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Article

Effects of the Novel Lacticaseibacillus paracasei K-68 Inoculant on Nutrient Content, Fermentation, and Microbial Dynamics Changes in Dacheongok Corn Silage

by
Ilavenil Soundharrajan
1,†,
Chang-Woo Min
1,2,†,
Jeong Sung Jung
1,* and
Ki Choon Choi
1,*
1
Forage Production System Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea
2
Division of Applied Life Science (BK21), IALS, Gyeongsang National University, Jinju 52828, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(6), 304; https://doi.org/10.3390/fermentation11060304
Submission received: 29 April 2025 / Revised: 17 May 2025 / Accepted: 21 May 2025 / Published: 23 May 2025
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Abstract

:
This study investigated the role of Lacticaseibacillus paracasei K-68 (LABK) and cocktail LAB (LABC) as silage inoculants to enhance corn silage fermentation quality and microbial stability. Silage spoilage is primarily caused by undesirable microbes such as Clostridium, Klebsiella, yeasts, and molds. The isolated LAB strain K-68 exhibited strong antibacterial and antifungal activity, particularly against spoilage organisms, and was identified as L. paracasei. Experimental silages inoculated with LABK or a LABC significantly improved fermentation profiles, with reduced pH and increased lactic acid levels. Microbial counts revealed that LAB-inoculated silages had higher LAB counts and significantly reduced yeast and mold populations. Furthermore, there were no significant differences in acetic acid, isobutyric acid, and propionic acid levels. High-throughput sequencing confirmed that LABK-treated silage was dominated by Lacticaseibacillus paracasei, whereas LABC-treated silage supported more diverse microbiota, including Pediococcus pentosaceus, Lacrimispora xylanolytica, and Levilactobacillus brevis. Both treatments suppressed spoilage-associated genera such as Clostridium and Klebsiella. Furthermore, correlation analysis showed that Lacticaseibacillus abundance was positively associated with lactic acid production and negatively correlated with pH and yeast levels. L. paracasei K-68 is a promising bio-inoculant for corn silage production since it promotes beneficial microbial dominance and suppresses spoilage organisms better than cocktail LAB.

1. Introduction

Whole crop corn silage is more nutritious for livestock, has a higher yield and provides more energy at a lower cost than other forages. The fermentation of corn silage is dominated by a variety of microbes that conserve the quality of the corn silage and improve animal performance [1]. The ensiling process involved in the fermentation of forage materials under anaerobic conditions is predominantly driven by lactic acid bacteria [2]. LAB can ferment water-soluble carbohydrates (WSCs) into organic acids, particularly lactic acid and marginal levels of acetic acid. The LAB-mediated fermentation process lowers pH via increasing organic acids, especially lactic acid, which inhibits spoilage microorganisms and preserves the nutritional content of forage [3,4]. However, undesirable microbes can still proliferate under suboptimal situations, which negatively impacts the silage quality, which leads to reduced animal performance and productivity [5].
Silage spoilage is primarily caused by undesirable microbial multiplications, which include bacteria, yeast and fungi, which cause nutrient loss, reduced digestibility and mycotoxin contamination, all of which affect silage quality. Clostridia, enterobacteria, and bacilli are the main bacterial groups responsible for spoilage. Clostridium spp. convert lactic acid into butyric acid, leading to an increase in pH, protein degradation, and the production of ammonia, resulting in increased pH and a lower quality of feed and unpalatable silage [6,7]. Enterobacteria such as klebsiella sp. and Escherichia coli strongly compete with lactic acid bacteria growth in the early stage of fermentation, causing increased pH and protein degradation, whereas bacillus spp. can contribute to spoilage by producing heat-resistant spores and degrading proteins [7,8]. Yeasts, particularly Candida, Pichia, and Saccharomyces spp., are among the first microorganisms to initiate aerobic spoilage and they metabolize lactic acid and residual sugars, leading to increased pH and ethanol production and reduced aerobic stability, which negatively impacts silage palatability [1,9]. Molds such as Aspergillus, Penicillium, and Fusarium spp. are major threats when silage is exposed to oxygen, leading to the loss of nutrient content and production of mycotoxins, which affect liver and kidney functions and cause immunosuppression and reproductive disorders in animals [10,11]. To mitigate spoilage, effective ensiling practices such as the use of biological inoculants, oxygen barrier films, and proper feedout management are essential to limit microbial growth and preserve silage quality [2,12,13].
Lactic acid bacteria (LAB) have been extensively used in silage production for their potential to accelerate the accumulation of lactic acid, reducing pH, preventing protein degradation, reducing dry matter loss and enhancing animal performance [5,14,15]. Mostly, LAB such as Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus buchneri, Lactococus lactis and Leuconostoc sp. have been studied for their ability to enhance fermentation, reduce spoilage, and prolong silage stability [16,17]. But Levilactobacillus casei has often not been used in silage production as its effects are not clear. Few studies have shown positive effects of L. paracasei on silage fermentation such as increased lactic acid and reduced acetic acid levels [18,19,20]. Another study reported that L. paracasei could not exert beneficial effects on silage fermentation [21]. This study aims to explore the potential of Levilactobacillus casei as a biological silage additive in corn silage production, especially in enhancing fermentative metabolites and microbial dynamics. The study objectives include assessing the effects of newly isolated L. paracasei on fermentative metabolites such as pH, lactic acid, acetic acid, butyric acid, and propionic acid levels, nutrients such as crude protein (CP), acid detergent fiber (ADF), and bacterial dynamics and to compare the efficiency of L. paracasei with a commercially available cocktail LAB inoculant to confirm whether L. paracasei can contribute to improving silage fermentation and prevent nutrient loss. Furthermore, heatmap correlation was used to examine interactions between bacterial communities and treatments or fermentative metabolites.

2. Methods and Materials

2.1. Isolation of Lactic Acid Bacteria from Corn Silages

Fermented bale corn silage was collected aseptically from the Grassland and Forage Farm located at the National Institute of Animal Science (NIAS), South Korea. Then, 10 g of corn samples was suspended in distilled water and then the sample was diluted by a ten-fold serial dilution method. The diluted samples were then spread onto MRS agar and incubated at 37 °C for 48 h. Fast growing colonies were picked and streaked onto BCP agar medium for LAB identity confirmation. Furthermore, the growth of selected colonies in different plant extracts such as Alfalfa, triticale and corn were investigated. Physiological and biochemical characteristics of isolated strains were confirmed. The extracellular enzyme production and fermentation of different carbohydrates were confirmed by API ZYM and APICH-50 kits, respectively [22].

2.2. Antimicrobial Activity of Selected LAB

2.2.1. Antifungal Activity of LAB by Pour Plate Method

Aspergillus sp. (KACC 40080) and Penicillium sp. (KACC 41354) were grown on potato dextrose agar (PDA) for 10 to 14 days. The spores were then collected using 0.05% Triton-X 100 for antifungal assay. Twenty-four-hour active LAB cultures were spotted in an MRS agar plate and incubated for 24 h at 37 °C. After sufficient LAB growth, the fungal spore in 0.8% PDA was poured onto LAB colonies and incubated for a week at 30 °C. Fungal growth was monitored regularly. The zone of inhibition was determined after a few days [23].

2.2.2. Antibacterial Activity of LAB by Well Method

Lactic acid bacteria were cultured in MRS broth for 48 h at 37 °C. The culture filtrate was collected by centrifugation at 4000 rpm at 4 °C for an hour and then filtered by a different pore size of filter paper (Whatman No 4, Whatman No 2 and 0.2 μm filters). Pathogenic bacteria such as Klebsiella sp. and E. coli were grown in nutrient broth at 37 °C for 24 h. The pathogens were spread onto a nutrient agar plate and made a well-by-well cutter. Then, 100 μLof the LAB culture filtrate was loaded into each well and incubated for 24–48 h at 37 °C. Then, the zone of inhibition was observed after 48 h incubation [22].

2.3. Corn Silage Production

2.3.1. Inoculum Preparation

The isolated LAB was grown in MRS (Conda, Madrid, Spain) for 36 h at 37 °C with mild shaking at 150 rpm in an orbital shaker in a microaerobic environment. The LAB cultures were then centrifuged for 45 min at 4000 rpm at 4 °C. Then, filter residue was washed twice with PBS, pH 7.4 (Gibco, Waltham, MA, USA). A logos Quantom Tx Microbial cell counter was used to count the total LAB density (Logos Biosystem, Biosystem, Anyang-si, Gyeonggi, Republic of Korea). LAB were then suspended in sterile distilled water at a density of 108/mL for further use.

2.3.2. Cultivation and Silage Production

The corn Dacheongok cultivar was cultivated in farmland at the National Institute of Animal Science, Cheonan, Korea, according to the Rural Development Administration guidelines. At the ripening stage, the whole crop corn was collected and chopped by a manual cutter. Three whole crop chopped corn samples/bag were placed in a polythene bag (Aostar Co., Ltd., Seoul, Republic of Korea). Furthermore, the packed samples were divided into three groups such as the control group (CONT), Inoculum I-treated group and Inoculum II-treated group (LABK). The control group: corn silage was produced without inoculum; the Inoculum I-treated group: corn silage was produced with Lacticaseibacillus paracasei at a density of 1 × 105/gram fresh weight (FW); and the Inoculum II-treated group: corn silage was made with commercial LAB cocktail 100 g/tonne, which includes P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19 (Top Silage, Republic of Korea). All silage bags were sealed by a vacuum machine (MK Corporation, Seoul, Republic of Korea, Food Saver V48802). All sample bags were stored at room temperature for 60 days in a laboratory [15].

2.3.3. Sampling and Organic Acid Analysis in Corn Silage

After 60 days’ fermentation, ten grams of corn silage was suspended in 90 mL of distilled water and shaken with an orbital shaker for 60 min and then placed in a cold room for 12 h. Then, a portion of samples was taken and filtered by different sizes of filters (Whatman No 2 and 0.2 μm membrane syringe filters). The pH of the sample filtrate was determined by a pH meter (Thomas Scientific, Logan Township, NJ, USA). For organic acid quantification, the sample’s pH was reduced to pH 2 with H2SO4 (50%) and it was frozen at −20 °C for high-performance liquid chromatography (HPLC; HP1100, Agilent Corporation, Santa Clara, CA, USA). To elute the sample, 0.1 M H2SO4 was used on a Hi-Plex Ligand exchange column from Agilent (300 × 7.7 mm). The wavelength was fixed at 220 nm and the flow rate was 0.6 mL/min. To determine the acetic acid and butyric acid content of silage, a CP7485 column fused with silica gel (length—25 cm, diameter—0.32, and film thickness—0.30) was used at temperatures ranging from 20 °C to 270 °C. Flow rates of 10 microliters per min were used [14].

2.3.4. Nutrient Content Analysis

After two months, the fermented samples were dried at 60 °C until they reached a constant weight in an oven to determine DM content and then the dried samples were ground and passed through a 1 mm sieve and used for the analysis of nutrient content. Acid detergent fiber (ADF) and crude protein [24] and neutral detergent fiber (NDF) [25] were measured in experimental silages.

2.3.5. Bacteria, Yeast and Mold Enumeration in Corn Silage

Another portion of samples from above was used to analyze the microbial population by MRS agar and 3M Pertrifilm methods. The sample was serially diluted with distilled water by a tenfold serial dilution method and then 100 μL of the diluted sample (104–105) was spread onto MRS agar and then all plates were incubated at 37 °C for 48 h. The bacterial colonies in the plates were counted and calculated. For yeast and mold enumeration, 1 mL of the serially diluted (103–104) sample was placed on a 3M Petrifilm and spread uniformly and kept at 32 °C for 72–120 h. Yeast and mold were then enumerated according to Jung et al. with slight modifications [15].

2.3.6. Bacterial Dynamics in Corn Silage by Metagenomics

Microbial community distribution in samples was determined using high-throughput sequencing technology. Using a Qiagen DNA extraction kit (Qiagen, Hilden, Germany), DNA was extracted from each experimental sample. The DNA level was quantified using the Quant-iT PicoGree Kit (Invitrogen, Waltham, MA, USA). A 16S Metagenomic Sequencing Library Kit from Illumina was used to prepare sequencing libraries according to the standard protocol. A MiSeqTM platform from Illumina (San Diego, CA, USA) was used for sequencing by Macrogen Pvt Ltd., Seoul, Republic of Korea [26]. After sequencing, Cutadapt-3.2 was used to remove adapter and primer sequences from the raw data. In addition, forward and reverse reads were trimmed to 250 bp and 200 bp by Cutadapt-3.2. Amplicon sequence variants (ASVs) were generated after an error correction, merging and denoising process with DADA2-1.18.0 [27]. Also, microbial community comparison and normalization was performed using QIIME-1.9 [28].

2.4. Statistical Analysis

Significant differences between treatments (n = 3) were calculated using SPSS 16. A one-way ANOVA, a t-test and multivariate analyses were performed with post hoc, Duncan’s, and descriptive tests. Statistical significance was defined as p < 0.05. To determine the interaction between bacterial taxonomic profiles at the phyla and genus level and different treatments, a heatmap method was used (Macrogen Pvt. Ltd., Seoul, Republic of Korea). A correlation coefficient was determined between microbial abundance and fermentative parameters. Based on microbial abundance in the phylum and genus, lactic acid, acetic acid, and butyric acid were dependent variables. A correlation coefficient measured at the p < 0.05 level is considered significant.

3. Results

3.1. Selection of Potent Lactic Acid Bacteria for Corn Silage Production

Primarily, several lactic acid bacteria were isolated from fermented whole crop corn and screened for their antibacterial and antifungal activity against common silage spoilage microorganisms including Aspergillus sp., Penicillium sp., Klebsiella sp. and E. coli. The data indicate that K-68 showed the strongest inhibition against all tested microorganisms with the highest inhibitory zone of 35.7 ± 2.1 mm against Aspergillus spp. and 25.5 ± 0.5 mm against Klebsiella sp. K-56, and K-50 also showed significant antifungal activity against Aspergillus sp. (28.7 ± 1.2 mm and 23.3 ± 1.4 mm, respectively). Other LAB strains such as K-23, K-28 and K-60 exhibited moderate inhibitory effects, whereas K-30 had the weakest inhibitory activity against Klebsiella sp. compared to the other LAB (Table 1). According to this finding, K-68 showed strong inhibitory activity against all tested microorganisms, suggesting K-68 could be effective in controlling fungal and bacterial spoilage in silage. Hence, K-68 was selected and screened for its physiochemical properties, carbohydrate fermentation ability and extracellular enzyme production (Supplementary Tables S1–S3). The 16srRNA sequence data revealed that K-68 is Lactobacillus paracasei-K68 and it was used for corn silage fermentation.

3.2. Impacts of L. paracasei and Cocktail LAB on Fermentation of Whole Crop Corn

Table 2 presents the impact of L. paracasei-K68 (LABK) and cocktail LAB (LABC) on pH and organic acid levels in corn silage after two months. The results show that corn silage developed without LAB inoculum showed the highest pH and the lowest lactic acid content compared to LAB-treated silages, suggesting a less efficient fermentation process. But LABK treatment significantly reduced the pH to 3.5 ± 0.02 and increased lactic acid content to 6.10 ± 0.25% compared to the control silage (p < 0.05). Similarly, LABC treatment also showed a lower pH (3.6 ± 0.01) and increased lactic acid levels (5.36 ± 0.31%) but not as efficiently as the LABK treatment. Levels of acetic acid, propionic acid, and isobutyric acid did not show significant differences between the groups. Overall, the data indicate that LABK enhances fermentation quality in silage by promoting lactic acid production and reducing pH.
Microbial composition including LAB, yeast and mold in fermented corn silage was evaluated after 2 months (Table 3). The data suggest that the LAB-treated group had significantly higher LAB counts and lower yeast/mold populations compared to the control group silage (p < 0.05). The cocktail LAB group had the highest LAB count (38.0 ± 8.2 × 10⁵ CFU/g), followed by the LABK-treated group (30.0 ± 4.6 × 10⁵ CFU/g), while the control had the lowest LAB population (17.0 ± 2.0 × 10⁵ CFU/g). Total yeast and mold counts were higher in control silages (529.3 ± 47.4 × 10⁴ CFU/g for yeast and 97.0 ± 28 × 103 CFU/g for mold) compared to LAB-treated groups. In contrast, LABK-treated silage had the lowest yeast counts (150.0 ± 10.0 × 10⁴ CFU/g) and mold (23.3 ± 1.7 × 103 CFU/g) compared to the control and LABC-treated silages.
Nutrient composition, including acid detergent fiber (ADF), neutral detergent fiber (NDF), and crude protein content were evaluated in the experimental silage after fermentation (Table 4). Silage produced with LABK or LABC showed no significant differences (p < 0.05) in ADF, NDF and CP content in corn silages after two months compared to the control silage.

3.3. Microbial Dynamics Changes in Experimental Silages

LAB treatments significantly improved silage quality by increasing lactic acid and reducing butyric acid levels. Then, bacterial dynamics changes in experimental silages were investigated to confirm the LAB addition in silage fermentations. Figure 1a–d present the alpha diversity index such as amplicon sequence variants (ASVs), the Shannon index, the Gini–Simpson index and PD whole tree in corn silage after two months of fermentation under the different treatments LABK, and LABC, suggesting silage developed with LABC exhibited the highest ASVs (139.6), Shannon index (2.73), Gini–Simpson index (0.67), and PD whole tree value (12.48). But silage developed with LABK had the lowest ASV (63), Shannon index (1.32), Gini–Simpson index (0.35), and PD whole tree value (7.34) compared to the LABC and control silage group.

3.4. Bacterial Dynamic Changes in Experimental Silages

At the phylum level, Bacillota was the dominant phyla in all experimental silages, followed by Pseudomonadota (Table 5 and Figure 1e). The control silage had Bacillota (91.90%), Pseudomonadota (7.55%), Actinomycetota (0.15%) and bacteroidota (0.11%). But the level of Bacillota increased in silage developed with LABK (99.77%) or LABC (96.76%). But the level of Pseudomonadota decreased to 0.17% and 2.83% in LABK- or LABC-treated silages, respectively.
At the genus level, the microbial composition in the experimental silages varied dramatically between treatments (Table 6 and Figure 1f). Control silage had the highest level of Levilactobacillus (28.22%), Lentilactobacillus (21.60%), Lacticaseibacillus (13.93%), Clostridium (8.00%), Klebsiella (5.07%) followed by Lactiplantibacillus, Paenibacillus, Leuconostoc and Lacrimispora etc. Silage developed with LABK predominantly comprised Lacticaseibacillus (93.26%) followed by Pediococcus (2.96%), Lacrimispora (1.58%) and Paenibacillus (0.79%). In addition, Lacticaseibacillus (28.41%), Pediococcus (23.47%), Lacrimispora (14.81%), Levilactobacillus (9.97%), Paenibacillus (9.45%), and Lactiplantibacillus (5.87%) were the dominant genera found in silage developed with LABC.
At the species, the microbial composition varied dramatically between treatments. The control silage had highest level of Levilactobacillus brevis (28.22%), Lentilactobacillus hilgardii (21.58%), Lacticaseibacillus paracasei (13.93%), Clostridium beijerinckii (7.87%), Lactiplantibacillus fabifermentans (3.86%), Klebsiella variicola (3.55%), and Leuconostoc holzapfelii (3.67%), whereas the silage developed with LABK showed the highest level of Lacticaseibacillus paracasei (93.25%) and Pediococcus pentosaceus (2.96%). LABC-treated silages comprised 28.41% of Lacticaseibacillus paracasei, 23.47% of Pediococcus pentosaceus, 14.63% of Lacrimispora xylanolytica, 9.9% of Levilactobacillus brevis and 8.16% of Paenibacillus azotifigens and 5.74% of Lactiplantibacillus fabifermentans (Table 7).
Figure 2a,b shows a heatmap study between the bacteria at the phylum/genus level for the experimental groups. The control silage had a strong positive relationship with Pseudomonadota, Planctomycetota Verrucomicrobiota, and Actinomycetota and a negative relationship with Bacillota, whereas silage developed with LABK had a strong relationship with bacillota followed by Acidobacteriota and a strong negative relationship with Pseudomonadota, Bacteroidota and Pseudomonadota. But silage developed with cocktail LAB (LABC), shows a strong positive relationship with Bdellovibrionota, Deinococcota, Thermomicrobiota, Myxococcota, Bacillota, and Bacteroidota (Figure 2a). At the genus level, the control group silage had a more positive interaction with Clostridium, Klebsiella, Leuconostoc, Levilactobacillus, and Lentilactobacillus, whereas silage developed with LABK had a strong relationship with Lacticaseibacillus and a negative relationship with Klebsiella, Clostridium Lactiplantibacillus, and Anaerocolumn etc. The genera Pediococcus, Paenibacillus, Lactiplantibacillus and Lacrimispora showed a positive relationship with LABC-treated silage (Figure 2b).
Furthermore, interactions between bacterial profiles at the phylum or genus level and fermentative metabolites such as pH, lactic acid (LA), acetic acid (AA), butyric acid (BA), propionic acid (PA) and isobutyric acid (IBA) were also studied by heatmap correlations. At the phylum level (Figure 3), pH and yeast were negatively correlated with Bacillota (p < 0.01), lactic acid was positively correlated with Bacillota. Pseudomonadota and Cyanobacteriota were positively correlated with pH and yeast growth (p < 0.01). At the same time, these phyla were negatively correlated with lactic acid production. Furthermore, the evidence correlation between bacteria at the genus level and fermentative parameters was investigated; it suggested that Lacticaseibacillus was positively correlated with LA (p < 0.07) and negatively correlated with pH and yeast (p < 0.05). Acetic acid showed a strong positive correlation with Levilactobacillus (p < 0.01) and negative correlation with Pediococcus (p < 0.01). LAB treatment showed a significant negative correlation with clostridium (Figure 3a,b).

4. Discussion

This study demonstrated that lactic acid bacteria (LAB) inhibit fungal and bacterial spoilage organisms in corn silage, improving fermentation quality and microbial populations after two months’ storage. The antibacterial and antifungal activity of the isolated strains, especially K-68, K-56, and K-50, were significant against all tested bacteria and fungi. Isolated LAB effectively suppresses fungi such as Aspergillus sp., Penicillium sp., and bacteria such as Klebsiella sp. and E. coli. These results aligned with previous studies indicating that LAB can produce various organic acids, hydrogen peroxide and bacteriocins, which exhibit strong antimicrobial properties against spoilage microorganisms [17,29]. The maximum zone of inhibition was observed against all tested spoilage microorganisms with K-68. This confirms that this strain possesses strong antibacterial and antifungal properties than other strains. The maximum inhibition zone was observed for all tested spoilage microorganisms with K-68. This confirms that this strain possesses stronger antibacterial and antifungal properties than others. A 16srRNA sequence analysis of the selected strain revealed that it belongs to Lacticaseibacillus casei.
Silage fermentation preserves forage nutrients. The ensiling method preserves plant nutrients through spontaneous lactic acid production in an anaerobic condition. The ensiling of forage has gained a great deal of attention as it offers ruminants a consistent, reliable and predictable supply of feed. Lactic acid bacteria (LAB) play a key role in the fermentation of forages. LAB can convert water-soluble carbohydrates (WSCs) in plants into lactic acid and acetic acid, resulting in a lower pH of ensiled forages. LAB typically occurs at low levels in plants, not sufficient to cause rapid fermentation. Thus, adding LAB as a biological additive during ensiling is crucial to induce rapid fermentation that increases lactic acid content and reduces acetic acid and butyric acid levels, as well as reducing proteolysis and increasing dry matter recovery [30,31]. In this study, corn silage was developed with Lacticaseibacillus paracasei- K68 (LABK) or cocktail LAB (LABC) and its fermentation efficiency was evaluated after two months. A reduction in pH and higher levels of lactic acid were observed after LABK treatment compared to the control silages. A reduction in pH and an increase in lactic acid are closely related to the preservation of ensiled silage. A rapid acidification process inhibits microbial growth during ensiling, reducing proteolytic degradation and inhibiting undesirable growth [12]. In general, pH is used to monitor silage quality; 3.8–4.2 is usually considered ideal for ensiling [32]. For silage with a high moisture content, pH 4.2 is also considered a benchmark for well-conserved silage [33]. In the present study, the pH was reduced below the desired pH (pH < 4) in all experimental silages. However, silage developed with LABK or LABC significantly lowered the pH of corn silage compared to control silage, suggesting an addition of LAB could have significant impacts on pH reduction.
In silage, lactic acid, and acetic are the main acids, and they are the most common acids produced by Lactobacillus from WSCs [9]. Lactic acid is present at high concentrations during fermentation, and it is 10–20 times stronger than other acids [9] and it reduces silage pH. In this study, silage treated with Lacticaseibacillus paracasei- K68 (LABK) showed a marked decrease in pH and an increase in lactic acid levels, indicating enhanced lactic acid fermentation. Additionally, cocktail LAB (LABC) also increased fermentation characteristics, though slightly less than LABK and greater than the control silage. The findings demonstrate the effectiveness of LABK in driving the production of lactic acid, which lowers the pH of the surrounding environment and creates unfavorable conditions for spoilage microorganism’s growth. In the control silage, lactic acid production was lower due to a lower number of LABs and a higher number of yeast and mold counts. The lack of LAB dominance over fermentation for corn silage was supported by the microbial enumeration, and silages produced with either LABK or LABC had significantly higher numbers of LABs and lower numbers of yeasts and molds counts compared to the control silage. Notably, LABK-treated silages had the lowest counts of yeast and mold, suggesting its dominant ability to reduce yeast and fungal multiplications in corn silages. The reduction in undesirable microbial activity is essential for extending the shelf life of fermented silage and maintaining its nutritional value for a long time. A higher LAB count in LAB-treated silages also indicates a healthier microbial environment dominated by beneficial bacteria. It has been shown that high acetic acid and butyric acid contents are the primary negative indicators of fermentation quality and reduce dry matter content and energy production during fermentation [31]. All experimental corn silages contained noticeable levels of acetic acid, but there were no significant differences between treatments. In terms of trend, LABK-treated silages had the lowest level of acetic acid, followed by LABC-treated silages. This indicates that the presence of LAB treatment modulated the level of acetic acid slightly. The negative indicator acid butyric acid was absent in all experimental silages. The levels of propionic acid and isobutyric acid were not significantly changed between treatments (p < 0.05). LABK- and LABC-treated silage did not differ in the amount of ADF, NDF, and CP compared to the control silage, indicating that neither LABK nor LABC had the ability to degrade the fiber content of corn silage.
The relative abundance and diversity of bacteria in experimental corn silages were investigated by the Illumina sequencing platform. It is a powerful method used to understand microbial diversity and composition in samples [34]. Corn silage after 2 months of fermentation under different treatments such as control, L. paracasei-K68 (LABK), and cocktail LAB (LABC) shows significant differences in microbial diversity and composition that affect silage quality, fermentation stability, and microbial resilience. Alpha diversity indices reveal that the LABC treatment maintained the highest microbial richness and evenness, as shown by the Shannon index, Gini–Simpson index, and PD whole tree value. This suggests that LABC promotes a more diverse and stable microbial community in fermented corn silage. In contrast, LABK significantly reduced microbial diversity (63 ASVs) compared to LABC and the control. The LABK group had the lowest Shannon index. This reduction in diversity indicates that LABK dominates other microbial growth and favors positive fermentation. The majority of bacteria involved in lactic acid fermentation in silage belong to the phylum Bacillota [35] and include Lactobacillus, Pediococcus, Lactococcus, Weissella and Leuconostoc [36]. The fermentation quality and microbial composition of corn silage are profoundly affected by lactic acid bacteria inoculants. A phylum-level analysis of bacterial populations revealed significant changes in dominant microbial communities under different treatment conditions such as control, LABK, and cocktail LAB (LABC). These shifts provide insight into the role that inoculation plays in silage fermentation and other bacterial growth suppression.
Bacillota constituted 91.90% of the bacterial population in the control group, while Pseudomonadota, which includes spoilage-associated and opportunistic pathogens, represented 7.55%. In addition, it had significant proportions of Actinomycetota, Bacteroidota, and Cyanobacteriota, which may reflect an undesirable fermentation process. Silage quality could be reduced due to the relatively high percent of Pseudomonadota in the control silages. However, silage treated with LABK or LABC showed higher bacillota dominance, increasing to 99.77% and 96.76%, respectively. Pseudomonadota abundance in silages treated with LABK or LABC dropped sharply to just 0.7% and 2.83%, respectively, compared to the control silage. The results suggest that LABK treatment effectively promotes beneficial fermentative bacteria growth in ensiled corn. The dominance of Bacillota, particularly members of the Lacticaseibacillus genus, is a desirable outcome of because these bacteria are key producers of lactic acid and play a central role in reducing the pH of silage, which inhibits pathogenic and spoilage organisms. Minor phyla such as Actinomycetota, Bacteroidota, and Cyanobacteriota remain present in all treatments but at much lower levels in LAB-treated silages.
Several studies have reported differences in the microbial dynamics in corn silage produced with LAB or without LAB. Xu et al. reported that the addition of L. buchneri combined with Saccharomyces cerevisiae did not affect total numbers of yeast and mold communities in corn silage during ensiling and aerobic exposure, whereas lactobacilli abundance in silage treated with L. acidophilus or L. plantarum was increased after 45 and 90 days of ensiling [37]. Another report claimed that silage produced with L. salivarius and L. rhamnosus could affect the microbial dynamics throughout the ensiling periods, resulting in a gradual shift in dominant bacterial genera from Pediococcus to Lactobacilli [38]. L. Plantarum treatment modulated the bacterial communities at the early stage of fermentation, but silage produced with L. buchneri modulated the bacterial dynamics at the later stage of fermentation [39,40]. In the present study, Lacticaseibacillus dominated at the genus level (93.26%), almost completely eliminating other bacterial genera in corn silage treated with L. paracasei K-68. Hence, L. paracasei K-68 is highly competitive, outcompeting other microorganisms rapidly and ensuring rapid lactic acid fermentation. The LABC treatment maintained a more balanced bacterial composition, with substantial proportions of Pediococcus (23.47%), Lacrimispora (14.81%), Paenibacillus (9.45%), and Lacticaseibacillus (28.14%). Control silage had a wider distribution of bacteria, including Levilactobacillus (28.22%), Lentilactobacillus (21.60%), Clostridium (8.00%) and Klebsiella (5.07%). Clostridium and Klebsiella genera include spoilage-associated species that cause butyric acid fermentation, leading to poor silage quality. According to these data, L. paracasei K-68 inoculum modulated bacterial communities after 2-month fermentation, resulting in rapid shifts in Levilactobacillus, Lentilactobacillus, Clostridium, and Klebsiella abundances into Lacticaseibacillus. In addition, LABC treatment also significantly modulates bacterial communities, and this results in shifts in Levilactobacillus, Lentilactobacillus, Clostridium and Klebsiella into Lacticaseibacillus Pediococcus and Lactiplantibacillus.
Species-level distributions provide insight into microbial dynamics. The L. paracasei K-68 (LABK)-treated silage was almost entirely dominated by Lacticaseibacillus paracasei (93.25%), demonstrating its ability to outcompete other bacterial species and drive fermentation. On the other hand, LABC treatment showed a more heterogeneous bacterial community, with high proportions of Pediococcus pentosaceus (23.47%), Lacrimispora xylanolytica (14.63%), and Lacticaseibacillus paracasei (28.41%). LABC treatment maintained a balance between efficient lactic acid fermentation and microbial diversity, which may improve fermentation stability and resilience. In the control silage, Levilactobacillus brevis (28.22%), Lentilactobacillus hilgardii (21.58%), and Clostridium beijerinckii (7.87%) were the most abundant bacteria. There is a noticeable presence of Clostridium beijerinckii, which is linked to butyric acid production, and it negatively affects silage quality and preservation. This highlights that LABK exhibits a more significant effect on the fermentation of corn silage and its bacterial dynamics than cocktail LAB and the control, which prevent the multiplication of spoilage microorganisms. Particularly, most of the 16srRNA sequences belong to Lacticaseibacillus paracasei (93.25%), evidence that L. paracasei K-68 outcompetes other bacterial growth and induces positive lactic acid fermentation within a short period of time. The application of L paracasei K-68 in corn silage production is similar to that of L Plantarum and differs from L. Buchneri [39,40].
The heatmap analysis provides valuable insights into the relationships between microbial communities and different treatments. The control silage showed a positive correlation with phyla such as Pseudomonadota, Planctomycetota, Verrucomicrobiota, and Actinomycetota and a negative correlation with Bacillota, which includes beneficial lactic acid bacteria. This pattern reflects an unfavorable environment for corn silage fermentation. In contrast, silage inoculated with L. paracasei K-68 showed a strong positive correlation with Bacillota, and a negative correlation with Pseudomonadota, Bacteroidota, and other spoilage-associated phyla. In LABC, there were positive associations with not only Bacillota but also Bdellovibrionota, Deinococcota, and Myxococcota, suggesting complex microbial interactions. At the genus level, control silage was positively associated with spoilage and undesirable microbes, while L. paracasei K-68 showed a strong association with Lacticaseibacillus, a key genus associated with lactic acid production. Pediococcus, Paenibacillus, Lactiplantibacillus, and Lacrimispora were positively associated with the LABC-treated group, indicating a broader range of genera, but still, these are favorable microbial compositions.
Heatmap correlations between bacterial dynamics and fermentation products further emphasized the role of Bacillota, which was negatively correlated with pH and yeast and positively correlated with lactic acid production. Contrary to this, Pseudomonadota and Cyanobacteriota were negatively associated with lactic acid and positively associated with pH. At the genus level, Lacticaseibacillus showed a strong positive relationship with lactic acid and a negative correlation with pH and yeast growth, which affirms its crucial role in effective silage fermentation and prevents an undesirable fermentation process. Interestingly, Levilactobacillus had a strong positive correlation with acetic acid, while Pediococcus was negatively associated with acetic acid production, indicating functional differences even among beneficial LAB. Importantly, LAB populations in silages were negatively correlated with Clostridium, a harmful genus associated with butyric acid production and silage spoilage. Overall, these findings highlight that targeted inoculation with L. paracasei K-68 not only enhances silage fermentation but also reshapes microbial interactions to favor preservation and feed quality compared to commercial cocktail LAB.

5. Conclusions

This study concludes that Lacticaseibacillus paracasei K-68 is an effective silage inoculant. In addition, it has antibacterial and antifungal properties against spoilage bacteria such as E. coli and Klebsiella sp. and fungi such as Penicillium sp. and Aspergillus sp. Also, it can lower silage pH, boost lactic acid production, and suppress spoilage organisms by shifting them to Lacticaseibacillus. This makes it a highly promising candidate for improving corn silage quality and stability. The cocktail LAB treatments provide a broad spectrum of applications in corn silage production. But L. paracasei K-68 appears to provide more consistent and robust outcomes, making it particularly useful for silage management. We conclude that L. paracasei K-68 can improve feed quality and safety and the predictability and resilience of silage fermentation systems, warranting further research into its broader applications and long-term effects.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11060304/s1, Table S1: A description of the taxonomic characteristics of L. paracasei-K68. Table S2: In vitro fermentation of carbohydrate substrates by L. paracasei-K68 Table S3: In vitro extracellular enzyme detection from L. paracasei-K68 using API-ZYM kit.

Author Contributions

Conceptualization, K.C.C., I.S. and J.S.J.; methodology, K.C.C., I.S. and J.S.J.; software, I.S.; formal analysis, I.S. and C.-W.M.; investigation, I.S., J.S.J. and C.-W.M.; writing, I.S.; writing—review and editing, J.S.J. and C.-W.M.; supervision, K.C.C.; project administration, K.C.C.; funding acquisition, K.C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by a grant from the National Institute of Animal Science, Rural Development Administration, Republic of Korea, titled “Efficient succession production of Domestic Maize and Triticale silage and feeding to Hanwoo cow and heifer for growth, reproduction and beef production (PJ017203)”. This study was supported by the 2025 RDA Fellowship Program of the National Institute of Animal Science, Rural Development Administration, Republic of Korea.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

This article contains all the experimented data.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00304 g001
Figure 1. The Alpha diversity index and the abundance of bacteria at the phylum and genus levels in corn silage produced using LABK or LABC. (a) Amplicon sequence variants (ASVs) in control and inoculum-treated corn silages; (b) the Shannon index in control and inoculum-treated corn silages; (c) the Gini–Simpson index in control and inoculum-treated corn silages; (d) the phylogenetic diversity whole tree index (PD whole tree) in control and inoculum-treated corn silages; (e) the abundance of bacteria at phylum levels in the corn silage produced; (f) the abundance of bacteria at genus levels in the corn silage produced. The data are presented as the mean ± std of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
Figure 1. The Alpha diversity index and the abundance of bacteria at the phylum and genus levels in corn silage produced using LABK or LABC. (a) Amplicon sequence variants (ASVs) in control and inoculum-treated corn silages; (b) the Shannon index in control and inoculum-treated corn silages; (c) the Gini–Simpson index in control and inoculum-treated corn silages; (d) the phylogenetic diversity whole tree index (PD whole tree) in control and inoculum-treated corn silages; (e) the abundance of bacteria at phylum levels in the corn silage produced; (f) the abundance of bacteria at genus levels in the corn silage produced. The data are presented as the mean ± std of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
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Figure 2. Heatmap analysis between bacteria and the experimental group of silages. (a) Heatmap interactions between bacteria at the phyla level and experimental group of silages; (b) Heatmap interactions between bacteria at the genus level and experimental group of silages. The data are presented as the mean of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
Figure 2. Heatmap analysis between bacteria and the experimental group of silages. (a) Heatmap interactions between bacteria at the phyla level and experimental group of silages; (b) Heatmap interactions between bacteria at the genus level and experimental group of silages. The data are presented as the mean of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
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Figure 3. Correlation studies between bacteria and fermentative parameters. (a) Correlation between bacteria at the phyla level and fermentative parameters of silages; (b) correlation between bacteria at the genus level and fermentative parameters of silages. The data are presented as the mean of three replicates. ** p < 0.05 and * p < 0.00 levels indicate significance. LA: lactic acid; AA: acetic acid; PA: propionic acid; IBA: isobutyric acid; LAB: lactic acid bacteria.
Figure 3. Correlation studies between bacteria and fermentative parameters. (a) Correlation between bacteria at the phyla level and fermentative parameters of silages; (b) correlation between bacteria at the genus level and fermentative parameters of silages. The data are presented as the mean of three replicates. ** p < 0.05 and * p < 0.00 levels indicate significance. LA: lactic acid; AA: acetic acid; PA: propionic acid; IBA: isobutyric acid; LAB: lactic acid bacteria.
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Table 1. Antifungal and antibacterial activity of LAB against common microorganisms associated with silage spoilage.
Table 1. Antifungal and antibacterial activity of LAB against common microorganisms associated with silage spoilage.
StrainsFungi Inhibition (mm)Bacterial Inhibition (mm)
Aspergillus sp.Penicillium sp.Klebsiella sp.E. coli
K-2314.0 ± 1.212.0 ± 1.021.3 ± 1.222.8 ± 0.8
K-2814.0 ± 3.613.3 ± 1.522.3 ± 0.922.5 ± 0.9
K-3015.7 ± 3.215.0 ± 0.02.21 ± 1.821.8 ± 2.4
K-5023.3 ± 1.414.7 ± 1.520.7 ± 0.521.5 ± 1.7
K-5628.7 ± 1.214.7 ± 2.120.7 ± 0.521.8 ± 1.9
K-6021.7 ± 1.415.7 ± 1.522.0 ± 0.821.3 ± 1.3
K-6835.7 ± 2.128.0 ± 1.325.5 ± 0.524.5 ± 0.5
Table 2. Corn silage pH and organic acids after 2 months of treatment with K-68 or cocktail LAB.
Table 2. Corn silage pH and organic acids after 2 months of treatment with K-68 or cocktail LAB.
GroupspHLA (DM%)AA (DM%)PA (DM%)IBA (DM%)
Control3.7 ± 0.01 a4.10 ± 0.06 c0.73 ± 0.12 a0.10 ± 0.06 a0.04 ± 0.04 a
LABK3.5 ± 0.02 c6.10 ± 0.25 a0.42 ± 0.28 a0.04 ± 0.00 a0.03 ± 0.02 a
LABC3.6 ± 0.01 b5.36 ± 0.31 b0.65 ± 0.35 a0.10 ± 0.05 a0.10 ± 0.01 a
LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19). LABK—L. paracasei-K68, LA: lactic acid; AA: acetic acid; PA: propionic acid; IBA: isobutyric acid; DM: dry matter content. The data are presented as the mean ± STD of three replicates (n = 3). Different alphabets (a, b, and c) within a column indicate significant differences between the experimental groups.
Table 3. Changes in microbial profiles in corn silage after 2 months in response to K-68 or cocktail LAB.
Table 3. Changes in microbial profiles in corn silage after 2 months in response to K-68 or cocktail LAB.
GroupsLAB (×105 CFU/g)Yeast (×104 CFU/g)Mold (×103 CFU/g)
Control17.0 ± 2.0 c529.3 ± 47.4 a97.0 ± 28 a
LABK30.0 ± 4.6 b150.0 ± 10.0 c23.3 ± 1.7 c
LABC38.0 ± 8.2 a227.0 ± 30.5 b35.5 ± 1.5 b
LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68, CFU: colony forming unit. The data are presented as the mean ± STD of three replicates (n = 3). Different alphabets (a, b, and c) within a column indicate significant differences between the experimental groups (p < 0.05).
Table 4. Nutrient content (%) of experimental corn silages after two months’ fermentation.
Table 4. Nutrient content (%) of experimental corn silages after two months’ fermentation.
GroupsCPNDFADF
Control8.19 ± 0.4 a41.86 ± 1.8 a22.21 ± 1.5 a
LABK8.04 ± 0.2 a43.91 ± 1.7 a22.93 ± 1.7 a
LABC7.71 ± 0.4 a40.22 ± 4.0 a23.10 ± 2.6 a
CP: crude protein; ADF: acid detergent fiber, NDF: neutral detergent fiber. The data are presented as the mean ± STD of three replicates (n = 3). a Different alphabets within a column indicate significant differences between the experimental groups.
Table 5. The abundance of bacteria in corn silages treated with LABC or LABK.
Table 5. The abundance of bacteria in corn silages treated with LABC or LABK.
PhylaControlLABKLABC
Bacillota91.90%99.77%96.76%
Pseudomonadota7.55%0.17%2.83%
Actinomycetota0.15%0.02%0.13%
Bacteroidota0.11%0.01%0.17%
Cyanobacteriota0.20%0.01%0.05%
Other0.06%0.01%0.04%
The data are presented as the mean of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
Table 6. The abundance of bacteria at the genus level in corn silages treated with LABC or LABK.
Table 6. The abundance of bacteria at the genus level in corn silages treated with LABC or LABK.
GenusControlLABKLABC
Lacticaseibacillus13.93%93.26%28.41%
Levilactobacillus28.22%0.19%9.97%
Pediococcus1.99%2.96%23.47%
Lentilactobacillus21.60%0.00%0.01%
Lacrimispora2.91%1.58%14.81%
Paenibacillus3.90%0.79%9.45%
Lactiplantibacillus4.30%0.06%5.87%
Clostridium8.00%0.05%0.02%
Klebsiella5.07%0.11%1.72%
Leuconostoc3.74%0.01%0.96%
Anaerocolumna1.78%0.30%2.51%
Konateibacter0.35%0.10%0.80%
Raoultella0.65%0.00%0.04%
Lactococcus0.35%0.00%0.20%
Serratia0.49%0.01%0.04%
Anaerospora0.10%0.26%0.00%
Acinetobacter0.04%0.00%0.31%
Enterobacter0.08%0.00%0.24%
Potamosiphon0.20%0.01%0.05%
Burkholderia0.21%0.00%0.02%
Others2.09%0.28%1.09%
The data are presented as the mean of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
Table 7. The abundance of bacteria at the species level in corn silages treated with LABC or LABK.
Table 7. The abundance of bacteria at the species level in corn silages treated with LABC or LABK.
Bacterial SpeciesControlLABKLABC
Lacticaseibacillus paracasei13.93%93.25%28.41%
Levilactobacillus brevis28.22%0.19%9.97%
Pediococcus pentosaceus1.99%2.96%23.47%
Lentilactobacillus hilgardii21.58%0.00%0.01%
Lacrimispora xylanolytica2.59%1.58%14.63%
Lactiplantibacillus fabifermentans3.86%0.03%5.74%
Paenibacillus azotifigens1.11%0.15%8.16%
Clostridium beijerinckii7.87%0.03%0.00%
Klebsiella variicola3.55%0.09%1.40%
Leuconostoc holzapfelii3.67%0.00%0.91%
Anaerocolumna chitinilytica1.68%0.30%2.51%
Konateibacter massiliensis0.35%0.10%0.80%
Paenibacillus larvae0.69%0.00%0.36%
Paenibacillus piscarius0.80%0.03%0.16%
Klebsiella pneumoniae0.70%0.02%0.21%
Raoultella ornithinolytica0.63%0.00%0.04%
Lactiplantibacillus garii0.43%0.03%0.13%
Paenibacillus turicensis0.36%0.15%0.09%
Klebsiella electrica0.51%0.00%0.07%
Lactococcus lactis0.35%0.00%0.20%
Others5.12%1.07%2.74%
The data are presented as the mean of three replicates. LABC—cocktail lactic acid bacteria (P. pentosaceus—KCC-23 and L. plantarum—KCC-10 + L. plantarum—KCC-19), LABK—L. paracasei-K68.
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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

AMA Style

Soundharrajan I, Min C-W, Jung JS, Choi KC. Effects of the Novel Lacticaseibacillus paracasei K-68 Inoculant on Nutrient Content, Fermentation, and Microbial Dynamics Changes in Dacheongok Corn Silage. Fermentation. 2025; 11(6):304. https://doi.org/10.3390/fermentation11060304

Chicago/Turabian Style

Soundharrajan, Ilavenil, Chang-Woo Min, Jeong Sung Jung, and Ki Choon Choi. 2025. "Effects of the Novel Lacticaseibacillus paracasei K-68 Inoculant on Nutrient Content, Fermentation, and Microbial Dynamics Changes in Dacheongok Corn Silage" Fermentation 11, no. 6: 304. https://doi.org/10.3390/fermentation11060304

APA Style

Soundharrajan, I., Min, C.-W., Jung, J. S., & Choi, K. C. (2025). Effects of the Novel Lacticaseibacillus paracasei K-68 Inoculant on Nutrient Content, Fermentation, and Microbial Dynamics Changes in Dacheongok Corn Silage. Fermentation, 11(6), 304. https://doi.org/10.3390/fermentation11060304

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