values statistically not different.
Regarding the chemical composition of Paredão grass silage containing fermented corn silage
juice, only the crude protein
Fig 3: Microorganisms count values in Paredão grass silage with inclusion of fermented corn
silage juice of different fermentation periods
LAB: lactic acid bacteria ¥FM: fresh material
Table 3: Chemical composition of Paredão grass silage with the inclusion of fermented corn
silage juice of different fermentation periods
Variables |
Fermented corn silage juice (days) |
Mean |
SEM¤ |
P-value |
||||
0 |
5 |
10 |
15 |
30 |
||||
DM†, |
188 |
174 |
193 |
183 |
202 |
188 |
86 |
0.27 ns |
CP‡ |
79ab |
731ab |
821a |
717ab |
612b |
734 |
382 |
0.03* |
NDF§ |
554 |
557 |
547 |
570 |
611 |
568 |
166 |
0.14ns |
Ashes |
455 |
532 |
492 |
524 |
492 |
499 |
237 |
0.25ns |
pH |
7.00 |
7.10 |
7.00 |
7.20 |
6.40 |
7.01 |
063 |
0.88ns |
Means followed by
different letters in the row are statistically different by the Tukey’s test with P < 0.05;
*significant at P < 0.05; nsnon-significant at P > 0.05; †, DM: dry matter, expressed as g/kg; ‡CP:
crude protein, expressed as g/kg DM; §NDF: Neutral Detergent Insoluble
Fiber, expressed as g/kg DM;, expressed as g/kg DM; ¤SEM: standard
error of the mean
Table 4: Volatile fatty acids (VFA) of Paredão grass silage with the inclusion of fermented corn
silage juice of different fermentation periods
VFA (g kg−1 DM) |
Fermented corn silage juice (days) |
Mean |
SEM† |
P-value |
||||
0 |
5 |
10 |
15 |
30 |
||||
Lactic acid |
88c |
00d |
53a |
128b |
00d |
149 |
22 |
< 001* |
Acetic acid |
00c |
133b |
83bc |
9bc |
354a |
133 |
17 |
< 001* |
Propionic acid |
03b |
34b |
16b |
1b |
91a |
31 |
06 |
< 001* |
Butyric acid |
69 |
107 |
71 |
91 |
84 |
76 |
25 |
033ns |
Means followed by
different letters in the row are statistically
different by the Tukey’s test with P <
0.05; *significant at P < 0.05; nsnon-significant at P > 0.05; †SEM: standard error of the mean
content were affected (P =
0.03). No effect (P > 0.05) of the inclusion of fermented corn silage juices
was observed on the dry matter (DM), neutral detergent fiber (NDF), Ashes, and
pH values (Table 3). The highest CP content value was observed in the silage
containing corn silage juice fermented for 10 days, showing 82.1±3.82 g/kg of
CP; while the lowest CP value was observed in the silage containing corn silage
juice fermented for 30 days, with 61.2±3.82 g/kg. The other treatments were not
statistically different, presenting values of 79±3.82, 73.1±3.82 and 71.7±3.82
g/kg of CP for the silages containing corn silage juice fermented for 0, 5 and
15 days, respectively.
Regarding the volatile fatty
acids (Table 4), the contents of lactic, acetic, and propionic acids were
affected (P < 0.01) by the inclusion of the bioinoculant. Non-significant
effects were observed only on the butyric acid content. The highest content of
lactic acid was observed in the treatment containing corn silage juice
fermented for 10 days, which presented 53±2.2 g/kg. The treatments containing
corn silage juice fermented for 0 and 15 days presented values of 8.8 and 12.8
g/kg, respectively. The acids acetic and propionic were found in greater
concentration in silages inoculated with the fermented corn silage juice of the
longest fermentation period, 30 days, showing 35.4±1.7 and 9.1±0.6 g/kg,
respectively. The other treatments did not present significant differences in the
content of propionic acid with the addition of fermented corn silage juice in
the silage of Paredão grass.
Microbial population
All treatments showed similar results for the
development of lactic acid bacteria,
enterobacteria, yeasts and molds in the Paredão grass
silages (Fig. 3). Regarding the presence of microorganisms in the corn plant
(6.43, 3.47, 7.15 and 7.13 log CFU/g Table 5: Analysis of aerobic stability of Paredão
grass silage with inclusion of fermented corn silage juice of different
fermentation periods
FP† (days) |
Air exposure of silage (h) |
Mean |
P-value |
|||||
0 |
12 |
24 |
36 |
48 |
x |
x2 |
||
Room temperature (°C) |
||||||||
|
252 |
248 |
258 |
252 |
248 |
|
|
|
Silage surface temperature (°C) |
||||||||
0 |
158 |
222 |
21 |
211 |
202 |
200 |
- |
- |
5 |
153 |
209 |
205 |
21 |
203 |
196 |
- |
- |
10 |
165 |
221 |
205 |
206 |
195 |
198 |
- |
- |
15 |
165 |
215 |
205 |
206 |
194 |
197 |
- |
- |
30 |
16 |
143 |
205 |
207 |
205 |
184 |
- |
- |
Mean |
16 |
202 |
206 |
208 |
20 |
- |
< 001* |
< 001* |
Silage internal temperature (°C) |
||||||||
0 |
268 |
231 |
24 |
23 |
24 |
242 |
- |
- |
5 |
235 |
225 |
226 |
23 |
24 |
231 |
- |
- |
10 |
258 |
224 |
233 |
223 |
23 |
233 |
- |
- |
15 |
271 |
216 |
233 |
24 |
246 |
241 |
- |
- |
30 |
261 |
208 |
226 |
223 |
233 |
23 |
- |
- |
Mean |
259 |
221 |
232 |
229 |
238 |
- |
< 001* |
< 001* |
Silage pH |
||||||||
0 |
7.90 |
7.10 |
7.00 |
6.40 |
6.90 |
7.00 |
- |
- |
5 |
7.00 |
7.10 |
7.30 |
6.70 |
7.50 |
7.10 |
- |
- |
10 |
7.40 |
7.00 |
7.00 |
7.70 |
7.00 |
7.00 |
|
- |
15 |
7.70 |
7.20 |
7.60 |
5.80 |
7.90 |
7.20 |
- |
- |
30 |
5.50 |
6.20 |
7.00 |
5.60 |
7.70 |
6.40 |
- |
- |
Mean |
7.10 |
6.90 |
7.20 |
6.20 |
7.40 |
- |
- |
- |
Analysis of variance |
P-value |
|||||||
FP¥ |
H |
FP† × h |
‡SEM |
|||||
Silage surface temperature |
046ns |
< 001* |
042ns |
067 |
||||
Silage internal temperature |
009ns |
< 001* |
059ns |
037 |
||||
Silage pH |
031ns |
009ns |
086ns |
029 |
†FP: corn silage fermentation
period for the production of fermented juice ‡SEM: standard error of
the mean *significant at P <
0.05; nsnon-significant at P > 0.05; x: linear effect; x2:
quadratic effect
AF, of LAB, enterobacteria,
yeasts and molds, respectively) when compared to the silages of Paredão grass containing fermented corn silage juice of
different fermentation periods, there was a reduction in the amount of LAB,
yeasts and molds, presenting averages of 5.84, 5.75 and 5.67 CFU/g AF,
respectively, in the silages of Paredão grass. An increase of 2.82 log CFU/g AF was also observed for enterobacteria
in the Paredão grass silages when compared to the corn
plant, with mean values of 5.75 and 3.47 log CFU/g AF, for the silages and corn
plant, respectively.
Aerobic stability and pH
There was no interaction (P >
0.05) of silage fermentation period for the production of the bioinoculant×hours of air exposure of the Paredão grass silage (Table 5). The surface and internal
temperatures of the Paredão grass silage were
affected (P < 0.01) only by the
hours of air exposure. There was no effect on the pH values, which averaged
6.96. There was an increasing linear effect of hours of air exposure on the
surface temperature of the silage, which increased by 4ºC after 48 h. However,
even with the increase in the surface temperature, it remained on average 4.8°C
below the room temperature. There was a linear decreasing effect on the
internal temperature of the silage, where the highest temperature was obtained
in the silage of Paredão grass with 0 h of air
exposure. There was a reduction of 2.1ºC after 48 h of air exposure in the Paredão grass silage.
Discussion
In general, our results do not
support our initial hypothesis that a longer storage period of corn silage for
the production of fermented juice as a bioinoculant would provide better
quality of Paredão grass silage. However, an issue of
tropical grass silage is inadequate fermentation due to the low content of
soluble carbohydrates and reduced presence of bacteria that actively reduce pH
(McDonald et al. 1991; Kung et al.
2018). In our study, we observed that the inclusion of fermented corn juice
with 10 days of fermentation acts as an enhancer for the presence of BAL in Paredão grass silages. These effects opposite to the ones
we hypothesized (that is, they are not the best in the longest fermentative
periods, but in the smallest ones) are positive because they indicate that the
production of fermented corn silage juice can enter the strategic planning of
rural properties with a period shorter fermentation, allowing the procedure to be
performed with a better window for collecting the material to be ensiled.
The Paredão
grass silages without fermented corn silage juice and with corn silage juice
fermented for 10 days provided lesser effluent losses. The silage with corn
silage juice fermented for 30 days provided 178.9 g/kg more dry matter recovery
(DMR) than the silage without fermented corn silage juice. The losses by
effluents observed in the silages of Paredão grass
containing fermented corn silage juice were lower than those observed by Moraes et al.
(2017), who added different mixtures microbial inoculants (Lactobacillus buchneri; Propionibacterium acidipropionici;
P. acidipropionici
+ L. plantarum; L. buchneri + P. acidipropionici; L. buchneri + P. acidipropionici + L. plantarum). This shows that the use of fermented corn silage
juice may be viable for the conservation of Paredão
grass silage, as they reduce the loss of nutritional components and green
matter.
The low values of effluent
losses observed in silages containing corn silage juice fermented for 10 days
may have been due to the microbiological composition of the corn silage during
the juice production (Fig. 2), since the active fermentation phase of the
silage occurs from 7 to 30 days (Muck and Pitt 1993). The presence of lactic
fermenting microorganisms that act as pH-reducing acid producers, allows the
conservation of the material and the stabilization of losses by inactivating
microorganisms such as enterobacteria and yeasts. Oliveira et al. (2010) observed in their studies that the volume of effluent
produced in a silo is mainly influenced by the DM content of the ensiled
species, and that the effluent losses are minimized when the DM content in the
silage reaches 300 g/kg. In fact that was not observed in the Paredão grass (Table 1).
The silage goes through 5 phases
of different durations and intensities of proliferation of microorganisms,
which vary according to the native fauna, processing quality and fermentation
period (McDonald et al. 1991; Tao et al. 2020). This shows that the
fermented corn silage juice with 0 and 5 days of fermentation have reduced
quality, since this period may be insufficient for the proliferation of
anaerobic microorganisms that conserve the silage. Different fermented corn
silage juices used as additives in the Paredão grass
silage had no effect on the chemical composition of these silages, as well as
the silage without fermented corn silage juice, except for the crude protein
(CP) content. This probably occurred due to the little influence in the change
promoted by the fermented juices in the silage on the components of dry matter,
neutral detergent fiber and ashes, resulting in less alteration of the chemical
composition due to the treatments applied.
The Paredão
grass silage containing corn silage juice fermented for 10 days showed the
highest CP content. This is due to the microbial activity related to this
fermentation period, when it presents consumption of soluble carbohydrates and
consequent increase of CP (Siqueira et al. 2011). Adequate levels of CP
serve as indicative of lower intensities of proteolysis during the fermentation
of ensiled material. This fact can be due to lower activity of Clostridiums
and, consequently, the lowest concentration of butyric acid (McDonald et al. 1991; Drahokoupil
and Patáková 2020). The CP content of the Paredão grass silages inoculated with fermented corn silage
juice were larger than those observed by Bezerra et al. (2019), who found silages of
grasses with contents ranging from 64.6 to 72.7 g/kg. In addition, treatments
with lower CP values presented greater losses, which indicate greater
proteolysis activity.
A greater amount of lactic acid
was observed in the silages containing corn silage juice fermented for 10 days
(Table 4). The greater amount of lactic acid reduces the proliferation of
bacteria of the genus Clostridium
(Muck et al. 2018) and the quality of the silage is maintained for a
longer period, as well as the better palatability and forage intake (Muck and Bolsen 1991; Sofyan et al. 2017). According to Bureenok et al.
(2005b) increase in the population of lactic acid bacteria and decrease in
other microorganisms occurs in the first days of fermentation, showing that
microbial succession occurs very quickly and very definitively. However, these
microorganisms were still present in the silage. This corroborates to the
present study that showed an increase in the lactic acid content in silages
inoculated with corn silage bioinoculant fermented for 10 days (Table 4), with
no reduction in the microorganism population (Fig. 3).
There was a greater amount of
acetic acid in the Paredão grass silage containing
corn silage juice fermented for 30 days, showing levels higher than those
recommended as ideal for silages, which is < 20 g/kg DM, a level that defines
a good quality silage (Rego et al. 2013). The increase in the acetic
acid values of the Paredão grass silages inoculated
with corn silage juice fermented for more than 5 days is likely owing to the
peak of enterobacterial development that occurred from the third day of
fermentation, as observed by Pinho et al.
(2013), since the final product of these microorganisms from the consumption of
glucose is one molecule of lactate and one molecule of acetate or ethanol (Mcdonald et al. 1991; Yang et al. 2020). According to Luis and Ramírez (1988), enterobacteria
usually multiply until approximately the seventh day of fermentation, when they
are replaced by lactic acid groups. At the beginning of fermentation,
enterobacteria compete with LABs for the available soluble carbohydrates, and
the greater competitive and antagonistic capacity arising from the bacteriocins
produced by LABs is crucial, an effect not observed in the present experiment
possibly due to the resistance in decreasing pH, which made it difficult the
development of lactic acid bacteria.
The results obtained on the
production of acids in the Paredão grass silage
inoculated with corn silage juice fermented for 10 days demonstrate that this
fermentation period was more efficient, probably for conserving and cultivating
the beneficial epiphytic micro fauna. Silages with 10 days of storage enhance
the actions of beneficial microorganisms, in addition to keeping the
proliferation of microorganisms that deteriorate the silage quality stable (Bureenok et al.
2005a). Likewise, Pinho et al. (2013) observed
peak of the development of LAB populations from the seventh day of
fermentation. The highest concentration of lactic acid in the silage inoculated
with corn silage juice fermented for 10 days helps to control the proliferation
of bacteria of the genus Clostridium, and contributes to better values of
silage dry matter recovery and acceptability of the silage by the animals (Denek et al. 2011;
He et al. 2020).
The presence of butyric acid in
the Paredão grass silage was probably due to high
moisture content (Table 1), combined with the high buffering capacity intrinsic
to grasses; the conditions that favor butyric fermentation instead of lactic
fermentation (Meeske et al. 1999). The fermentation periods of 0, 5, 10, 15 and 30 days
of the corn silage for the production of fermented juice had no influence on
the number of microorganisms (LAB, enterobacteria, yeasts and molds) in the Paredão grass silages. These data indicated that there was
stabilization of microorganisms in the Paredão grass
silage 120 days after ensiling, regardless of the use of corn silage
bioinoculant.
A difference was observed between
the population of microorganisms of the Paredão grass
plant and its silage, and this occurred because the microbial population of the
plant at the time of harvest is different from the one found during the
fermentation process as well as in the silage (Bureenok
et al. 2011). The development of
these microbial populations in the ensiled material is related to the
conditions of the environment, which will naturally select the microbial groups
that may develop (Bezerra et al. 2019), mainly
due to the chemical characteristics of the plant. It can also be stated that
the concentration of lactic acid in all treatments, was possibly not sufficient
to prevent the development of molds and yeasts. However, the population of
lactic acid bacteria and enterobacteria observed in the present study were
similar to those found in previous studies that used corn silage as a bioinoculant
(Silva et al. 2018; 2020).
Regarding the aerobic stability
of the Paredão grass silage, an increase in the
surface temperature was observed in all treatments, but none exceeded the room
temperature, with no break in the aerobic stability, thus deducing that this
increase in the surface temperature occurred due to exogenous factors and not
by intrinsic factors of the silage itself (Silva et al. 2020). Increase
in temperature observed after opening the silo may be the result of reactions
promoted by undesirable microorganisms, such as yeasts, filamentous fungi and
aerobic bacteria (Amaral et al. 2008), however, the exposure of the Paredão grass silages, regardless of the treatment, did not
cause increase of the temperature in relation to the environment. This may have
occurred mainly due to the low amount of soluble carbohydrates and low number
of endogenous bacteria present in the grass (Bureenok et al. 2005a), which keep the
activity of microorganisms at low levels even after opening the silo.
Higher pH levels found in the Paredão grass silage, which obtained an average of 7.0
regardless of treatment, can be explained by high buffering capacity that
grasses have and low content of soluble carbohydrates in the plant. The
consumption of carbohydrates by microorganisms is vital for the reduction of pH
(Pinho et al. 2013). The authors found
decreasing carbohydrates content from values close to 3% (%DM), up to values of
0.5%, for the pH decrease from 5.4 to 4.4.
Conclusion
The inclusion of fermented corn
silage juice influences the quality of Paredão grass
silage. The addition of corn silage juice fermented for 10 days is recommended
because it promotes greater recovery of the silage dry matter, higher crude
protein content and greater amount of beneficial acids. However, it is
important to carry out further studies regarding the storage of fermented
juices and their use as a bioinoculant, especially regarding the aerobic
stability of silages.
Acknowledgements
This work was financially
supported by Federal University of Piauí.
Author Contributions
RLE, MJA and LRB conceived and
designed the experiments, KSN, MAS and RRN carried out the experiments and
analyzed the data, KSN and SJAV wrote the manuscript with the help of DMAB. All
authors read, edited, and approved the manuscript.
Conflicts of Interest
The authors reported no
potential conflict of interest.
Data Availability
Data presented in this study
will be available on a fair request to the corresponding author.
Ethics Approval
Not applicable in this paper.
References
Alvares CA, JL Stape, PC Sentelhas, JLM Gonçalves, G Sparovek
(2013). Köppen's climate classification map for Brazil. Meteorol Z 22:711‒728
Amaral RC, TF Bernardes, GR
Siqueira, RA Reis (2008). Aerobic stability of marandu
grass silages submitted to different intensities of compaction in silage. Rev Bras Zootec 37:977‒983
Association of Official Analytical Chemists (AOAC) (1990). Official
Methods of Analysis, 15th edn,. AOAC,
Arlington, Texas, USA
Bezerra HFC, EM Santos, JS Oliveira,
GGP Carvalho, RMA Pinho, TC Silva, AM Zanine (2019). Fermentation characteristics and chemical
composition of elephant grass silage with ground maize and fermented juice of
epiphytic lactic acid bacteria. S Afr J Anim Sci 49:522‒533
Bolsen KK, DG Tiemann, RN Sonon,
RA Hart, B Dalke, JT Dickerson, C Lin (1992).
Evaluation of inoculant-treated corn silages. Kansas Agric Exp Stn Res Rep 1:104‒107
Borjas‐Ventura R, LR Alves, R
Oliveira, CA Martínez, PL Gratão (2019). Impacts of
warming and water deficit on antioxidant responses in Panicum maximum. Physiol Plantarum
165:413‒426
Bureenok S, T Namihira,
Y Kawamoto, T Nakada (2005a). Additive effects of fermented juice of epiphytic
lactic acid bacteria on the fermentative quality of guineagrass
(Panicum maximum Jacq) silage. Grassl Sci 51:243‒248
Bureenok S, T Namihira,
M Tamaki, M Mizumachi, Y Kawamoto, T Nakada (2005b).
Fermentative quality of guineagrass silage by using
fermented juice of the epiphytic lactic acid bacteria (FJLB) as a silage
additive. Asian-Aust J Anim Sci
18:807‒811
Bureenok S, W Suksombat,
Y Kawamoto (2011). Effects of the fermented juice of epiphytic lactic acid
bacteria (FJLB) and molasses on digestibility and rumen fermentation
characteristics of ruzigrass (Brachiaria ruziziensis) silages. Livest Sci 138:266‒271
Cardoso LL, KG Ribeiro, MI Marcondes,
OG Pereira, K Weiβ (2019). Chemical
composition and production of ethanol and other volatile organic compounds in
sugarcane silage treated with chemical and microbial additives. Anim Prod Sci 59:721‒728
Costa DM, BF Carvalho, TF Bernardes,
RF Schwan, CLA Silva (2021). New epiphytic strains of lactic acid bacteria
improve the conservation of corn silage harvested at late maturity. Anim Feed Sci Technol 274:48‒52
Denek N, A Can, M Avci,
M Aksu (2011). The effect of fresh and frozen pre-fermented juice on the
fermentation quality of first-cut lucerne silage. Sci Commun Fac Anim
Sci 54:785‒790
Drahokoupil M, P Patáková
(2020). Production of butyric acid at constant pH by a solventogenic strain of Clostridium beijerinckii.
Czech J Food Sci 38:185‒191
González G, AA Rodríguez (2003). Effect of storage
method on fermentation characteristics, aerobic stability, and forage intake of
tropical grasses ensiled in round bales. J
Dairy Sci 86:926‒933
Hang YD, EE Woodams, LE Hang
(2003). Use of corn juice by Kluyveromyces marxianus. Bioresour
Technol 3:305‒307
He L, C Wang, Y Xing, W Zhou, R Pian, X Chen, Q Zhang
(2020). Ensiling characteristics, proteolysis and bacterial community of
high-moisture corn stalk and stylo silage prepared
with Bauhinia variegata
flower. Bioresour Technol 296; Article 122336
Kung
Jr L, RD Shaver, RJ Grant, RJ Schmidt (2018). Silage review: Interpretation of
chemical, microbial, and organoleptic components of silages. J Dairy Sci 101:4020‒4033
Ludovico A, EH Hoshi, LC Silva, FCAR Grecco,
LFV Cunha Filho, MR Baran (2014). Chemical composition and losses of nutrients
involved in sugarcane ensiling with microbial and chemical additives. Arch
Latinoamer Prod Anim 22:33‒42
Luis
L, M Ramírez (1988). Evolución de la flora microbiana en un ensilaje de king
grass. Pastos For 11:3‒12
Martha
Júnior GB, LA Vilela, DM Sousa (2007). Cerrado:
Uso eficiente de corretivos e fertilizantes em cerrados. Embrapa Cerrados, Planaltina, Brazil
McDonald P, AR Henderson, SJ
Heron (1991). The Biochemistry of Silage, 2nd edn. Chalcombe Publications,
Marlow, Buckinghamshire, England
Meeske R, HM Basson,
CW Cruywagen (1999). The effect of a lactic acid
bacterial inoculant with enzymes on the fermentation dynamics, intake and
digestibility of Digitaria eriantha
silage. Anim Feed Sci Technol 81:237‒248
Moraes RLD, KG Ribeiro, OG Pereira, MI
Marcondes, LL Cardoso (2017). Silage from sugarcane
treated with microbial inoculants and mixtures thereof. Rev Bras Agropec Sustentável 7:76‒83
Muck RE, KK Bolsen (1991). Silage
preservation and silage additive products. In: Field Guide for Hay and
Silage Management in North America,
pp:105‒126 National Feed Ingredients Association Des Moines, Iowa,
USA
Muck RE, RE Pitt (1993).
Ensiling and its effect on crop quality. In:
Proceedings of the National Silage Production Conference, pp:57‒66.
Syracuse, New York NRAES-67 Northeast Reg Agric
Ext Service, Ithaca, New York, USA
Muck RE, EMG Nadeau, TA
McAllister, FE Contreras-Govea, MC Santos, L Kung Jr
(2018) Silage review: Recent advances and future uses of silage additives. J Dairy Sci 101:3980‒4000
Oliveira
LB, AJV Pires, GGP Carvalho, LSO Ribeiro, VV Almeida, CAM Peixoto (2010).
Perdas e valor nutritivo de silagens de milho, sorgo-sudão, sorgo forrageiro e
girassol. Rev Bras Zootec 39:61‒67
Pinho
RMA, EM Santos, GGPD Carvalho, APGD Silva, TCD Silva, FS Campos, CHO Macedo
(2013). Microbial and fermentation profiles, losses and chemical composition of
silages of buffel grass harvested at different cutting Heights. Rev Bras Zootec 42:850‒856
Rego
FCA, A Ludovico, LC Silva, LD Lima, EW Santana, MC Françozo (2013).
Fermentation profile, chemical composition and dry matter losses of orange pulp
silage with different microbial inoculants. Semina: Ciências
Agrárias 33:3411‒3420
Senger CC, GV Kozloski, LMB Sanchez, FR Mesquita, TP Alves, DS Castanino (2008). Evaluation of autoclave procedures for fibre
analysis in forage and concentrate feedstuffs. Anim
Feed Sci Technol 146:169‒174
Silva LD, OG Pereira, TC Silva,
ES Leandro, RA Paula, SA Santos, KG Ribeiro, SCV Filho (2018) Effects of Lactobacillus
buchneri
isolated from tropical maize silage on fermentation and aerobic
stability of maize and sugarcane silages. Grass Forage Sci 73:660‒670
Silva MA, RL Edvan, HN Parente, AM Zanine, JM Pereira
Filho, EM Santos, LR Bezerra (2020). Addition of
fermented corn juice as bioinoculant improved quality of Saccharum officinarum silage. Intl J Agric
Biol 23:349‒356
Siqueira
GR, RA Reis, RP Schocken-Iturrino, APDTP Roth, MDTP Roth, FD Resende (2011).
Perfil fermentativo de silagens de cana-de-açúcar in natura ou queimada e
tratadas ou não com Lactobacillus
buchneri. Rev Bras Zootec 40:1651‒1661
Sofyan
A, Y Widyastuti, R Utomo, LM Yusiati (2017). Improving physico-chemical
characteristic and palatability of king grass (pennisetum hybrid) silage by
inoculation of Lactobacillus plantarum-Saccharomyces cerevisiae consortia and
addition of rice bran. Buletin Peternakan
41:61‒71
Tao
Y, D Niu, F Li, S Zuo, Q Sun, C Xu (2020). Effects of ensiling Oxytropis glabra with whole-plant corn
at different proportions on fermentation quality, alkaloid swainsonine content,
and lactic acid bacteria populations. Animals
10:1733‒1741
Taylor CC, L Kung Jr (2002). The Effect of Lactobacillus
buchneri
40788 on the fermentation and aerobic stability of high moisture corn in
laboratory silos. J Dairy Sci 85:1526‒1532
Van Soest
PJ, JB Robertson, BA Lewis (1991). Methods for dietary fiber, neutral detergent
fiber, and nonstarch polysaccharides in relation to
animal nutrition. J Dairy Sci 74:3583‒3597
Vrátný P, Z Mudřík
(1985). Liquid chromatography of organic acids in silage extracts using dual
detection. J Chromatogr
A 322:352‒357
Weiss WP (1999). Energy prediction equations for
ruminant feeds In: Proceedings from the Cornell Nutrition Conference for
Feed Manufacturers, pp:176‒185 Cornell University, Ithaca, New York,
USA
Yang Y, Z Zhang, X Lu, J Gu, Y Wang, Y Yao, J Hao (2020). Production of
2, 3-dihydroxyisovalerate by Enterobacter
cloacae. Enz Microb Technol
140; Article 109650
Zanine AM, EM Santos, JRR Dórea, PAS Dantas, TCS Silva, OG
Pereira (2010) Evaluation of elephant grass silage with the addition of cassava
scrapings. Rev Bras Zootec 39:2611‒2616