Isolation of lactic acid bacteria and quantification of Lentilactobacillus buchneri using qPCR in sorghum silage inoculated with native strains in tropical conditions

Abstract

The objective of this study was to isolate lactic bacteria, quantify the population of Lentilactobacillus buchneri by qPCR, and evaluate the effects of the inoculation of native strains of L. buchneri in sorghum silage. The treatments were arranged in a 4 × 5 factorial scheme, with four inoculants (I) and five fermentation periods (P) (7, 14, 28, 45, and 90), in a completely randomized design, with three replicates. Forty-seven LAB strains were isolated from control silage, and L. buchneri was the predominant species at 45 and 90 days of fermentation. The qPCR data showed that L. buchneri predominated during all fermentation periods in the inoculated silages. There was an effect (P<0.05) of I × P interaction on all studied variables of the fermentative profile and microbial population. There was an effect (P<0.05) of I × P interaction on the dry matter (DM), neutral detergent fiber (NDF), in vitro DM digestibility, and in vitro NDF digestibility. Inoculation with L. buchneri resulted in silages with better fermentative quality and digestibility and a lower yeast population. The native LB.1 and LB.4 strains have potential to be used as inoculants in sorghum silage production, with effects on fermentation quality at 45 days of storage.

Key words
16S rRNA gene; fermentation; molecular technique; tropical grass

INTRODUCTION

Globally, sorghum (Sorghum bicolor (L.) Moench) is an important forage used to produce silage, especially in tropical and subtropical climates. In addition to the suitable characteristics for the fermentation process, sorghum has a high yield of dry matter, can tolerate water deficit, high temperatures, and low soil fertility, and allows the harvesting of plant regrowth (Weinberg et al. 2011, Amer et al. 2012, Costa et al. 2016).

Lactic acid-producing bacteria (LAB) are the main components of interest in the microbiota of forage crops intended for silage production. Thus, in tropical regions, studies have been developed with the aim of isolating, characterizing, and identifying epiphytic microorganisms that have potential for use as an inoculant (Ávila et al. 2009, Silva et al. 2018, Agarussi et al. 2022), as the majority of commercial inoculants are composed of strains of bacteria isolated from temperate forages (Heinritz et al. 2012).

Lentilactobacillus buchneri (LB) is an obligate heterolactic LAB often used as an inoculant to improve the fermentative profile and increase the aerobic stability in silages (Da Silva et al. 2018, Gallo et al. 2018, Arriola et al. 2021a, Kung et al. 2021). This microorganism metabolizes lactic acid and produces moderate amounts of acetic acid (AA), which, under low pH conditions, has antifungal activity (Moon, 1983). The highest anaerobic conversion of lactic acid into AA occurs mainly after 45 days of fermentation, as demonstrated previously (Oude Elferink et al. 2001, Kleinschmit & Kung 2006, Schmidt et al. 2008, Arriola et al. 2021a).

However, it is necessary to carry out studies to better understand the growth of L. buchneri in silages, mainly using autochthonous strains isolated from tropical conditions, as well as to quantify the presence of LB during fermentation and its relationship with fermentation products, thus facilitating the more efficient use of these microorganisms to improve the quality of silages. In this sense, molecular techniques, such as the quantitative polymerase chain reaction (qPCR), are specific and sensitive for the rapid detection of bacteria based on the sequences of the 16S rRNA gene in different environments, such as silages (Schmidt et al. 2008, Lynch et al. 2012, Tran et al. 2018, Xu et al. 2019) and rumen (Ding et al. 2014).

Therefore, the objective was to isolate and identify LAB in sorghum silage to quantify the population of L. buchneri by qPCR and to evaluate the changes in the fermentation profile and chemical composition of these silages inoculated with autochthonous strains. We hypothesized that using LAB strains isolated from sorghum silage enhances its fermentation under tropical conditions.

MATERIALS AND METHODS

Location and silage preparation

The experiment was carried out at the Department of Animal Science of the Universidade Federal de Viçosa (UFV, Viçosa, MG, Brazil). Viçosa is located at 20°45’ south latitude, 42°51’ west longitude, and 657 m above sea level, with a mean annual rainfall of 1341 mm.

Forage sorghum Volumax (Agroceres®) was harvested manually, 120 days after planting, with grains in the milky to farinaceous stage, and chopped in a stationary forage machine (model PN Plus 2000, Nogueira® S.A., São João da Boa Vista-SP, Brazil) to an average particle size of 1.5 cm. Three piles (replicates) were prepared, containing 10 kg of forage for each inoculant, totaling 12 piles, which were inoculated individually: without inoculant (CTRL, only distilled water), with L. buchneri strain 50.1 (LB.1), with L. buchneri strain 50.4 (LB.4) and with LALSIL AS, L. buchneri CNCM I-4323 (Lallemand Animal Nutrition®, Patos de Minas, Brazil) (LAS). The inoculants were diluted in 100 mL of water and sprayed onto the forage at an application rate of 10⁶ cfu/g of forage. After homogenization, 500 g of the forage was ensiled in bags (25 cm x 35 cm, Doug Care Equipment, Springville, CA, USA), and anaerobic conditions inside the mini-silo were obtained using a vacuum sealer (Eco vacum 1040, Turin, Italy). Five bags were prepared from each pile, referring to the fermentation periods, and stored in the laboratory at room temperature. Cultures of L. buchneri strains 50.1 and 50.4 were obtained from the collection of microorganisms of the Silage Microbiology Laboratory of the Department of Animal Science - UFV, previously isolated from forage sorghum silages under tropical conditions at 56 days of fermentation.

A 4 × 5 factorial scheme was used, with four inoculants, namely control (CTRL), L. buchneri strain 50.1 (LB.1), L. buchneri strain 50.4 (LB.4), and LALSIL AS, L. buchneri CNCM I-4323, Lallemand (LAS), and five fermentation periods (7, 14, 28, 45, and 90 days) in a completely randomized design, with three replications.

Fermentation profile

Water extracts from the silage or plant samples were prepared by homogenizing 25 g of sample in 225 mL of sterile solution (Ringer Solution®, Oxoid, Hampshire) in an industrial blender (Model-LB10S, Waring Laboratory®, Toronto, Canada) for 1 min. Subsequently, the extract was filtered through a double layer of sterile gauze, and the pH was measured with the aid of a potentiometer (Tecnal®, São Paulo-SP, Brazil).

A 15-mL aliquot of the extract was filtered through Whatman 54 filter paper (Whatman®, Florham, NJ) and placed in tubes containing 100 μL of H₂SO₄ 50% for the analysis of ammoniacal nitrogen (NH₃-N) (Okuda et al. 1965), water-soluble carbohydrates (WSC) (Nelson, 1944), lactic acid (LA), acetic acid (AA), propionic acid (PA), butyric acid (BA), and ethanol (ETA) by high-performance liquid chromatography (HPLC, Dionex Corporation, Sunnyvale, CA, USA) (Siegfried et al. 1984).

Microbial population

The populations of LAB, enterobacteria (ENT), molds, and yeasts in the forage before ensiling and in the respective silages were quantified. An aliquot (10 mL) of the water extract (25 g of forage/225 mL of sterile saline solution) was subjected to serial dilution (10^-1 to 10^-8), and the microorganisms were cultured on sterile Petri plates on De Man, Rogosa, and Sharpe agar (MRS) (Merck®, Darmstadt, GER) for LAB, violet red bile (Oxoid®, Basingstoke, UK) for ENT, and potato dextrose agar (Difco®, Detroit, USA), supplemented with 1.5% of 10% tartaric acid (wt/vol), for molds and yeasts, using the spread-plate plating technique for LAB and the pour-plate technique for the other microorganisms. The plates were incubated in an oven, with the temperature and period determined for each group of microorganisms as follows: ENT, 37°C/24 h; LAB, 37°C/48 h; yeast and molds, 25°C/72 and 120 h, respectively. Plates containing between 30 and 300 colony-forming units (cfu) were analyzed.

Isolation and identification of lactic acid bacteria

After 45 and 90 days of fermentation, silage samples from the CTRL treatment were used to isolate the LAB. After counting, colonies of different sizes, shapes, and colors were selected (Holt et al. 1994). Subsequently, they were streaked in MRS agar medium with 0.04 g/L of bromocresol purple and 5.0 g/L of calcium carbonate. The plates were then incubated in anaerobic jars in a BOD oven (TE 371, Tecnal®, Piracicaba, Brazil) at 37°C for 48 hours, and subsequently, the catalase test was performed with 3% hydrogen peroxide (H₂O₂). The isolates with acid production capacity and negative catalase were stored in MRS medium with 20% glycerol at -20°C and -80°C for later DNA extraction.

The DNA of the isolates was extracted using a commercial kit (Wizard Genomic DNA Purification Kit®; Promega, Madison, WI, USA). The 16S rRNA gene sequences were amplified by PCR using P027F primers (5’-GAGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGG(C/T)TACCTTGTTACGACTT-3’) (Heuer et al. 1997). The PCR reaction was performed in microtubes containing 50 µL of the following mixture: 4 µL of genomic DNA (20 ng/µL), 10 µL of GoTaq reaction buffer (5X), 3 µL of MgCl₂ (25 mmol/L), 1 µL dNTP (10 mmol/L) (Promega, Madison, WI, USA), 0.4 µl of Taq DNA polymerase (5U/µL) (Promega, Madison, WI, USA), 0.6 µL of each primer (10 µmol/L); autoclaved ultrapure water was used to reach the final volume. Amplification took place in a thermocycler (Thermal Cycler®, Techne TC-512, Analytical, Brazil) under the following conditions: initial denaturation 94°C/5 min, 30 cycles (denaturation, 94°C/30s; annealing, 60°C/30s; polymerization, 72°C/2 min), and final extension at 72°C/5 min. The PCR product was analyzed by agarose gel electrophoresis (12 g/L) in Tris borate EDTA buffer (0.5X TBE). The gel was stained with 0.5 μg/mL of ethidium bromide, and the bands were visualized under UV illumination (UVB Transilluminator, model LTB-20X20 HE, Loccus Biotecnologia, Brazil). The PCR product of approximately 1,500 bp was sent to Macrogen© (Seoul, South Korea) for purification and sequencing. Sequence similarity searches were performed using the GenBank DNA database, and the sequences were aligned using the BLASTn (Basic Local Alignment Search Tool) algorithm for nucleotides (http://www.ncbi.nlm.nih.gov/BLAST). The sequences of the 16S rRNA gene that showed similarity equal to or greater than 97% were considered as belonging to the same operational taxonomic unit (OTU) (Altschul et al. 1990).

The sequences of the isolates obtained in the present study were deposited in the GenBank database, as described in Tables IV and V.

Table IV
Taxonomic identity of isolates obtained from control treatment of sorghum silage at 45 days of fermentation.

Identification	Speciesa	Similarity	Access codeb
SS45.1	Lacticaseibacillus casei	99%	MH936275.1
SS45.2	Companilactobacillus farciminis	97%	MH936276.1
SS45.3	Lentilactobacillus buchneri	98%	MH936277.1
SS45.4	Lentilactobacillus buchneri	98%	MH936278.1
SS45.5	Lentilactobacillus buchneri	97%	MH936279.1
SS45.6	Lentilactobacillu buchneri	98%	MH936280.1
SS45.7	Lentilactobacillus buchneri	98%	MH936281.1
SS45.8	Lentilactobacillus buchneri	98%	MH936282.1
SS45.10	Lentilactobacillus buchneri	98%	MH936283.1
SS45.12	Fufurilactobacillus rossiae	98%	MH936284.1
SS45.13	Lentilactobacillus buchneri	98%	MH936285.1
SS45.14	Fufurilactobacillus rossiae	98%	MH936286.1
SS45.15	Fufurilactobacillus rossiae	98%	MH936287.1
SS45.17	Lentilactobacillus buchneri	98%	MH936288.1
SS45.18	Lentilactobacillus buchneri	98%	MH936289.1
SS45.19	Lentilactobacillus buchneri	98%	MH936290.1
SS45.21	Lentilactobacillus buchneri	98%	MH936291.1
SS45.22	Lentilactobacillus buchneri	98%	MH936292.1
SS45.23	Lentilactobacillus buchneri	98%	MH936293.1
SS45.24	Lentilactobacillus buchneri	98%	MH936294.1
SS45.25	Lentilactobacillus buchneri	99%	MH936295.1
SS45.26	Lentilactobacillus buchneri	98%	MH936296.1
SS45.27	Lentilactobacillus buchneri	98%	MH936297.1
SS45.28	Companilactobacillus farciminis	98%	MH936298.1
SS45.29	Secundilactobacillus kimchicus	98%	MH936299.1
SS45.31	Lentilactobacillus buchneri	98%	MH936300.1
SS45.32	Lentilactobacillus buchneri	98%	MH936301.1
SS45.33	Secundilactobacillus kimchicus	98%	MH936302.1
SS45.34	Lacticaseibacillus paracasei	99%	MH936303.1
SS45.35	Lentilactobacillus buchneri	98%	MH936304.1
SS45.36	Lentilactobacillus buchneri	98%	MH936305.1
SS45.37	Lentilactobacillus buchneri	98%	MH936306.1
SS45.38	Lentilactobacillus buchneri	98%	MH936307.1
SS45.40	Lentilactobacillus buchneri	98%	MH936308.1
SS45.41	Lentilactobacillus buchneri	97%	MH936309.1

• ^aNomenclature proposed by Zheng et al. (2020). ^bAccess code from the isolated on GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

Table V
Taxonomic identity of isolates obtained from control treatment of sorghum silage at 90 days of fermentation.

Identification	Speciesa	Similarity	Access codeb
SS90.7	Pediococcus pentosaceus	98%	MH936310.1
SS90.9	Enterococcus sp.	98%	MH936311.1
SS90.11	Pediococcus pentosaceus	97%	MH936312.1
SS90.13	Lentilactobacillus buchneri	98%	MH936313.1
SS90.14	Lentilactobacillus buchneri	98%	MH936314.1
SS90.16	Pediococcus pentosaceus	98%	MH936315.1
SS90.17	Pediococcus pentosaceus	98%	MH936316.1
SS90.25	Lacticaseibacillus paracasei	99%	MH936317.1
SS90.26	Lentilactobacillus buchneri	98%	MH936318.1
SS90.27	Lentilactobacillus buchneri	97%	MH936319.1
SS90.28	Lentilactobacillus buchneri	97%	MH936320.1
SS90.31	Lentilactobacillus buchneri	99%	MH936321.1

• ^aNomenclature proposed by Zheng et al. (2020). ^bAccess code from the isolated on GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

Characterization of L. buchneri and Fufurilactobacillus rossiae

Physiological, biochemical, and antimicrobial tests were used to characterize L. buchneri and F. rossiae isolated in the present study. Strains were cultured in MRS broth for 16 h at 37°C. Subsequently, they were reactivated, and the inoculum was standardized to an optical density (OD) between 0.4 and 0.5, using a spectrophotometer (630 nm). The inoculum (400 µL) was transferred to 40 mL of MRS broth, which was incubated at 37°C. At 0, 6, 12, 24, 30, 48, and 72 h, samples were collected for analysis of microbial growth, using the microdrop plating technique (Morton 2001), and pH measurement. Strains were cultivated at different temperatures (15, 37, and 45°C) and pH values (3.5, 4.5, and 8.5). Growth was monitored by reading the OD in a spectrophotometer (630 nm) after incubation in MRS broth for 24 hours (Silva et al. 2018). Antimicrobial activity was evaluated according to the method proposed by Tagg et al. (1976). After OD standardization (0.4–0.5), the isolates were plated and incubated in an anaerobic jar at 37°C/24 h. The yeasts Candida glabrata, C. tropicales, Torulaspora delbrueckii, Pichia kudriavzevii, and Saccharomyces cerevisiae were used as indicator cultures and inoculated onto the plates at a cell concentration of 10⁵ cfu/mL.

Quantitative PCR

Bacterial DNA was extracted from the plant aqueous extract and silages by the phenol: chloroform: isoamyl alcohol purification method. The primers used in the quantitative real-time reaction (qPCR) specific for L. buchneri were LBF2 (5’-GAAACAGG TGCTAATACCGTATAACAACCA-3’) and the reverse primer LBR1 (5’-CGCCTTGGTA GGCCGTTACCTTACCAACA-3’) (Integrated DNA Tech., Coralville, IA, USA), which produce a fragment of approximately 130 bp, according to Schmidt et al. (2008). The samples, in triplicate, were analyzed in a thermocycler (CFX 96 Real Time System; Bio-Rad, Hercules, CA, USA) under the following conditions: denaturation, 95°C/15 s; annealing/extension, 60°C/60 s for 40 cycles. The reaction mixture contained 6 μL of the enzyme GoTaq® qPCR Master Mix (A6001, Promega), 2 μL of DNA (50 ng/μL), 0.3 μL of each primer (10 μmol/L), and water (Nuclease free water, Promega) to adjust the final volume to 12 μL. Strain Lentilactobacillus buchneri SS45.25 was used to construct the standard curve. The isolate was cultivated in MRS broth medium at 37°C/16 h. Subsequently, the cells were collected to count the colony-forming units. The DNA was extracted as previously described, quantified in Nanodrop 2000 (Thermo Scientific), and submitted to serial dilutions (base 10) for the construction of the standard curve. Microsoft Excel 2010 was used to construct the standard curve, plotting the number of cells (y) with the value of the threshold cycle (x); y = -0.2918x + 11.982, R² = 0.99.

Chemical composition and in vitro digestibility

Forage samples before ensiling and silages were dried in a forced ventilation oven at 55°C for 72 h and then ground in a Willey mill with a 1-mm sieve. The DM (934.01 method), crude protein, CP (984.13 method), acid detergent fiber, ADF (973.18 method), and ash (942.05 method) concentrations were analyzed according to the AOAC (1990), and the neutral detergent fiber (NDF) level was determined according to Mertens (2002).

The in vitro dry matter digestibility (IVDMD) and in vitro neutral detergent fiber digestibility (IVNDFD) levels were estimated in replicated assays at different times, using the Daisy incubator (ANKOM® Technology Corp., Macedon, NY, USA), and following the method proposed by Tilley & Terry (1963) and adapted by Holden (1999). The inoculum was collected from three rumen-fistulated lactating cows fed a diet based on corn silage (60%) and concentrate (40%), on a DM basis.

Statistical analysis

Data were analyzed in a factorial scheme in a completely randomized design. The inoculants, the fermentation periods, and the interaction between the factors were considered fixed effects, according to the following model:

$$Y_{ijk}=\mu +I_i+P_j+(IP{)}_{ij}+e_{ijk},$$

where Y_ijk = response variable; µ = general constant; I_i = effect of inoculant i; P_j = effect of period j; (IP)_ij = interaction of inoculant i and period j; and e_ijk = random error assuming an independent normal distribution, NID (0, σ²). After analysis of variance, the significant interactions among the factors were unfolded and compared using the Tukey test; 0.05 was adopted as the critical level of probability for type I error, using the PROC MIXED of SAS version 9.4 (SAS 2013).

RESULTS

Plant composition and fermentation profile of silages

The chemical composition and microbial population of sorghum plant before ensiling are shown in Table I.

Table I
Chemical composition (g/kg DM), microbial population (log cfu/g NM) and pH of sorghum before ensiling.

Itema
DM (g/kg NMb)	262.97
CP	58.92
NDF	507.91
ADF	280.26
WSC	106.40
Ash	51.02
LAB	7.08
Enterobacteria	7.45
Yeast	6.29
Molds	5.39
pH	5.76

• ^aCP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; WSC = water-soluble carbohydrates; LAB = latic acid bacteria. ^bNM = natural matter.

There was an effect of the I × P interaction (P<0.05) on all evaluated silage characteristics (Table II). On the seventh day of fermentation, the CTRL silage presented a higher pH (P=0.005) compared to the other silages (Table III). However, at 90 days of fermentation, a lower pH (3.55) was recorded in the CTRL silage compared to those treated with L. buchneri (Table III). The CTRL silage showed higher (P<0.05) concentrations of LA at 28, 45, and 90 days of fermentation compared to the inoculated silages. During fermentation, the inoculated silages showed higher (P<0.05) concentrations of LA at 45 and 90 days of fermentation compared to 7, 14, and 28 days, except for the LAS treatment (Table III).

Table II
Effect of inoculant (I), period (P) and interaction (I × P) factors on the evaluated variables of sorghum silages.

Itema	P-value
	I	P	I × P	SEM b
DM	0.126	<0.001	<0.001	1.326
WSC	<0.001	<0.001	<0.001	2.583
pH	0.002	<0.001	0.002	0.008
Latic acid	<0.001	<0.001	<0.001	1.329
Acetic acid	<0.001	<0.001	<0.001	1.966
Propionic acid	<0.001	<0.001	<0.001	0.089
Ethanol	<0.001	<0.001	<0.001	0.797
Ammonia N	<0.001	<0.001	0.003	0.170
LAB	<0.001	<0.001	<0.001	0.108
Yeast	<0.001	<0.001	<0.001	0.267
Molds	<0.001	<0.001	<0.001	0.074
LBq	<0.001	<0.001	<0.001	0.214

• ^aWSC = water-soluble carbohydrates; LAB = latic acid bacteria; LBq = population of L. buchneri estimated by qPCR. ^bSEM = mean standard error.

Table III
Mean dry matter (g/kg NM^a), water-soluble carbohydrates (g/kg DM), fermentative profile (g/kg DM) and microbial populations (log cfu/g NM) of sorghum silages with or without inoculant in different fermentation periods.

Itemb	Fermentation period (days)
	7				14				28				45				90
	CTRL	LB.1	LB.4	LAS	CTRL	LB.1	LB.4	LAS	CTRL	LB.1	LB.4	LAS	CTRL	LB.1	LB.4	LAS	CTRL	LB.1	LB.4	LAS
DM	251.20aA	250.23aA	252.51aA	254.16aA	247.80aAB	238.61aB	243.73aAB	243.83aB	237.13aBC	234.30aBC	229.83aC	232.30aCD	229.61aC	233.52aBC	235.71aBC	235.71aC	215.01bD	229.37aC	230.63aC	228.76aD
WSC	62.20bA	58.82bA	54.62bA	70.95aA	46.33aB	29.80bB	27.83bB	50.00aB	24.43aC	13.30bC	11.40bC	16.70bC	18.83aC	14.20bC	10.41cC	14.80bC	18.90aC	14.80bC	10.90cC	9.60cC
pH	3.86aA	3.79bA	3.80bB	3.79bA	3.74aB	3.72aB	3.73aC	3.70aB	3.8aAB	3.78aAB	3.81aAB	3.80aA	3.77aAB	3.80aA	3.85aA	3.79aA	3.55bC	3.80aC	3.82aD	3.79aC
LA	37.67aC	36.40aC	37.92aD	35.23aC	42.73abC	40.19bBC	40.93abCD	44.49aB	63.73aB	45.30bB	47.55bBC	46.31bB	66.41aAB	54.19bA	55.20bA	50.11bAB	71.29aA	54.09bA	53.93bAB	55.42bA
AA	2.39cD	26.20abD	27.80aB	20.77bC	4.11cC	30.62aC	30.12aB	24.59bC	7.49cB	33.68aB	25.62bB	25.08bC	8.61cB	47.28aA	46.41aA	40.65bB	12.34bA	48.61aA	48.99aA	47.99aA
PA	ND	1.16aD	1.17aC	0.42bD	ND	1.30aCD	1.27aBC	0.44bD	ND	1.80aA	1.56bABC	0.60cC	ND	1.53aB	1.64aAB	1.76aA	ND	1.42bCB	1.90aA	1.24cB
BA	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Ethanol	1.32cC	2.28bA	5.82aA	2.21bB	5.87aB	1.89bAB	2.13bB	1.21cC	18.71aA	1.49cBC	1.29cC	3.35bA	18.61aA	ND	ND	ND	18.87aA	ND	ND	ND
NH3-N (% TN)	4.44bB	4.00bC	4.65abB	5.54aB	5.27aB	4.37aBC	4.25aB	4.98aB	5.66aB	4.85abB	4.36bB	4.95abB	5.60aB	4.93aB	4.71aB	5.00aB	8.35aA	7.49aA	7.48aA	7.86aA
LAB	9.36aA	9.71aA	9.52aA	9.36aB	8.42bAB	9.80aA	9.80aA	9.66aAB	7.82cBC	9.01bB	9.23bB	9.82aA	7.12aC	8.86bBC	8.93bB	9.10bC	7.20bC	8.43aC	8.34aC	8.84aC
Yeast	4.97aD	2.49bA	3.95aA	3.60abA	5.41aC	2.53bA	2.49bB	3.14bA	6.73aB	3.52bA	ND	3.04bA	7.16aA	3.34bA	ND	2.75bA	5.62aC	2.95bA	ND	3.16bA
Molds	2.61aA	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
ENT	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND

• ^aNM = natural matter. ^bWSC = water-soluble carbohydrates; LA = lactic acid; AA = acetic acid; PA = propionic acid; BA = butiric acid; NH₃-N = ammoniacal nitrogen; LAB = lactic acid bacteria; ENT = enterobacteria. ND = not detected at the lowest dilution performed (10^-1). CTRL = control;
  LB.1 = L. buchneri strain 50.1; LB.4 = L. buchneri estrain 50.4; LAS = Lalsil AS (L. buchneri CNCM I – 4323, Lallemand). Means followed by the same lowercase letter, inoculant unfolding in each period; and uppercase, period unfolding in each inoculant in the row do not differ (P>0.05) from each other by the Tukey test.

In all fermentation periods, the CTRL silage showed lower AA concentrations (P<0.05) than the treated silages. Silages inoculated with LB.1 and LB.4 strains showed higher AA concentrations (P<0.05) than LAS-treated silages at 14 and 45 days of fermentation (Table III). However, at 90 days of fermentation, the AA concentrations were similar among the silages inoculated with L. buchneri (Tabe III). During fermentation, silages treated with LAS showed higher concentrations (P<0.05) of AA at 90 days of fermentation compared to other days, whereas the concentrations of AA in silages inoculated with LB.1 and LB.4 strains at 45 days of fermentation were similar (P>0.05) to those at 90 days of fermentation (Table III).

Higher concentrations (P<0.05) of PA were observed in silages inoculated with LB.1 and LB.4 strains compared to LAS, except at 45 days of fermentation. The presence of PA was not detected in the CTRL silage during fermentation, and BA was not detected in any of the evaluated silages (Table III). The CTRL silage showed a higher concentration (P<0.05) of ethanol compared to the silage inoculated with L. buchneri in all fermentation periods, and at 45 and 90 days of fermentation, ethanol was not detected in the inoculated silage (Table III). In all evaluated silages, higher concentrations of NH₃-N (P<0.05) were observed at 90 days of fermentation (Table III).

Microbial population of silages

Inoculation with L. buchneri provided a higher (P<0.05) LAB population when compared to CTRL silage at 14, 28, 45, and 90 days of fermentation (Table III). Silages inoculated with LB.1 and LB.4 showed higher LAB populations (P<0.05) at 7 and 14 days of fermentation, with a subsequent reduction until day 90. For the LAS inoculant, lower populations were verified at 45 and 90 days (P<0.05) when compared to other days of fermentation (Table III).

At 14, 28, 45, and 90 days of fermentation, higher (P<0.05) yeast counts were recorded in the CTRL silage compared to the inoculated silages (Table III). After 28 days of fermentation, yeasts were not detected in the silages inoculated with the LB.4 isolate. There was no effect (P>0.05) for yeast population in silages treated with LB.1 and LAS during fermentation, whose mean values were 2.97 and 3.14 log cfu/g NM, respectively (Table III). Mold counts were verified only in the CTRL silage, after 7 days of fermentation. Enterobacteriaceae were not detected in the evaluated silages (Table III).

Isolation and identification of lactic acid bacteria

Forty-seven cultures were isolated at 45 and 90 days of fermentation, with a predominance of the genus Lentilactobacillus (Tables IV and V). The predominant species were L. buchneri and F. rossiae, which represented 74.29% and 8.57% of the total isolates at 45 days of fermentation, respectively (Table IV). At 90 days of fermentation, in addition to bacteria belonging to the genus Lentilactobacillus, species of the genera Pediococcus, Enterococcus, and Lacticaseibacillus were identified. The species L. buchneri and P. pentosaceus represented 50% and 33.34% of the total number of isolates after 90 days of fermentation, respectively (Table V).

Characterization of L. buchneri and F. rossiae strains

The strains of L. buchneri showed exponential growth in the batch culture medium, with an approximate increase of two log cycles, up to 24 hours, whereas the pH of the medium decreased, with final values varying between 4.0 and 4.5 (Figure 1). Isolates SS45.27, SS45.32, and SS45.38 showed less growth at pH 3.5. The maximum growth of most isolates was observed at pH 4.5 and at temperatures of 37 and 45°C (Table VI).

Figure 1
Growth curves of L. buchneri strains (a, b, c, d, e, f, g, and h) and medium pH at different in vitro batch cultivation times.

Table VI
Growth at different pH and temperatures measured by optical density (630 nm) and antagonist activity of L. buchneri and F. rossiae strains isolated from sorghum silage.

L. buchneri a
Isolated		pH		Temperature (°C)				Yeast c
	3.5	4.5	8.5	15	37	45	SC	CT	CG	th	PK
SS45.3	++	+++	+++	+	+++	+++	+++	-	-	+	-
SS45.4	++	+++	+++	+	+++	+++	+	-	-	+	-
SS45.5	++	+++	++	+	+++	++	+	-	-	+	-
SS45.6	++	+++	++	+	+++	++	+	-	-	+	-
SS45.7	++	+++	+++	+	+++	++	++	-	-	++	-
SS45.8	++	+++	+++	+	+++	++	-	-	-	+	-
SS45.10	++	+++	+++	+	+++	++	+	-	-	+	-
SS45.13	++	+++	+++	+	+++	+++	+	-	-	+	-
SS45.17	++	+++	++	+	+++	++	+	-	-	+	-
SS45.18	++	+++	++	+	+++	++	+	-	-	+	-
SS45.19	++	+++	++	+	+++	+++	+	-	-	-	-
SS45.21	++	+++	++	+	+++	+++	+	-	-	+	-
SS45.22	++	+++	++	+	+++	++	+	-	-	-	-
SS45.23	++	+++	+++	+	+++	+++	+	-	-	+	-
SS45.24	++	+++	+++	+	+++	+++	+	-	-	-	-
SS45.25	++	+++	+++	+	+++	++	+	-	-	-	-
SS45.26	++	+++	+++	+	+++	+++	+	-	-	-	-
SS45.27	+	+++	++	+	+++	+++	+	-	-	+	-
SS45.31	++	+++	++	+	+++	+++	+	-	-	+	-
SS45.32	+	++	+++	+	+++	+++	+	-	-	+	-
SS45.35	++	+++	++	+	+++	+++	+	-	-	+	-
SS45.36	++	+++	++	+	+++	+++	+	-	-	+	-
SS45.37	++	+++	++	+	+++	+++	+++	-	-	+++	-
SS45.38	+	+++	+++	+	+++	+++	+++	-	-	+++	-
SS45.40	++	+++	++	+	+++	+++	+++	-	-	+++	-
SS45.41	++	+++	++	+	+++	+++	+	-	-	+	-
SS90.13	++	+++	++	+	+++	+++	+	-	-	+	-
SS90.14	++	+++	++	+	+++	++	+	-	-	+	-
SS90.26	++	+++	++	+	+++	+++	+	-	-	+	-
SS90.27	++	+++	+++	+	+++	+++	+++	-	-	+++	-
SS90.28	++	+++	++	+	+++	+++	+++	-	-	+++	-
SS90.31	++	+++	+++	+	+++	+++	+++	-	-	+++	-
F. rossiae b
SS45.12	+	+++	+++	+	+++	++	-	-	-	+	-
SS45.14	+	+++	+++	+	+++	++	+	-	-	+	-
SS45.15	+	+++	+++	+	+++	++	-	-	-	+	-

• ^apH = 0.4–0.9 = +; >0.9–1.10 = ++; >1.10 = +++; temperature = 0.10–0.5 = +; >0.5–1.10 = ++; >1,10 = +++. ^bpH = 0.25–1 = +; >1 = +++; temperature = 0.06–0.09 = +; >0.09–0.2 = ++; >1 = +++. ^cIndicator cultures: SC = Saccharomyces cerevisiae; CT = Candida tropicalis; CG = Candida glabrata; TD = Torulaspora delbrueckii; PK = Pichia kudriavzevii. Inhibition radius in mm: >2 to ≤6 mm = +; >6 to ≤8 mm = ++; >8mm= +++; - lack of inhibition.

The F. rossiae strains grew up to 12 hours of fermentation, and their pH varied between 4.3 and 4.4. Subsequently, a reduction in viable cells was observed for up to 72 hours, with final pH values of 4.1–4.2 (Figure 2). The growth of F. rossiae strains was most pronounced at 37°C and at pH 4.5 and 8.5 (Table VI).

Figure 2
Growth curve of F. rossiae strains and medium pH at different times of in vitro batch culture.

Only Saccharomyces cerevisiae and Torulaspora delbrueckii were inhibited in the presence of L. buchneri and F. rossiae strains, whose largest inhibition halos were verified for the SS45.3 isolates SS45.37, SS45.38, SS45.40, SS90.27, SS90.28, and SS90.31 (Table VI).

Quantitative PCR

The qPCR data showed that the natural population of L. buchneri in the forage before ensiling was 2.54 log cfu/g, with an increase of approximately 2.4 log cycles at 90 days of fermentation (5.02 log cfu/g) (Figure 3). Silages inoculated with L. buchneri showed a higher population (P<0.05) than the CTRL silage, irrespective of the fermentation period (Figure 3). The highest population, 8.01 log cfu/g, was found in the silage treated with LB.4 at 7 days of fermentation. At 90 days of fermentation, there was a greater (P<0.05) population of L. buchneri in the silage inoculated with LAS when compared to the silage inoculated with LB.1 and LB.4, with values of 7.13, 6.52, and 6.51 log cfu/g, respectively (Figure 3).

Figure 3
Population of L. buchneri estimated by qPCR of sorghum silages in different fermentation periods. NM = natural matter; CTRL= control; LB.1 = L. buchneri strain 50.1; LB.4 = L. buchneri strain 50.4; LAS = Lalsil AS (L. buchneri CNCM I - 4323, Lallemand). Error bars indicate the SEM. Means followed by the same letter, in each period, do not differ by the Tukey test (P>0.05).

Regression analysis between the population of L. buchneri estimated by qPCR and the concentration of AA in silages showed a positive linear correlation (R² = 0.54, P<0.001) during fermentation. However, there was an increase in this correlation (R² = 0.79, P<0.001) when evaluating the final fermentation periods (45 and 90 days) (Figure 4b).

Figure 4
Correlation between acetic acid concentration and L. buchneri population estimated by qPCR throughout fermentation (a) and at 45 and 90 days of fermentation (b). NM = natural matter.

Chemical composition and in vitro digestibility

The DM and WSC variables were affected by the I × P interaction (Table II). Inoculated silage showed higher DM values (P<0.05) when compared to CTRL silage at 90 days of fermentation (Table III and VII). In all treatments, reductions (P<0.05) in the DM content were verified when comparing the periods from 7 to 90 days of fermentation (Table III) and 45 to 90 days of fermentation (Table VII).

Table VII
Means of chemical composition (g/kg DM), in vitro digestibility of dry matter (IVDMD) and neutral detergent fiber (IVNDFD) (g/kg DM) of sorghum silages with or without inoculant at 45 and 90 days of fermentation.

Itema		Fermentation period (days)								P-valued
	45				90
	CTRL	LB.1	LB.4	LAS	CTRL	LB.1	LB.4	LAS	SEM c	I	P	I × P
DM (g/kg NMb)	229.61aA	233.52aA	235.71aA	235.71aA	215.01bB	229.37aA	230.63aA	228.76aB	1.430	0.003	<0.001	<0.001
CP	57.43	53.82	57.12	56.70	58.70	56.08	58.33	57.30	0.384	<0.001	0.037	0.793
NDF	559.26aB	554.21aA	548.54aA	544.33aA	593.97aA	547.75bA	542.98bA	559.83bA	3.795	<0.001	0.066	0.024
ADF	319.01	315.41	318.28	309.44	342.13	317.31	313.04	316.23	2.612	0.040	0.136	0.149
IVDMD	685.31aA	693.72aA	685.09aB	701.85aA	654.74bB	694.15aA	708.97aA	709.29aA	3.837	<0.001	0.944	0.002
IVNDFD	462.90bB	497.17abA	476.03abB	501.60aB	485.90bA	503.57bA	514.13bA	559.30aA	6.070	<0.001	<0.001	0.021
Ash	56.29	56.05	55.46	53.47	57.60	54.49	58.07	56.94	0.440	0.256	0.074	0.143

• ^aCP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber. ^bNM = natural matter. ^cSEM = mean standard error. ^dI = inoculant; P = period; I × P = interaction. CTRL = control; LB.1 = L. buchneri strain 50.1; LB.4 = L. buchneri strain 50.4; LAS = Lalsil AS (L. buchneri CNCM I – 4323, Lallemand). Means followed by the same lowercase letter on the line, inoculant unfolding in each period, do not differ (P>0.05) from each other by the Tukey test. Means followed by the same uppercase letter on the line, period unfolding in each inoculant, do not differ (P>0.05) from each other by the F test.

The residual WSC concentration observed in the CTRL silage was higher (P<0.05) compared to those of the inoculated silages at 28, 45, and 90 days of fermentation. A reduction (P<0.05) in WSC contents was observed up to 28 days in all silages (Table III). When evaluating the chemical composition of the silages at 45 and 90 days of fermentation, effects of the I × P interaction (P<0.05) on DM, NDF, IVDMD, and IVNDFD were verified (Table VII). The CP content was affected by I (P<0.001) and P (P=0.037), whereas the ADF content was affected (P=0.040) only by I, and mineral matter was not affected (P>0.05) by the factors studied (Table VII). At 90 days of fermentation, the silage inoculated with L. buchneri showed lower values (P=0.004) of NDF compared to the CTRL silage (Table VII). Inoculation with LB.1, LB.4, and LAS provided a reduction (P=0.040) in ADF contents compared to CTRL silage, with values of 316.36, 315.66, 312.84, and 330.57 g/kg DM, respectively.

The IVDMD of silages at 45 days of fermentation was similar (P=0.203). However, at 90 days, the silages treated with L. buchneri showed higher (P=0.005) values compared to the CTRL. When comparing the fermentation days, silages inoculated with LB.1 (P=0.956) and LAS (P=0.438) strains showed similar values for IVDMD (Table VII). For IVNDFD, inoculation with LAS resulted in a higher (P=0.004) value for this variable compared to the CTRL silages LB.1 and LB.4 at 90 days of fermentation (Table VII). Similar values (P=0.391) were observed for this variable in the silage inoculated with the LB.1 strain at 45 and 90 days of fermentation. However, CTRL silages (P=0.045), LB.4 (P=0.003), and LAS (P=0.028) showed higher values for IVNDFD at 90 days of fermentation compared to 45 days (Table VII).

DISCUSSION

Plant composition and fermentation profile of silages

The WSC contents and the LAB population of the sorghum before ensiling were adequate to ensure good fermentation of the ensiled mass. Generally, WSC contents between 60 and 80 g/kg DM are adequate to produce good-quality silages, according to McDonald et al. (1991). The higher residual WSC concentrations in the CTRL silages at 28, 45, and 90 days of fermentation, compared to those of the inoculated silages, indicate that the application of L. buchneri increased the LAB population in these silages, resulting in a higher consumption of WSC during fermentation as it is the main substrate used by bacteria during metabolism (Table III).

The pH values from 3.55 to 3.86 indicate adequate fermentation of the evaluated silages. The pH is directly affected by the concentration of organic acids produced by LAB, with LA being most effective in reducing the pH during fermentation (Kung et al. 2018). Generally, LA is found in dissociated form (Kung et al. 2018), and in higher concentrations in ensiled mass compared to other acids (Ferrero et al. 2019a, Fernandes et al. 2020, Arriola et al. 2021a), as verified in our study. The higher pH values at 90 days of fermentation in silages treated with L. buchneri are due to the lower concentration of LA and the higher production of AA in these silages (Rabelo et al. 2017, Da Silva et al. 2018, Arriola et al. 2021b).

The reduction in the concentration of WSC in the fermentation process and the change in the pH of the medium, as verified in the present study, condition L. buchneri to the need to metabolize new substrates (Johanningsmeier & McFeeters 2015). Lactic acid is metabolized into AA and alcohol, thus reducing the concentration of non-dissociated acid in the medium, which favors its survival (Oude Elferink et al. 2001); this explains the increases in AA concentrations in the silage inoculated in the present study. Acetic acid at a pH lower than its pKa remains in an undissociated form, which has antifungal activity (Moon 1983), providing a reduction of molds and yeasts in the silage and an increase in the aerobic stability of the silages inoculated with L. buchneri (Arriola et al. 2021a, b, Agarussi et al. 2022), as observed in another study carried out in our laboratory using the same strains (Pinho RMA, unpublished data). Heinl et al. (2012) studied the genome of L. buchneri CD034 isolated from stable grass silage and identified a set of genes involved in the synthesis of exopolysaccharides. The production of exopolysaccharides is positively correlated with resistance to low pH conditions in Lactobacillus species (Sabir et al. 2010), which probably contributes to the resistance of L. buchneri in an acidic environment, as occurs in the silo.

The final PA concentration in well-fermented silages is low (<1 g/kg DM) (Kung et al. 2018). This acid also has antifungal activity, which facilitates the production of more aerobically stable silages (Muck et al. 2018, Ferrero et al. 2019b). In the present study, PA was not found in the CTRL silage, as observed by Ferrero et al. (2019a). However, silage inoculated with L. buchneri showed PA values lower than those reported by these authors in sorghum silage after 100 days of fermentation. The inoculation of L. buchneri in silages can provide increases in propionic acid concentrations due to the conversion of lactic acid into 1,2-propanediol (Oude Elferink et al. 2001), followed by the conversion of 1,2-propanediol into propionic acid by other microorganisms present in the medium, including strains of L. buchneri (Zielińska et al. 2014, 2017). An interesting finding of our study is that silage inoculated with LB.1 and LB.4 strains obtained higher PA concentrations compared to LAS treatment silages, except at 45 days of fermentation, which may contribute to the production of more aerobically stable silages in shorter storage times.

The absence of BA in the silages evaluated in the present study indicates that there was no significant metabolic activity of undesirable microorganisms, which corroborates the other fermentative characteristics observed in the silages inoculated or not with L. buchneri (Table III).

Microbial population of silages

During fermentation, the total LAB population is commonly reduced since, in addition to the low pH, the lack of substrate and fermentation products inhibits the growth of LAB (McDonald et al. 1991, Pahlow et al. 2003), as verified in the present study.

The success in using LAB as an inoculant depends on the capacity of the inoculated bacteria to grow rapidly in the ensiled mass, the presence of an adequate fermentable substrate, and the population of inoculated bacteria in relation to the forage epiphyte population (McDonald et al. 1991, Muck 2010, Lynch et al. 2012). The largest LAB population at 14, 28, 45, and 90 days of fermentation shows that inoculation with L. buchneri surpassed the natural LAB population, an important fact in the selection of inoculants for silage (Muck 2010).

The non-detection of enterobacteria in the different fermentation periods is probably due to the rapid acidification of the ensiled mass since these microorganisms are sensitive to pH decrease, as suggested by Pahlow et al. (2003).

The lower yeast population in silages treated with L. buchneri after 14 days of fermentation can be attributed to the higher production of AA and PA in these silages and, therefore, their antifungal activity (Moon 1983, Danner et al. 2003, Ferrero et al. 2019b), which can provide aerobically stable silages since yeasts are the main microorganisms responsible for the beginning of silage deterioration when exposed to air (Kung & Stanley 1982).

During ensiling, molds develop at the beginning of the process, when there is still oxygen remaining inside the silo. However, during fermentation, the absence of oxygen associated with the presence of organic acids inhibits the growth of these microorganisms (Woolford 1984, McDonald et al. 1991), which may explain the detection of molds only in the control silage, in the first period of fermentation, and after exposing the silage to air.

Isolation, characterization, and quantification of LAB

The isolation of LAB in MRS medium, grown on plates and under anaerobic conditions, with the subsequent analysis of the 16S rDNA gene fragment, allows the identification of different species in silages (Ni et al. 2015, Silva et al. 2018). The predominance of isolates of the genus Lactobacillus in our study, at 45 and 90 days of fermentation, corroborates the results reported by other studies, such as Dunière et al. (2017), Silva et al. (2018), Agarussi et al. (2018), Puntillo et al. (2020), and You et al. (2021), for small-grain silages, corn, alfalfa, sorghum, and native grasses, respectively. As fermentation progresses, there is a reduction in the pH of the ensiled mass, which selects groups that are more resistant to acidic conditions, such as species of the genus Lactobacillus (Woolford, 1984). Ogunade et al. (2018) demonstrated that the pH of corn silages was negatively correlated (R² = -0.59) with the genus Lactobacillus.

The percentages of 74.29% and 50% of the total isolates identified as L. buchneri at 45 and 90 days of fermentation, respectively, in the control silage, are probably due to the fact that these microorganisms are more effective in the anaerobic conversion of LA into AA between 45 and 60 days of fermentation (Oude Elferink et al. 2001). Corroborating our data, Silva et al. (2018) isolated 16 strains of L. buchneri in corn silage at 56 days of fermentation, corresponding to 60% of the total LAB population.

The quantification of higher populations of L. buchneri by the qPCR technique in the treated silages (Figure 3), as well as the higher concentrations of AA in these silages, demonstrate that the added strains dominated the epiphytic population in all fermentation periods. Furthermore, regression analysis showed a positive correlation between the population of L. buchneri estimated by qPCR and the concentrations of AA during fermentation, with an increase in this correlation when only the final fermentation periods, 45 and 90 days, were analyzed (Figure 4). This demonstrates a greater metabolic effectiveness of L. buchneri after 45 days of fermentation (Oude Elferink et al. 2001), as previously reported. Corroborating the assertion, Ferrero et al. (2019a), when inoculating heterofermentative LAB, including L. buchneri, in sorghum silage, observed a significant effect of inoculant and fermentation period, with higher AA values obtained at 250 days of fermentation.

An interesting finding of the present study is that silages inoculated with LB.1 and LB.4 strains showed stability in AA production after 45 days of fermentation, despite the reduction in the population estimated by qPCR at 90 days of fermentation (Table III, Figure 3). Thus, it is possible to infer that these isolates provide aerobically stable silage after 45 days of fermentation. Additional investigations are, however, needed to evaluate the genetic and metabolic characteristics of strains isolated under tropical conditions, with the aim to understand their effects when inoculated since there may be intraspecific variability between strains (Tanizawa et al. 2020).

As for the population of L. buchneri estimated by qPCR, as in the present study, Schmidt et al. (2009) also observed a reduction in the population of L. buchneri in alfalfa silages inoculated after 45 days of fermentation. Oude Elferink et al. (2001) emphasized that LA can freely enter microbial cells, decreasing the internal pH. Therefore, the bacteria, to remove the proton, hydrolyze ATP, which causes energy expenditure. Thus, when using LA as substrate, L. buchneri may show reduced growth during fermentation.

The highest population of L. buchneri, 5.02 log cfu/g of forage, estimated by qPCR at 90 days of fermentation in the CTRL silage (Figure 3), followed by an increase in AA concentration in this silage (Table III), indicates that sorghum ensilage without the use of L. buchneri requires a longer fermentation time to increase the AA concentration. That is, despite the presence of L. buchneri, from the epiphytic flora of this silage, the addition of microbial inoculants containing strains of this species resulted in higher concentrations of AA and lower yeast populations, which may provide a greater aerobic stability of silages, as already reported.

Regarding the F. rossiae isolates identified at 45 days of fermentation in sorghum silages, this species is an obligate heterofermentative bacterium whose main characteristics are the production of vitamin B₁₂ and the activity as a cofactor of other B vitamins (riboflavin and folic acid), along with the ability to use multiple carbon sources, providing metabolic flexibility in terms of nutrient availability (De Angelis et al. 2014). In addition, the high number of genes correlated with regulatory systems indicates that F. rossiae is a versatile species with a high potential for colonizing different environments (De Angelis et al. 2014) and producing different antifungal compounds (Garofalo et al. 2012, Crowley et al. 2013). However, under the conditions of the present study, in an in vitro test, F. rossiae showed moderate inhibition only for the yeasts S. cerevisiae and T. delbrueckii (Table VI) Recently, Tian et al. (2022) identified F. rossiae in silages produced with a Pennisetum purpureum hybrid (Pennisetum americanum × Pennisetum purpureum) after 120 days of storage.

The greater tolerance of L. buchneri strains to acidic conditions compared to that of F. rossiae strains can be attributed to the genetic characteristics of L. buchneri, resulting in greater metabolic effectiveness under acidic conditions (Oude Elferink et al. 2001, Heinl et al. 2012).

Chemical composition and in vitro digestibility

During the fermentation process, due to the activity of microorganisms, there is a reduction in the DM content of the ensiled forage (Roth et al. 2016, Michel et al. 2017, Rabelo et al. 2017). However, by controlling undesirable microorganisms during ensiling, such as yeasts, reductions in the DM content can be minimized (Roth et al. 2016). In the present study, silages inoculated with L. buchneri showed lower yeast populations compared to the CTRL silage, which was reflected in lower DM reductions in these silages at 90 days of fermentation.

The lower NDF content at 90 days of fermentation in the silages inoculated with L. buchneri can be attributed to the degradation of the cell wall content, which, according to Junges et al. (2013), can be considered positive in the process of supplying soluble carbohydrates to microorganisms and increasing silage intake by animals.

The higher IVDMD at 90 days of fermentation in silages treated with L. buchneri may be associated with the production of the enzyme ferulato esterase, as suggested by Kang et al. (2009). This enzyme breaks the binding of ferulic acid in the plant cell wall and my therefore enhance fiber digestion or increase the cell wall’s susceptibility to cellulolytic hydrolysis (Kang et al. 2009). These authors, when inoculating L. buchneri in corn silage, observed an increase of 4.1 percentage points in DM digestibility and 5.7 in NDF digestibility when compared to the control silage. Weinberg et al. (2007) reported an increase in IVDMD and IVNDFD when inoculating L. buchneri in wheat silage.

In the present study, although higher IVNDFD values were recorded at 90 days compared to 45 days of fermentation in CTRL silages, for LB.4 and LAS, some studies showed a reduction in IVNDFD and highlighted that this variable is only slightly affected by the ensiling time (Der Bedrosian et al. 2012, Young et al. 2012). An interesting finding of our study is that the silages inoculated with the LB.1 strain showed no difference in DM and NDF digestibilities in the different fermentation periods, which allows us to infer that the nutritive value of the silage would not be compromised when opening the silo after 45 days of fermentation.

In addition to improving silage quality, L. buchneri has a potential probiotic effect as there is a greater synthesis of microbial protein in the rumen, consequently increasing the performance of animals that consume inoculated silage (Basso et al. 2014, Rabelo et al. 2016).

CONCLUSIONS

Lentilactobacillus buchneri was the predominant epiphytic species in sorghum silages at 45 and 90 days of fermentation and the quantitative polymerase chain reaction (qPCR) was considered a suitable to quantify these microorganisms in silages.

Inoculation with L. buchneri provided silages with a higher AA content and a lower yeast population after 14 days of fermentation, which may result in silage with greater aerobic stability. The autochthonous strains LB.1 and LB.4 have the potential to be used as inoculants for silage production, with positive effects on fermentation after 45 days of storage.

ACKNOWLEDGMENTS

We thank to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Instituto Nacional de Ciência e Tecnologia em Ciência Animal (INCT-CA) for their invaluable financial support and assistance.

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Publication Dates

• Publication in this collection
  31 Mar 2025
• Date of issue
  2025

History

• Received
  15 July 2024
• Accepted
  9 Dec 2024