Effects of Lactic Acid Bacterial Inoculants on Fermentation Quality, Bacterial Community, and Mycotoxins of Alfalfa Silage under Vacuum or Nonvacuum Treatment

To investigate the effects of lactic acid bacterial (LAB) inoculants and vacuuming on the fermentation quality and bacterial community, alfalfas were ensiled with or without a commercial LAB YX or Lactobacillus plantarum strain ZZUA493 for 10, 30, 60, and 90 days while undergoing either vacuum (V) or nonvacuum (NV) treatment. At 90 days, analysis of the microbial community by high-throughput sequencing was performed, and contents of aflatoxin B1 and deoxynivalenol (DON) mycotoxins in alfalfa silage were determined. In all inoculated alfalfa silage, irrespective of V or NV treatment, lactic acid (LA) content increased, pH (p < 0.05), and ammonia nitrogen (p < 0.05) content decreased, and no butyric acid was detected. Lactobacillus or Pediococcus became the dominant genus, and the abundance of Garciella decreased in alfalfa silage with the addition of either inoculant. The LAB inoculants YX and ZZUA493 helped reduce the mycotoxin content in alfalfa silage. The abundance of Garciella in the control and DON content in all alfalfa silage groups were higher (p < 0.05) in NV than V. In summary, LAB inoculants and vacuuming had a positive influence on alfalfa silage quality, and LAB inoculants were effective in reducing mycotoxins in silage alfalfa.

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Effects of Class IIa Bacteriocin-Producing Lactobacillus Species on Fermentation Quality and Aerobic Stability of Alfalfa Silage

Animals ◽

10.3390/ani10091575 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1575

Author(s):

Fuhou Li ◽

Zitong Ding ◽

Adegbola T. Adesogan ◽

Wencan Ke ◽

Yun Jiang ◽

...

Keyword(s):

Lactic Acid ◽

Lactobacillus Plantarum ◽

Crude Protein ◽

Ammonia Nitrogen ◽

Water Soluble ◽

Neutral Detergent Fiber ◽

Lactobacillus Delbrueckii ◽

Aerobic Stability ◽

Fermentation Quality ◽

Alfalfa Silage

The effects of two strains of class IIa bacteriocin-producing lactic acid bacteria, Lactobacillus delbrueckii F17 and Lactobacillus plantarum (BNCC 336943), or a non-bacteriocin Lactobacillus plantarum MTD/1 (NCIMB 40027), on fermentation quality, microbial counts, and aerobic stability of alfalfa silage were investigated. Alfalfa was harvested at the initial flowering stage, wilted to a dry matter concentration of approximately 32%, and chopped to 1 to 2 cm length. Chopped samples were treated with nothing (control, CON), Lactobacillus delbrueckii F17 (F17), Lactobacillus plantarum (BNCC 336943) (LPB), or Lactobacillus plantarum MTD/1 (NCIMB 40027) (LPN), each at an application rate of 1 × 106 colony-forming units/g of fresh weight. Each treatment was ensiled in quadruplicate in vacuum-sealed polyethylene bags packed with 500 g of fresh alfalfa per bag and ensiled at ambient temperature (25 ± 2 °C) for 3, 7, 14, 30, and 60 days. The samples were then subjected to an aerobic stability test after 60 days of ensiling. Compared with the CON silage, the inoculants reduced the pH after 14 days of ensiling. After 60 days, pH was lowest in the LPB-treated silage, followed by the F17 and LPN-treated silages. Inoculation of F17 increased concentrations of lactic acid in silages fermented for 7, 14, 30, and 60 days relative to other treatments, except for the LPN-treated silages ensiled for 30 and 60 days, in which the lactic acid concentrations were similar to that of F17 silage. Application of F17 and LPB decreased the number of yeast and mold relative to CON and LPN-treated silages. Compared with the CON silage, inoculant-treated silages had greater aerobic stability, water-soluble carbohydrate, and crude protein concentrations, and lower neutral detergent fiber, amino acid nitrogen, and ammonia nitrogen concentrations. The LPB-treated silage had the greatest aerobic stability followed by the F17-treated silage. Both class IIa bacteriocin producing inoculants improved alfalfa silage fermentation quality, reduced the growth of yeasts and molds, and improved the aerobic stability of the ensiled forage to a greater extent than the proven LPN inoculant. However, higher crude protein concentration and lower ammonia nitrogen concentration were observed in LPN-treated silage relative to other treatments.

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Enzyme additives influence bacterial communities of Medicago sativa silage as determined by Illumina sequencing

10.21203/rs.3.rs-29755/v1 ◽

2020 ◽

Author(s):

Zongfu Hu ◽

Deying Ma ◽

huaxin Niu ◽

Jie Chang ◽

Jianhua Yu ◽

...

Keyword(s):

Lactic Acid ◽

Bacterial Community ◽

High Throughput ◽

Bacterial Communities ◽

Crude Protein ◽

High Throughput Sequencing ◽

Ammonium N ◽

Enzyme Combination ◽

Fermentation Quality ◽

Dominant Bacteria

Abstract This study aimed to evaluate the effects of enzymes (cellulase combined with galactosidase),, and the combination of these enzymes with Lactobacillus plantarum (LP) on bacterial diversity using high-throughput sequencing. Alfalfa forages were treated without or with cellulase + ɑ-galactosidase (CEGA), cellulase + LP (CELP), ɑ-galactosidase + LP (GALP). After 56 days of ensiling, All the treated silages exhibited improved fermentation quality as reflecting by decreased pH, ammonium-N and increased lactic acid levels compared to the control silage. Enzymatic treatment improved nutrients value by increased the level of crude protein and decreased the neutral detergent fibre (NDF) level. Treatment of the silage significantly changed the bacterial community, as determined by the PCoA test. LAB dominated the bacterial community of the treated silage after ensiling. The dominant bacteria from Garciella, Enterococcus, Lactobacillus and Pediococcus in control silage changed to Lactobacillus and Pediococcus in CEGA silage, and Lactobacillus in CELP and GALP silages. Collectively, enzymes and enzyme in combination with inoculants both greatly increased the abundance of LAB, with Enterococcus, Lactobacillus and Pediococcus in enzymes only silge (CEGA) and Lactobacillus in enzyme combination with inoculants silage (CELP and GALP).

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Dynamics of Bacterial Community and Fermentation Quality during Ensiling of Wilted and Unwilted Moringa oleifera Leaf Silage with or without Lactic Acid Bacterial Inoculants

mSphere ◽

10.1128/msphere.00341-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 3

Author(s):

Yi Wang ◽

Liwen He ◽

Yaqi Xing ◽

Yanting Zheng ◽

Wei Zhou ◽

...

Keyword(s):

Lactic Acid ◽

Bacterial Community ◽

Dry Matter ◽

Fermentation Process ◽

Moringa Oleifera ◽

Content Type ◽

Bacterial Inoculants ◽

Dry Matter Loss ◽

Fermentation Quality

ABSTRACT To investigate the effects of wilting and lactic acid bacterial inoculants on the fermentation quality and bacterial community of Moringa oleifera leaf silage, fresh and wilted M. oleifera leaves were ensiled with or without Lactobacillus farciminis LF or Lactococcus lactis LL for 1, 7, 14, 30, and 60 days. The results showed that wilting, inoculants, and their interaction exerted significant (P < 0.05) effects on the fermentation characteristics covering dry matter loss, pH value, lactic acid bacterial number, the ratio of lactic acid to acetic acid, and the relative abundances of bacteria, like for species of Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Leuconostoc, and Enterobacter. Both LF and LL improved the fermentation quality of wilted and unwilted M. oleifera leaf silage by accelerating lactic acid production and pH decline, decreasing dry matter loss, and inhibiting yeast and coliform bacterial growth through the whole fermentation process. During ensiling, the abundances of Lactococcus, Enterococcus, and Leuconostoc spp. increased from day 1 to day 7 and then declined sharply from day 7 to day 14. Members of these genera and Enterobacter were inhibited, whereas Lactobacillus spp. were enhanced by these two lactic acid bacterial inoculants. The relative abundances of Enterobacter, Enterococcus, and Pediococcus spp. in inoculated silages were relatively low during the whole ensiling process. A lower abundance of Enterobacter spp. was observed in wilted silages than in unwilted silages. In summary, wilting and lactic acid bacterial inoculants had an influence on bacterial community and the fermentation process; LF and LL improved the fermentation quality of wilted and unwilted M. oleifera leaf silage. IMPORTANCE Moringa oleifera leaf is a high-quality feed source for livestock and is increasingly used all over the world. Ensiling might be an effective method for preservation of the leaves. In the practice of silage making, lactic acid bacterial inoculants and wilting are commonly used to improve nutrition preservation. Monitoring the changes in a bacterial community during fermentation gives an insight into understanding and improving the ensiling process. Our results suggest that wilting and lactic acid bacterial inoculants had an influence on the bacterial community and fermentation process of M. oleifera leaf silage. Wilting showed positive effects on silage fermentation by decreasing the abundance of Enterobacter spp., while LF and LL improved the fermentation quality by inhibiting Enterobacter spp. and enhancing Lactobacillus spp. Both LF and LL accelerated the ensiling process from cocci (like Lactococcus, Enterococcus, and Leuconostoc spp.) to lactobacilli.

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Metagenome analyses reveal the role of Clostridium perfringens in alfalfa silage anaerobic deterioration

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa052 ◽

2020 ◽

Vol 367 (8) ◽

Author(s):

Mingli Zheng ◽

Dongze Niu ◽

Di Jiang ◽

Rongrong Li ◽

Lin Meng ◽

...

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

Clostridium Perfringens ◽

Relative Abundance ◽

Dominant Role ◽

Ammonia Nitrogen ◽

Fermentation Products ◽

Microbial Community Analyses ◽

Alfalfa Silage

ABSTRACT The clostridial fermentation caused by the outgrowth of Clostridia was mainly responsible for the silage anaerobic deterioration. Our previous results showed that Clostridium perfringens dominated the clostridial community in poor-fermented alfalfa silage. This study was conducted to further examine the role of C. perfringens in silage anaerobic deterioration through fermentation products and the microbial community analyses. Direct-cut alfalfa was ensiled with C. perfringens contamination (CKC) or with the addition of Lactobacillus plantarum, sucrose and C. perfringens (LSC). Contamination with C. perfringens enhanced the clostridial fermentation in CKC silage, as indicated by high contents of butyric acid, ammonia nitrogen and Clostridia, while LSC silage was well preserved. The genera Bifidobacterium, Garciella and Clostridium dominated the bacterial community in CKC silage, while predominate genus was replaced by Lactobacillus in LSC silage. The clostridial community in CKC silage was dominated by Garciella sp. (26.9 to 58.1%) and C. tyrobutyricum (24.4 to 48.6%), while the relative abundance of C. perfringens was below 5.0%. Therefore, the effect of Clostridia contamination on ensiling fermentation was dependent on the ensilability of the silage material. Garciella sp. and C. tyrobutyricum, rather than C. perfringens, played dominant role in the clostridial fermentation in CKC silage.

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Improvement of Fermentation Quality in the Fermented Total Mixed Ration with Oat Silage

Microorganisms ◽

10.3390/microorganisms9020420 ◽

2021 ◽

Vol 9 (2) ◽

pp. 420

Author(s):

Hong Yang ◽

Bing Wang ◽

Qing Zhang ◽

Hui Cheng ◽

Zhu Yu

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Bacterial Community ◽

Ammonia Nitrogen ◽

Water Soluble ◽

Soluble Carbohydrates ◽

Nonprotein Nitrogen ◽

Total Mixed Ration ◽

Spoilage Yeast ◽

Fermentation Quality

The use of the fermented total mixed ration (FTMR) is a promising approach for the preservation of homogeneous feed, but changes during fermentation and links with the bacterial community of FTMR are not fully understood. This study investigated the effects of adding oat silage (OS) to the fermented total mixed ration (FTMR) in terms of fermentation, chemical composition, and the bacterial community. The fermentation quality of FTMR with 22% OS was greatly improved, as demonstrated by decreases in the butyric acid concentration, a lower lactic acid/acetic acid ratio, a larger population of lactic acid bacteria (LAB), and quicker spoilage yeast death. Further examination of the effects of various ensiling days on nutritive values showed stable crude protein and nonprotein nitrogen (NPN) contents. The concentrations of acetic acid, propionic acid, and ammonia–nitrogen (NH3–N) were increased following all FTMR treatments after 15 d, while the concentration of water-soluble carbohydrates (WSC) was decreased. More heterofermentative LAB, such as Lentilactobacillus buchneri, Lentilactobacillus brevis, and Companilactobacillus versmoldensis were found after adding 11% and 22% OS. Moreover, the addition of 22% OS caused a marked increase in both bacterial richness and diversity, dominated by the Lactobacillus genus complex. Among species of the Lactobacillus genus complex, the occurrence of Loigolactobacillus coryniformis was positively correlated with lactic acid, NPN, and NH3–N concentrations, suggesting its potential role in altering the fermentation profiles.

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Dynamics of the Fermentation Products, Residual Non-structural Carbohydrates, and Bacterial Communities of Wilted and Non-wilted Alfalfa Silage With and Without Lactobacillus plantarum Inoculation

Frontiers in Microbiology ◽

10.3389/fmicb.2021.824229 ◽

2022 ◽

Vol 12 ◽

Author(s):

Fengyuan Yang ◽

Yanping Wang ◽

Shanshan Zhao ◽

Changsong Feng ◽

Xiaomiao Fan

Keyword(s):

Bacterial Community ◽

Lactobacillus Plantarum ◽

Bacterial Communities ◽

High Throughput Sequencing ◽

Bacterial Community Composition ◽

Water Soluble ◽

Soluble Carbohydrates ◽

Fermentation Products ◽

Fermentation Quality ◽

Alfalfa Silage

The aim of this study was to investigate effects of wilting and Lactobacillus plantarum inoculation on the dynamics of the fermentation products, residual non-structural carbohydrates, and bacterial communities in alfalfa silage. Fresh and wilted alfalfa were ensiled with and without L. plantarum for 10, 30, 60, and 90 days. A high-throughput sequencing method for absolute quantification of 16S rRNA was adopted to determine the bacterial community composition at different ensiling periods. For the wilted silage, the bacterial community, pH value, and ammonia nitrogen concentration remained stable in the silage at 30 days. L. plantarum inoculation accelerated lactic acid fermentation and altered the predominant genus in the wilted silage as compared with the non-inoculated group. For the non-wilted group, fast consumption of water-soluble carbohydrates (WSCs) was observed at 10 days in the non-inoculated silage along with rapid growth of undesirable Hafnia. L. plantarum inoculation inhibited growth of Hafnia at 10 days in the non-wilted silage. Clostridia fermentation occurred in the non-wilted silage at 90 days, as indicated by an increased pH, formation of butyric acid (BA), and apparent abundance of genera belonging to Clostridia. L. plantarum inoculation inhibited BA accumulation and growth of Garciella in the non-wilted silage at 90 days as compared with the non-wilted silage without inoculation, but had little effect on the growth of Clostridium sensu stricto. Overall, the high moisture content of the non-wilted alfalfa silage led to rapid consumption of WSCs and growth of harmful microorganisms at the early stage of ensiling, resulting in poor fermentation quality. Wilting and L. plantarum inoculation both improved fermentation quality and inhibited the growth of spoilage microorganisms in alfalfa silage, while L. plantarum inoculation alone failed to achieve optimum fermentation quality of non-wilted alfalfa silage.

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Enzyme additives influence bacterial communities of Medicago sativa silage as determined by Illumina sequencing

AMB Express ◽

10.1186/s13568-020-01158-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zongfu Hu ◽

Deying Ma ◽

Huaxin Niu ◽

Jie Chang ◽

Jianhua Yu ◽

...

Keyword(s):

Lactic Acid ◽

Bacterial Community ◽

Bacterial Communities ◽

Crude Protein ◽

High Throughput Sequencing ◽

Protein Levels ◽

Nutrient Value ◽

Ammonium N ◽

Fermentation Quality ◽

Dominant Bacteria

AbstractThe goal of the present study was to evaluate the effects of enzymes (cellulase combined with galactosidase) and their combination with Lactobacillus plantarum (LP) on bacterial diversity in alfalfa silages using high-throughput sequencing. Alfalfa forages were treated with or without cellulase + ɑ-galactosidase (CEGA), cellulase + LP (CELP), or ɑ-galactosidase + LP (GALP). After 56 days of ensiling, all treated silages exhibited improved fermentation quality, as reflected by decreased pH, ammonium-N and increased lactic acid levels compared to the control silage (P < 0.05). Enzymatic treatment improved nutrient value by increasing crude protein levels and decreasing neutral detergent fibre (NDF) levels (P < 0.05). Silage treatment significantly altered the bacterial community, as determined by PCoA (P < 0.05). Lactic acid bacteria (LAB) dominated the bacterial community of the treated silage after ensiling. The dominant bacteria changed from Garciella, Enterococcus, Lactobacillus and Pediococcus in the control silage to Lactobacillus and Pediococcus in the CEGA silage and Lactobacillus in the CELP and GALP silages. Collectively, these results suggest that treatment with both enzymes alone and in combination with inoculants greatly increased the abundance of LAB, with Enterococcus, Lactobacillus and Pediococcus observed in the silage treated with enzymes alone (CEGA) and Lactobacillus observed in the silage treated with a combination of enzymes and inoculants (CELP and GALP).

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Effect of Mixing Alfalfa with Whole-Plant Corn in Different Proportions on Fermentation Characteristics and Bacterial Community of Silage

Agriculture ◽

10.3390/agriculture11020174 ◽

2021 ◽

Vol 11 (2) ◽

pp. 174

Author(s):

Musen Wang ◽

Run Gao ◽

Marcia Franco ◽

David B. Hannaway ◽

Wencan Ke ◽

...

Keyword(s):

Acetic Acid ◽

Lactic Acid ◽

Bacterial Community ◽

Ammonia Nitrogen ◽

Weight Basis ◽

Fresh Weight ◽

Fresh Weight Basis ◽

Silage Fermentation ◽

Whole Plant ◽

Fermentation Quality

The influence of mixing alfalfa with whole-plant corn in different proportions on the fermentation characteristics and bacterial community of silage was investigated. Alfalfa and whole-plant corn, harvested at dry matter content of 276.47 and 328.43 g/kg fresh weight, accordingly, were chopped to approximately 2 cm and mixed at ratios of 100:0 (C0, control), 80:20 (C20), 60:40 (C40), 40:60 (C60), 20:80 (C80) and 0:100 (C100) on a fresh weight basis, respectively. Silos of each treatment were produced in triplicate and anaerobically fermented in darkness for 100 days at room temperature (20–21 °C). At silo opening, silage fermentation characteristics and bacterial composition and diversity were analyzed. The C0 silage was weakly preserved, evidenced by a low lactic acid concentration and a high value of pH, acetic acid, propionic acid, butyric acid and ammonia nitrogen. With corn proportion in the mixture increasing from 0% to 40%, silage pH, acetic acid, butyric acid and ammonia nitrogen level decreased, whereas the value of lactic acid and lactic acid to acetic acid ratio increased. The C40, C60, C80 and C100 silages’ Flieg score, used to evaluate the overall fermentation quality, was above 80 and higher than C0 (25) and C20 (61) silages. The C0 silage contained a complex bacterial community at the genus level, consisting mainly of Enterococcus (38.86%), Enterobacteria (20.61%), Rhizobium (8.45%), Lactobacillus (8.15%), Methylobacterium (5.54%) and Weissella (5.24%). As corn percentage increased from 0% to 40%, the relative abundance of desirable Lactobacillus increased and undesirable Rhizobium and Methylobacterium population reduced. With corn proportion in the mixture increasing from 0% to 40%, inclusion of corn to alfalfa at ensiling significantly improved silage fermentation quality and shifted the bacterial community for better silage preservation. Overall, high quality silage was produced when alfalfa was combined with at least 40% whole-plant corn on a fresh weight basis.

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The Potential Effects on Microbiota and Silage Fermentation of Alfalfa Under Salt Stress

Frontiers in Microbiology ◽

10.3389/fmicb.2021.688695 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qiang Lu ◽

Zhen Wang ◽

Duowen Sa ◽

Meiling Hou ◽

Gentu Ge ◽

...

Keyword(s):

Lactic Acid ◽

Salt Stress ◽

Chemical Composition ◽

Bacterial Community ◽

Natural Fermentation ◽

Silage Fermentation ◽

Lactic Acid Content ◽

Fermentation Quality ◽

Alfalfa Silage

This study investigated the fermentation quality of alfalfa grown in different salt stress regions in China. Following the production of silage from the natural fermentation of alfalfa, the interplay between the chemical composition, fermentation characteristics, and microbiome was examined to understand the influence of these factors on the fermentation quality of silage. The alfalfa was cultivated under salt stress with the following: (a) soil content of <1%0 (CK); (b) 1–2%0 (LS); (c) 2–3%0 (MS); (d) 3–4%0 (HS). The pH of the silage was high (4.9–5.3), and lactic acid content was high (26.3–51.0 g/kg DM). As the salt stress increases, the NA+ of the silages was higher (2.2–5.4 g/kg DM). The bacterial alpha diversities of the alfalfa silages were distinct. There was a predominance of desirable genera including Lactococcus and Lactobacillus in silage produced from alfalfa under salt stress, and this led to better fermentation quality. The chemical composition and fermentation characteristics of the silage were closely correlated with the composition of the bacterial community. Furthermore, NA+ was found to significantly influence the microbiome of the silage. The results confirmed that salt stress has a great impact on the quality and bacterial community of fresh alfalfa and silage. The salt stress and plant ions were thus most responsible for their different fermentation modes in alfalfa silage. The results of the study indicate that exogenous epiphytic microbiota of alfalfa under salt stress could be used as a potential bioresource to improve the fermentation quality.

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Effects of ferulic acid esterase-producing Lactobacillus fermentum and cellulase additives on the fermentation quality and microbial community of alfalfa silage

PeerJ ◽

10.7717/peerj.7712 ◽

2019 ◽

Vol 7 ◽

pp. e7712 ◽

Cited By ~ 4

Author(s):

Rina Su ◽

Kuikui Ni ◽

Tianwei Wang ◽

Xiaopan Yang ◽

Jie Zhang ◽

...

Keyword(s):

Lactic Acid ◽

Microbial Community ◽

Ferulic Acid ◽

Plant Cell Wall ◽

Water Soluble ◽

Neutral Detergent Fiber ◽

Lactobacillus Fermentum ◽

Ferulic Acid Esterase ◽

Fermentation Quality ◽

Alfalfa Silage

Background Alfalfa (Medicago sativa) is an important forage material widely used for animal feed production. Ensiling is an effective method for preserving alfalfa, but it has shown some limitations in the production of high-quality alfalfa silage due to its low water soluble carbohydrates (WSC) content and high buffering capacity. Lactic acid bacteria (LAB) and cellulase are often used as silage additives to promote the ensiling process and enhance fermentation quality. Methods Experiments were conducted to investigate the effects of ferulic acid esterase (FAE)-producing Lactobacillus fermentum 17SD-2 (LF) and cellulase (CE) on the fermentation quality and microbial community of alfalfa silage. After 60 days of ensiling, analysis of fermentation quality and bacterial diversity in alfalfa silages were conducted using high-performance liquid chromatography and high-throughput sequencing methods. Results Alfalfa was ensiled with additives (LF, CE, and LF+CE) or without additives for 60 days. All additives increased lactic acid and decreased pH values and ammonia-N contents compared to control. Among all treatments, the combined addition of LF and CE showed lowest pH (4.66) and ammonia-N (NH3-N, 0.57% DM) content, highest contents of lactic acid (LA, 10.51% DM), dry matter (DM, 22.54%) and crude protein (CP, 24.60% DM). Combined addition of LF and CE performed better in reducing neutral detergent fiber (NDF, 29.76% DM) and acid detergent fiber (ADF, 22.86% DM) contents than the addition of LF (33.71, 27.39% DM) or CE (32.07, 25.45% DM) alone. Moreover, the microbial analysis indicated that LF+CE treatments increased the abundance of desirable Lactobacillus and inhibited the growth of detrimental Enterobacter and Clostridia in alfalfa silage. Discussion Combined addition of FAE-producing LF and CE is more effective than treatments of LF or CE alone in improving fermentation quality and nutrition values of alfalfa silage. This is likely due to a synergistic effect of CE and FAE produced by LF on plant cell wall degradation, indicating that these additives promote each other to improve fiber degradation and silage fermentation. In conclusion, combined addition of FAE-producing LF and CE could be a feasible way to improve alfalfa silage quality.

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