Role of lactic acid bacteria inoculants in optimizing fermentation dynamics and nutrient retention in alfalfa silage at different moisture conditions

Ilavenil Soundharrajan; Jeong Sung Jung; Jae Hyuk Kim; Jae-Hoon Woo; Ki Won Lee; Min Gon Kim; Seung Min Jeong; Ki Choon Choi

doi:10.25259/JKSUS_1116_2025

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Research Article

2025

:37;

11162025

doi:

10.25259/JKSUS_1116_2025

Role of lactic acid bacteria inoculants in optimizing fermentation dynamics and nutrient retention in alfalfa silage at different moisture conditions

Ilavenil Soundharrajan^a,†, Jeong Sung Jung^a,†, Jae Hyuk Kim^b, Jae-Hoon Woo^a, Ki Won Lee^a, Min Gon Kim^c, Seung Min Jeong^a, Ki Choon Choi^a,*

aDepartment of Forage Production System Division, National Institute of Animal Science, Seonghwan-eup, Seobuk, Chungnamdo, 31000, Korea, Republic of Korea

bDepartment of Agriculture, Korea National Open University, Seoul 03087, Korea, Seoul, 03087, Korea, Republic of Korea

cDepartment of Animal Science, Chonnam National University, 77, Yongbong-ro, Buk-gu, Gwangju, 61186, Korea, Republic of Korea

† These authors contributed equally to this work.

* Corresponding author E-mail address: choiwh@korea.kr (KC Choi)

Received: 2025-07-03, Accepted: 2025-09-09, Epub ahead of print: 2025-10-14, Published: 2025-10-18

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Abstract

The use of lactic acid bacteria (LAB) inoculants significantly improved the fermentation quality of silage by inhibiting undesirable microbial growth. The present study evaluated the beneficial role of various LAB on the fermentation characteristics, microbial profiles, and nutrient content of alfalfa silage under different moisture conditions after 3 and 6 months of ensiling. LAB strains, including Leuconostoc citreum - KCC-57, L. citreum- KCC-58, Lactococcus lactis-RWP-3, L. lactis-RWP-7, and a cocktail inoculum, were applied and ensiled. Inoculated silages exhibited a significant reduction in pH and an increase in lactic acid (LA) content, particularly under high-moisture conditions. L. citreum- KCC-58 and the cocktail LAB showed the most notable improvements in LA production and undesirable microbial suppression. Also, LAB treatments slightly altered the crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) levels compared with control silages. Total bacterial and LAB counts were elevated in treated silages, while yeast and mold populations were markedly suppressed. This study demonstrated that different LAB inoculants, particularly L. citreum- KCC-58 and the cocktail combination can be effectively used to improve microbial activity, silage preservation quality and nutritional content over extended storage periods.

Keywords

Alfalfa

Different moistures

Ensiling

Lactic acid bacteria

Storage periods

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1. Introduction

Medicago sativa (Alfalfa) is a high crude protein (CP)-containing legume species widely cultivated for ruminant feeding due to its excellent nutritional value, particularly its high CP and digestible fiber content. However, effective ensiling of alfalfa remains challenging because of its high buffering capacity and relatively low concentrations of water-soluble carbohydrates (WSCs), which limit the natural proliferation of lactic acid bacteria (LAB) and hinder rapid acidification during fermentation (McDonald Henderson 1962, Muck et al., 2018, Wang et al., 2025). Consequently, poor fermentation can lead to nutrient losses, mold and yeast proliferation, and reduced aerobic stability.

Recently, microbial additives have attracted more attention for improving silage fermentation and enhancing the digestibility rate of forage silages (Okoye et al., 2023). LAB additives include both homo-lactic and hetero-lactic bacteria. Homofermentative LAB utilize WSCs in plants and convert them into essential organic acids, particularly LA, which accelerates pH reduction (Hashemzadeh-Cigari et al., 2014). Therefore, greater emphasis has been placed on homolactic inoculants for silage production to minimize dry matter (DM) content losses compared with heterolactic bacteria. Heterolactic bacteria ferment sugars in forages and convert them into LA and carbon dioxide, as well as ethanol or acetic acid as by-products. In these situations, the DM content and feed value of silage could be reduced (Borreani et al., 2018; Carvalho et al., 2021).

Although heterolactic bacteria do produce significantly valuable by-products during ensiling periods that enhance aerobic stability via moderate levels of acetic acid (AA) production, this acetic acid has the potential to inhibit the growth of microbes such as yeast, mold, and other undesirable bacteria (Ferrero et al., 2021, Soundharrajan et al., 2025). Heterolactic bacteria are also used in silage production due to improvements in aerobic stability and reduced losses at feed-out, and they compensate for the modest losses of DM content (Borreani et al., 2018). Several LAB have been used in silage production, including Lactiplantibacillus plantarum, L. acidophilus, Pediococcus pentosaceus, Lactococcus lactis, and emerging strains like Leuconostoc citreum, which have demonstrated variable effectiveness depending on strain specificity, forage type, and ensiling conditions (Li et al., 2020, Zhao et al., 2025).

Recent advancements in strain isolation and selection have highlighted the potential of indigenous and multifunctional LAB strains in enhancing silage fermentation across different moisture conditions. In this context, evaluating the performance of novel LAB strains, including L. citreum KCC-57 and KCC-58, L. lactis RWP3 and RWP7, and a cocktail inoculant composed of P. pentosaceus and several L. plantarum offers critical insights into their potential to regulate silage fermentation dynamics during ensiling. The present study investigates the effect of these LAB inoculants on fermentation quality (pH, lactic and acetic acid production), microbial community shifts (total bacteria, LAB, yeast, and mold counts), and nutritional attributes (ADF, NDF, and CP) of alfalfa silage under high and low-moisture conditions after 3 and 6 months of ensiling. Understanding the interaction between moisture content and inoculant type is essential, as moisture plays a decisive role in microbial succession and acidification kinetics during silage fermentation (Yi et al., 2023, Liu et al., 2025). The study’s findings not only provide evidence for strain-specific effects on silage quality but also reinforce the value of tailored inoculant selection to optimize fermentation outcomes in variable field conditions, supporting improved feed quality and animal productivity.

2. Materials and Methods

2.1 Inoculants preparation

The inoculums used in the present study (Lactococcus lactis-RWP3, L. lactis-RWP-7, Leuconostoc citreum-KCC-57, and L. citreum-KCC-58) were isolated from triticale forage (Soundharrajan et al., 2021, Muthusamy et al., 2023). All LAB strains were grown in (De Man, Rogosa, and Sharpe broth (MRS) broth for 32 h at 37°C, and then collected by centrifugation at 4000 rpm for 45 min at 4°C. The pellets were then washed three times with sterilized distilled water and diluted in the same solvent. The bacterial colonies were enumerated using a QUANTOM™ live-cell staining kit (Logos Biosystems, Gyeonggi-do, South Korea)(Soundharrajan et al., 2020). All strains were suspended in distilled water at a final concentration of 10⁷/CFU/mL and used for silage production.

2.2 Alfalfa cultivation and silage production

Alfalfa was grown at the Grassland and Forage System Field of the National Institute of Animal Science in Cheonan, South Korea, following guidelines from the Rural Development Administration (Jung et al., 2024). Alfalfa was harvested (10% flowering stage) and wilted under field conditions for 40 to 48 h until the target moisture levels of 40-45% and 55-60% were achieved. Once the desired moisture content was reached, the forage was chopped using forage cutters into a theoretical cutting length between 1.5 and 2.5 cm. The chopped alfalfa was divided into two major groups: non-inoculated (control) and inoculated (treatment). The inoculated group was further divided into five subgroups, each group treated with a different inoculant: L. lactis RWP3, L. lactis RWP7, L. citreum-KCC-57, L. citreum KCC-58, and a LAB cocktail containing P. pentosaceus KCC-23, L. plantarum KCC-10, and L. plantarum KCC-19. All LAB strains were used for silage production at a concentration of 10⁵ CFU/g of forage during ensiling.

2.3 Sampling after fermentation

Sampling was conducted on days 90 and 180 to analyze the nutrient composition, organic acids, and microbial profiles of the alfalfa silages.

2.4 Determination of nutrient composition.

DM content of silage was determined using the oven drying method at 60°C until a constant weight was reached. Samples were subsequently powdered and passed through a 1 mm sieve to ensure uniform particle size. The content of ADF and NDF was determined in silage after the ensiling periods (Van Soest et al., 1991). The CP level in experimental silage was analyzed according to the AOAC guideline (AOAC 1990).

2.5 Organic acids quantification

For this, 10 g of fermented alfalfa silage were homogenized in 90 mL of sterile distilled water and agitated on an orbital shaker for 1 h. The resulting suspension was filtered sequentially through multiple layers of cheesecloth and a 0.2 µm membrane filter. The pH of the silage extract was then measured using a calibrated pH meter (Inolab, Thomas Scientific, NJ, USA). The pH was subsequently adjusted to 2.0 using 50% sulfuric acid, and the samples were stored at –20 °C for organic acid analysis(Arasu et al., 2014, Jung et al., 2024)

2.6 Microbial counting in experimental alfalfa silages

The microbial profiles of the experimental silages, including total bacteria, LAB, mold, and yeast, were enumerated. Then, 100 µL of diluted silage samples were poured onto an MRS agar plate (De Man, Rogosa, and Sharpe) plate for LAB enumeration and 1mL onto 3M Petri films (3M microbiology Products, St. Paul, USA) for mold and yeast detection. Total bacterial counts were enumerated using the QUANTOM Tx™ fluorescence staining method (Jung et al., 2024).

2.7 Statistical analysis

SPSS-16 was used to perform statistical analysis for all experimental data. The “One-way ANOVA” option was used to find out significant differences between experimental silages (p < 0.05 level).

3. Results

3.1 Influence of lactic acid bacteria on pH and organic acid profiles in experimental silages

Tables 1-2 represent the impact of different LAB inoculants, such as L. citreum KCC-57, L. citreum KCC-58, L. lactis-RWP3, L. lactis-RWP7, and cocktail LAB (P.pentosaceus KCC-23, L.plantarum KCC-10, and L.plantarum KCC-19), on the pH, LA, and acetic acid levels of silage across all moisture conditions after 3 and 6 months of ensiling. In high-moisture silage, after 3 months, all inoculated treatments reduced pH with values to around 4.7 compared to the control silages (5.3 ± 0.08). Silage treated with L. citreum KCC-58 produced the highest level of LA (10.0 ± 0.09 DM%) followed by L. lactis-RWP7 (9.70 ± 0.61 DM%), L. lactis-RWP3 (9.12 ± 0.19 DM%), L. citreum KCC-57 (8.95 ± 0.27 DM%), and cocktail LAB (8.02 ±0.83 DM%). L. citreum KCC-58 and L. lactis-RWP7 produced the highest level of acetic acid in alfalfa silage compared to other strains and the control. Under low moisture conditions, after 3-month fermentation, the control maintained a higher pH (6.2 ± 0.05) and extremely lower LA compared to silage produced with LA inoculants. However, alfalfa silage treated with inoculants showed a slight reduction in pH values, with around 5.9 to 6.1. Cocktail LAB-treated silages showed the lowest pH (5.9 ± 0.09) with the highest LA (0.95 ± 0.08 DM%) compared to other LAB-treated alfalfa silage. Alfalfa silage developed with L. citreum-KCC-57 showed a higher acetic acid content compared to the control silage (p< 0.05). There were no significant differences in acetic acid among LAB treatments.

Table 1. Effects of LAB inoculants on pH and organic acid level of alfalfa silage at varying moisture levels after 3 months of ensiling.

Groups	High-Moisture
	pH	LA (DM%)	AA(DM%)	LA/AA ratio
Control	5.33 ± 0.08^a	4.77 ± 0.81c	1.74 ± 0.13^ab	2.80 ± 0.96^b
L. citrum-KCC-57	4.75 ± 0.01^b	8.95 ± 0.27^ab	1.68 ± 0.14^ab	5.36 ± 0.42^a
L. citrum- KCC-58	4.73 ± 0.02^b	10.0 ± 0.09^a	2.05 ± 0.19^a	4.96 ± 0.72^ab
L. lactis-RWP3	4.74 ± 0.00^b	9.12 ± 0.19^ab	1.70 ± 0.19^ab	5.44 ± 0.85^a
L. lactis -RWP7	4.74 ± 0.05^b	9.70 ± 0.61^ab	2.11 ±0.08^a	4.62 ± 0.67^ab
Cocktail LAB	4.72 ± 0.10^b	8.02 ± 0.83^b	1.47 ± 0.14^b	5.56 ± 0.89^a
Low-Moisture
Control	6.2 ± 0.05^a	0.04 ± 0.05^c	0.27 ± 0.07^b	0.00 ± 0.00^c
L. citrum-KCC-57	6.0 ± 0.02^ab	0.57 ± 0.01^ab	0.43 ± 0.01^a	1.32 ±0.01^b
L. citrum- KCC-58	6.1 ± 0.02^a	0.27 ± 0.01^b	0.38 ± 0.02^ab	0.70 ± 0.31^b
L. lactis-RWP3	6.1 ± 0.04a	0.42 ± 0.17^ab	0.32 ± 0.00^ab	1.28 ± 0.37^b
L. lactis -RWP7	6.1 ± 0.02^a	0.21 ± 0.01^b	0.38 ± 0.05^ab	0.57 ± 0.15^b
Cocktail LAB	5.9 ± 0.09^c	0.95 ± 0.08^a	0.40 ±0.02^ab	2.36 ± 0.47^a

AA: acetic acid; Cocktail LAB (P. pentosaceus –KCC-23 and L.plantarum - KCC-10 + L.plantarum - KCC-19). Data are represented as mean ± STD of three replicates (n=3). Different letters within a column indicate significant differences between treatments.

Table 2. Effects of LAB inoculants on pH and organic acid level of alfalfa silage at different moisture levels after 6 months of ensiling.

Groups	High-Moisture
	pH	LA (DM%)	AA(DM%)	LA/AA ratio
Control	5.5 ± 0.10^a	4.2 ± 0.27^c	3.15 ± 0.28^ab	1.36 ±0.21^c
L. citrum-KCC-57	4.7 ± 0.02^c	9.1 ± 0.13^a	2.41 ± 0.30^b	3.84±0.75^a
L. citrum- KCC-58	4.7 ± 0.00^c	9.4 ± 0.50^a	2.54 ± 0.18^a	3.75±0.66^a
L. lactis-RWP3	4.9 ± 0.06^b	8.4 ± 0.37^ab	3.48 ± 0.45^a	2.46±0.29^b
L. lactis -RWP7	4.9 ± 0.02^b	7.3 ± 0.04^b	3.27 ± 0.02^ab	2.23±0.00^bc
Cocktail LAB	4.6 ± 0.02^c	9.2 ± 0.19^a	2.70 ±0.07^b	3.43±0.23^a
Low-Moisture
Control	6.2 ± 0.21^a	0.12 ± 0.01^b	0.55 ± 0.06^a	0.22±0.05^b
L. citrum-KCC-57	6.0 ± 0.06^a	0.30 ± 0.00^b	0.48 ± 0.08^a	0.64±0.14^b
L. citrum- KCC-58	5.7 ±0.01^b	2.01 ± 0.25^a	0.53 ± 0.01^a	3.78±0.54^a
L. lactis-RWP3	5.9 ±0.01^ab	0.28 ± 0.03^b	0.51 ± 0.07^a	0.56±0.18^b
L. lactis -RWP7	5.9 ± 0.04^ab	0.28 ± 0.03^b	0.53 ± 0.04^a	0.52±0.01^b
Cocktail LAB	5.8 ± 0.04^ab	0.21 ± 0.09^b	0.39 ± 0.02^a	0.55±0.36^b

AA: acetic acid. Cocktail LAB (P. pentosaceus –KCC-23 and L.plantarum - KCC-10 + L.plantarum - KCC-19). Data are represented as mean ± STD of three replicates (n=3). Different letters within a column indicate significant differences between treatments.

After 6 months of fermentation, similar trends were noted in experimental alfalfa silages (Table 2). Under high-moisture conditions, LAB inoculants significantly lowered the pH of alfalfa silage to 4.6- 4.9 compared to the control silage (5.5 ± 0.10) with higher LA content, particularly in silage produced with L. citreum and cocktail LAB than the other strains treated silage. Silage produced with L. lactis had the highest acetic acid compared to the other strains and control, whereas inoculants L. citreum KCC-57, L. citreum- KCC-58, and cocktail LAB significantly reduced acetic acid level compared to the other treatments and control. In low-moisture, control silage had high pH and low LA, while inoculants moderately improved fermentation parameters. L.citreum-KCC-58-treated silage had the highest level of LA content (2.01 ± 0.25 DM%) and reduced pH (5.7) compared to other strains as well as control silage.

3.2 Impact of LAB on microbial composition after 3 and 6 months

The microbial composition, including yeast, mold, LAB, and total bacteria, was counted in inoculum and non-inoculum treated silage at different moisture conditions after fermentation periods (Tables 3 and 4). In high-moisture silage, inoculant treatments elevated LAB, total bacterial counts, and reduced yeast and mold counts. Notably, higher total bacterial counts were noticed in silage treated with L. citreum KCC-58 (7.7 ±0.2 Log10g-1) and cocktail LAB (7.6 ±0.1 Log10g-1), followed by L. citreum KCC-57 and L. lactis compared to the control. Silage treated with L. lactis -RWP7 and L. lactis -RWP7 had the highest LAB population compared to other treatments. Reduced yeast and mold population in alfalfa silage produced with different LAB inoculants compared to control silage, particularly L. lactis and L. citreum, showed the lowest count of yeast and mold than the other treatments. In low-moisture conditions, inoculant treatment raised LAB and TB counts and lowered yeast & mold counts than the control. L. lactis and cocktail LAB showed the highest LAB counts in alfalfa silage than the other treatments. Drastically reduced mold and yeast population in alfalfa silage after 3 months of fermentation of LAB inoculants (Table 3)

Table 3. Impacts of LAB inoculants on microbial composition (log10g^-1) of alfalfa silage at varying moisture after 3 months.

Groups	High-Moisture
Groups	TB (Log10g^-1	LAB (Log10g^-1)	Yeast (Log10g^-1)	Mold (Log10g^-1)
Control	7.1 ± 0.0^b	6.1 ± 0.1^b	5.4 ± 0.1^a	3.8 ± 0.2^a
L. citrum-KCC-57	7.5 ±0.2^a	6.8 ± 0.1^a	5.3 ± 0.3^a	3.4 ± 0.1^b
L. citrum- KCC-58	7.7 ±0.2^a	6.9 ± 0.0^a	3.5 ± 0.1^b	3.3 ± 0.0^b
L. lactis -RWP7	7.5 ±0.0^a	7.0 ± 0.0^a	3.7 ± 0.1^b	3.5 ± 0.1^b
L. lactis -RWP7	7.5 ±0.0^a	7.3 ± 0.0^a	3.8 ± 0.1^b	3.5 ± 0.0^b
Cocktail LAB	7.6 ±0.1^a	7.1 ± 0.1^a	4.9 ± 0.1^b	3.3 ± 0.2^b
Low-Moisture
Control	6.4 ± 0.1^a	5.1 ± 0.0^b	5.3 ± 0.0^a	4.1 ± 0.1^a
L. citrum-KCC-57	6.7 ± 0.4^a	5.8 ± 0.1^ab	5.0 ± 0.1^b	3.6 ± 0.8^b
L. citrum- KCC-58	7.0 ± 0.4^a	5.9 ± 0.0^a	4.9 ± 0.0^bc	3.3 ± 0.3^b
L. lactis -RWP7	6.7 ± 0.3^a	6.0 ± 0.1^a	4.7 ± 0.1^c	3.5 ± 0.1^b
L. lactis -RWP7	6.5 ± 0.2^a	6.3 ± 0.0^a	4.7± 0.2^b	3.5 ± 0.0^b
Cocktail LAB	6.9 ± 0.3^a	6.1 ± 0.1^a	5.0 ± 0.0^b	3.3 ± 0.2^b

TB: total bacteria; Cocktail LAB (P. pentosaceus –KCC-23 and L.plantarum - KCC-10 + L.plantarum - KCC-19). Data are represented as mean ± STD of three replicates (n=3). Different letters within a column indicate significant differences between treatments.

Table 4. Impacts of LAB inoculants on microbial composition of alfalfa silage at varying moisture after 6 months.

Groups	High-Moisture
Groups	TB (Log10g^-1)	LAB (Log10g^-1)	Yeast (Log10g^-1)	Mold (Log10g^-1)
Control	7.5 ± 0.7^b	6.3 ± 0.2^d	5.4 ± 0.4^a	4.9 ± 0.1^a
L. citrum-KCC-57	7.8 ± 0.9^b	7.5 ± 0.1^b	4.3 ± 0.2^b	4.2 ± 0.2^b
L. citrum- KCC-58	8.4 ± 0.6^a	7.4 ± 0.1^c	4.7 ± 0.0^b	4.6 ± 0.0^b
L. lactis-RWP3	8.5 ± 0.5^a	7.5 ± 0.5^b	4.7 ± 0.1^b	4.5 ± 0.1^b
L. lactis -RWP7	8.4 ± 0.6^a	7.6 ± 0.6^b	4.8 ± 0.1^b	4.6 ± 0.0^b
Cocktail LAB	8.4 ± 0.5^a	7.8 ± 0.5^a	4.5 ± 0.1^b	4.6 ± 0.1^b
Low-Moisture
Control	7.4 ± 0.5^a	6.1 ± 0.1^b	5.1 ± 0.0^a	4.3 ± 0.0^a
L. citrum-KCC-57	7.4 ± 0.6^a	6.7 ± 0.3^a	4.5 ± 0.1^b	3.7 ± 0.1^b
L. citrum- KCC-58	7.6 ± 0.5^a	6.9 ± 0.1^a	4.4 ± 0.2^b	3.9 ± 0.2^ab
L. lactis-RWP3	7.6 ± 0.6^a	6.8 ± 0.0^a	4.5 ± 0.3^b	4.0 ± 0.3^ab
L. lactis -RWP7	7.6 ± 0.6^a	6.7 ± 0.2^a	4.6 ± 0.0^b	4.2 ± 0.0^ab
Cocktail LAB	7.4 ± 0.5^a	7.0 ± 0.1^a	4.5 ± 0.1^b	3.7 ± 0.1^b

TB: total bacteria; Cocktail LAB (P. pentosaceus –KCC-23 and L.plantarum - KCC-10 + L.plantarum - KCC-19).). Data are represented as mean ± STD of three replicates (n=3). Different letters within a column indicate significant differences between treatments.

After 6 months, in high moisture, total bacterial counts were highest in inoculum-treated alfalfa silages, particularly L. citreum - KCC-58 (8.4 ± 0.6 Log10g-1), L. lactis-RWP3 (8.5 ± 0.5 Log10g-1), L. lactis-RWP7 (8.4 ± 0.6 Log10g-1), and cocktail LAB (8.4 ± 0.5 Log10g-1) compared to other treatments and controls. In addition, LAB inoculants treatment showed the highest LAB colonies and the lowest mold and yeast counts compared to control silage. In low moisture silage, LAB inoculum treated silage had the highest TB and LAB counts and lowest counts of yeast and mold compared to the control silage, especially, L. citreum KCC-57 (3.7 ± 0.1 Log10g-1), L. citreum - KCC-58 (3.9 ± 0.2 Log10g-1), and cocktail LAB (3.7 ± 0.1 Log10g-1) treated sharply reduced mold counts compared to the other strains. L. citreum - KCC-58 and L. lactis strains notably suppressed molds and maintained favorable LAB populations.

3.3 Effects of LAB on nutrient profiles after 3 and 6 months

This table evaluates fiber components, including ADF, NDF, and CP. In high moisture, most LAB inoculants slightly reduced ADF and NDF levels while improving CP content compared to the control. L. citreum strains and L. lactis-RWP3 were especially more effective on fiber digestibility than the other treatments (Table 5). In low moisture, the nutrient differences in alfalfa were minimal between experimental silages. But LAB treatment helped maintain protein and slightly lower fiber, especially with RWP7, which showed the highest CP (20.3 ± 1.4%). After 6 months, the trends in fiber and protein content were similar to those of the 3-month silage. Most LAB-treated silages exhibited a slight reduction in ADF and NDF content compared to the control, with L. citreum - KCC-58 and cocktail LAB being consistently effective across moisture conditions. CP levels were slightly higher in inoculated groups, especially in L. lactis-RWP3 and L. citreum - KCC-58 under high moisture, and in, KCC-57, RWP7, and cocktail LAB under low moisture (Table 6).

Table 5. Effects of LAB on nutrient profiles in alfalfa silage under different moisture condition after 3-month storage periods.

Treatments	High-moisture
Treatments	ADF%	NDF%	CP%
Control	32.2 ± 0.1^a	42.7 ± 0.7^a	19.6 ± 0.1^a
L. citrum-KCC-57	30.0 ± 0.1^bcd	39.7 ± 0.2^bc	20.2 ± 0.1^ab
L. citrum- KCC-58	29.8 ± 0.7^e	39.9 ± 0.8^bc	20.3 ± 0.2^a
L. lactis-RWP3	31.4 ± 0.5^ab	41.7 ± 1.0^ab	20.0 ± 0.4^ab
L. lactis -RWP7	30.8 ± 0.3^cd	41.1 ± 0.6^ab	19.6 ± 0.5^ab
Cocktail LAB	31.2 ± 0.6^abc	41.0 ± 0.8^ac	19.2 ± 0.7^b
Low-moisture
Control	30.9 ± 0.5^a	44.0 ± 0.5^a	19.7 ± 0.2^a
L. citrum-KCC-57	30.6 ± 1.7^a	43.4 ± 1.1^a	19.8 ± 1.4^a
L. citrum- KCC-58	31.3 ± 0.3^a	43.1 ± 0.5^a	19.1 ± 0.2^a
L. lactis-RWP3	30.9 ± 0.6^a	44.0 ± 0.1^a	19.6 ± 1.1^a
L. lactis -RWP7	30.2 ± 1.5^a	43.2 ± 1.4^a	20.3 ± 0.4^a
Cocktail LAB	31.1 ± 0.9^a	44.2 ± 0.4^a	19.4 ± 0.1^a

Cocktail LAB (P. pentosaceus –KCC-23 and L. plantarum - KCC-10 + L. plantarum - KCC-19).). Data are represented as mean ± STD of three replicates (n=3). Different alphabets within a column indicate significant differences between treatments.

Table 6. Effects of LAB on nutrient content in alfalfa silage at different moisture condition after 6-month storage period.

Treatments	High-moisture
Treatments	ADF	NDF	CP
Control	31.3 ±0.3^a	42.9 ±0.1^a	18.8 ±0.0^a
L. citrum-KCC-57	30.9 ±0.7^a	40.8 ±0.8^a	19.2 ±0.2^a
L. citrum- KCC-58	29.9 ±1.9^a	40.6 ±2.5^a	19.9 ±1.2^a
L. lactis-RWP3	30.3 ±0.3^a	41.9 ±1.0^a	20.2 ±0.0^a
L. lactis -RWP7	31.7 ±0.7^a	42.4 ±0.3^a	19.0 ±0.9^a
Cocktail LAB	30.8 ±0.2^a	41.5 ±0.6^a	19.4 ±0.2^a
Low-Moisture
Control	31.4 ±0.1^a	43.6 ±0.3^a	19.5 ±0.8^a
L. citrum-KCC-57	30.2 ±0.9^a	43.4 ±1.3^a	19.8 ±0.6^a
L. citrum- KCC-58	30.5 ±0.4^a	43.0 ±0.4^a	19.2 ±0.0^a
L. lactis-RWP3	30.4 ±0.6^a	43.1 ±0.3^a	19.1 ±0.6^a
L. lactis -RWP7	29.6 ±0.1^a	42.5 ±0.4^a	19.8 ±0.4^a
Cocktail LAB	29.8 ±0.5^a	41.8 ±1.8^a	19.9 ±0.7^a

Cocktail LAB (P. pentosaceus –KCC-23 and L.plantarum - KCC-10 + L.plantarum - KCC-19. Data are represented as mean ± STD of three replicates (n=3). Different alphabets within a column indicate significant differences between treatments.

4. Discussion

The utilization of LAB as biological inoculants has shown clear and sustained significant benefits in improving the forage-based silage quality via improving the fermentation profile, microbial composition and content of nutrients in silage under different moistures after 3 and 6 months of storage. Across both 3 and 6 months of ensiled periods, the results consistently demonstrated that alfalfa silage produced with different LAB reduced silage pH more effectively than the controls, which reflects that LAB treatment more efficiently stimulates the fermentation process during ensiling periods. These effects were more pronounced in high moisture silage, which may be LAB rapidly utilized available WSCs to produce LA and other organic acids (Muck et al., 2018). LA is a major acid that stabilizes silage quality by lowering the pH and inhibiting spoilage organisms (Guo et al., 2023).

pH is a key indicator used to assess silage quality (Restelatto et al., 2019), with values ranging from 3.8 to 4.2 typically considered for effective ensiling processes (Lv et al., 2020, Bao et al., 2023). For silages produced with high moisture, a pH of 4.2 is regarded as a benchmark for well-preserved silage material (Cui et al., 2022). In the current study, control at both moistures exhibited higher pH values, increased yeast and mold counts, and reduced LAB colonies across both ensiling periods. The elevated pH levels suggest a suboptimal fermentation process, maybe due to a low initial native LAB and more unwanted microorganisms naturally present on the plant material. But LAB treatments significantly reduced pH of alfalfa silage compared to non-inoculated silages. Especially, Leuconostoc citreum- KCC-58 and the cocktail LAB exhibited faster acidification capacity than the other treatments under high and low moisture conditions after 3 and 6 months of storage periods. In all storage periods, adding LAB inoculum strongly reduced pH levels of silages with high moisture levels. However, a slight reduction in pH was observed in silages with low moisture content across all storage durations. Achieving a pH below 5 is generally considered difficult for legume silages because alfalfa contains a relatively high DM content(Kung et al., 2018). In the present study, a pH below 5 was achieved across all storage periods in high-moisture silages. However, this threshold was not reached in low-moisture silages, possibly due to more restrictive fermentation conditions and limited availability of WSCs, which are essential for LAB growth and activity.

The primary organic acids in fermented silages are LA, acetic acid, and butyric acid, with LA typically being the most predominant acid in fermented silage (Kung et al., 2018). Of these, LA plays a crucial role during the ensiling process due to its high concentration and strong acidifying effect, which contributes significantly to the rapid reduction in silage pH. Its concentration is generally more than 10 fold higher compared to other organic acids, underscoring its importance in effective fermentation (Naidu et al., 2025). Alfalfa silage produced without inoculum addition showed lower LA content at both moisture conditions after 3 and 6 months reflected that native bacteria in the alfalfa are not ability to induce the fermentation process vigorously whereas, LAB treatment exhibited that the LA level strongly increased in alfalfa silage at both moisture conditions after 3 and 6 months of ensiling, suggesting that the additional inoculum could induce silage fermentation sharply as evidenced by higher LA content. Among the LAB, L. citreum- KCC-58 consistently exhibited the highest LA production, suggesting it possesses strong fermentative capabilities in both experimental storage periods. LA is a stronger acid than acetic acid and is more efficient in reducing pH quickly during the early fermentation phase (Bai et al., 2021). Moreover, the presence of moderate acetic acid levels, especially with L citreum, L. lactis, or cocktails, contributes to aerobic stability by inhibiting yeast and mold (Wang et al., 2021, Wang et al., 2025), a similar effect also observed in the present study.

LAB inoculation led to significantly higher LAB populations and reduced counts of spoilage organisms like yeasts and molds, particularly in high-moisture silage after both 3 and 6 months of storage periods. This microbial shift is essential for maintaining aerobic stability and extending the storage life of silage (Guo et al., 2023, Jatkauskas et al., 2024). Suppression of spoilage microorganisms is crucial during feed out, when it exposure to oxygen can lead to rapid deterioration of silage quality. L. citreum - KCC-58 and cocktail LAB were particularly effective due to their ability to produce higher amounts of lactic and acetic acids, which offers a dual mode of action against spoilage organisms. Production of alfalfa silage with LAB significantly affects the composition and succession of microbial communities during ensiling periods (Guo et al., 2023). Addition of LAB strains during alfalfa silage production significantly affects the microbial environment, enhancing beneficial bacterial dominance and limiting undesirable microbial growth in ensiled silage.

Apart from fermentation and microbial stabilization, LAB treatments significantly improve forage digestibility by preserving valuable nutrients, particularly CP. The present findings reveal that LAB-treated silages maintained slightly higher CP and exhibited lower fiber content compared with the control, with notable performance by L. lactis-RWP7 and L. citreum - KCC-58. The improved protein preservation likely results from the rapid pH drop, which inhibits proteolytic enzymes and clostridial activity, which degrade amino acids and produce ammonia. The decline in structural fiber components in forages, suggests partial cell wall degradation, potentially aided by microbial enzymatic activity or synergistic effects of LAB with endogenous plant enzymes. Ruminants tend to have higher feed intake and rumination rates when consuming feed with low ADF and NDF content. Silage treated with LAB inoculants have been shown to reduce the ADF and NDF levels in alfalfa (Wang et al., 2025), which aligns with the findings of the present study.

Selection of LAB strains can also influence fiber degradability by modulating microbial groups that enhance cellulolytic activity (Chukwuma et al., 2020, Okoye et al., 2023). Overall finding strongly supported the use of LAB inoculants to optimize the alfalfa silage quality under different moisture conditions. Silage inoculants improve not only fermentation characteristics, but also nutritional and microbial stability during ensiling periods. Treatments that are most effective are L. citreum- KCC-58 and cocktail LAB, which exhibit multifunctional roles in both high- and low-moisture silages, suggesting their practical utility for farmers seeking reliable silage preservation. LAB strain selection is vital for forage preservation and livestock nutrition, depending on the environmental conditions and ensiling goals. The present study demonstrated that L. citreum- KCC-58 and cocktail LAB significantly improved alfalfa silage fermentation during the ensiling process. However, it has several limitations that must be acknowledged. The experiments were executed under controlled conditions; large-scale farm production together with aerobic stability assessments are required to validate the effectiveness of used LAB inoculants in diverse environments with different forages. Hence, future research should focus on farm scale validation across different forage crops, extended storage durations and feeding trial to assess its impacts on animal health and productivity.

5. Conclusions

LAB inoculants significantly improved alfalfa silage fermentation quality, microbial stability, and nutrient preservation under high- and low-moisture conditions after 3 and 6 months of ensiling. In particular, Leuconostoc citreum-KCC-58 and the cocktail LAB consistently performed better by reducing silage pH, increasing LA production, and suppressing undesirable microorganisms. In addition, these treatments reduced fiber content (ADF and NDF) while maintaining or improving CP content. Among the tested strains, L. lactis-RWP3 and L. lactis-RWP7 also showed potential for improving silage quality, particularly in terms of microbial composition and nutrient retention. Overall, the use of LAB inoculants, especially L. citreum- KCC-58 and the cocktail combination, represent an effective strategy to enhance the fermentation profile, stability, and feed value of alfalfa silage under different moisture conditions.

CRediT authorship contribution statement

Ilavenil Soundharrajan: Concepts, design, literature search, experimental studies, data analysis, manuscript preparation. Jeong Sung Jung: Concepts, design, data acquisition, data analysis, literature search, manuscript editing and review. Jae Hyuk Kim: Concepts, design, data acquisition, data analysis, statistical analysis, experimental studies manuscript editing and review. Jae-Hoon Woo: Literature search, experimental studies, data analysis, statistical analysis manuscript editing and review. Ki Won Lee: Literature search, experimental studies, data acquisition, data analysis, statistical analysis, manuscript editing and review. Min Gon Kim: Experimental studies, data acquisition, data analysis, statistical analysis, manuscript editing and review. Seung Min Jeong: Experimental studies, data acquisition, data analysis, statistical analysis, manuscript editing and review, and literature search. Ki Choon Choi: Concepts, design, literature search, experimental studies, data acquisition, data analysis, manuscript editing and review, and project administrator.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.

Data availability

All experimental data were available in the original manuscript as well as in the supplementary file. Raw data will be provided on request

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of Artificial Intelligence (AI)-Assisted Technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Acknowledgement

A Cooperative Research Program for Agriculture Science and Technology Development supported this work (Project Name: Technique development for manufacturing high-quality legume silage; Project No. PJ01358902), RDA-NIAS, Republic of Korea. This study was supported by 2025 the RDA Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea.

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Show Sections

[1] AOAC. Official method of analysis (15thed). 1990.

[2] Arasu M.V., Jung M.W., Kim D.H., Ilavenil S., Jane M., Park H.S., Al-Dhabi N.A., Jeon B.T., Choi K.C.. Enhancing nutritional quality of silage by fermentation with lactobacillus plantarum. Indian J Microbiol. 2014;54:396-402. https://doi.org/10.1007/s12088-014-0473-9
[Google Scholar]

[3] Bai J., Ding Z., Ke W., Xu D., Wang M., Huang W., Zhang Y., Liu F., Guo X.. Different lactic acid bacteria and their combinations regulated the fermentation process of ensiled alfalfa: Ensiling characteristics, dynamics of bacterial community and their functional shifts. Microb Biotechnol. 2021;14:1171-1182. https://doi.org/10.1111/1751-7915.13785
[Google Scholar]

[4] Bao X., Guo G., Huo W., Li Q., Xu Q., Chen L.. Ensiling pretreatment fortified with laccase and microbial inoculants enhances biomass preservation and bioethanol production of alfalfa stems. Sci. Total Environ.. 2023;857:159442. https://doi.org/10.1016/j.scitotenv.2022.159442
[Google Scholar]

[5] Borreani G., Tabacco E., Schmidt R.J., Holmes B.J., Muck R.E.. Silage review: Factors affecting dry matter and quality losses in silages. J Dairy Sci. 2018;101:3952-3979. https://doi.org/10.3168/jds.2017-13837
[Google Scholar]

[6] Carvalho B.F., Sales G.F.C., Schwan R.F., Ávila C.L.S.. Criteria for lactic acid bacteria screening to enhance silage quality. J Appl Microbiol. 2021;130:341-355. https://doi.org/10.1111/jam.14833
[Google Scholar]

[7] Chukwuma O.B., Rafatullah M., Tajarudin H.A., Ismail N.. Lignocellulolytic enzymes in biotechnological and industrial processes: A Review. Sustainability. 2020;12:7282. https://doi.org/10.3390/su12187282
[Google Scholar]

[8] Cui X., Yang Y., Zhang M., Jiao F., Gan T., Lin Z., Huang Y., Wang H., Liu S., Bao L., Su C., Qian Y.. Optimized ensiling conditions and microbial community in mulberry leaves silage with inoculants. Front Microbiol. 2022;13:813363. https://doi.org/10.3389/fmicb.2022.813363
[Google Scholar]

[9] Ferrero F., Tabacco E., Piano S., Casale M., Borreani G.. Temperature during conservation in laboratory silos affects fermentation profile and aerobic stability of corn silage treated with Lactobacillus buchneri, Lactobacillus hilgardii, and their combination. J Dairy Sci. 2021;104:1696-1713. https://doi.org/10.3168/jds.2020-18733
[Google Scholar]

[10] Guo X., Xu D., Li F., Bai J., Su R.. Current approaches on the roles of lactic acid bacteria in crop silage. Microb Biotechnol. 2023;16:67-87. https://doi.org/10.1111/1751-7915.14184
[Google Scholar]

[11] Hashemzadeh-Cigari F., Khorvash M., Ghorbani G.R., Ghasemi E., Taghizadeh A., Kargar S., Yang W.Z.. Interactive effects of molasses by homofermentative and heterofermentative inoculants on fermentation quality, nitrogen fractionation, nutritive value and aerobic stability of wilted alfalfa (Medicago sativa l) silage. J Anim Physiol Anim Nutr (Berl). 2014;98:290-299. https://doi.org/10.1111/jpn.12079
[Google Scholar]

[12] Henderson P.A.R.M.D.. Buffering capacity of herbage samples as a factor in ensilage. J Sci Food Agric. 1962;13:395-400. https://doi.org/10.1002/jsfa.2740130709
[Google Scholar]

[13] Jatkauskas J., Vrotniakiene V., Amaral R.C.D., Witt K.L., Cappellozza B.L.. Influence of ensiling timing and inoculation on whole plant maize silage fermentation and aerobic stability (Preliminary research) Plants (Basel). 2024;13:2894. https://doi.org/10.3390/plants13202894
[Google Scholar]

[14] Jung J.S., Wong J.W.C., Soundharrajan I., Lee K.W., Park H.S., Kim D., Choi K.C., Chang S.W., Ravindran B.. Changes in microbial dynamics and fermentation characteristics of alfalfa silage: A potent approach to mitigate greenhouse gas emission through high-quality forage silage. Chemosphere. 2024;362:142920. https://doi.org/10.1016/j.chemosphere.2024.142920
[Google Scholar]

[15] Kung L., Shaver R.D., Grant R.J., Schmidt R.J.. Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. J Dairy Sci. 2018;101:4020-4033. https://doi.org/10.3168/jds.2017-13909
[Google Scholar]

[16] Li R., Jiang D., Zheng M., Tian P., Zheng M., Xu C.. Microbial community dynamics during alfalfa silage with or without clostridial fermentation. Sci Rep. 2020;10:17782. https://doi.org/10.1038/s41598-020-74958-1
[Google Scholar]

[17] Liu Y., Wang Z., Sun L., Zhang Y., Zhao M., Hao J., Liu M., Ge G., Jia Y., Du S.. Effects of moisture content gradient on alfalfa silage quality, odor, and bacterial community revealed by electronic nose and GC-MS. Microorganisms. 2025;13:381. https://doi.org/10.3390/microorganisms13020381
[Google Scholar]

[18] Lv J., Fang X., Feng G., Zhang G., Zhao C., Zhang Y., Li Y.. Effects of sodium formate and calcium propionate additives on the fermentation quality and microbial community of wet brewers grains after short-term storage. Animals (Basel). 2020;10:1608. https://doi.org/10.3390/ani10091608
[Google Scholar]

[19] Muck R.E., Nadeau E.M.G., McAllister T.A., Contreras-Govea F.E., Santos M.C., Kung L.. Silage review: Recent advances and future uses of silage additives. J Dairy Sci. 2018;101:3980-4000. https://doi.org/10.3168/jds.2017-13839
[Google Scholar]

[20] Muthusamy K., Han H.S., Soundharrajan I., Jung J.S., Valan Arasu M., Choi K.C.. A novel strain of probiotic leuconostoc citreum inhibits infection-causing bacterial pathogens. Microorganisms. 2023;11:469. https://doi.org/10.3390/microorganisms11020469
[Google Scholar]

[21] Naidu T., Sanusi I.A., Nouadjep N.S., Sewsynker-Sukai Y., Mambili-Mamboundou H., Beukes L.S., Gueguim-Kana E.B.. Enhanced lactic acid production from potato peel waste via MnO₂ nanoparticle-assisted simultaneous saccharification and fermentation: Process optimization and kinetic studies. Bioresour Technol. 2025;435:132884. https://doi.org/10.1016/j.biortech.2025.132884
[Google Scholar]

[22] Okoye C.O., Wang Y., Gao L., Wu Y., Li X., Sun J., Jiang J.. The performance of lactic acid bacteria in silage production: A review of modern biotechnology for silage improvement. Microbiol Res. 2023;266:127212. https://doi.org/10.1016/j.micres.2022.127212
[Google Scholar]

[23] Restelatto R., Novinski C.O., Pereira L.M., Silva E.P.A., Volpi D., Zopollatto M., Schmidt P., Faciola A.P.. Chemical composition, fermentative losses, and microbial counts of total mixed ration silages inoculated with different Lactobacillus species. J Anim Sci. 2019;97:1634-1644. https://doi.org/10.1093/jas/skz030
[Google Scholar]

[24] Soundharrajan I., Min C.-W., Jung J.S., Choi K.C.. Effects of the novel lacticaseibacillus paracasei k-68 Inoculant on nutrient content, fermentation, and microbial dynamics changes in dacheongok corn silage. Fermentation. 2025;11:304. https://doi.org/10.3390/fermentation11060304
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[25] Soundharrajan I., Muthusamy K., Han O.-K., Lee H.J., Purushothaman S., Kim D., Choi K.C.. Effects of microbial inoculants on the fermentation and preservation of triticale silages at high and low moisture levels. Appl Sci. 2020;10:7855. https://doi.org/10.3390/app10217855
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[26] Soundharrajan I., Yoon Y.H., Muthusamy K., Jung J.S., Lee H.J., Han O.K., Choi K.C.. Isolation of lactococcus lactis from whole crop rice and determining its probiotic and antimicrobial properties towards gastrointestinal associated bacteria. Microorganisms. 2021;9:2513. https://doi.org/10.3390/microorganisms9122513
[Google Scholar]

[27] Van Soest P.J., Robertson J.B., Lewis B.A.. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991;74:3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
[Google Scholar]

[28] Wang S.Y., Jing Y.Y., Yang G., Liu B., Gao F.Q.. Effects of inoculants on the quality of alfalfa silage. Front Microbiol. 2025;16:1541454. https://doi.org/10.3389/fmicb.2025.1541454
[Google Scholar]

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[Google Scholar]

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Role of lactic acid bacteria inoculants in optimizing fermentation dynamics and nutrient retention in alfalfa silage at different moisture conditions

Abstract

Keywords

Alfalfa

Different moistures

Ensiling

Lactic acid bacteria

Storage periods

1. Introduction

2. Materials and Methods

2.1 Inoculants preparation

2.2 Alfalfa cultivation and silage production

2.3 Sampling after fermentation

2.4 Determination of nutrient composition.

2.5 Organic acids quantification

2.6 Microbial counting in experimental alfalfa silages

2.7 Statistical analysis

3. Results

3.1 Influence of lactic acid bacteria on pH and organic acid profiles in experimental silages

3.2 Impact of LAB on microbial composition after 3 and 6 months

3.3 Effects of LAB on nutrient profiles after 3 and 6 months

4. Discussion

5. Conclusions

CRediT authorship contribution statement

Declaration of competing interest

Data availability

Declaration of Generative AI and AI-assisted technologies in the writing process

Acknowledgement

References

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