Investigating the mechanisms of Lactiplantibacillus plantarum-mediated optimization of alfalfa silage fermentation and enhancement of antioxidant capacity based on 16 S sequencing technology.

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Learn more: PMC Disclaimer | PMC Copyright Notice BMC Microbiol . 2026 Mar 4;26:330. doi: 10.1186/s12866-026-04879-y Search in PMC Search in PubMed View in NLM Catalog Add to search Investigating the mechanisms of Lactiplantibacillus plantarum -mediated optimization of alfalfa silage fermentation and enhancement of antioxidant capacity based on 16 S sequencing technology Yuqian Liu Yuqian Liu 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Yuqian Liu 1 , Lin Guo Lin Guo 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Lin Guo 1 , Zishang Li Zishang Li 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Zishang Li 1 , Wenting Gou Wenting Gou 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Wenting Gou 1 , Jia Fu Jia Fu 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Jia Fu 1 , Mengxue Liu Mengxue Liu 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Mengxue Liu 1 , Ze Wang Ze Wang 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Ze Wang 1 , Nanyi Zhang Nanyi Zhang 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Nanyi Zhang 1 , Qiang Si Qiang Si 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China Find articles by Qiang Si 1, ✉ , Hongmei Shang Hongmei Shang 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China 2 Jilin Provincial Key Laboratory of Tree and Grass Genetics and Breeding, Jilin Agricultural University, Changchun, 130118 China Find articles by Hongmei Shang 1, 2, ✉ Author information Article notes Copyright and License information 1 College of Forestry and Grassland Science, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun, 130118 China 2 Jilin Provincial Key Laboratory of Tree and Grass Genetics and Breeding, Jilin Agricultural University, Changchun, 130118 China ✉ Corresponding author. Received 2025 Sep 30; Accepted 2026 Feb 19; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13067481  PMID: 41781850 Abstract Background Lactiplantibacillus plantarum ( L. plantarum ) is a safe and eco-friendly additive capable of improving the yield and quality of silage. Currently, research on the use of antioxidative lactic acid bacteria in silage production remains limited. Silage inoculated with antioxidative L. plantarum retains more nutrients and exhibits relatively high antioxidant capacity, demonstrating significant potential for enhancing animal health and improving the quality of animal-derived products. Methods The objective of this research was to assess how four strains of L. plantarum with antioxidant properties impact the fermentation characteristics, bacterial composition, and antioxidant capacity of alfalfa silage. Freshly chopped alfalfa was subjected to five different treatments: I.) no inoculation (control); II.) inoculation with L. plantarum S2; III.) inoculation with L. plantarum LP1; IV.) inoculation with L. plantarum LP8; and V.) inoculation with L. plantarum H8. The additives were all added with a dosage of 1 × 10 6 cfu/g fresh weight and stored for 90 days for sampling and analysis. Results The research results indicated that, compared with the control group, the addition of four L. plantarum strains had a significant impact on both the quality and microbial community of silage ( P < 0.05). The crude protein content in silage inoculated with L. plantarum was significantly higher than that in the control group ( P < 0.05). Among all the L. plantarum treatment groups, the LP1 group exhibited the largest increase in crude protein content, with a rise of 10.06%. Compared with the control group, the total phenolic content in silage supplemented with L. plantarum LP8 significantly increased ( P < 0.05), with an increase of 13.98%. The silage supplemented with L. plantarum H8 had significantly lower pH and ammonia nitrogen content than the control group ( P < 0.05), with decreases of 6.21% and 120%, respectively. Its lactic acid and flavonoid contents significantly increased, with rises of 18.21% and 18.47%, respectively. Moreover, the contents of 1,1-diphenyl-2-picrylhydrazyl radical and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities in the silage supplemented with L. plantarum H8 were significantly higher than those in the control group ( P < 0.05). In the treatment groups supplemented with L. plantarum , the relative abundance of the Lactobacillus genus significantly increased ( P < 0.05), inhibiting the growth and reproduction of undesirable microorganisms (such as Chryseobacterium and Stenotrophomonas ) ( P < 0.05). The addition of L. plantarum H8 and LP8 simplified the microbial network of alfalfa silage, while the addition of L. plantarum S2 and LP1 increased the complexity of the microbial network structure. Metabolic function prediction showed that the addition of L. plantarum promoted carbohydrate and amino acid metabolism ( P < 0.05). Conclusion In conclusion, the addition of all four L. plantarum species improved alfalfa silage quality, but L. plantarum H8 was more effective at improving fermentation quality and antioxidant activity. Keywords: Silage additive, Fermentation, Antioxidant activity, Microbial community Introduction Alfalfa ( Medicago sativa L.) dominates global legume cultivation, with production reaching 450 million tons annually from 30 million hectares of farmland. The importance of alfalfa stems from its universal utilization, exceptional nutritional content, high digestibility, and valuable feed properties [ 1 , 2 ]. On a global scale, alfalfa is cultivated across a variety of climatic zones ranging from temperate to subtropical regions [ 3 ]. In some areas, extreme weather conditions, such as prolonged droughts, heavy rainstorms, high temperatures, or low temperatures, can severely impact the growth and quality of alfalfa. When using this uneven-quality alfalfa as raw material for silage, it introduces uncertainties to the silage-making process. This is because alfalfa of different qualities exhibits variations in its initial microbial communities, nutritional components, and buffering capacities [ 4 ]. The global shortage of high-quality forage has led to a persistent imbalance between forage grass and livestock, seriously hampering the development of the animal husbandry industry [ 5 ]. Thus, to preserve nutrients and improve palatability, it can be made into alfalfa silage [ 6 ]. Alfalfa’s natural limitations, including the low water-soluble carbohydrate (WSC) levels and strong buffering capacity, complicate silage production without additives or complementary forages [ 7 – 9 ]. Silage serves as a nutritionally balanced and palatable feed resource, ensuring year-round stable feed supply for ruminant livestock [ 10 , 11 ]. During silage fermentation, anaerobic fermentation is usually carried out by lactic acid bacteria (LAB) [ 12 ]. LAB are used as a natural green additive because of their high safety, nonpollution and noncorrosive properties [ 13 , 14 ]. The incorporation of LAB as feed additives has become widespread in animal husbandry to optimize feed utilization, prevent diseases, and bolster the animal’s immune response [ 15 ]. Lactiplantibacillus plantarum ( L. plantarum ), which has high antioxidant activity, significantly modulates oxidative stress in organisms and cells [ 16 , 17 ]. Numerous studies have documented the antioxidant capabilities of Lactobacillus both in laboratory settings and within living organisms. Wu et al. [ 18 ] reported that L. plantarum ZJ316 was effective at reducing oxidative stress and reducing inflammation. Xia et al. [ 19 ] reported that L. plantarum AR113 has a favourable inhibitory effect on oxidative stress and affects the antioxidant capacity of the host in addition to its own antioxidant function. The antioxidant capacity of alfalfa silage was significantly enhanced by L. plantarum J17 inoculation, as shown by Zhang et al. [ 20 ]. Thus, pre-ensiling inoculation with antioxidant-active Lactobacillus strains improves both fermentation quality and antioxidant capacity of silage. Few studies have been performed on the application of the antioxidant L. plantarum in silage. Silage inoculated with the antioxidant L. plantarum is proposed to have a relatively high antioxidant capacity, which in turn has beneficial potential for improving animal health and animal product quality [ 21 , 22 ]. We hypothesized that adding antioxidant-active L. plantarum S2、LP1、LP8 and H8 to silage would improve both fermentation quality and antioxidant capacity of the forage. Consequently, in this experimental work, we investigated the effects of four L. plantarum strains with high antioxidant properties on silage quality, the microbial community and the antioxidant activity of alfalfa silage for 90 d to screen L. plantarum strains that can be applied to alfalfa silage and thus provide a theoretical basis and practical reference for the development of functional forage products. Materials and methods Silage preparation Alfalfa ( Medicago sativa L. JiMu No.1 alfalfa) was collected in October 2023 from the experimental field of Jilin Agricultural University (longitude 125.410385, latitude 43.810433, altitude: 222 m), leaving a stubble height of 2–5 cm. Alfalfa has been bred and authenticated by Xu Bo’s team, with a variety registration number of cpzsj00716. The harvested alfalfa was transported to the laboratory, allowed to wilt indoors for approximately 24 h, and then chopped to a length of 2–3 cm using a chopper. Table 1 displayed the nutrient composition of the raw alfalfa material. L. plantarum was added at 1 × 10 6 cfu/g FM. L. plantarum LP1 and LP8 were screened from the intestinal tract of geese and were gifted by the College of Animal Science and Technology, Jilin Agricultural University. L. plantarum S2 and H8 were provided by the College of Food Science, Jilin Agricultural University. The experiment included the following treatment groups: (1) additive-free control (CON); (2) L. plantarum S2 (S2); (3) L. plantarum LP1 (LP1); (4) L. plantarum LP8 (LP8); and (5) L. plantarum H8 (H8). Approximately 200 g of alfalfa forage was thoroughly mixed, placed in polyethylene silage bags (28 × 35 cm) and sealed with a vacuum sealing machine. A total of 30 silage bags (5 treatments × 6 replicates) were used in this study, and the ambient temperature (20 ± 2 °C) was maintained. The 90 d silage samples were used for the analysis of dynamic changes in nutrient composition, fermentation indices, antioxidant activity, and microbial composition. Table 1. Nutritional quality of alfalfa silage material Item Content DM (g/kg FW) 415.3 ± 2.79 CP (g/kg DM) 204.1 ± 1.28 WSC (g/kg DM) 70.5 ± 1.10 NDF (g/kg DM) 482.9 ± 5.74 ADF (g/kg DM) 290.9 ± 3.25 Open in a new tab FW fresh weight, DM dry matter, WSC water-soluble carbohydrates, NDF neutral detergent fiber, ADF acid detergent fiber Determination of the chemical composition and fermentation quality of silage After unsealing the silage bags, a 10 g aliquot of silage from each bag was blended with 90 mL of distilled water. The mixture was refrigerated at 4 °C for 24 h before filtration through four-layer medical gauze. The filtrate was divided into three equal parts. Real-time pH measurements of the filtrate were conducted using a calibrated FE28 pH meter (Mettler Toledo Instruments Co., Ltd., Shanghai, China); one portion was frozen at -20 °C for the determination of ammoniacal nitrogen (NH 3 -N) content; and the third aliquot of the filtrate was used for the analysis of lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) contents. The phenol-sodium hypochlorite colorimetric method was employed to measure NH 3 -N concentration [ 23 ]. Before the determination of LA, AA, PA, and BA, the filtrate was filtered through a 0.22 μm pore size membrane, and then the organic acid content was determined using a Waters Iclass high-performance liquid chromatograph (column: KC-811 column, Shimadzu, Japan; mobile phase: 3 mmol/L perchloric acid; flow rate, 1 mL/min; column temperature, 50 °C; detection wavelength, 210 nm; and injection volume, 10 µL). To determine the dry matter (DM) content, the alfalfa silage was first opened, dried at 65 °C, then shredded using a grinder, and stored at -20 °C for subsequent analysis. The Kjeldahl method was employed to determine the crude protein (CP) content [ 24 ]. The Paradigm detergent fiber method was employed to quantify neutral detergent fiber (NDF) and acid detergent fiber (ADF) [ 25 ]. The anthrone-sulfuric acid method was used to determine the water-soluble carbohydrate (WSC) content [ 26 ]. Determination of the antioxidant activity of silage One gram of alfalfa silage grass powder was weighed, 25 mL of 95% ethanol extract was added, the mixture was ultrasonicated at 20 °C and 100 W for 60 min, and the filtrate was taken as the extract. The total phenols, flavonoids, 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) of the extract were determined. A modified approach based on Sytar et al.‘s method was employed to determine the total phenol content [ 27 ]. Initially, 0.1 mL of the extract was transferred into a 10 mL volumetric flask with 6 mL of water and thoroughly mixed. Subsequently, 0.5 mL of Folin’s reagent was introduced and the solution was thoroughly mixed. After a 60 s incubation, 1.5 mL of 20% Na 2 CO 3 solution was introduced into the mixture and well blended. The mixture was diluted to volume with distilled water, incubated in a 75 °C water bath for 10 min, allowed to cool to room temperature, and finally the absorbance was read at 765 nm. The flavonoid content was determined according to a modified protocol adapted from Le et al.‘s method [ 28 ]. To 1 mL extract in a 10 mL test tube, anhydrous ethanol was added to make 5 mL total volume. Following mixing, 0.3 mL of 5% NaNO 2 was added, the solution was shaken, and incubated for 6 min. Following this, 0.3 mL of 10% Al (NO 3 ) 3 solution was introduced into the reaction system. The resulting mixture was homogenized by vigorous shaking and subsequently maintained under quiescent conditions for a 6-minute period. The reaction was terminated by adding 4 mL of 1 M NaOH solution, followed by dilution with anhydrous ethanol. After 10 min of incubation, the absorbance was measured at 510 nm. The DPPH radical scavenging capacity was evaluated according to a modified method adapted from Li et al. [ 29 ] and He et al. [ 30 ]. The assay system was prepared by combining 0.1 mL of the extract with 3.9 mL of 0.1 mM DPPH solution in a 10 mL test tube. After 30 min of dark incubation, the absorbance was recorded at 515 nm. The ABTS + free radical scavenging activity was measured using a slightly modified method of Li et al. [ 29 ] and Zhang et al. [ 31 ]. The working solution was prepared by mixing 5 mL of 7 mM ABTS + with 88 µL of 140 mM potassium persulfate, followed by 12-hour reaction at room temperature in the dark. An ABTS stock solution was obtained, and when used, it was diluted into a working solution with 10 mmol/L phosphate buffer (pH of 7.4) to obtain the absorbance at 734 nm (0.70 ± 0.02) at room temperature. The assay was performed by adding 0.3 mL of the diluted extract (1:3, v/v) to the ABTS reaction mixture, incubating for 30 min at room temperature, and measuring the absorbance at 734 nm. Microbial community analysis Unfermented (fresh) and 90-d silage samples were selected for bacterial community analysis. PCR amplification targeting the V3-V4 hypervariable region of bacterial 16 S rDNA genes was performed using manufacturer-recommended protocols, succeeded by library preparation. The constructed libraries were quantified using both Qubit fluorometry and quantitative PCR (qPCR). Following quality control assessment, paired-end 250 bp sequencing was performed on the NovaSeq 6000 platform. The raw reads were multiplexed, merged, quality filtered, deduplicated and chimaera removed using QIIME2, followed by denoising to amplicon sequence variants (ASVs) using DADA2 as a plug-in in QIIME2 [ 32 ]. ASVs were assigned to taxonomic classifications using the Silva 138.1 gene database. Bioinformatics analyses of bacterial microbial communities were performed using QIIME. PCoA analysis was conducted, with calculations and plotting performed using the ade4 package and ggplot2 package in R software (Version 4.0.3). In addition, we calculated multi-abundance correlations based on genus-level matrices to establish symbiotic patterns using the RStudio toolkit version 1.1.453. Symbiotic network visualization was performed using Gephi software (version 0.9.2). Heatmaps based on Spearman’s correlation were created to visualize the relationships between bacterial genera, silage quality, and antioxidant activity. The functions of the bacterial microbiota were predicted via PICRUSt software. Statistical analysis The raw experimental data were initially processed and organized using Microsoft Excel. All the statistical analyses were conducted utilizing the Statistical Package for the Social Sciences software (SPSS version 19.0; Chicago, IL, USA). Statistical comparisons were performed using one-way analysis of variance. Values are reported as means ± standard errors of the mean, with a P-value less than 0.05 considered statistically significant. Results Nutritional qualities of alfalfa silage According to the data presented in Table 2 , the nutritional characteristics of alfalfa silage were altered by the addition of four L. plantarum species. No statistically significant difference was detected in the dry matter content between each L. plantarum group and the CON group. Relative to the CON group, the four L. plantarum species significantly reduced the soluble carbohydrate content ( P < 0.05) and increased the crude protein content ( P < 0.05) of the silage. The LP1 and LP8 groups exhibited relatively high crude protein concentrations, reaching 204.5 and 202.3 g/kg DM respectively. The LP8 and H8 groups exhibited significantly reduced NDF content compared to the CON group ( P < 0.05). While the groups supplemented with L. plantarum S2 and LP1 exhibited no significant differences compared to the control group, both the LP1 and LP8 groups demonstrated significantly reduced ADF contents relative to the CON group ( P < 0.05). Table 2. Effects of four L. plantarum strains on the nutritional quality of alfalfa silage after 90 days of ensiling Item Treatment CON L. Plantarum S2 L. Plantarum LP1 L. Plantarum LP8 L. Plantarum H8 DM (g/kg FW) 370.3 ± 3.25 a 371.5 ± 3.54 a 371.2 ± 4.46 a 365.8 ± 2.35 a 371.8 ± 4.27 a CP (g/kg DM) 185.8 ± 1.69 c 200.5 ± 1.53 ab 204.5 ± 1.65 a 202.3 ± 1.22 a 196.8 ± 1.58 b WSC (g/kg DM) 23.8 ± 0.46 a 21.7 ± 0.45 b 22.1 ± 0.46 b 21.5 ± 0.46 b 22.1 ± 0.21 b NDF (g/kg DM) 486.0 ± 5.64 a 487.7 ± 6.01 a 477.1 ± 10.85 a 449.4 ± 4.85 b 446.7 ± 2.19 b ADF (g/kg DM) 313.5 ± 7.42 a 302.6 ± 2.49 ab 298.1 ± 4.30 b 293.6 ± 3.67 b 302.0 ± 4.99 ab Open in a new tab FW fresh weight, DM dry matter, WSC water-soluble carbohydrates, NDF neutral detergent fiber, ADF acid detergent fiber, Means with different superscripts in the same row (a-c) are significantly different from each other ( P < 0.05) Fermentation characteristics of alfalfa silage The effects of the four L. plantarum species on the pH and NH 3 -N, LA, AA, PA and BA contents of the alfalfa silage are shown in Table 3 . Compared with the CON group, all L. plantarum treatment groups exhibited significantly lower pH values ( P < 0.05), ranging from 4.3 to 4.7. The H8 and LP1 groups showed the most pronounced acidification with a pH of 4.35. Compared to the control group, all L. plantarum -supplemented treatments significantly reduced NH 3 -N content, with LP1 and H8 groups demonstrating the most pronounced effects. The addition of any of the four L. plantarum strains resulted in a statistically significant elevation in LA content relative to the CON group ( P < 0.05). It is particularly noteworthy that the AA content in all treatment groups treated with L. plantarum is lower than that in the CON group, with the AA concentrations in the LP1 group and the S2 group being even lower compared to the other L. plantarum treatment groups ( P < 0.05). The PA content did not differ significantly across treatment groups. No BA was detected in any treatment group. Table 3. Effects of four different L. plantarum strains on fermentation characteristics of alfalfa silage after 90 days Item Treatment CON L. Plantarum S2 L. Plantarum LP1 L. Plantarum LP8 L. Plantarum H8 pH 4.62 ± 0.04 a 4.48 ± 0.01 b 4.38 ± 0.01 cd 4.44 ± 0.02 bc 4.35 ± 0.04 d NH 3 -N (% TN) 2.20 ± 0.03 a 1.76 ± 0.02 b 1.02 ± 0.03 d 1.36 ± 0.02 c 1.00 ± 0.02 d LA (% DM) 3.02 ± 0.12 b 3.53 ± 0.09 a 3.57 ± 0.09 a 3.40 ± 0.15 a 3.57 ± 0.11 a AA (% DM) 1.91 ± 0.03 a 1.61 ± 0.13 b 1.60 ± 0.06 b 1.85 ± 0.14 ab 1.80 ± 0.03 ab PA (% DM) 0.09 ± 0.00 a 0.11 ± 0.01 a 0.08 ± 0.03 a 0.09 ± 0.00 a 0.07 ± 0.02 a BA (% DM) ND ND ND ND ND Open in a new tab DM dry matter, LA lactic acid, AA acetic acid, PA propionic acid, BA butyric acid, NH 3 –N ammonia nitrogen, TN total nitrogen, ND not detected, Means with different superscripts in the same row (a-d) are significantly different from each other ( P < 0.05) Antioxidant activities of alfalfa silage According to the data presented in Table 4 , the four L. plantarum species affected the antioxidant properties of alfalfa silage, including total phenols, flavonoids, and both DPPH and ABTS + radical scavenging activities. L. plantarum LP8 supplementation significantly increased total phenolic content relative to the control group ( P < 0.05). However, the total phenolic content in the S2 group decreased significantly ( P < 0.05). The flavonoid content of the alfalfa silage with the addition of the four L. plantarum strains was significantly greater ( P < 0.05) than that of the CON group. All four L. plantarum strains significantly enhanced ( P < 0.05) the DPPH radical scavenging capacity of alfalfa silage compared to the untreated CON group. Compared with the LP1, S2 and LP8 groups, the H8 group presented greater DPPH radical scavenging activity, with values of 8.49 µmol/g and 8.05 µmol/g, respectively. In comparison with the CON group, the ABTS + radical scavenging activity in the H8, LP1, and LP8 groups was significantly greater ( P < 0.05), with values of 67.42 µmol/g, 66.46 µmol/g, and 61.84 µmol/g, respectively. These findings indicate that the addition of L. plantarum H8 and LP1 had a more pronounced effect on enhancing the flavonoid content and free radical scavenging activity of the silage. In contrast, the ABTS + radical scavenging activity in the S2 group was significantly lower than that in the CON group ( P < 0.05). Table 4. Effects of four L. plantarum strains on the antioxidant activity of alfalfa silage after 90 days of ensiling Item Treatment CON L. Plantarum S2 L. Plantarum LP1 L. Plantarum LP8 L. Plantarum H8 Total phenols (%) 0.93 ± 0.02 b 0.74 ± 0.03 c 0.95 ± 0.02 b 1.06 ± 0.03 a 0.98 ± 0.02 b Flavonoid (%) 0.59 ± 0.01 b 0.62 ± 0.01 a 0.64 ± 0.01 a 0.63 ± 0.01 a 0.64 ± 0.01 a DPPH (µmol/g) 5.98 ± 0.12 d 7.32 ± 0.11 b 8.05 ± 0.20 a 6.76 ± 0.19 c 8.49 ± 0.22 a ABTS + (µmol/g) 56.74 ± 0.56 c 54.29 ± 0.75 d 66.46 ± 1.28 a 61.84 ± 0.75 b 67.42 ± 0.66 a Open in a new tab DPPH DPPH free radical scavenging activity, ABTS + ABTS free radical scavenging activity, Means with different superscripts in the same row (a-d) are significantly different from each other ( P < 0.05) Effects of four L. plantarum species on the microbial community of alfalfa silage Figure 1 shows that the alpha diversity analysis of bacterial communities in alfalfa silage showed coverage values exceeding 0.99 for all samples, confirming the sequencing data adequately captured the bacterial microbiota characteristics. The Chao1 index serves as a metric for microbial community richness, representing the estimated species number within the community. Higher Chao1 values correspond to greater species abundance. The CON group had the highest Chao1 index, suggesting the greatest number of species, whereas the LP8 group had lower Chao1 index, indicating a smaller number of species. The Shannon and Simpson indices serve as indicators of microbial community diversity, reflecting both the number of species types present and the degree of evenness or homogeneity among those species within the community. The reduced Shannon and Simpson indices observed in the LP8 group suggested that inoculation with L. plantarum LP8 decreased both the diversity and evenness of bacterial populations in the alfalfa silage. PCoA analysis using Bray-Curtis distance revealed distinct clustering patterns. PCoA analysis revealed differences in the bacterial communities among the treatment groups after 90 days of silage. A significant difference in the bacterial community composition between fresh alfalfa and silaged alfalfa was found. Fig. 1. Open in a new tab Bacterial community diversities of fresh and ensiled alfalfa with different additives. A – D Alpha diversities (Shannon, Chao1, Simpson, and coverage indexes) of bacterial community. E The principal coordinates analysis (PCoA) of the samples conducted based on weighted UniFrac distance. FM, fresh alfalfa; CON, control silage, no additive; S2, L. plantarum S2; LP1, L. plantarum LP1; LP8, L. plantarum LP8; H8, L. plantarum H8; * indicates a significant difference Figure 2 A presents the phylum-level changes in bacterial community composition during alfalfa fermentation after addition of four L. plantarum strains. The silage bacterial communities were predominantly composed of Firmicutes, Cyanobacteria, and Proteobacteria at the phylum level. The dominant phyla before silage were Cyanobacteria and Proteobacteria, with relative abundances of 82.55% and 17.22%, respectively. Following silage fermentation, Firmicutes emerged as the predominant phylum. Comparative analysis revealed the LP8 group exhibited reduced Proteobacteria abundance relative to CON, while both LP8 and S2 groups showed decreased Actinobacteria levels compared to CON ( P < 0.05). In Fig. 2 B, the alterations in the bacterial community structure (genus level) of alfalfa silage induced by four different L. plantarum strains are presented. The dominant genera of the alfalfa bacterial community before silage were unidentified Chloroplast and unidentified Mitochondria , and the microorganisms in each group after silage were mainly Lactiplantibacillus , unidentified Chloroplast , Chryseobacterium , unidentified Mitochondria , Levilactobacillus , Methylobacterium-Methylorubrum , Sphingomonas , Stenotrophomonas , Allorhizobium-Neorhizobium Pararhizobium Rhizobium , and Pediococcus . Relative to the CON group, all treatment groups exhibited increased Lactobacillus abundance, with LP8 demonstrating the most pronounced enhancement. Moreover, the H8 group presented decreases in the relative abundances of Chryseobacterium and Stenotrophomonas . Compared to the CON group, group S2 showed a higher relative abundance of Pediococcus , while group H8 had a greater relative abundance of Levilactobacillus . Additionally, groups S2, LP1, and H8 all exhibited higher relative abundances of Sphingomonas than the CON group. Fig. 2. Open in a new tab The relative abundance of bacterial communities in alfalfa and silage at the phylum ( A ) and genus ( B ) levels under different additive treatments. FM, fresh alfalfa; CON, control silage, no additive; S2, L. plantarum S2; LP1, L. plantarum LP1; LP8, L. plantarum LP8; H8, L. plantarum H8 Significant differences in bacterial communities between treatments (LDA > 4) were identified using LEfSe, and the results for fresh samples versus treatment groups are shown in Fig. 3 . In the FM group, unidentified Chloroplast and unidentified mitochondria were more highly classified. The genus Aeromicrobium was more common in the CON group, the genus Lactiplantibacillus was more common in the LP8 group, and Levilactobacillus was more common in the H8 group. Compared with the CON group and other L. plantarum treatment groups, no single bacterial genus exhibited unique enrichment in the LP1 treatment group. Fig. 3. Open in a new tab Comparison of microbial community changes in silage treated with alfalfa and different additives after 90 days of ensiling based on Linear Discriminant Analysis Score. FM, fresh alfalfa; CON, control silage, no additive; S2, L. plantarum S2; LP1, L. plantarum LP1; LP8, L. plantarum LP8; H8, L. plantarum H8 Correlation-based symbiotic networks partially reveal interactions among microbial community members. We constructed bacterial co-occurrence networks using Spearman correlations for both fresh and additive-treated silage samples. To evaluate the intricacy of the bacterial network in alfalfa silage, we employed network topology indicators, including the number of nodes, number of edges, and degree of mediation. The interaction network between alfalfa silage and bacteria under different L. plantarum treatments after 90 days of ensiling is shown in Fig. 4 . The number of nodes in the bacterial network was ranked in the following order: CON > LP1 > S2 > LP8 > FM > H8. In addition, S2 had the highest number of edges, with 291, followed by LP1 (132) and CON (88). Fig. 4. Open in a new tab The interaction network between alfalfa and silage bacteria treated with different additives. Calculate the bacterial associated network based on 16 S rRNA gene for alfalfa and silage treated with different additives using all bacterial species. The node size is scaled based on the total abundance of each bacterial species. The correlation strength between the edge and each metabolite systemic pair is proportional (measured by correlation), with red edges indicating positive correlation and green edges indicating negative correction Figure 5 shows the correlation between antioxidant activity and the bacterial community. Cellulomonas abundance showed a positive correlation with both NH 3 -N concentration and pH levels. Chryseobacterium abundance positively correlated with WSC content, whereas Lactobacillus showed a negative correlation with WSC (Fig. 5 A). Cellulomonas was negatively correlated with ABTS + , while Pediococcus was negatively correlated with ABTS+. Massilia was positively correlated with DPPH. Pantoea was negatively correlated with total phenols (Fig. 5 B). Fig. 5. Open in a new tab Correlation analysis between dominant bacterial microbiota in alfalfa silage and silage quality ( A) and antioxidant activities ( B) . Levels of significance are shown as follows: * P < 0.05; ** P < 0.01. DM, dry matter; WSC, water-soluble carbohydrates; NDF, neutral detergent fiber; ADF, acid detergent fiber; LA, lactic acid; AA, acetic acid; PA, propionic acid; NH 3 -N, ammonia nitrogen; DPPH, free radical DPPH scavenging activity; ABTS + , radical ABTS + scavenging activity As shown in Fig. 6 , the KEGG functional mapping at pathway level 1 for fresh and additive - treated alfalfa silage was related mainly to metabolism (Fig. 6 A), and the KEGG functional mapping at pathway level 2 was related mainly to carbohydrate and amino acid metabolism, followed by cofactor and vitamin metabolism (Fig. 6 B). At pathway level 3 (Fig. 6 C), all additive-treated groups showed enhanced carbohydrate metabolism compared to CON, including key pathways like TCA cycle, pyruvate metabolism, and various sugar metabolic processes. In contrast, inositol phosphate metabolism, as well as glyoxylate and dicarboxylic acid metabolism, were reduced. The additive groups exhibited significantly enhanced amino acid metabolism pathways—including arginine biosynthesis; glycine, serine, and threonine metabolism; arginine and proline metabolism; alanine, aspartate, and glutamate metabolism; histidine metabolism; and cysteine and methionine metabolism—compared to the CON group ( P < 0.05). In contrast, compared with the CON group, valine, leucine, and isoleucine degradation; phenylalanine, tyrosine, and tryptophan biosynthesis; lysine degradation; tryptophan metabolism; phenylalanine metabolism; tyrosine metabolism; and valine, leucine, and isoleucine degradation were lower in abundance in the additive group than in the CON group. Fig. 6. Open in a new tab Functional characteristics of alfalfa silage bacteria treated with different additives. A 16 S rRNA gene predicts KEGG functional profile at pathway level (1). B 16 S rRNA gene predicts KEGG functional profile at pathway level (2). C 16 S rRNA gene predicts KEGG functional profile at pathway level (3). FM, fresh alfalfa, CON, control silage, no additive; S2, L. plantarum S2; LP1, L. plantarum LP1; LP8, L. plantarum LP8; H8, L. plantarum H8 Discussion Chemical properties of fresh material prior to silage Research suggested that wilting fresh alfalfa until the dry matter content exceeded 300 g/kg FM was necessary to curb the growth of some unwanted microorganisms [ 33 ]. In this study, the DM content of the alfalfa silage feedstock was 415 g/kg FM, indicating that the conditions for successful silage fermentation were met in this experiment. In silage fermentation, WSC is a vital substrate. For effective fermentation, silage components need to have more than 5% DM of WSCs [ 34 , 35 ]. The WSC content of alfalfa in this study was 70.5 g/kg DM. Lin et al. [ 36 ] determined the content of soluble sugars in different varieties of fresh alfalfa and reported that the WSC content of the XJD variety was 66.1 g/kg DM. This may be due to the varying soluble sugar contents caused by different alfalfa varieties. In this study, the CP, NDF and ADF contents of the alfalfa silage feedstocks were 204.1 g/kg DM, 482.9 g/kg DM and 290.9 g/kg DM, respectively, but were within the reasonable range reported by Jung et al. [ 37 ]. Nutritional qualities of silage and indicators of fermentation The DM content is a key indicator for evaluating the efficiency of pasture utilization [ 38 ]. After 90 days of ensiling, no statistically significant differences were detected among the treatment groups. Bai et al. [ 39 ] reported no difference in DM content between silage inoculated with antimicrobial peptide-producing Bacillus subtilis and Lactobacillus buchneri silage and control silage. WSCs function as essential substrates for lactic acid bacterial fermentation [ 40 ]. LAB convert WSCs to organic acids (mainly LA) and rapidly reduce the pH to preserve silage. Kleinschmit. et al. [ 41 ] reported that silage treated with L. plantarum had a lower WSC content than did control silage. This is consistent with our results, which may be due to the addition of L. plantarum in the treatment group, where LAB utilized WSCs as a substrate for fermentation. The CP is a key measure of the nutritional value of forage [ 42 ]. In this experiment, more CP was retained in the alfalfa silage in each additive group than in the CON group. Liu et al. [ 43 ] reported that a relatively high CP content was retained in alfalfa after the addition of silage. During the silage process, L. plantarum grows and multiplies to produce lactic acid to lower the pH, which directly reduces protein hydrolysis through the active inhibition of proteases in low-pH environments [ 44 ]. NDF and ADF are key feed quality parameters, where lower levels indicate higher nutritional value [ 45 ]. This study revealed that the NDF content was significantly reduced in the L. plantarum LP8 and H8 groups compared to the CON group, while the ADF content was significantly decreased in all treatment groups. The lower NDF and ADF contents in the alfalfa silage may be due to microbial acid hydrolysis or the production of fibrinolytic enzymes during silage fermentation [ 46 ]. A lower pH results in more digestible plant cells being acid hydrolysed and, therefore, lower NDF and ADF contents [ 47 ]. Both the velocity and magnitude of pH decline represent essential biomarkers for evaluating silage fermentation efficiency. Legume silages are usually stabilized when their pH falls below 4.50 [ 48 ]. After 90 days of silage, the pH decreased to less than 4.5 in all additive treatment groups (the lowest values were observed in the LP8 and H8 groups). Silage NH 3 -N concentration indicates protein degradation extent, where elevated levels signify greater proteolysis by undesirable microbes and poorer fermentation quality [ 49 ]. Liu et al. [ 50 ] reported a reduction in ammoniacal nitrogen in silage inoculated with L. plantarum LP3-27. Compared with the untreated CON group, all additive treatments resulted in statistically significant reductions in NH 3 -N content ( P < 0.05). These findings indicate that the addition of L. plantarum had an inhibitory effect on the accumulation of ammoniacal nitrogen and that the protein depletion of alfalfa during silage was lower. The LA content in the groups treated with L. plantarum was significantly higher than that in the CON group, while the AA content was significantly lower. This indicates that homofermentative L. plantarum utilizes WSC for growth and reproduction, producing LA and thereby lowering the pH. Antioxidant activities of silage Studies conducted previously have shown that phenolic compounds serve as both reducing agents and free radical scavengers and have significant antioxidant activity [ 51 , 52 ]. Flavonoids play crucial roles in inhibiting lipid oxidation and protecting vitamins and enzymes present in plants [ 53 ]. The increase in total phenol and flavonoid contents plays a crucial role in the antioxidant activity of silage. The results of this study demonstrated that silage inoculated with L. plantarum LP1, LP8 and H8 showed a significant increase in total phenol content, while all additive-treated groups exhibited significantly higher flavonoid content compared to the CON group. Wang et al. [ 54 ] reported that Lactobacillus fermentation significantly increased the total phenol and flavonoid contents in kiwifruit juice. Phenolic substances can be interconverted to improve the antioxidant activity of fermentation broths [ 55 ]. Nevertheless, during L. plantarum fermentation, certain phenolic acid decarboxylase enzymes are generated. These enzymes modify phenolic compounds through glycosylation, methylation, and other substitution reactions, resulting in the transformation of phenolic profiles in the samples and consequently changing their antioxidant properties [ 56 ]. DPPH and ABTS⁺ radical scavenging assays are among the most frequently employed methods for evaluating antioxidant capacity [ 57 , 58 ]. DPPH is a stable organic radical that can be used to assess the hydrogen-donating capacity of antioxidants [ 30 ]. ABTS + is commonly used as an oxidant to assess the Trolox equivalent antioxidant capacity of natural and artificial antioxidants in foods, beverages, and other nutrients in aqueous solutions [ 59 ]. The results demonstrated that all inoculated groups exhibited significantly higher DPPH radical scavenging capacity compared to the CON group. Furthermore, the LP1, LP8, and H8 strains of L. plantarum significantly enhanced ABTS + radical scavenging activity in alfalfa silage. Different strains of L. plantarum exhibit variations in the types and contents of metabolites, as well as in their ability to degrade and transform substrate components during the ensiling process of alfalfa. This, in turn, leads to differences in the retention or production levels of antioxidant substances (such as polyphenols, flavonoids, etc.) in the silage, ultimately resulting in variations in ABTS⁺ radical scavenging activity [ 60 – 62 ]. These improvements in antioxidant activities were likely associated with the elevated levels of total phenolics and flavonoids produced by these four L. plantarum strains during fermentation. Microbial communities in silage To delve deeper into the interaction between L. plantarum and microorganisms in silage, the bacterial community was examined. To evaluate the diversity and abundance of the bacterial communities, the Chao1, Shannon, and Simpson indices were utilized. Microbial community analysis revealed that alfalfa silage treated with L. plantarum exhibited significantly lower species richness than the CON group after 90 days of ensiling ( P < 0.05). Ogunade et al. [ 63 ] reported that the diversity of silage microbial communities was lower when the dominant bacteria dominated after inoculation with Lactobacillus buchneri or L. plantarum . A series of physiological, biochemical and microflora changes occur during the fermentation and storage of silage materials, which in turn affect the quality of silage [ 63 , 64 ]. Therefore, compared with that of silage material, the microbial diversity of silage is lower after fermentation. PCoA is a nonbinding method for downscaling data. Differences in the bacterial communities between the treatment groups after 90 days of silage were analysed by PCoA, and in this study, a significant separation of the bacterial community composition was observed between fresh and post-silage alfalfa. Similarly, Su et al. [ 65 ] reported a significant separation of microbial communities between raw material and post-silage. Silage is a process of microbial interaction, and understanding the structure of the microbial community may help to gain insight into silage fermentation. This study investigated the effects of adding S2, LP1, LP8 and H8 to alfalfa silage on the bacterial community. The alfalfa feedstock was dominated by the phylum Cyanobacteria. In this study, after 90 days of fermentation, the dominant microorganisms in the alfalfa silage were all in the phylum Firmicutes, which was attributed to the anaerobic and acidic environment that led to microbial community succession from Proteobacteria to Firmicutes in the alfalfa silage [ 66 ]. In environments with a low oxygen content and effective sealing ability, the activity of most aerobic microorganisms is inhibited, and Lactobacillus spp . gradually grow and multiply to become the dominant species [ 67 ]. In this study, after 90 days of fermentation, Lactobacillus spp. became dominant in the alfalfa silage. Consequently, silage microbial diversity decreases substantially post-fermentation. Spearman correlation analysis in this study additionally uncovered relationships between bacteria community composition and both silage quality parameters and antioxidant capacity. A positive relationship was found between Cellulomonas and both NH 3 -N and pH, while Cellulomonas was negatively related to ABTS + . This may be because Cellulomonas is a poorly acid-tolerant bacterium that grows vigorously in the early stages of silage. Zafar et al. [ 68 ] reported that Cellulomonas can breakdown complex compounds. The WSC content was found to be significantly positively associated with Chryseobacterium and negatively associated with Lactobacillus , suggesting that the reduction in WSCs in silage was due mainly to the increase of L. plantarum rather than the depletion of undesirable microorganisms. Massilia was positively correlated with DPPH. This bacterial strain produces diverse secondary metabolites potentially enhancing alfalfa silage’s antioxidant properties [ 69 ]. In the present study, Pantoea was found to be negatively correlated with total phenols, and Bijelic et al. [ 70 ] reported that microorganisms of Pantoea consume some of the nutrients to promote their own growth and reproduction. Bacterial co-occurrence networks were analyzed to investigate L. plantarum’s impact on silage microbial communities during anaerobic fermentation. An increase in nodes and edges points to a more complicated network structure [ 71 , 72 ]. Network analysis demonstrated that both LP8 and H8 strains of L. plantarum simplified the bacterial interaction networks in silage, exhibiting significantly fewer nodes and edges compared to the untreated CON group. It indicates that L. plantarum LP8 and H8 have a competitive edge over other microorganisms during the fermentation process of alfalfa silage. The process of silage involves microbial pathways that convert metabolites or decompose substrates. As an innovative strategy for understanding cellular and organismal functions, KEGG can be utilized to evaluate the effects of bacterial communities on metabolic pathway alterations in silage by predicting bacterial roles [ 73 ]. Therefore, the KEGG pathway database of PICRUSt2 was used in this study to predict the function of the alfalfa silage bacterial community in response to the addition of different L. plantarum strains. In this study, the pathways at the level of the first pathway were related mainly to metabolism, suggesting that, during silage fermentation, bacteria utilize fermentable substrates for conversion to different metabolites, resulting in a greater abundance of metabolic pathways. L. plantarum inoculation enhanced metabolic activity in silage compared to the untreated CON group. At pathway level 2, microbial functional analysis revealed dominant roles in carbohydrate and amino acid metabolism during both pre-ensiling and fermentation stages. The carbohydrate metabolism pathway was more abundant in the silage with L. plantarum than in the CON group, which may be attributed to the dominance of LAB in the fermentation process of silage, where they ferment carbohydrates into lactic acid without oxygen [ 65 , 74 ]. Amino acids, as essential substances for living organisms, significantly contribute to the advancement of primary metabolism and protein production [ 40 ]. The L. plantarum -treated group showed enriched amino acid metabolic pathways compared to the CON group, likely attributable to bacterial amino acid utilization and metabolic regulation, while LAB dominance in fermentation potentially drove this metabolic shift [ 72 ]. To further elucidate the functional shifts in the alfalfa feedstock and silage bacterial communities, we specifically analysed carbohydrate metabolism and amino acid metabolism at the pathway 3 level. The silage process promotes ascorbate and aldarate metabolism; pentose and glucuronate interconversion; butanoate metabolism; fructose and mannose metabolism; the citrate cycle (TCA cycle); galactose metabolism; and pyruvate metabolism. The L. plantarum -supplemented mixture had the highest carbohydrate metabolism pathway activity. These results correspond with LAB’s characteristic ability to metabolize diverse carbohydrate substrates through anaerobic fermentation, demonstrating that L. plantarum inoculation stimulates carbohydrate metabolic pathway expression. Phenylalanine, tyrosine, and tryptophan biosynthesis; arginine biosynthesis; tryptophan metabolism phenylalanine metabolism; arginine and proline metabolism; valine, leucine, and isoleucine biosynthesis; and alanine, aspartate, and glutamate metabolism pathways were significantly inhibited after silage. A previous study showed that the acidic environment established by silage inhibited undesirable microbial-induced amino acid metabolism [ 75 ]. Conclusion By adding four species of L. plantarum , we thoroughly investigated their effects on alfalfa silage quality, fermentation indices, antioxidant activity, and the microbial community during fermentation. Results demonstrated that L. plantarum inoculation enhanced alfalfa silage quality, with strain H8 particularly improving flavonoid content and antioxidant capacity. L. plantarum inoculation modifies microbial composition, reduces diversity, and restructures interaction networks in alfalfa silage. This study emphasizes the potential of the addition of L. plantarum to improve silage quality and antioxidant activity, with positive implications for microbial-driven strategies to increase animal health and productivity. Acknowledgements No. Declaration of Generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors did not use any AI and AI-assisted technologies. Authors’ contributions Yuqian Liu: Writing – original draft, Visualization, Software, Investigation, Data curation, Conceptualization. Lin Guo: Methodology, Investigation, Data curation. Zishang Li: Methodology, Investigation, Data curation. Wenting Gou: Methodology, Investigation, Data curation. Jia Fu: Methodology, Investigation, Data curation. Mengxue Liu: Methodology, Investigation. Ze Wang: Methodology, Investigation, Data curation. Nanyi Zhang: Methodology, Investigation. Qiang Si: Writing – review & editing, Data curation. Hongmei Shang: Supervision, Project administration, Funding acquisition, Data curation, Conceptualization. Funding This work was financially supported by the Science and Technology Department of Jilin Province of China (20260102170JC). Data availability All the necessary data have been included in the manuscript. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Contributor Information Qiang Si, Email: [email protected]. Hongmei Shang, Email: [email protected]. References 1. Zhang W, Grimi N, Jaffrin MY, Ding L, Tang B. A short review on the research progress in alfalfa leaf protein separation technology. J Chem Technol Biotechnol. 2017;92:2894–900. 10.1111/jctb.5364. [ Google Scholar ] 2. Barros J, Temple S, Dixon RA. Development and commercialization of reduced lignin alfalfa. Curr Opin Biotechnol. 2019;56:48–54. 10.1016/j.copbio.2018.09.003. [ DOI ] [ PubMed ] [ Google Scholar ] 3. Zhang Y, Wang L. 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