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Article

Dynamic Effects of Exogenous and Epiphytic Pediococcus pentosaceus on Quality and Bacterial Community Succession of Silage Mulberry Leaves

by
Chen Zhang
1,2,†,
Gangqin Shu
1,3,4,†,
Zhigang Zhu
1,2,
Yusen Li
1,5,
Zhenyu Fang
1,3,
Liyuan Chen
2,
Fachun Wan
3,
Yunhua Zhang
5,
Dingfu Xiao
3,* and
Lijuan Chen
1,*
1
The Biological Feedstuff Laboratory, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2
Anhui Key Laboratory of Livestock and Poultry Product Safety Engineering, Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei 230011, China
3
Yuelushan Laboratory, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
4
Guidong County Animal Husbandry and Aquatic Affairs Center, Chenzhou 423500, China
5
Anhui Key Laboratory of Farmland Ecological Conservation and Pollution Prevention, College of Resources and environment, Anhui Agricultural University, Hefei 230036, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(16), 1726; https://doi.org/10.3390/agriculture15161726
Submission received: 30 May 2025 / Revised: 19 July 2025 / Accepted: 4 August 2025 / Published: 11 August 2025
(This article belongs to the Section Farm Animal Production)
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Abstract

The aim of this study is to investigate the dynamic changes in nutritional components, fermentation parameters, and microbial communities of mulberry leaves during silage fermentation with exogenous and epiphytic Pediococcus pentosaceus (P. pentosaceus). P. pentosaceus P (Pp) and P. pentosaceus M (Pm), isolated from the epiphytic microbiota of paper mulberry and mulberry leaves, respectively, were used as fermentation inoculants (OD600 = 0.6). Fresh mulberry leaves were treated with the inoculants at 1% (mL/g) of leaf weight and ensiled for 60 days. Three groups were established: T1 (exogenous Pp), T2 (epiphytic Pm), and CK (control, sterile water). Samples were collected on days 1, 3, 5, 7, 15, 30, and 60 to analyze chemical composition, fermentation characteristics, and bacterial communities. Redundancy analysis was conducted to explore relationships between fermentation characteristics and bacterial communities. The results showed that T2 had significantly higher dry matter content from day 30 (p < 0.05) and lower neutral detergent fiber content from day 3 (p < 0.05) compared to T1. Additionally, T2 exhibited faster water-soluble carbohydrate consumption and more rapid pH decline during the early fermentation phase (days 1–7). Lactic acid (LA) content in T2 was significantly higher during days 1–7 (p < 0.05), while acetic acid (AA) content was significantly lower from day 3 (p < 0.05). T2 consistently showed higher crude protein and lower ammonia nitrogen (NH3-N) levels than T1 throughout fermentation. Microbial analysis revealed higher abundance of Firmicutes in T2 during days 1–15 and greater relative abundance of Pediococcus from day 1 to 30. Kosakonia was more abundant in T2, whereas Escherichia-Shigella was less abundant. During days 3–15, bacterial communities in T1 and T2 correlated positively with LA, with stronger effects in T2, driven by Pediococcus. In the later stages (days 30 and 60), bacterial communities were influenced by AA, NH3-N, and propionic acid, with Enterobacter, Lactobacillus, and Enterococcus as key contributors. This study demonstrates that supplementing epiphytic P. pentosaceus improves fermentation efficiency and nutritional quality of mulberry leaf silage compared to exogenous P. pentosaceus.

1. Introduction

Mulberry (Morus alba L.) is a deciduous tree or shrub of the genus Morus of Moraceae, which is potential feed resource as a natural woody plant because of its strong reproductive ability, good stress resistance, high yield, and wide distribution [1]. Srivastava et al. [2] found that 100 g of mulberry leaves (dry matter, DM) contains 15.31 to 30.91 g of crude protein, 2.09 to 4.93 g of crude fat, 9.70 to 29.64 g of carbohydrates, and 113 to 224 kcal of energy. Mulberry leaves can be used as a functional feed source or feed supplement in the diets of ruminants and monogastric animals [3], and replacing part of the traditional feed in livestock diets with mulberry leaves is an effective strategy to alleviate feed shortages and reduce feed costs [4].
Using microbial additives for ensiling mulberry leaves can improve the microbial community and fermentation quality of the silage and can avoid the waste of leaves due to seasonal growth. This is considered an ideal method for preparing and storing mulberry leaf feed [1]. Research shows that ensiling fermentation can improve the nutritional quality of leaves, soften lignin or plant fiber, reduce protein loss [5], enhance palatability, and increase the nutrient utilization of livestock feed [6]. However, mulberry leaf silage is usually difficult to ensile due to its high crude protein concentration and strong buffering capacity for acidic conditions.
The inoculation of lactic acid bacteria (LAB) can accelerate the ensiling process by promoting lactic acid production, increasing beneficial bacteria abundance, and reducing harmful microorganisms, thereby improving nutritional quality and fermentation characteristics [7]. Among LAB strains, Pediococcus pentosaceus (P. pentosaceus) is gradually attracting attention as a promising candidate for silage production [8]. Vadopalas et al. demonstrated that P. pentosaceus LUHS183 is involved in the acidification process during feed fermentation [9]. Inoculation with P. pentosaceus S22 significantly reduced the pH of legume silage [10]. Compared to commercial silage inoculant, P. pentosaceus Q6 could lower the pH of silages at low temperature (10 °C or 15 °C) and improve fermentation quality [11]. In addition, as a major advance in the application of animal husbandry, several species of P. pentosaceus have been successively confirmed by the European Food Safety Authority (EFSA) to be safe for use in silage production. The species P. pentosaceus has been shown to be safe for livestock, consumers of animal products fed with treated silage, and the environment, such as P. pentosaceus DSM 32291 [12] and P. pentosaceus IMI 5070255 [13], and P. pentosaceus DSM 16244 has been certified by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) as a safe additive for all animal species [14].
Despite these advantages, there are few reports on the fermentation of mulberry leaves ensiled with P. pentosaceus. Furthermore, the use of LAB inoculants does not always guarantee successful regulation of silage fermentation. Even the same species of LAB may exhibit differences in adaptive preferences towards silage due to differences in their living environment, silage raw materials, and the content and types of carbohydrates available [15,16]. Current research has primarily focused on exogenous P. pentosaceus strains while neglecting the potential of epiphytic (native) P. pentosaceus populations naturally associated with mulberry leaves. No studies have systematically compared the performance of epiphytic versus exogenous P. pentosaceus in mulberry silage systems. This study addresses critical research gaps by investigating the efficacy of both epiphytic and exogenous P. pentosaceus strains in mulberry leaf ensiling, specifically evaluating their comparative effects on nutritional preservation, fermentation dynamics, and microbial community succession. By providing foundational data for developing optimized, strain-specific inoculation strategies, our findings significantly advance the understanding of microbial ecology in silage systems while offering practical insights to enhance mulberry leaf utilization in animal feeding programs.

2. Materials and Methods

2.1. Determination of Growth Ability and Acid Production Capacity

For this experiment, two strains of P. pentosaceus were selected as fermentation inoculants: P. pentosaceus M (Pm, mulberry leaves epiphytic P. pentosaceus; isolated, identified, and preserved from mulberry leaves) and P. pentosaceus P (Pp, paper mulberry leaves epiphytic P. pentosaceus; isolated, identified, and preserved from paper mulberry leaves). Following OD600 adjustment to 0.6 for both seed solutions [17], the seed solutions were separately inoculated (2% v/v) into MRS broth. The inoculated broths were incubated at 37 °C with continuous monitoring for 24 h. At 2 h intervals, samples were collected for (1) growth measurement via OD600 using a UV–visible spectrophotometer (UV-55-00PC, Shanghai Metash Instruments Co., Ltd., Shanghai, China), and (2) acid production assessment through pH determination using a calibrated pH meter (FE, Mettler Toledo International Trading Co., Ltd., Shanghai, China) [18].

2.2. Silage Preparation

Mulberry (Morus alba L.) plants were planted on experimental farms of the Anhui Agricultural University (31°51′ N; 117°15′ E, Hefei, Anhui, China) and cultivated without herbicides or fertilizers. On September 20, 2022, fresh mulberry leaves were harvested (The chemical composition of fresh mulberry leaves is presented in Appendix A), cut into 2–3 cm2, and air-dried to a moisture level of 65%. Two seed solutions of P. pentosaceus were adjusted to OD600 = 0.8 using sterile distilled water. Each solution was then uniformly inoculated onto fresh mulberry leaves at 1% (v/w, mL inoculant per g fresh matter) for ensiling, with single-strain application maintained per batch.
The Pp treatment was recorded as the T1 group (fermentation of exogenous P. pentosaceus), and the Pm treatment was recorded as the T2 group (fermentation of epiphytic P. pentosaceus). Control groups (CK) received an equivalent volume of sterile water following identical application procedures. All materials were immediately packed into fermentation bags (lab-level silo bags, 20 × 30 cm2; Dongguan Bojia Packaging, Dongguan China), which were sealed with a vacuum sealer (Lvye DZ280, Yijian Packaging Machinery Co. Ltd., Dongguan, China). In total, 45 bags (3 treatments × 5 time × 3 replicates) were prepared and stored at room temperature (25–30 °C). The mulberry leaves underwent 60 days of ensiling, samples were taken at 1, 3, 5, 7, 15, 30, and 60 d of fermentation, and their chemical composition, fermentation characteristics, and bacterial communities were analyzed.

2.3. Analyses of Chemical Composition

Fresh and fermented mulberry leaves were dried at 105 °C for 15 min, then immediately reduced to 65 °C and dried to constant weight to determine the DM content, and they were then ground with a mill for nutritional analysis [19]. Crude protein (CP) was measured according to the Association of Official Analytical Chemists International procedures [20]. Neutral and acid detergent fibers (NDF and ADF) were analyzed by the method of Van Soest et al. [21]. Water-soluble carbohydrates (WSCs) were measured by the anthrone method [22].

2.4. Analyses of Fermentation Quality

Samples (25 g) were mixed with 225 mL of distilled water and stored at 4 °C for 24 h and filtered to determine their fermentation characteristics. The pH was measured with a pH meter, the ammonia-N (NH3-N) content was measured using the phenol-sodium hypochlorite colorimetry method described by Broderick et al. [23], and lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were analyzed by high-performance liquid chromatography [HPLC; column, Agilent TC-C18(2) gel C (250 × 4.6 mm, 5 μm; Agilent Technologies, Everett, WA, USA)] with the following parameters: oven temperature, 30 °C; mobile phase, 0.01 mol/L C2H3N; flow rate, 0.6 mL/min; injection volume, 20 μL; and detector, SPD-M10AVP. The remaining samples were oven-dried and ground for chemical analysis.

2.5. Sequencing-Based Bacterial Community Analysis

Bacterial DNA was extracted from fresh and silage mulberry leaves using a E.Z.N.A.® Stool DNA Kit (D4015, Omega, Inc., Norcross, GA, USA) according to the manufacturer’s instructions. All bacterial DNA samples were immediately sent to LC-Bio Technology Co., Ltd. (Hangzhou, China) for PCR amplification and bioinformatic analysis. The V3–V4 region(s) of the 16S rRNA gene was amplified with primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) [24]. The amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and with a Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced on the Nova Seq PE250 platform.

2.6. Statistical Analysis

The IBM SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) statistical software package for Windows was used to analyze the effects of LAB addition on the fermentation characteristics of mulberry leaves silage. The effects were evaluated using one-way ANOVA, with Duncan’s multiple range tests. Statistical significance was determined at the <0.05 level. OmicStudio tools (https://www.omicstudio.cn/home, accessed on 27 May 2024) was used to analyze the sequencing data of the bacterial community.

3. Results

3.1. Growth Ability and Acid Production Capacity of Epiphytic and Exogenous P. pentosaceus

The growth and acid production curves of epiphytic and exogenous P. pentosaceus in mulberry leaves are shown in Figure 1. Although Pm and Pp come from different sources, their growth ability (Figure 1A) and acid production capacity (Figure 1B) are consistent in MRS broth under laboratory cultivation conditions.

3.2. Chemical Compositions of Silage Mulberry Leaves

The effects of P. pentosaceus from different sources on the chemical composition of silage mulberry leaves are shown in Table 1. From day 1 to day 3 of ensiling, there were no differences in DM among the groups (p > 0.05). However, from day 5 to day 60, the DM content in the T1 and T2 groups was significantly higher than that in the CK group (p < 0.05). From day 1 to day 15, there was no difference in DM content between the T1 and T2 groups (p > 0.05), but from day 30 to day 60, the DM content in the T2 group was significantly higher than that in the T1 group (p < 0.05). As ensiling progressed, the DM content in all groups decreased, but the decline in DM content from days 1 to 3 was not significant (p > 0.05). After day 5, the DM content in the CK group continued to decrease, while in the T1 and T2 groups, it tended to stabilize after day 30.
On day 1 of ensiling, there was no difference in CP content among the groups (p > 0.05). On day 3, the CP content in the T2 group was significantly higher than that in the CK group (p < 0.05). From day 5 to day 60, the CP content in the T1 and T2 groups was significantly higher than that in the CK group (p < 0.05). From day 3 to day 60, the CP content in the T2 group was significantly higher than that in the T1 group (p < 0.05). As ensiling progressed, the CP content in all groups decreased, but the T2 group retained more CP.
The NDF content in the T1 group was significantly lower than that in the CK group from day 7 to day 60 (p < 0.05). In the T2 group, the NDF content was significantly lower than that in the CK group after day 3 (p < 0.05). From day 3 to day 60, the NDF content in the T2 group was significantly lower than that in the T1 group (p < 0.05). As ensiling progressed, the NDF content in all groups showed an initial decrease followed by stabilization.
As ensiling progressed, the ADF content in all groups showed an initial decrease followed by stabilization, but this trend was more pronounced in the T1 and T2 groups. From day 1 to day 3, there was no difference in ADF content among the groups (p > 0.05). After day 5, the ADF content in the T1 and T2 groups was significantly lower than that in the CK group (p < 0.05), with no significant difference between the T1 and T2 groups (p > 0.05).
As ensiling progressed, the WSC content in all groups decreased, but the consumption of WSCs was higher in the groups inoculated with P. pentosaceus. Throughout the ensiling period, the WSC content in the T1 and T2 groups was significantly lower than that in the CK group (p < 0.05). From day 5 to day 60, the WSC content in the T2 group was significantly lower than that in the T1 group (p < 0.05).

3.3. Fermentation Quality of Silage Mulberry Leaves

The fermentation quality of silage mulberry leaves in each silage period stage is shown in Figure 2 and Table 2. As shown in Figure 2A, the pH of each group exhibited a decreasing trend as the ensiling process progressed. The pH in the T1 and T2 groups sharply decreased from day 1 to day 15 and then tended to stabilize after 15 days. Throughout the entire ensiling period, the pH in the T1 and T2 groups was significantly lower than that in the CK group (p < 0.05) during the same ensiling period. From day 1 to day 7 of ensiling, the pH in the T2 group was significantly lower than that in the T1 group (p < 0.05), but there was no difference in pH between the two groups from day 15 to day 60 (p > 0.05). As the ensiling time increased, the NH3-N content in each group increased (Figure 2B). On all ensiling days, the NH3-N content in the T1 and T2 groups was lower than that in the CK group (p < 0.05). On day 1, there was no significant difference in NH3-N content between the T1 and T2 groups (p > 0.05), but from day 3 to day 60, the NH3-N content in the T2 group was lower than that in the T1 group (p < 0.05).
The LA and AA content in each group increased as ensiling progressed. Only the CK group produced a small amount of PA after day 7, and no BA was detected in any group throughout the entire experimental period. At all stages of ensiling, the LA and AA content in the T1 and T2 groups was significantly higher than that in the CK group (p < 0.05). During the same silage period, the LA content in the T2 group was consistently higher than that in the T1 group, with a significant difference from day 1 to day 7 (p < 0.05) and no difference from day 15 to day 60 (p > 0.05). Regarding AA content, there was no significant difference between the T1 and T2 groups on the first day of ensiling (p > 0.05), but after day 3, the AA content in the T2 group was significantly lower than that in the T1 group (p < 0.05).

3.4. Effects of P. pentosaceus from Different Sources on the Bacterial Community of Silage Mulberry Leaves

Bacterial community diversity is shown in Figure 3. In the first 7 days of silage, there were differences in α diversities (Shannon’s index) among the three groups (p < 0.05), with the CK group having the highest and the T2 group having the lowest. After 15 to 60 days of silage, the α diversities of the T1 and T2 groups showed no difference (p > 0.05), but they were significantly lower than that of the CK group (p < 0.05) (Figure 3A). From the principal component analysis (β diversities), component 1 and component 2 could explain 60.69% and 27.2% of the total variance, respectively (Figure 3B). In addition, significant differences and consistent changes in bacterial community composition were observed in mulberry leaves silage at different fermentation stages, the bacterial community in each group was clearly separated from ML (fresh mulberry leaves), and the bacterial community in the T1 and T2 groups was clearly separated from that in the CK group. Meanwhile, the T2 group was not clearly separated from the T1 group, and compared with T2, the bacterial community of T1 was more dispersed during different silage periods.
The bacterial community dynamics in silage mulberry leaves are shown in Figure 4A (phylum level) and Figure 4B (genus level). The main phylum of epiphytic bacteria at the level of fresh mulberry leaves was Proteobacteria, while the proportion of Firmicutes was relatively lower. After ensiling, the main phyla in each group were Firmicutes and Proteobacteria. At different silage days, the abundance of Firmicutes in silage mulberry leaves after inoculation with Pp or Pm was higher than that in the CK group. The relative abundance of Firmicutes in the T1 and T2 groups increased rapidly within 1–5 days of silage, while the abundance of Firmicutes in the T2 group was higher than that in the T1 group on 1–15 days. After 15 days, the abundance of Firmicutes in the T1 group surpassed that in the T2 group (Figure 4A).
At the genus level, the predominant genus in fresh mulberry leaves was Pantoea. From day 1 to day 60 of ensiling, the relative abundance of the genus Pantoea in both the T1 and T2 groups during the same silage period was lower than that in the CK group, and the predominant genus in silage mulberry leaves of the T1 and T2 groups were Lactobacillus and Pediococcus, and their relative abundance was significantly higher than that in the CK group at different silage periods. From day1 to 30 day, the relative abundance of Pediococcus in the T2 group was higher than that in the T1 group, but on day 60 of ensiling, the abundance of Pediococcus in the T1 group surpassed that of the T2 group. Throughout the entire experimental period, the relative abundance of Kosakonia in the T1 and T2 groups was higher than that in the CK group during the same silage period, and the relative abundance of Kosakonia in the T2 group was higher than that in the T1 group during the same silage period. The relative abundance of Escherichia-Shigella in the T1 and T2 groups was lower than that in the CK group during the same silage period, while the relative abundance of Escherichia-Shigella in the T2 group was lower than that in the T1 group during the same silage period.

3.5. Redundancy Analysis Between Fermentation Characteristics and Microbial Community

Redundancy analysis (RDA) was used to evaluate the relationship between fermentation characteristics and bacterial communities; the result is shown in Figure 5. The eigenvalues of the first two canonical axes in the RDA explained 32.57% and 21.98%, respectively. LA, AA, NH3-N, PA, WSC, and pH were positively correlated with the first canonical axis (RAD1), while WSC and pH were negatively correlated with the RAD1. At the initial stage of ensiling (1 day), the bacterial communities in each group had a negative correlation with WSC and pH, with Weissella being the main influencing factor. In the early stage of ensiling (3 to 15 days), the bacterial communities had a positive correlation with LA in the T1 and T2 groups, with a greater impact on LA in the T2 group compared to the T1 group, and with Pediococcus being the main influencing factor. In the middle and late stages of ensiling (30 and 60 days), the bacterial communities mainly influenced AA, NH3-N, and PA, with Enterobacter, Lactobacillus, Kosakonia, and Enterococcus being the main factors. In the CK group, the impact of the dominant bacterial communities on the fermentation characteristics of mulberry leaves ensiled from 3 to 60 days was significantly different from that in the T1 and T2 groups. This was mainly reflected in the correlation of Lactococcus, Escherichia-Shigella, and Klebsiella with NH3-N and PA, with Escherichia-Shigella being negatively correlated with Pediococcus.

4. Discussion

4.1. The Influence of Different Sources of P. pentosaceus on the Chemical Composition of Silage Mulberry Leaves

DM is the basis for nutrient presence, and the DM content of silage is an important factor in ensuring the success of silage production [25]. In the early stages of fermentation, DM provides sufficient substrate for LAB to convert into organic acids, leading to a decrease in the pH of the fermentation environment, which inhibits the growth of spoilage microbes [26]. Additionally, DM is the part of silage that animals can ultimately consume. CP is a major nutrient component in silage, and the degradation of CP affects the nutritional value of the feed, which, in turn, impacts the DM intake of livestock [27]. In this study, as ensiling progressed, the content of DM and CP in mulberry leaves inoculated with P. pentosaceus (T1 and T2 groups) gradually shifted from no significant difference to being significantly higher than that of the CK group at the same period. This indicates that P. pentosaceus treatment better preserved the CP and DM of mulberry leaves. Meanwhile, the NDF and ADF contents gradually shifted from no significant difference to being significantly lower than those of the CK group at the same period, which might be due to P. pentosaceus promoting microbial proliferation, enhancing microbial respiration and fermentation of fiber components [28]. Compared to the T1 group, as ensiling progressed, the DM and CP contents in the T2 group gradually shifted from no significant difference to being significantly higher than those in the T1 group at the same period, while the NDF and ADF contents gradually shifted from no significant difference to being significantly lower than those in the T1 group at the same period. This indicates that, compared to exogenous P. pentosaceus, epiphytic P. pentosaceus better preserved the CP and DM of mulberry leaves during the middle and late stages of ensiling. During the silage process, a large amount of organic acids produced by LAB fermentation degrades structural carbohydrates such as cellulose and hemicellulose into WSCs [29], resulting in a decrease in the content of NDF and ADF in mulberry leaves. WSCs further promote microbial proliferation and stability of the microbial community and provide more substrate for lactic acid fermentation [26,28]. As ensiling progressed, the WSC content in the T2 group gradually shifted from no significant difference to being significantly lower than that in the T1 group at the same period. This decrease may be due to the different utilization and consumption abilities of WSCs in mulberry leaves by P. pentosaceus from different sources.

4.2. Effect of Different Sources of P. pentosaceus on the Fermentation Quality of Mulberry Leaves

NH3-N production is related to CP degradation, and its content reflects the extent of proteolysis in silage [30]. During ensiling, LAB produce a large amount of lactic acid and acetic acid, promoting a rapid decrease in the pH of the silage to around 4.0, inhibiting the activity of harmful microorganisms and their ability of protein decomposition [31]. In the early stages of fermentation (1 to 7 days), the pH continuously decreases, but the acid accumulation is not sufficient to inhibit harmful microbial activity, leading to intense protein degradation and causing a rapid increase in NH3-N content in all groups [30]. The addition of P. pentosaceus to the T1 and T2 groups resulted in significantly lower pH compared to the CK group throughout the experimental period. This acidification effect led to corresponding reductions in NH3-N content in both inoculated groups relative to the control. While the growth and acid production kinetics of epiphytic and exogenous P. pentosaceus in MRS medium showed similar patterns, the T2 (epiphytic) group maintained consistently lower pH values than T1 (exogenous). Consequently, NH3-N content in T2 was significantly reduced compared to T1 at all time points, demonstrating the superior acidogenic potential of the epiphytic strain. This may be attributed to the stronger adaptability and metabolism of epiphytic P. pentosaceus to mulberry leaves than exogenous P. pentosaceus
LA is the most important acid in silage and can directly reflect the pH of silage [32]. The content of LA in the treatment groups was higher than that in the CK group, which proved that the addition of LAB can improve the silage nutrition of mulberry leaves, which was consistent with Yahaya et al. [33]. However, the LA in the T1 group was significantly lower than that in the T2 group at same silage period, indicating that in mulberry leaf silages, the adaptive ability and the ability to use nutrients from mulberry leaves for lactate production of exogenous P. pentosaceus are weaker than mulberry leaves epiphytic P. pentosaceus. The content of AA in all groups increased, and the results were consistent with Wang et al. [34]. The content of AA in the T1 group was significantly higher than that in the T2 group at the same period. The reason may be that the abundance of Enterobacter in the T1 group is higher than in the T2 group, and lactic acid is fermented into AA and other substances [35]; PA and BA are usually undetectable in well-fermented silages. The presence of these acids indicates metabolic activity from clostridial organisms, which leads to large losses of DM and poor recovery of energy [36]. In this study, only the CK group produced a small amount of PA (≤2 g/kg) after 7 d of ensiling, and no BA was detected in any group, indicating that P. pentosaceus helps reduce the loss of DM and energy during the process of ensiling mulberry leaves.

4.3. Bacterial Diversity of Fermentated Mulberry Leaves with P. pentosaceus from Different Sources

The addition of LAB can rapidly produce acid and create an acidic silage environment and reduce bacterial diversity [37]. Compared with the CK group, the addition of Pm or Pp reduced the microbial diversity in mulberry silage, and the Shannon index of the T2 group was significantly lower than that of the T1 group, further indicating that epiphytic P. pentosaceus has a stronger impact on the microbial diversity in silage mulberry leaves than exogenous P. pentosaceus. At the phylum level, bacterial communities were altered by the addition of P. pentosaceus, while Firmicutes and Proteobacteria remained the dominant phyla; the results were consistent with those reported by Zhao et al. [38]. Firmicutes can degrade biological macromolecules such as fiber and protein by secreting a variety of cellulases and proteases [39]. As the silage progressed, the Firmicutes gradually increased in the P. pentosaceus treatment groups, which suggests that P. pentosaceus addition promoted Firmicutes growth, further supporting their established role in silage fermentation. During the early silage period (1–15 d), the relative abundance of Firmicutes was higher in the T2 group than in the T1 group, indicating that epiphytic P. pentosaceus was more beneficial to the growth of Firmicutes than exogenous P. pentosaceus in the early ensiling stages. Enterobacteriaceae are generally considered undesirable in ensiling processes [35]. Enterobacteriaceae can survive in low-pH environments, compete with LAB for WSC [40], promote the production of ammonia nitrogen [41], and can ferment LA into AA and other products, resulting in nutritional loss [35]. The presence of Pantoea and Escherichia-Shigella was negatively correlated with silage quality. These bacteria degrade silage through proteolytic reduction in CP content and by elevating biogenic amine levels [42,43]. Such alterations collectively diminish the feed nutritional value while increasing livestock physiological risks, ultimately compromising silage quality [44]. Li et al. found that the addition of LAB can inhibit the growth of harmful microorganisms, such as Escherichia and Enterobacter, and increase the count of desirable LAB [28]. Therefore, the abundance of harmful microorganisms such as Pantoea and Escherichia-Shigella were reduced after treatment with P. pentosaceus, while the abundance of beneficial bacteria such as Lactobacillus and Pediococcus were increased, which is consistent with Jiang et al. [45]. Lactobacillus and Pediococcus play a critical role in silage fermentation, with strong abilities to produce LA and reduce pH [41,46]. Due to their ideal functions, they greatly improve the quality of silage mulberry leaves. Kosakonia has been observed by many researchers in silage [47,48,49,50]. It is considered to have an ability to reduce NH3-N [47] and to be positively related with many volatile chemicals in silage [51], and it can also secrete tannase [52]. Xiong et al. found that the addition of LAB can increase the abundance of Kosakonia in forage oat silage [50], which is consistent with the results of this study. In this study, mulberry leaves, as a protein resource rich in tannins [53], were fermented by epiphytic P. pentosaceus, which increased the abundance of Kosakonia and reduced the levels of NH3-N and tannins. This may be another reason for the improvement in the quality of silage mulberry leaves.
While this study demonstrates the effectiveness of epiphytic P. pentosaceus inoculants in improving mulberry leaf silage quality at the laboratory scale, several limitations must be acknowledged. First, lab-scale experiments may not fully replicate farm conditions due to differences in packing density and temperature control. Second, comprehensive animal feeding trials will be required to validate practical benefits. Future large-scale validation is warranted to evaluate practical applicability.

5. Conclusions

In conclusion, both exogenous and epiphytic P. pentosaceus improved the chemical composition and fermentation quality of mulberry leaf silage. Notably, the epiphytic strain demonstrated superior efficacy, attributable to its native adaptation to the phyllosphere environment. This adaptation conferred enhanced metabolic efficiency and microbial community modulation, leading to significantly better fermentation characteristics compared to its exogenous counterpart. These findings highlight the potential of utilizing naturally occurring LAB as sustainable silage inoculants. Our study provides both mechanistic insights into ensiling microbial dynamics and practical strategies for optimizing forage preservation in livestock production systems.

Author Contributions

C.Z.: Conceptualization, Methodology, Writing—original draft. G.S.: Formal analysis, Investigation, Writing—original draft. Z.Z.: Data curation, Software. Y.L.: Data curation, Software. Z.F.: Investigation, Validation. L.C. (Liyuan Chen): Supervision, Writing—review and editing. F.W.: Resources, Writing—review and editing. Y.Z.: Conceptualization, Resources, Writing—review and editing. D.X.: Funding acquisition, Project administration, Writing—review and editing. L.C. (Lijuan Chen): Funding acquisition, Resources, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32172769), Earmarked Fund for China Agriculture Research System (CARS-37), Regional Key R&D Program of Ningxia Hui Autonomous Region (2024BBF02016).

Data Availability Statement

Dataset is available on request from the authors. The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

AA, acetic acid; ADF, acid detergent fibers; BA, butyric acid; CK group, control group; CP, crude protein; DM, dry matter; EFSA, European Food Safety Authority; FEEDAP, panel on additives and products or substances used in animal feed; LA, lactic acid; LAB, lactic acid bacteria; ML, fresh mulberry leaves before ensiling; NDF, neutral detergent fibers; NH3-N, ammonia-N; PA, propionic acid; PCA, principal component analysis; P. pentosaceus, Pediococcus pentosaceus; Pm, mulberry leaves epiphytic P. pentosaceus; Pp, paper mulberry leaves epiphytic P. pentosaceus; RDA, redundancy analysis; T1 group, fermented by exogenous P. pentosaceus (Pp); T2 group, fermented by epiphytic P. pentosaceus (Pm); WSCs, water-soluble carbohydrates.

Appendix A

Characteristics of fresh mulberry leaves before ensiling.
Table A1. Chemical composition of mulberry leaves before ensiling.
Table A1. Chemical composition of mulberry leaves before ensiling.
ItemsEstimated Value
Dry matter (% FM)33.32 ± 0.14
Crude protein (% DM)17.43 ± 0.01
Neutral detergent fiber (% DM)27.76 ± 0.02
Acid detergent fiber (% DM)16.87 ± 0.02
Water-soluble carbohydrates (% DM)12.15 ± 0.04
NH3-N (g/kg TN)4.00 ± 0.01
Note: FM, fresh matter; DM, dry matter.

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Agriculture 15 01726 g001
Figure 1. Growth and acid production curves of P. pentosaceus from two sources. (A) Growth curve; (B) Acid production curve. Pm: P. pentosaceus isolated from mulberry leaves; Pp: P. pentosaceus isolated from paper mulberry leaves.
Figure 1. Growth and acid production curves of P. pentosaceus from two sources. (A) Growth curve; (B) Acid production curve. Pm: P. pentosaceus isolated from mulberry leaves; Pp: P. pentosaceus isolated from paper mulberry leaves.
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Figure 2. Changes in pH and NH3-N content of mulberry leaves during silage. (A) pH values; (B) NH3-N concentrations. NH3-N, ammonia-N. T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different little letters show significant differences among treatments in the same ensiling day (p < 0.05).
Figure 2. Changes in pH and NH3-N content of mulberry leaves during silage. (A) pH values; (B) NH3-N concentrations. NH3-N, ammonia-N. T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different little letters show significant differences among treatments in the same ensiling day (p < 0.05).
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Figure 3. Microbial diversity of mulberry leaves silage. (A) principal component analysis (PCA) of bacterial communities in mulberry leaf silage with different kinds of LAB; (B) alpha diversity of bacterial communities in mulberry leaf silage with different kinds of LAB. ML, fresh mulberry leaves before ensiling; T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different capital letters show significant differences among treatments in the same ensiling day (p < 0.05).
Figure 3. Microbial diversity of mulberry leaves silage. (A) principal component analysis (PCA) of bacterial communities in mulberry leaf silage with different kinds of LAB; (B) alpha diversity of bacterial communities in mulberry leaf silage with different kinds of LAB. ML, fresh mulberry leaves before ensiling; T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different capital letters show significant differences among treatments in the same ensiling day (p < 0.05).
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Figure 4. Relative abundance of bacterial communities at the phylum and genus level. (A) the phylum-level relative abundance of bacterial communities; (B) the genus-level relative abundance of bacterial communities. ML, fresh mulberry leaves before ensiling; T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water.
Figure 4. Relative abundance of bacterial communities at the phylum and genus level. (A) the phylum-level relative abundance of bacterial communities; (B) the genus-level relative abundance of bacterial communities. ML, fresh mulberry leaves before ensiling; T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water.
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Figure 5. Correlation analysis of the bacterial communities with fermentation characteristics. Redundancy analysis (RDA) plot showing the correlations between fermentation characteristics and bacterial community composition. The canonical axes are labelled with the percentage of total variance explained (%). Red arrow lengths indicate the variance explained by fermentation characteristics; blue arrow lengths indicate the variance explained by bacterial communities at genus level. Different inoculant treatments at different fermentation times are presented as individual data points. Arabic numbers indicate days of ensiling.
Figure 5. Correlation analysis of the bacterial communities with fermentation characteristics. Redundancy analysis (RDA) plot showing the correlations between fermentation characteristics and bacterial community composition. The canonical axes are labelled with the percentage of total variance explained (%). Red arrow lengths indicate the variance explained by fermentation characteristics; blue arrow lengths indicate the variance explained by bacterial communities at genus level. Different inoculant treatments at different fermentation times are presented as individual data points. Arabic numbers indicate days of ensiling.
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Table 1. Changes in chemical composition of mulberry leaves during silage.
Table 1. Changes in chemical composition of mulberry leaves during silage.
IndexTreatmentEnsiling Days
1357153060
DM (% FM)CK32.55 ± 0.08 a32.38 ± 0.26 a31.49 ± 0.01 Bb30.81 ± 0.06 Bc30.49 ± 0.18 Bd29.91 ± 0.56 Cd29.09 ± 0.12 Ce
T132.83 ± 0.16 a32.51 ± 0.23 a32.18 ± 0.08 Ab31.57 ± 0.12 Ac31.05 ± 0.25 Ad30.79 ± 0.04 Bde30.63 ± 0.09 Be
T232.77 ± 0.03 a32.54 ± 0.11 ab32.34 ± 0.26 Ab31.68 ± 0.09 Ac31.33 ± 0.1 Acd31.02 ± 0.13 Ad31.05 ± 0.28 Ad
CP (% DM)CK16.98 ± 0.28 a16.56 ± 0.13 Ba15.93 ± 0.14 Cb15.60 ± 0.11 Cbc15.23 ± 0.16 Ccd15.06 ± 0.16 Cd14.48 ± 0.17 Ce
T117.04 ± 0.22 a16.62 ± 0.14 Bb16.30 ± 0.13 Bbc16.13 ± 0.10 Bc15.73 ± 0.11 Bd15.52 ± 0.10 Bd15.49 ± 0.22 Bd
T217.27 ± 0.14 a17.06 ± 0.15 Aab16.77 ± 0.19 Abc16.53 ± 0.14 Ac16.11 ± 0.17 Acd16.11 ± 0.17 Ad16.17 ± 0.13 Ad
NDF (% DM)CK24.29 ± 0.37 a23.65 ± 0.35 Ab22.67 ± 0.46 Ac21.98 ± 0.20 Acd21.66 ± 0.43 Ad21.56 ± 0.11 Ad21.41 ± 0.12 Ad
T124.11 ± 0.36 a23.13 ± 0.36 Ab21.90 ± 0.35 Ac21.28 ± 0.33 Bcd20.77 ± 0.27 Bd20.56 ± 0.20 Bd20.57 ± 0.21 Bd
T223.67 ± 0.31 a22.37 ± 0.22 Bb21.14 ± 0.29 Bcd20.58 ± 0.41 Cd19.87 ± 0.28 Cd19.72 ± 0.34 Cde19.61 ± 0.16 Ce
ADF (% DM)CK16.54 ± 0.17 a16.21 ± 0.16 ab15.81 ± 0.15 Ab15.66 ± 0.19 Abc15.35 ± 0.25 Acd15.13 ± 0.17 Ad15.07 ± 0.25 Ad
T116.52 ± 0.32 a16.13 ± 0.20 ab15.43 ± 0.21 Bb15.10 ± 0.25 Bb14.71 ± 0.25 Bc14.41 ± 0.25 Bc14.26 ± 0.15 Bc
T216.50 ± 0.24 a15.93 ± 0.20 b15.17 ± 0.18 Bc14.85 ± 0.20 Bcd14.64 ± 0.31 Bd14.44 ± 0.29 Bd14.33 ± 0.33 Bd
WSCs (% DM)CK11.23 ± 0.29 Aa10.43 ± 0.18 Ab9.95 ± 0.10 Ac9.29 ± 0.19 Ad8.58 ± 0.28 Ae7.22 ± 0.13 Af6.71 ± 0.22 Ag
T110.64 ± 0.37 Ba9.12 ± 0.26 Bb7.28 ± 0.12 Bc6.39 ± 0.13 Bd5.88 ± 0.12 Be5.64 ± 0.13 Be5.57 ± 0.13 Be
T210.34 ± 0.26 Ba8.76 ± 0.15 Bb6.87 ± 0.20 Cc5.68 ± 0.15 Cd5.39 ± 0.14 Cde5.20 ± 0.15 Ce5.11 ± 0.11 Ce
Note: FM, fresh matter; DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSCs, water-soluble carbohydrates. T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different little letters show significant differences among ensiling days in the same treatment, and values with different capital letters show significant differences among treatments in the same ensiling day (p < 0.05).
Table 2. Changes in organic acid content of mulberry leaves during silage.
Table 2. Changes in organic acid content of mulberry leaves during silage.
ItemTreatmentEnsiling Days
1357153060
LA (g/kg DM)CK9.37 ± 1.05 Cc9.9 ± 0.29 Cc8.56 ± 0.33 Cc9.45 ± 1.01 Cc13.41 ± 1.33 Bb15.33 ± 0.93 Cb18.78 ± 0.96 Ba
T122.06 ± 1.44 Bf27.73 ± 1.13 Be32.7 ± 0.61 Bd35.48 ± 0.48 Bc41.33 ± 1.28 Ab44.85 ± 0.66 Aa45.05 ± 1.89 Aa
T224.92 ± 0.96 Af33.67 ± 0.97 Ae36.58 ± 0.35 Ad41.2 ± 0.73 Ac43.53 ± 0.59 Ac46.52 ± 0.65 Ab47.63 ± 0.75 Aa
AA (g/kg DM)CK3.16 ± 0.14 Be3.4 ± 0.16 Ce5.55 ± 0.01 Cd10.31 ± 0.40 Cc17.2 ± 1.05 Cb19.66 ± 0.83 Ca20.08 ± 0.89 Ca
T112.28 ± 0.86 Ae16.41 ± 0.38 Ac15.91 ± 0.76 Ac23 ± 0.77 Ab23.76 ± 1.23 Ab26.05 ± 0.37 Aa26.56 ± 0.41 Aa
T211.99 ± 1.64 Ae15.02 ± 0.47 Bd13.5 ± 0.55 Bd18.92 ± 0.28 Bc22.06 ± 0.74 Bb23.81 ± 0.77 Ba25.35 ± 0.45 Ba
PA
(g/kg DM)		CKNDNDND0.53 ± 0.02 c0.91 ± 0.05 b1.14 ± 0.07 a1.18 ± 0.03 a
T1NDNDNDNDNDNDND
T2NDNDNDNDNDNDND
Note: LA, lactic acid; AA, acetic acid; PA, propionic acid; Butyric acid (BA) was not detected in any treatment across all sampling days; ND, not detected (PA < 0.02 g/kg DM; BA < 0.001 g/kg DM). T1 group, fermented by exogenous P. pentosaceus; T2 group, fermented by epiphytic P. pentosaceus; CK group, control group, fermented by equal amount of sterile water. Values with different little letters show significant differences among ensiling days in the same treatment, and values with different capital letters show significant differences among treatments in the same ensiling day (p < 0.05).
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MDPI and ACS Style

Zhang, C.; Shu, G.; Zhu, Z.; Li, Y.; Fang, Z.; Chen, L.; Wan, F.; Zhang, Y.; Xiao, D.; Chen, L. Dynamic Effects of Exogenous and Epiphytic Pediococcus pentosaceus on Quality and Bacterial Community Succession of Silage Mulberry Leaves. Agriculture 2025, 15, 1726. https://doi.org/10.3390/agriculture15161726

AMA Style

Zhang C, Shu G, Zhu Z, Li Y, Fang Z, Chen L, Wan F, Zhang Y, Xiao D, Chen L. Dynamic Effects of Exogenous and Epiphytic Pediococcus pentosaceus on Quality and Bacterial Community Succession of Silage Mulberry Leaves. Agriculture. 2025; 15(16):1726. https://doi.org/10.3390/agriculture15161726

Chicago/Turabian Style

Zhang, Chen, Gangqin Shu, Zhigang Zhu, Yusen Li, Zhenyu Fang, Liyuan Chen, Fachun Wan, Yunhua Zhang, Dingfu Xiao, and Lijuan Chen. 2025. "Dynamic Effects of Exogenous and Epiphytic Pediococcus pentosaceus on Quality and Bacterial Community Succession of Silage Mulberry Leaves" Agriculture 15, no. 16: 1726. https://doi.org/10.3390/agriculture15161726

APA Style

Zhang, C., Shu, G., Zhu, Z., Li, Y., Fang, Z., Chen, L., Wan, F., Zhang, Y., Xiao, D., & Chen, L. (2025). Dynamic Effects of Exogenous and Epiphytic Pediococcus pentosaceus on Quality and Bacterial Community Succession of Silage Mulberry Leaves. Agriculture, 15(16), 1726. https://doi.org/10.3390/agriculture15161726

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