1Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Silage quality and bacterial diversity of silages inoculated with 
Listeria monocytogenes and Lacticaseibacillus paracasei_6714

Natalia Barboza1,2*;  https://orcid.org/0000-0003-0881-6867
Laura Brenes-Guillén3;  https://orcid.org/0000-0002-7185-4084
Lidieth Uribe4,5;  https://orcid.org/0000-0002-8276-7824
Rodolfo WingChing-Jones6, 7;  https://orcid.org/0000-0002-8009-2210

1. Escuela de Tecnología de Alimentos, Universidad de Costa Rica, San José, Costa Rica, natalia.barboza@ucr.ac.cr 
(Correspondencia*).

2. Centro Nacional en Ciencia y Tecnología de Alimentos (CITA), Universidad de Costa Rica, San José, Costa Rica.
3. Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, San José, Costa Rica, 

laubregui2603@gmail.com
4. Centro de Investigaciones Agronómicas (CIA), Universidad de Costa Rica, San José, Costa Rica, 
 lidieth.uribe@ucr.ac.cr
5. Escuela de Agronomía, Universidad de Costa Rica, San José, Costa Rica.
6. Escuela de Zootecnia, Universidad de Costa Rica, San José, Costa Rica, rodolfo.wingching@ucr.ac.cr
7. Centro de Investigación en Nutrición Animal (CINA), Universidad de Costa Rica, San José, Costa Rica.

Received 09-VI-2022.        Corrected 07-XII-2022.       Accepted 10-II-2023.

ABSTRACT
Introduction: King grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum purpuphoides) and 
pineapple peel (Ananas comosus) silages are food alternatives for livestock in conditions of feed shortage. 
Objective: To describe the dynamics of the microbiota present in king grass and pineapple silage during 
the fermentation process using next generation sequencing (NGS) and to evaluate the protective effect of 
Lacticaseibacillus paracasei_6714 as a silage inoculum against Listeria monocytogenes. 
Methods: We used an unrestricted randomized design to characterize the microbiota present in silages made 
from king grass harvested 70 days after regrowth and pineapple peel. We inoculated mixtures of grass and peel 
with L. paracasei_6714 or L. monocytogenes, or both, with a non-inoculated treatment as control. The nutritional 
and fermentative profile was evaluated after 30 days. After 15 and 30 days of fermentation, we used 16S rRNA 
analysis to determine the dynamics and diversity of the microbiota in the inoculated and control silages. 
Result: Dry matter content and digestibility did not differ significantly; however, there were differences in crude 
protein, pH and organic acids. We obtained 4432 amplicon sequence variants of Proteobacteria, Firmicutes, 
Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes and Patescibacteria. The relative abundance 
of each phylum varied depending on the material and fermentation period. Phylum similarity was over 70 % (but 
not greater than 50 % with Bray-Curtis at the species level). 
Conclusion: These bacterial communities seem to have an important role during silage fermentation. Proper 
management of silage processing can reduce or eliminate pathogenic bacteria.

Key words: lactic acid bacteria; pathogens; agroindustrial residual; silage; nutritional value, next generation 
sequencing.

https://doi.org/10.15517/rev.biol.trop..v71i1.50692

BIOMEDICINE



2 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

INTRODUCTION

Animal feed systems in the tropics. Live-
stock systems in tropical conditions are affect-
ed by variations in pasture productivity due 
to seasonal rainfall distribution and climatic 
phenomena such as tropical storms and hur-
ricanes (Alfaro & Quesada, 2010). The use of 
conserved forage (hay, haylage and silage) and 
agro-industrial by-products as alternative food 
sources can help maintain the productivity of 
the system and the welfare of livestock dur-
ing periods of the year when the demand for 
dry matter exceeds the forage supply (Pulido 
et al., 2020). Preservation of forage as silage 
is limited by the moisture content of the for-
age used, the soluble carbohydrate content, 
the compaction of the material (particle size 
and oxygen availability), the use of additives 
and the natural population of microorganisms 
in the material or mixture at the beginning of 
the process (Ávila & Carvalho, 2019). Non-
compliance with the minimum requirements 
for an adequate fermentation process can lead 

to deterioration of the material to be preserved 
(loss of dry matter) and the growth of microor-
ganisms that can affect the health of livestock 
and humans related to the production system 
(Auerbach & Nadeau, 2020). In addition, loss-
es during cultivation, harvesting, transfer and 
compaction of the material to be ensiled (Kim 
et al., 2020) increase production costs (Villalo-
bos-Villalobos et al., 2015). Undesirable micro-
organisms such as Clostridium, fungi, molds, 
yeasts, enterobacteria, Salmonella, and Listeria 
monocytogenes, are associated with the produc-
tion of secondary metabolites (as mycotoxins 
and toxins) (Ávila & Carvalho, 2019); in addi-
tion, these microorganisms promote deamina-
tion, decarboxylation, and oxidation/reduction 
processes that lower the nutritional value of the 
material and reduce voluntary consumption by 
livestock (Queiroz et al., 2018). In particular, L. 
monocytogenes causes the food-borne disease 
listeriosis, which is associated with septicemia, 
encephalitis, meningitis, meningoencephali-
tis, rhombencephalitis, abortions, fetal death, 

RESUMEN
Calidad de ensilado y diversidad bacteriana de los silos inoculados con 

Listeria monocytogenes y Lacticaseibacillus paracasei_6714

Introducción: Los ensilajes del pasto king grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum 
purpuphoides) y cáscaras de piña (Ananas comosus) son alternativas de alimento para ganado en condiciones de 
escasez alimentaria. 
Objetivo: Describir las dinámicas de la microbiota presente en los ensilajes de king grass y piña durante el pro-
ceso de fermentación usando secuenciación de próxima generación (NGS) y evaluar el efecto de protección de 
Lacticaseibacillus paracasei_6714 como inoculante de ensilaje ante Listeria monocytogenes. 
Métodos: Usamos un diseño aleatorio no restringido para caracterizar la microbiota presente en ensilajes de king 
Grass cosechados 70 días después de rebrote y de cáscaras de piña. Inoculamos mezclas de pasto y cáscara con 
L. paracasei_6714 o L. monocytogenes, o ambos, con un tratamiento control sin inocular. El perfil nutricional y 
de fermentación fue evaluado luego de 30 días. Después de 15 y 30 días de fermentación, usamos un análisis de 
para determinar la dinámicas y diversidad de la microbiota en los ensilajes inoculados y control. 
Resultado: Los contenidos de materia seca y digestibilidad, no difirieron significativamente; sin embargo, 
hubo diferencias en proteína cruda, pH y ácidos orgánicos. Obtuvimos 4 432 secuencias variantes de ampli-
con de Proteobacteria, Firmicutes, Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes y de 
Patescibacteria. La abundancia relativa de cada filo vario dependiendo del material y periodo de fermentación. 
Similitudes de filo fueron mayores al 70 % (pero no mayor que 50 % con Bray-Curtis a nivel de especie). 
Conclusión: Estas comunidades bacterianas parecen cumplir un papel importante durante la fermentación del 
ensilaje. Un manejo apropiado del proceso de ensilaje puede reducir o eliminar baterías patogénicas. 

Palabras clave: bacterias ácido lácticas; patógeno; residuo agroindustrial; ensilaje; valor nutricional; secuen-
ciación de nueva generación.



3Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

perinatal infections, and gastroenteritis in live-
stock (Dhama et al., 2015). L. monocytogenes 
can remain on dairy cattle farms for months, 
even when good hygienic practices are applied. 
Poor quality silage is the main source of infec-
tion by this pathogenic bacterium (Idland et al., 
2022; Yoshida et al., 1998).

Additives can be used to improve the fer-
mentation process and reduce material losses 
by stimulating (bacterial culture and carbohy-
drate sources) or inhibiting fermentation (acids 
and salts of weak acids), inhibiting aerobic 
spoilage (propionic acid), and improving nutri-
ent supply (urea) (McDonald, 1981). The addi-
tion of lactic acid bacteria (LAB) increases 
the initial number of these microorganisms 
and prevents the growth of enterobacteria and 
Clostridium (McDonald, 1981) by promoting 
the transformation of soluble carbohydrates 
to lactic acid (LA), which stabilizes the fer-
mentation process. Because of their probiotic 
characteristics, LAB consumed in silage reduce 
the production of enteric methane (CH4), a 
greenhouse gas produced during the digestive 
process in ruminants (Huyen et al., 2020).

Microbiota present in silage made from 
agroindustrial residuals have not been evaluat-
ed and characterized in Costa Rica. Therefore, 
the objective of this research was to describe 
the dynamics of the microbiota present in king 
grass and pineapple silage during the fermenta-
tion process using next generation sequencing 
(NGS). The potential protective effect of L. 
paracasei_6714 against L. monocytogenes was 
also evaluated in inoculated silages.

MATERIALS AND METHODS

Microbial strains and inoculum prepa-
ration: L. paracasei_6714, an isolate from 
pineapple peel silage (WingChing-Jones et 
al., 2021) with antagonistic activity against 
pathogens was used in this assay (Wu-Wu et al., 
2021). A 48 h culture of L. paracasei_6714 on 
De Man, Rogosa, and Sharpe agar (MRS) cul-
ture medium (Thermo Scientific™ Oxoid™, 
MA, USA) was used to prepare a suspension of 
1 x 109 CFU in 100 ml of deionized water. L. 

monocytogenes strain ATCC 19116 was grown 
for 48 h on Tryptone Soy Agar (ATS) medium 
(Merck, NJ, USA) and inoculum was prepared 
as described for LAB.

Silage treatments: Silage was made from 
king grass (Cenchrus purpureus (Schumach.) 
Morrone, syn. Pennisetum purpuphoides) har-
vested after 70 days of regrowth and pineapple 
peel at a ratio of 90:10 (w/w). A total of 2 ml 
was added to forage and pineapple peel mixture 
(90:10, w/w), placed in transparent bags with a 
2 kg capacity and a thickness of 0.0063 mm. 
The air was extracted with a vacuum pump and 
bags were sealed with elastic tape. The samples 
were kept on shelves in a laboratory under con-
trolled conditions for 30 days at 25 ± 2 °C. The 
inoculation and collection of samples treated 
with L. monocytogenes was performed in a type 
two biosafety chamber (Labconco. MO, USA).

Three inoculated silage treatments and a 
non-inoculated control (CK) were used. Treat-
ments were inoculated with either L. paraca-
sei_6714 (CKLA), L. monocytogenes (CKLi), 
or a mixture of L. paracasei_6714 and L. 
monocytogenes (1:1) (CKLL) in the concen-
trations described above. Eight replicates of 
each treatment were prepared for a total of 32 
bagged silages. At days 15 and 30 after the start 
of the fermentation process, two bags for each 
treatment and the control were opened and the 
dynamics of the microbiota were determined.

Nutritional value of silage after fermen-
tation: After the fermentation period, a one kg 
sample of ensiled material was taken from each 
treatment and the dry matter (DM), crude pro-
tein (CP) (AOAC, 1998) and in vitro dry matter 
digestibility (IVDMD) were determined using 
the methodology of Van-Soest and Robertson, 
(1985). The pH was measured using a potenti-
ometer with a hydrogen electrode and ammo-
niacal nitrogen was measured as described by 
Tobía et al. (2004), using 20 g of ensiled mate-
rial in 80 ml of distilled water. Organic acids 
were extracted by ultrasound-assisted aque-
ous extraction under acidic conditions using 
a 1 g dry sample following the methodology 



4 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

described in AOAC with some modifications 
(Leiva & Granados-Chinchilla, 2020).

Statistical analyses: An unrestricted ran-
domized design was used to evaluate the nutri-
tional value of each silage. An analysis of 
variance (ANOVA) was performed with the sta-
tistical program SAS (SAS Institute Inc., 2011). 
When significant differences were determined 
as a result of the main effects, Duncan’s test (P 
< 0.05) was applied to separate treatments.

16S rRNA sequencing: Forage and pine-
apple peels were sampled prior to fermentation. 
After 15 and 30 days of fermentation, 10 g 
composite samples containing material from 
three depths within the bag (5, 20 and 30 cm) 
were collected from each silage treatment.

Samples were homogenized with H20dd 
and DNA was extracted using the protocol 
described by Birnboim and Doly, (1979). The 
DNA was run in 1 % agarose gels. Samples 
were stored at -20 °C until further analysis. 
The DNA samples were sent to Macrogen Inc. 
(Korea) for the construction of genomic librar-
ies. The 16S rRNA was amplified using the 
V3-V4 hypervariable region with the primers 
Bakt_341F (5’-CCTACGGGGGNGGGGC-
WGCAG-3’) and Bakt_805R (5’-GAC-
TACHACHVGGGGGTATCTAATCC-3’) 
(Klindworth et al., 2013). Sequencing was 
performed using the MiSeq platform (Illumina.
Inc), which yielded 300 bp fragments.

Bioinformatic analysis: Raw sequence 
data obtained from the Illumina sequencing 
platform were processed using QIIME2 (ver-
sion 2018.11) (Bolyen et al., 2019; Caporaso 
et al., 2010) and its plugins. The ‘dada2′ plugin 
(Callahan et al., 2016) was applied to trun-
cate reads (--p-trim-left 0, --p-trunc-len 260). 
The vsearch uchime_ref method was used 
to identify chimeric feature sequences. Mito-
chondria sequences were removed. Taxonomy 
of the Amplicon Sequence Variant (ASV) was 
assigned using the SILVA 16S/18S rDNA non-
redundant reference dataset (SSURef 132 NR) 
(Quast et al., 2013) with the feature-classifier 

classify-sklearn. Taxonomic assignments of the 
remaining ASVs were manually checked by 
comparing them with sequences in the database 
using a combination of initial BLASTN-based 
searches and an extension of the EzbioCloud 
(Yoon et al., 2017) which stores 16S rRNA 
gene sequences of type strains of validly pub-
lished names.

All of the ASVs (4 432) were considered 
for graphics construction and diversity analy-
sis. Shannon (S), Tajima (d) and Fisher indices 
were determined. A shadeplot of the biotic data 
(after square-root transformation of the origi-
nal data-sheet) was created through the matrix 
display of the wizard tool, where real data are 
shown with samples and variables arranged and 
clustered according to their similarity (Bray 
Curtis similarity for samples and Whitaker 
index of association for ASVs).

Phylogeny analysis: A phylogeny analysis 
was performed using all LAB isolates belong-
ing to the family Lactobacillaceae that were 
present in the samples, considering the new 
classification proposed by Zheng et al. (2020) 
for the Lactobacillus group. The MUSCLE 
tool (Tamura et al., 2013) was used to align the 
sequences. The phylogeny was constructed by 
Bayesian analysis (Ronquist & Huelsenbeck, 
2003) using 10 million generations, eight Mar-
kov chains and sampling every 1 000 genera-
tions. The sequences of Lactococcus formensis 
NR114366 and L. formensis (obtained in this 
research) were used as the outgroup.

RESULTS

Silage quality - Nutritional parame-
ters: Dry matter content and digestibility of 
silages did not differ significantly (P > 0.05) 
among treatments after 30 days of fermentation 
(Table 1). Crude protein content was highest in 
silage inoculated with L. monocytogenes alone 
and lowest in silage inoculated with both L. 
paracasei_6714 + L. monocytogenes. The CP 
content of silage inoculated with L. paraca-
sei_6714 was not significantly different from 
that of the non-inoculated control. 



5Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Fermentation parameters: The pH was 
below 4.5 in all silage treatments. The pH in the 
treatment inoculated with L. monocytogenes 
was the highest and was statistically different 
from the control. Ammonia nitrogen values in 
treatments inoculated with L. monocytogenes 
were five to six times higher than in the control 
and in treatments inoculated with L. paraca-
sei_6714 (P < 0.05). The LA and isobutyric 
acid concentrations were significantly higher in 
treatments inoculated with L. monocytogenes. 
In contrast the concentration of acetic acid 
(AA) was up to 14 times higher in treatments 
without L. monocytogenes. Total acid content 
in the non-inoculated control and the treatment 
with L. paracasei_6714 were 1.7 to 2.3 times 
higher than in the treatments inoculated with L. 
monocytogenes (Table 1).

Microbiota diversity of the silages: A 
total of 4 432 ASVs were obtained. Proteobac-
teria, Firmicutes, Bacterioidetes, Actinobac-
teria, Verrucomicrobia, Planctomycetes and 
Patescibacteria were the most abundant phyla. 
The relative abundance of each phylum varied 
depending on the material used and fermenta-
tion period. In the pineapple peel samples, the 

most abundant phyla were Firmicutes (80 %) 
and Proteobacteria (14 %), while the relative 
abundance of the other groups was less than 
3 %. Proteobacteria (70 %) and Bacteroidetes 
(15 %) comprised up to 85 % of the phyla in 
king grass (Fig. 1). Despite the higher propor-
tion of king grass in the silages and the impor-
tant load of Proteobacteria, the abundance of 
Firmicutes was approximately 45 % at both 
sampling periods (15 and 30 days) in the treat-
ments inoculated with L. paracasei_6714 and 
the non-inoculated control. In silage inoculated 
with L. monocytogenes, the abundance of Fir-
micutes was approximately 28 % at 30 days. 
The abundance of Proteobacteria remained 
near 40 % at both sampling times in all treat-
ments (Fig. 1).

Structure of the bacterial community: 
The similarity of the communities at the spe-
cies level using Bray-Curtis analysis was not 
greater than 50 %. In contrast, similarity at the 
phylum level was greater than 70 %. The main 
genera present in the pineapple peel were Weis-
sella, Fructobacillus and Pseudomonas; spe-
cies belonging to the Acetobacteraceae family 
were also present (Fig. 2). In king grass, several 

TABLE 1
Nutritional and fermentative parameters of king grass and pineapple peel treatments after 30 days of storage

Treatments (%)

Nutritional parameters non-inoculated L. paracasei_6714
L. paracasei_6714 

+ L. monocytogenes 
L. monocytogenes 

Total dry matter 20.17 (± 0.21) 19.86 (± 0.49) 19.63 (± 0.55) 20.16 (± 0.51)

Crude protein 10.78 (± 0.49)ab 10.61 (± 0.12)ab 10.01 (± 0.30)b 11.21 (± 0.79)a

In vitro digestibility of dry matter 64.97 (± 1.37) 65.73 (± 1.06) 63.90 (± 0.52) 65.27 (± 1.06)

Fermentation parameters

pH 4.31 (± 0.12)b 4.40 (± 0.06)ab 4.43 (± 0.03)ab 4.49 (± 0.04)a

Ammonia nitrogen /N-total 4.90 (± 0.10)b 5.33 (± 0.35)b 30.40 (± 1.42)a 26.83 (± 3.84)a

Lactic acid 0.00 (± 0.00)b 0.00 (± 0.00)b 4.43 (± 0.36)a 4.16 (± 0.61)a

Acetic acid 7.94 (± 0.74)a 6.30 (± 0.80)b 0.50 (± 0.04)c 0.49 (± 0.04)c

Propionic acid 2.25 (± 0.25)a 2.26 (± 0.34)a 1.17 (± 0.17)b 1.44 (± 0.26)b

Isobutyric acid 0.39 (± 0.03)b 0.35 (± 0.06)b 0.58 (± 0.14)a 0.63 (± 0.04)a

Butyric Acid 5.14 (± 0.09)a 3.10 (± 0.05)b 0.11 (± 0.02)c 0.15 (± 0.01)c

Total Acids 15.72 (± 0.79)a 12.01 (± 1.13)b 6.69 (± 0.29)c 6.87 (± 0.35)c

Mean values (± standard deviation, n = 5). Different letters in the same row indicate significant differences of P < 0.05, 
according to Duncan’s multiple range test.



6 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Pseudomonas species were found, including 
P. flevescens, P. fluorescens, and P. synxantha. 
Analysis of the bacterial community showed no 
treatment effect. After 15 and 30 days, genera 
not detected in the initial samples (pineapple 
and king grass) were present in the silages. 
These included Lactobacillus, Lacticaseibacil-
lus, Levilactobacillus, Paucilactobacillus, Rhi-
zobium and Leuctonostoc. L. monocytogenes 
was not found in any of the treatments inocu-
lated with this bacterium or in the controls 
(Fig. 2).

Diversity analysis: Diversity values for 
the pineapple samples were lower than those of 
the king grass and fermented materials (Table 
2). Average ASV values were highest in the 
control treatment (30 days post-inoculation 
(dpi)), followed by silage inoculated with L. 
monocytogenes (15 dpi). Shannon (S), Tajima 
(d) and Fisher indices of diversity were highest 
for the initial king grass material, followed by 
the control silage evaluated at 15 and 30 days 
(Table 2). 

Phylogenetic analysis of LAB species: A 
total of 53 ASVs were selected and deposited in 
GenBank with accession numbers MZ504795-
MZ504853. A total of 33 sequences represent-
ing the main genera found in this research 
(marked in bold) were considered. The phylo-
genetic analysis confirmed the taxonomic clas-
sification of the ASV previously obtained using 
the NCBI and Ez Bio databases. There was a 
high level of support for most of the branches. 
ASVs corresponding to L. paracasei found 
in this research were affiliated with GenBank 
sequences of the same genus and with the close 
group of L. casei. These species are close to 
Levilactobacillus spicheri. Species more dis-
tant from L. paracasei in phylogeny were also 
identified and include Leuconostoc, Fructoba-
cillus, and several species of Weissella (Fig. 3).

DISCUSSION

King grass and pineapple residues are used 
for silage production in Costa Rica and other 
tropical countries (López-Herrera et al., 2019). 

Fig. 1. Relative abundance of microbiota by phylum found in initial samples of pineapple and king grass and in inoculated 
and control silage treatments incubated for 15 and 30 days. CK: non-inoculated king-grass: pineapple peel (90:10) silage, 
CKLA: king grass: pineapple peel silage inoculated with L. paracasei_6714, CKLi: king grass: pineapple peel silage 
inoculated with L. monocytogenes (CKLi), CKLL: king-grass: pineapple peel silage inoculated with a mixture of L. 
paracasei_6714 and L. monocytogenes (1:1).



7Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

In this research, physicochemical properties of 
these residues during the ensiling process were 
analyzed. Dry matter contains the main nutri-
ents for animal nutrition and their loss during 
fermentation is a problem for animals (Ni et al., 
2015; NRC 2001). The DM contents of LAB-
inoculated materials in this work were similar 
to those reported by Santoso et al. (2015) and 
higher than those of Zi et al. (2021). The differ-
ences may be associated with the age at harvest 
of the forage material. High dry matter content 
indicates a high concentration of nutrients in 
the ensiled material and therefore a greater 
contribution to the livestock diet.

In our study, inoculation with LAB did not 
affect the DM content or the in vitro digest-
ibility of the preserved material. This may have 
been due to the natural presence of LAB in 
the pineapple and king grass, which served as 
inoculum in the silage process (López-Herrera 
et al., 2014). Other studies have reported that 
inoculation with LAB increased DM content 
in LAB-inoculated materials (da Silva et al., 
2018), increased in vitro digestibility (Cao et 
al., 2011), or had no effect on DM content 
(Santoso et al., 2015; Zi et al., 2021) of LAB 
inoculated materials.

The higher CP content of the silages 
treated L. monocytogenes may be due to the 

Fig. 2. Shadeplot of the main species present in the initial residues and treatments. CKLA: king grass: pineapple peel silage 
inoculated with L. paracasei_6714, CKLi: king grass: pineapple peel silage inoculated with L. monocytogenes (CKLi), 
CKLL: king grass: pineapple peel silage inoculated with a mixture of L. paracasei_6714 and L. monocytogenes (1:1). 15 
and 30 days post inoculation (dpi) process. 



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high ammoniacal nitrogen (a product of the 
degradation of amino acids during the fermen-
tation process), and low total organic acids (a 
product of a less intense fermentation process). 
The pH values in all the treatments were lower 
than 4.5. A pH lower than 4.5 can inhibit the 
growth of undesirable microorganisms such 
as spoilage bacteria, yeasts, and pathogens 
(Rahman et al., 2014; Thierry et al., 2011). 
The lower pH of the control silage is the result 
of higher acid production during fermentation 
(Contreras-Govea et al., 2013). Both treatments 
inoculated with L. monocytogenes had higher 
pH values than the treatments not inoculated 
with the pathogenic bacteria. This result could 
be due to the presence of six times more 
ammonia nitrogen, which generates a buffer 
effect that reduces acidification of the silage 
(Gutierrez et al., 2003).

The high AA production in the silage 
treated with L. paracasei_6714 and the control 
could be explained by a fermentation pathway 
that promoted the transformation of sugars 
to AA. A heterofermentative pathway during 
ensiling stimulates the production of AA from 
the available carbohydrates (McDonald, 1981). 
In other trials, inoculation with heterofermenta-
tive bacteria, such as L. paracasei (Driehuis et 

al., 2002; Kleinschmit & Kung, 2006) improved 
the aerobic stability of silages due to the pro-
duction of AA, which also inhibits the growth 
of yeasts (Guan et al., 2020). Comparison of 
the fermentative parameters showed silages 
with characteristics of an adequate tropical 
fermentative process. Fermentation is a spon-
taneous and continuous process; changes in 
the composition of the microbiota occur at dif-
ferent times, especially at the beginning when 
changes in pH occur (Muyzer et al., 1993; Ni 
et al., 2015).

 The dominant bacteria present in pineap-
ple peel were members of the genera Weissella 
and Fructobacillus, especially W. ghanensis 
and F. trapeoli, and members of the family Ace-
tobacteraceae. Bacteria of the Weissella spp. 
have an important function at the onset of 
fermentation (Ni et al., 2015). These bacteria 
utilize water-soluble carbohydrates and convert 
them to CO2, water, and heat. During fer-
mentation of whole-crop wheat, LA-producing 
bacteria, such as Weissella, initiate lactate 
fermentation and create anaerobic conditions 
that are suitable for the development of LAB 
(Ni et al., 2015). Pseudomonas was present 
in the initial king grass material. The role of 
some genera, including Pseudomonas, in silage 

TABLE 2
Diversity indices determined for the Amplicon Sequence Variants (ASV) of the initial sample of pineapple, king grass and 
the silage treatments, 15 and 30 days post inoculation (dpi) process

Treatments N S d Fisher

pineapple 42 871 (± 10 838) 187 (± 46) 19 (± 1,7) 25 (± 6)

king grass 71 485 (± 10 471) 647 (± 114) 58 (± 9) 98 (± 18)

CK 15dpi 84 702 (± 899) 618 (± 7) 54 (± 0,57) 90 (± 1)

CK 30 dpi 88 062 (± 10 989) 622 (± 146) 54 (± 12) 90 (± 23)

CKLA15 dpi 80 893 (± 2 970) 575 (± 27) 51 (± 2) 83 (± 4)

CKLA 30 dpi 68 798 (± 13 009) 430 (± 148) 38 (± 13) 61 (± 22)

CKLI 15 dpi 87 577 (± 8 062) 609 (± 44) 53 (± 3) 88 (± 6)

CKLI 30 dpi 76 109 (± 2 827) 557 (± 72) 49 (± 6) 81 (± 12)

CKLL 15 dpi 80 808 (± 1 734) 553 (± 114) 49 (± 10) 80 (± 19)

CKLL 30 dpi 77 280 (± 1 179) 533 (± 35) 47 (± 3) 77 (± 6)

N: number of ASV, S: Shannon, d: Tajima. Mean values (± standard deviation, n = 2). Abbreviations: CKLA: king grass: 
pineapple peel silage inoculated with L. paracasei_6714, CKLi: king grass: pineapple peel silage inoculated with L. 
monocytogenes (CKLi), CKLL: king grass: pineapple peel silage inoculated with a mixture of L. paracasei_6714 and L. 
monocytogenes (1:1).



9Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Fig. 3. Phylogeny analysis using MrBayes for lactic acid bacteria (LAB) species found in this research (marked in bold) and 
compared with reference species available in databases.



10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

processes has not been studied in detail (Yang 
et al., 2019). The presence of Pseudomonas in 
silages is undesirable due to their potential to 
produce biogenic amines (Ren et al., 2018). 
In the present study and in other research, the 
abundance of Pseudomonas in silage decreased 
over time (He et al., 2020).

The strain L. paracasei_6714 (used in this 
work) inhibited L. monocytogenes growth (Wu-
Wu et al., 2021). Adding LAB inoculum has 
been shown to improve the ensiling process, 
protect ensiled material against the growth and 
survival of undesirable bacteria (Queiroz et al., 
2018), and ensure silage quality (Driehuis et 
al., 2002, Wang et al., 2006). 

In this research, NGS was used to examine 
the microbiota present in the ensiling of tropi-
cal forages and agro-industry residues. The 
use of this technique to characterize these pro-
cesses is a relatively recent technology (Keshri 
et al., 2018; Keshri et al., 2019; Muraro et al., 
2021; Wang et al., 2022; Zi et al., 2021) that 
allows a better understanding of the complex 
dynamics of microbial populations in silages 
(Keshri et al., 2019).

Our results suggest that inoculation with 
L. paracasei_6714 and L. monocytogenes can 
reduce the diversity of bacterial populations in 
silage. Diversity decreased over time (15 vs. 30 
days) in inoculated silages but not in the non-
inoculated control. Similar studies have shown 
lower diversity in silage treated with a lactoba-
cillus compared with untreated silage (Duniere 
et al., 2017; Keshri et al., 2018; Romero et 
al., 2017; Wang et al., 2016). The decrease in 
diversity is attributed to the high concentra-
tion of the added inoculum and the inhibitory 
effect of the inoculated species on the growth 
and multiplication of other microorganisms 
(Keshri et al., 2018).

The presence of LAB during the fermen-
tation process is important for the control 
of pathogenic microorganisms. When oxygen 
is consumed at the beginning of fermenta-
tion, anaerobic bacteria such as Lactobacillus 
grow and decrease the pH of the silage dur-
ing early stages of fermentation (Wang et al., 
2006). This could explain the presence of these 

microorganisms in the inoculated and control 
silages evaluated at 15 dpi. Most of the spe-
cies found in this study have been previously 
reported in silages used for animal feed. For 
example, L. sakei was the dominant species in 
brachiaria grass (Brachiaria decumbes) during 
the first 10 days, while L. plantarum was pres-
ent at all stages of storage and dominated in 
silage at 30 and 45 days (Santos et al., 2013). 
In other studies, L. brevis and L. buchneri 
predominated in maize grains stored for a pro-
longed period (Carvalho et al., 2017), L. para-
casei produces a high concentration of AA that 
inhibits the growth of molds, and yeasts and 
helps to improve the aerobic stability of silages 
(Blajman et al., 2018).

Although two treatments were inoculated 
with L. monocytogenes, this microorganism 
was not detected in the sequencing analyses. 
Listeria has been reported in more than 50 % of 
fecal samples from animals without symptoms 
of listeriosis (Nightingale et al., 2004). This 
is attributed mainly to the ingestion of con-
taminated silage with high loads of this patho-
gen, although other causes have been reported 
(Meng & Doyle, 1997).

Silage of pineapple residues and king grass 
forage are used for animal feed in Costa Rica 
and other countries in the tropics (Araya & 
Boschini, 2005). To the best of our knowledge, 
this is the first study to evaluate the potential 
of LAB isolated in Costa Rica from agroin-
dustrial pineapple silages to inhibit the growth 
of pathogenic microorganisms. The absence 
of L. monocytogenes suggests that growth 
of opportunistic pathogenic bacteria such as 
Listeria could be prevented with appropriate 
management of the silage process in the field. 
The risk of contamination is likely to increase 
when silages are exposed to air for feeding; 
multiplication of undesirable microorganisms 
may occur, and nutritional values may decrease 
(Driehuis & Oude-Elferink, 2000).

Proper management of silage processing 
can reduce and/or eliminate pathogenic bacte-
ria. Inoculation with L. paracasei_6714 did not 
improve the physicochemical characteristics or 



11Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

the associated microbiota of pineapple peel and 
king grass silage. 

This study highlights the importance of 
nutritional and microbiological characteriza-
tion of residues used for animal feed. In 
addition, the correct preparation of silages 
can help control pathogenic microorganisms 
in the field. Future research should consider 
other storage conditions, such as temperature 
and humidity, and evaluate potential protec-
tive effects of inoculated LAB against myco-
toxin-producing fungi.

Ethical statement: the authors declare 
that they agree with this publication and that 
each made significant contributions; that there 
is no conflict of interest of any kind; and that 
we followed all pertinent ethical and legal pro-
cedures and requirements. All financial sources 
are fully and clearly stated in the acknowledg-
ment section. A signed document has been 
filed in the journal archives.

ACKNOWLEDGMENTS

The work was supported by a grant from 
MICITT (Grant No. 735-B5-505) and Univer-
sity of Costa Rica (Grant No. 735-B9-457). 
The authors thank the producers who donated 
king grass and pineapple residues for the 
silage preparation.

REFERENCES

Alfaro, E., & Quesada, A. (2010). Ocurrencia de ciclones 
tropicales en el Mar Caribe y sus impactos sobre 
Centroamérica. Intersedes, 11, 136–153.

Araya, M., & Boschini, C. (2005). Producción de forraje 
y calidad nutricional de variedades de Pennisetum 
purpureum en la Meseta Central de Costa Rica. Agro-
nomía Mesoamericana, 16, 37–43.

Association of Official Analytical Chemist (AOAC). 
(1998). Official Methods of Analysis of AOAC Inter-
national (16th ed). AOAC.

Auerbach, H., & Nadeau, E. (2020). Effects of additive 
type on fermentation and aerobic stability and its 
interaction with air exposure on silage nutritive value. 
Agronomy, 10, 1229. 

Ávila, C., & Carvalho, B. (2019). Silage fermentation-
update focusing on the performance of micro-
organisms. Journal of Applied Microbiology, 128, 
966–984. 

Birnboim, H. C., & Doly, J. (1979). A rapid alkaline extrac-
tion procedure for screening recombinant plasmid 
DNA. Nucleic Acids Research, 7, 1513–1523. 

Blajman, J. E., Páez, R. B., Vinderola, C. G., Lingua, M. 
S., & Signorini, M. L. (2018). A meta-analysis on 
the effectiveness of homofermentative and heterofer-
mentative lactic acid bacteria for corn silage. Journal 
Applied Microbiology, 125, 1655–1669. 

Bolyen, E., Rideout, J. R., Dillon, M. R., Bokulich, N. A., 
Abnet, C. C., Al-Ghalith, G. A., Alexander, H., Alm, 
E. J., Arumugam, M., Asnicar, F., Bai, Y., Bisanz, J. 
E., Bittinger, K., Brejnrod, A., Brislawn, C. J., Brown, 
C. T., Callahan, B. J., Caraballo-Rodríguez, A. M., 
Chase, J., ... & Caporaso, J. G. (2019). Reproducible, 
interactive, scalable and extensible microbiome data 
science using QIIME 2. Nature Biotechnology, 37(8), 
852–857.

Callahan, B., McMurdie, P., Rosen, M., Han, A., Johnson, 
A. J., & Holmes, S. (2016). DADA2: High-resolution 
sample inference from Illumina amplicon data. Natu-
re Methods, 13, 581–583. 

Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., 
Bushman, F. D., Costello, E. K., Fierer, N., Gonzales 
Peña, A., Goodrich, J. K., Gordon, J. I., Huttley, G. 
A., Kelley, S. T., Knights, D., Koenig, J. E., Ley, R. 
E., Lozupone, C. A., McDonald, B. D., Pirrung, M., 
... & Knight, R. (2010). QIIME allows analysis of 
high-throughput community sequencing data. Nature 
Methods, 7(5), 335–336. 

Carvalho, B. F., Ávila, C. L., Bernardes, T. F., Pereira, M. 
N., Santos, C., & Schwan, R. F. (2017). Fermentation 
profile and identification of lactic acid bacteria and 
yeasts of rehydrated corn kernel silage. Journal of 
Applied Microbiology, 122, 589–600. 

Cao, Y., Cai, Y., Takahashi, T., Yoshida, N., Tohno, M., 
Uegaki, R., Nonaka, K., & Terada, F. (2011). Effect of 
lactic acid bacteria inoculant and beet pulp addition 
on fermentation characteristics and in vitro ruminal 
digestion of vegetable residue silage. Journal Dairy 
Science, 94, 3902–3912.

Contreras-Govea, F., Muck, R. E., Broderick, G. A., & 
Weimer, P. J. (2013). Lactobacillus plantarum effects 
on silage fermentation and in vitro microbial yield. 
Animal Feed Science Technology, 179, 61–68. 

Dhama, K., Karthik, K., Tiwari, R., Shabbir, M. Z., 
Barbuddhe, S., Veer, S., & Singh, R. K. (2015). 
Listeriosis in animals, its public health significance 
(food-borne zoonosis) and advances in diagnosis and 
control: a comprehensive review. Veterinary Quar-
terly, 35, 211–235. 



12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Driehuis, F., & Oude-Elferink, S. J. W. H. (2000). The 
impact of the quality of silage on animal health 
and food safety: a review. Veterinary Quarterly, 22, 
212–216. 

Driehuis, F., Oude-Elferink, S. J. W. H., & Van Wikselaar, 
P. G. (2002). Fermentation characteristics and aerobic 
stability of grass silage inoculated with Lactobacillus 
buchheri, with or without homofermentative lactic 
acid bacteria. Grass Forage Science, 56, 330–343. 

Duniere, L., Xu, S., Long, J., Elekwachi, C., Wang, Y., Tur-
kington, K., Forster, R., & McAllister, T. A. (2017). 
Bacterial and fungal core microbiomes associated 
with small grain silages during ensiling and aerobic 
spoilage. BMC microbiology, 17(1), 1–16.

Guan, H., Ke, W., Yan, Y., Shuai, Y., Li, X., Ran, Q., Yang, 
Z., Wang, X., Cai, Y., & Zhang, X. (2020). Screening 
of natural lactic acid bacteria with potential effect on 
silage fermentation, aerobic stability and aflatoxin B1 
in hot and humid area. Journal of Applied Microbiolo-
gy, 128, 1301–1311. 

Gutiérrez, F., Rojas-Bourrillón, A., Dormond, H., Poore, 
M., & WingChing-Jones, R. (2003). Características 
nutricionales y fermentativas de mezclas ensiladas de 
desechos de piña y avícolas. Agronomía Costarrin-
cense, 27(1), 79–89.

He, L., Lv, H., Xing, Y., Wang, C., You, X., Chen, X., & 
Zhang, Q. (2020). The nutrients in Moringa oleifera 
leaf contribute to the improvement of stylo and alfalfa 
silage: Fermentation, nutrition and bacterial commu-
nity. Bioresource Technology, 301, 122733. 

Huyen, N., Martínez, I., & Pellikaan, W. (2020). Using 
lactic acid bacteria as silage inoculants or direct-fed 
microbials to improve in vitro degradability and 
reduce methane emissions in dairy cows. Agronomy, 
10, 1482. 

Idland, L., Granquist, E. G., Aspholm, M., & Lindbäck, 
T. (2022). The prevalence of Campylobacter spp., 
Listeria monocytogenes and Shiga toxin‐producing 
Escherichia coli in Norwegian dairy cattle farms: A 
comparison between free stall and tie stall housing 
systems. Journal of Applied Microbiology, 132(5), 
3959–3972.

Keshri, J., Chen, Y., Pinto, R., Kroupitski, Y., Weinberg, Z. 
G., & Sela-Saldinger, S. (2018). Microbiome dyna-
mics during ensiling of corn with and without Lacto-
bacillus plantarum inoculant. Applied Microbiology 
and Biotechnology, 102, 4025–4037. 

Keshri, J., Chen, Y., Pinto, R., Kroupitski, Y., Weinberg, Z. 
G., & Sela-Saldinger, S. (2019). Bacterial dynamics 
of wheat silage. Frontiers in Microbiology, 10, 1532. 

Kim, D., Lee, K., & Choi, K. (2020). Role of LAN in silage 
fermentation: Effect on nutritional quality and orga-
nic acid production-An overview. AIMS Agriculture 
and Food, 6, 216–234.

Kleinschmit, D. H., & Kung, L. J. (2006). A meta-analysis 
of the effects of Lactobacillus buchneri on the fer-
mentation and aerobic stability of corn and grass 
and small-grain silages. Journal of Dairy Science, 
89, 4005–13. 

Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, 
C., Horn, M., & Glöckner, F. O. (2013). Evaluation 
of general 16S ribosomal RNA gene PCR primers 
for classical and next-generation sequencing-based 
diversity studies. Nucleic Acids Research, 41(1), 
e1–e1.

Leiva., A., & Granados-Chinchilla, F. (2020). Fatty acid 
profiling in animal feed and related food matrixes 
using a fast GC/MS method and in situ derivatization. 
International Journal of Agriculture, Environment 
and Food Sciences, 4, 70–89. 

López-Herrera, M., Rojas-Bourrillón, A., & Castillo-Uma-
ña, M. (2019). Efecto de la sustitución de King grass 
(Cenchrus purpureus) por yuca (Manihot esculenta 
crantz) sobre la calidad del ensilaje. Nutrición Animal 
Tropical, 13, 21–42. 

López-Herrera, M., WingChing-Jones, R., & Rojas-Bourri-
llón, A. (2014). Meta análisis de los subproductos de 
piña (Ananas comosus) para la alimentación animal. 
Agronomía Mesoamericana, 25(2), 383–392.

McDonald, P. (1981). The biochemistry of silage. John 
Wiley Ltd.

Meng, J., & Doyle, M. P. (1997). Emerging issues in micro-
biological food safety. Annual Review of Nutrition, 
17, 255–275. 

Muraro, G. B., de Almeida-Carvalho-Estrada, P., de Oli-
veira-Pasetti, M. H., Santos, M. C., & Nussio, L. 
G. (2021). Bacterial dynamics of sugarcane silage 
in the tropics. Environmental Microbiology, 23(10), 
5979–5991.

Muyzer, G., de Waal, E. C., & Uitterlinden, A. G. (1993). 
Profiling of complex microbial populations by dena-
turing gradient gel electrophoresis analysis of poly-
merase chain reaction-amplified genes coding for 
16S rRNA. Applied and Environmental Microbiolo-
gy, 59(3), 695–700. 

National Research Council (NRC). (2001). Nutrient requi-
rements of dairy catlle (7th rev. ed.). National aca-
demy press.

Ni, K., Wang, Y., Cai, Y., & Pang, H. (2015). Natural lactic 
acid bacteria population and silage fermentation of 
whole-crop wheat. Asian-Australasian Journal of 
Animal Sciences, 28, 1123–1132.

Nightingale, K. K., Schukken, Y. H., Nightingale, C. R., 
Fortes, E. D., Ho, A. J., Her, Z., Grohn, Y. T., & 
Wiedmann, M. (2004). Ecology and transmission of 
Listeria monocytogenes infecting ruminants and in 



13Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

the farm environment. Applied and Environmental 
Microbiology, 70(8), 4458–4467. 

Pulido, R., García, E., Beltrán, I., Daza, J., Keim, J., & 
Castillo, I. (2020). Effect of a homofermentative 
lactic acid bacteria additive on the quality of sugar-
beet pulp silage and animal response of dairy cows 
with restricted access to pasture. Chilean Journal of 
Agricultural Research, 80(2), 234–240. 

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., 
Yarza, P., Peplies, J., & Glöckner, F. O. (2013). The 
SILVA ribosomal RNA gene database project: Impro-
ved data processing and web‐based tools. Nucleic 
Acids Research, 41(D1), D590–D596.

Queiroz, O., Ogunade, I., Weinberg, Z., & Adesogan, T. 
(2018). Silage review: Foodborne pathogens in silage 
and their mitigation by silage additives. Journal of 
Dairy Science, 101, 4132–4142. 

Rahman, M. M., Abdullah, R. B., Khadijah, W. E., Naka-
gawa, T., & Akashi, R. (2014). Feed intake and 
growth performance of goats supplemented with 
soy waste. Pesquisa Agropecuária Brasileira, 49, 
554–558. 

Ren, H., Wang, C., Fan, W., Zhang, B., Li, Z., & Li, D. 
(2018). Effects of formic acid or acetic acid on the 
mixed-storage quality of air-dried maize stover and 
cabbage wastes and microbial community analysis. 
Food Technolgy and Biotechnology, 56, 71–82. 

Romero, J. J., Zhao, Y., Balseca-Paredes, M. A., Tiezzi, F., 
Gutiérrez-Rodríguez, E., & Castillo, M. S. (2017). 
Laboratory silo type and inoculation effects on nutri-
tional composition, fermentation, and bacterial and 
fungal communities of oat silage. Journal of Dairy 
Science, 100, 1812–1828. 

Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes 3: 
Bayesian phylogenetic inference under mixed models. 
Bioinformatics, 19, 1572–1574.

SAS Institute Inc. (2011). Base SAS Procedures Guide: 
Statistical Procedures. SAS Institute Inc.

Santos, A. O., Ávila, C. L. S., & Schwan, R. F. (2013). 
Selection of tropical lactic acid bacteria for enhancing 
the quality of maize silage. Journal of Dairy Science, 
96, 7777–7789.

Santoso, B., Hariadi, B. T., & Jeni, J. (2015). Fermenta-
tion quality of king grass silage treated with liquid 
or dried inoculants of lactic bacteria. Journal of 
the Indonesian Tropical Animal Agriculture, 40(4), 
208–214.

da Silva, N. C., Nascimento, C. F., Nascimento, F. A., 
de Resende, F. D., Daniel, J. L. P., & Siqueira, G. 
R. (2018). Fermentation and aerobic stability of 
rehydrated corn grain silage treated with different 
doses of Lactobacillus buchneri or a combination of 

Lactobacillus plantarum and Pediococcus acidilacti-
ci. Journal Dairy Science, 101, 4158–4167.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., & 
Kumar, S. (2013). MEGA6: Molecular evolutionary 
genetics analysis version 6.0. Molecular Biology and 
Evolution, 30(12), 2725–2729. 

Thierry, A., Deutsch, S. M., Falentin, H., Dalmasso, M., 
Cousin, F. J., & Jan, G. (2011). New insights into 
physiology and metabolism of Propionibacterium 
freudenreichii. International Journal of Food Micro-
biology, 149(1), 19–27.

Tobía, C., Rojas, A., Villalobos, E., Soto, H., & Uribe, L. 
(2004). Use of soybean silage to partially substitute 
balanced concentrate and its effects on dairy milk 
production and quality, in the wet tropics of Costa 
Rica. Agronomía Costarricense, 28, 27–35.

Van-Soest, P. J., & Robertson, J. B. (1985). Analysis of 
forages and fibrous foods: a laboratory manual for 
animal science. Cornell University.

Villalobos-Villalobos, L., Arce-Cordero, J., WingChing-
Jones, R. (2015). Costos de producción de ensilados 
de pastos tropicales elaborados en lecherías de Costa 
Rica. Nutrición Animal Tropical, 9, 27–48.

Wang, X., Haruta, S., Wang, P., Ishii, M., Igarashi, Y., 
& Cui, Z. (2006). Diversity of a stable enrichment 
culture which is useful for silage inoculant and its 
succession in alfalfa silage. FEMS Microbiology 
Ecology, 57(1), 106–115. 

Wang, S., Shao, T., Li, J., Zhao, J., & Dong, Z. (2022). 
Fermentation profiles, bacterial community compo-
sitions, and their predicted functional characteristics 
of grass silage in response to epiphytic microbiota 
on legume forages. Frontiers in Microbiology, 13, 
830888. 

Wang, Y. S., Shi, W., Huang, L. T., Ding, C. L., & Dai, C. 
C. (2016). The effect of lactic acid bacterial starter 
culture and chemical additives on wilted rice straw 
silage. Animal Science Journal, 87(4), 525–535.

WingChing-Jones, R., Redondo-Solano, M., Usaga, J., 
Uribe, L., & Barboza, N. (2021). Tipificación con 
secuencias multilocus en Lactobacillus casei proce-
dentes de ensilados de cáscara de piña. Agronomía 
Mesoamericana, 32, 508–522.

Wu-Wu, J., Redondo-Solano, M., Uribe, L., WingChing-
Jones, R., Usaga, J., & Barboza, N. (2021). First cha-
racterization of the probiotic potential of lactic acid 
bacteria isolated from Costa Rican pineapple silages. 
PeerJ, 9, e12437.

Yang, L., Yuan, X., Li, J., Dong, Z., & Shao, T. (2019). 
Dynamics of microbial community and fermentation 
quality during ensiling of sterile and nonsterile alfalfa 
with or without Lactobacillus plantarum inoculant. 
Bioresource Technology, 275, 280–287. 



14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, 
H., & Chun, J. (2017). Introducing EzBioCloud: a 
taxonomically united database of 16S rRNA gene 
sequences and whole-genome assemblies. Internatio-
nal Journal of Systematic and Evolutionary Micro-
biology, 67(5), 1613–1617.

Yoshida, T., Kato, Y., Sato, M., & Hirai, K. (1998). Sources 
and routes of contamination of raw milk with Listeria 
monocytogenes and its control. The Journal of Veteri-
nary Medical Science, 60, 1165–1168.

Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M., Harris, H. 
M., Mattarelli, P., O’Toole, P. W., Pot, B., Vandamme, 

P., Walter, J., Wantanabe, K., Wuyts, S., Felis, G. E., 
Gänzle, M. G., & Lebeer, S. (2020). A taxonomic 
note on the genus Lactobacillus: Description of 23 
novel genera, emended description of the genus Lac-
tobacillus Beijerinck 1901, and union of Lactobaci-
llaceae and Leuconostocaceae. International Journal 
of Systematic and Evolutionary Microbiology, 70(4), 
2782–2858. 

Zi, X., Li, M., Chen, Y., Lv, R., Zhou, H., & Tang, J. (2021). 
Effects of citric acid and Lactobacillus plantarum on 
silage quality and bacterial diversity of king grass 
Silage. Frontiers in Microbiology, 12, 631096.