Revista 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

2Revista 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.

Resultados: 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.

Revista 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

4Revista 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.

Revista 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)b11.21 (± 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)b4.40 (± 0.06)ab 4.43 (± 0.03)ab 4.49 (± 0.04)a

Ammonia nitrogen /N-total 4.90 (± 0.10)b5.33 (± 0.35)b30.40 (± 1.42)a26.83 (± 3.84)a

Lactic acid 0.00 (± 0.00)b0.00 (± 0.00)b4.43 (± 0.36)a4.16 (± 0.61)a

Acetic acid 7.94 (± 0.74)a6.30 (± 0.80)b0.50 (± 0.04)c0.49 (± 0.04)c

Propionic acid 2.25 (± 0.25)a2.26 (± 0.34)a1.17 (± 0.17)b1.44 (± 0.26)b

Isobutyric acid 0.39 (± 0.03)b0.35 (± 0.06)b0.58 (± 0.14)a0.63 (± 0.04)a

Butyric Acid 5.14 (± 0.09)a3.10 (± 0.05)b0.11 (± 0.02)c0.15 (± 0.01)c

Total Acids 15.72 (± 0.79)a12.01 (± 1.13)b6.69 (± 0.29)c6.87 (± 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.

6Revista 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).

Revista 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.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50692, enero-diciembre 2023 (Publicado Mar. 01, 2023)

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).

Revista 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

Revista 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.

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