Сосуществование Лактиплантацилла и Виккерхамомиц в верхнем слое сахарного тростника стимулирует потенциальное подавление патогенов и аэробную стабильность за счет увеличения производства лактической кислоты и уксусной кислоты.

Introduction

Silage is the most prevalent feed in animal husbandry, primarily for ruminants such as cattle and sheep. Fermentation quality and aerobic stability of silage directly affect the nutritional value of the feed and animal performance [ 1 , 2 ]. Notably, these key parameters of silage are governed by the microbial community structure and the biochemical processes driven by its metabolic activities during ensiling (such as the generation of organic acids) [ 3 , 4 ]. However, despite years of development in silage, challenges remain, particularly in the management of microbial contamination [ 1 , 5 ]. Among them, potential pathogens in silage, such as Enterobacteriaceae , not only lead to nutrient losses of silage, but produce toxins that threaten animal safety [ 4 , 6 ]. While, when silage is exposed to aerobic conditions, it is more susceptible to microbial contamination, including the rapid resurgence and activity of aerobic microorganisms (such as yeast and mold), which can lead to aerobic deterioration, resulting in decreased nutritional value and accumulation of mycotoxins [ 3 ]. Studies have shown that the accumulation of organic acids such as lactic acid (LA) and acetic acid (AA) in silage can improve fermentation quality, inhibit pathogenic bacteria, and promote aerobic stability [ 5 , 7 ].

Lactic acid bacteria (LAB) as core initiators, mainly fermenting sugar substrates to produce LA and AA in silage [ 8 ]. Yeasts, in contrast, are often considered undesirable under aerobic or poorly sealed conditions, where they consume organic acids, increase pH, and cause spoilage [ 2 ]. However, under strictly anaerobic and well-managed ensiling conditions, certain yeast species can exert beneficial effects. For example, some yeasts (e.g., Saccharomyces and Wickerhamomyces ) can produce various antimicrobial compounds, including killer toxins (proteinaceous toxins active against spoilage fungi and bacteria), siderophores (which chelate iron and limit pathogen growth), and organic acids (e.g., succinic and malic acid) that directly lower pH and inhibit pathogens such as Aspergillus [ 9 – 12 ]. Others, when certain yeasts (e.g., S accharomyces cerevisiae , Pichia anomala ) are co‑inoculated with LAB (e.g., Lactobacillus spp., Leuconostoc citreum ), these yeasts enhance LA and AA accumulation by promoting LAB growth through synergistic effects (e.g., supplying amino acids and vitamins) [ 13 – 16 ], reduce the mold counts [ 13 ], and extend the aerobic preservation time of silage [ 17 ]. This suggests that the accumulation of organic acids, improved aerobic stability, and inhibition of pathogenic microorganisms in silage can be achieved through the colonization and coexistence of LAB and yeast.

As a major byproduct accounting for about 20% of sugarcane's total output, sugarcane tops have an estimated annual production of around 20 million tons in China, primarily concentrated in Guangxi, China [ 18 ]. While the high-WSC content of sugarcane tops promotes LAB enrichment (e.g., Lactiplantibacillus ), the concomitant proliferation of yeasts compromises silage quality (e.g., butyric acid accumulation) and aerobic stability (e.g., proliferation of undesirable aerobic microorganisms such as Enterobacter) [ 19 ]. Previous studies have also shown that the LAB and yeast were also consistently present in well-preserved or poor sugarcane top silage under all tested conditions, including ensiling time [ 20 ], nitrogen application levels and varieties [ 21 ]. However, the key coexisting yeasts and LAB that determine the quality of sugarcane top silage have not yet been identified. Specifically, two key knowledge gaps still exist: (i) which specific species of LAB and yeasts coexist as the core functional microbiota in sugarcane top silage? and (ii) whether these yeasts mainly play a spoilage role or can positively contribute to fermentation quality and pathogen inhibition? So, the present study aimed to reveal and isolate the key coexisting LAB and yeasts that determine the fermentation quality and safety of sugarcane top silage, and offering innovative microbial agent strategies for developing next-generation silage additives.

Materials and methods

Raw materials and ensiling

Sugarcane tops (Sugarcane variety: Zhongzhe Funong No. 48) were collected from the forage breeding ground of Fusui Experimental Base of Guangxi University. The collection of sugarcane tops was authorized by the College of Life Sciences and Technology, Guangxi University. The chemical compositions of the fresh sugarcane tops are listed in Table S1 . Fresh sugarcane tops were chopped to a length of approximately 2 cm with a forage cutter (model zfd5570, Zheng Feng Machinery Company, Jining, China).

According to the methods of Mogodiniyai Kasmaei et al. [ 22 ], 30 kg fresh sugarcane tops was added to 120 L of Ringer’s solution, mixing and standing for 30 min at room temperature; Then, after thorough stirring, the mixture was filtered through four layers of gauze to obtain a microbial suspension; Microbial suspension was centrifuged at 15 500 × g for 90 min (AVANTIJ-E, Beckman, USA); Discard the supernatant, resuspension in physiological saline to 90 mL, divide into two equal portions, and store at 4 °C for later use.

The treatment in this study were as is shown in the Table 1 . Specifically, following the method described by Malek et al. [ 23 ], fresh sugarcane tops were sterilized at a total dose of 30 kGy (exposure time: 210 min) from a ⁶⁰Co source at Guangxi Nanxiang Environmental Protection Co., Ltd., Nanning, China. For the antimicrobial treatment, 45 mL of the resuspended epiphytic suspension was mixed with 6.75 g of antifungal agent or antibacterial agent, homogenized and allowed to stand for 5 min, after which the mixture was evenly sprayed onto the γ‑sterilized sugarcane tops at an application rate of 15 mL per kg of fresh weight. Table 1 The treatment groups in this study Treatment Abbreviation Description Untreated silage CK Fresh sugarcane tops ensiled directly (natural epiphytic microbiota) γ-ray sterilized control NA γ‑sterilized sugarcane tops without reinoculation Antifungal treatment AF γ-sterilized sugarcane tops + reinoculated with antifungal-pretreated suspension (15 mL/kg) Antibacterial treatment AB γ-sterilized + reinoculated with antibacterial-pretreated suspension (15 mL/kg)

The antibacterial agent mainly consists of penicillin streptomycin, penicillin G sodium salt, cefotaxime, tetracycline hydrochloride, and ciprofloxacin in a 1:1:1:1 ratio, all purchased from Solarbio Lifesciences, Beijing, China. The antifungal agent mainly consists of natamycin, fluconazole, itraconazole, and voriconazole in a 1:1:1:1 ratio, all purchased from Solarbio Lifesciences, Beijing, China. The selection of antibiotics and antifungal agents was based on their known selective inhibitory effects: penicillin and streptomycin inhibit bacterial cell wall synthesis and protein synthesis, respectively [ 24 , 25 ]; cefotaxime, tetracycline and ciprofloxacin provide broad‑spectrum coverage against Gram‑negative bacteria and potential resistant strains [ 25 ]; natamycin inhibits fungal growth via ergosterol‑dependent inhibition of membrane transport proteins [ 26 ]; fluconazole, itraconazole and voriconazole inhibit ergosterol biosynthesis via 14α‑sterol demethylase [ 27 ]. Preliminary experiments indicated that adding the antibacterial agent and antifungal agent to the microbial suspension at the provided dosage resulted in not detected epiphytic lactic acid bacteria (LAB) and yeast using typical microbial culture, respectively. Also, no countable microorganisms were detected after γ-irradiation. After mixing, 200 g of fresh material was packaged in polyethylene bags (200 mm × 250 mm; Shijiazhuang Xilong Packaging Co., Ltd., Shijiazhuang, China) and vacuum-sealed using a vacuum sealer (Model 680; Fujian Anxi Anshengke Machinery Co., Ltd., Quanzhou, China). Each treatment was prepared in triplicate. The bags were then stored at ambient temperature (10–35 °C, which reflects the actual natural conditions of Guangxi, China) for 3, 7, 12, 30, and 60 days. Ambient temperature was continuously monitored using a data logger (RC-5 +, Jingchuang Co., Ltd. Shanghai, China). After the ensiling period, the bags were opened and samples were collected for analysis of aerobic stability, fermentation quality, microbial counts, and microbial community structure.

Aerobic stability test

The aerobic stability of sugarcane top silage was determined following the method described by Nishino and Touno [ 28 ], with some modifications. Briefly, a 100-g silage sample from each treatment was taken and placed into a 500-mL plastic bottle. Simultaneously, a temperature data logger (RC-5 +, Jingchuang Co., Ltd. Shanghai, China) programmed to record every 2 h, was inserted. The plastic bottle was then covered with a layer of gauze (To reduce cross-contamination of the silage and ensure air circulation) and stored at ambient temperature (10–35 °C) for 8 days. After 8 days of aerobic exposure, 20 g of silage sample were taken for the determination of fermentation parameters, microbial counts, and microbial community structure.

Chemical composition, fermentation profile, and microbial population analysis

A 20-g fresh silage sample was combined with 180 mL of Ringer's solution and mixed thoroughly. After standing for 30 min at 4 °C, the mixture was filtered through a double-layer gauze. The collected filtrate was then used for the determination of fermentation parameters and microbial counts. The pH of the filtrate was measured directly using a pH meter (Mettler-Toledo Delta 320; Mettler-Toledo, Greifensee, Switzerland). A 10 mL of filtrate were centrifuged (12 000 × g , 15 min, 4 °C), and the supernatant was used to determine ethanol, organic acids [lactic acid (LA), acetic acid (AA), propionic acid (PA), butyric acid (BA)], and ammonia nitrogen (AN). LA and AN content were determined according to Zhang et al. [ 20 ], using the p-hydroxybiphenyl and phenol–hypochlorite reaction methods, respectively. The contents of AA, PA, BA, and ethanol were determined by gas chromatography (GC) according to Erwin et al. [ 29 ]. Analysis was performed on an Agilent 7890 A system (Agilent Technologies, CA, USA) equipped with a flame ionization detector and an HP-INNOWax column (19091N-213; 30.0 m × 320 μm × 0.5 μm). The injection volume was 1 μL with a split ratio of 10:1.

For microbial counts, filtrates (1 mL) were serially diluted (10 –1 to 10 –6 ) according to Gu et al. [ 19 ]. LAB were quantified on De Man, Rogosa, and Sharpe agar (MRS, Beijing Land Bridge Technology, Beijing, China) under anaerobic incubation at 35 °C for 48 h. Yeasts and molds were grown on potato dextrose agar (PDA, Beijing Land Bridge Technology, Beijing, China) at 25 °C for 72 h, and Enterobacteriaceae (EB) on violet red bile agar (Beijing Land Bridge Technology, Beijing, China) at 37 °C for 48 h.

DNA extraction and PCR amplification

Microbial DNA was extracted from silage samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, USA) according to manufacturer’s protocols. The extracted microbial DNA was sent to Shanghai Biozeron Biological Technology Co., Ltd. (Shanghai, China) for 16S RNA and ITS1 gene amplicon sequencing on a NovaSeq 6000 platform. Amplification sequencing of bacterial 16S RNA was performed using primers 341 F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplification of fungal ITS1 was performed using primers ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′-GCTGCGTTCTTCATCGATGC-3′). Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) according to the manufacturer’s instructions. Purified polymerase chain reaction (PCR) products were quantified by Qubit®3.0 (Life Technologies Corporation, Carlsbad, USA) and every twenty-four amplicons whose barcodes were different were mixed equally. The pooled DNA product was used to construct Illumina Pair-End library following Illumina’s genomic DNA library preparation procedure.

Raw FASTQ files were demultiplexed using Trimmomatic [ 30 ] and custom Perl scripts based on barcode sequences with the following criteria: (i) 300 bp reads were trimmed using a 10 bp sliding window (average quality < 20); reads < 50 bp were discarded. (ii) Exact barcode matches and ≤ 2 mismatches in primers were allowed; reads with ambiguous bases were removed. (iii) Reads with > 10 bp overlap were assembled; unassembled reads were discarded. The sequencing depth have shown in Table S2. OTUs were clustered with 97% similarity cutoff using UPARSE (version 10, http://drive5.com/uparse/ ) [ 31 ] and chimeric sequences were identified and removed using UCHIME. OTU clustering at 97% similarity was chosen to maintain comparability with our previous studies on sugarcane top silage, and because our research focuses on genus level community shifts and BugBase phenotypes, where OTU and ASV results are highly consistent [ 32 – 34 ]. Analysis of the alpha diversity (Shannon index) for bacterial and fungal communities was performed with QIIME 2 (v2024.10) based on the sequencing data. The SILVA database ( http://www.arb-silva.de/ ) and the UNITE database ( https://unite.ut.ee/ ) were used to annotate bacterial and fungal sequencing data, respectively, with a confidence threshold of 0.8 [ 35 ], thereby obtaining taxonomic information and calculating the community composition of each sample at each taxonomic level. BugBase algorithm ( https://bugbase.cs.umn.edu/index.html ) was used to predict the phenotypes of bacterial communities.

LAB and yeast isolation, identification

After microbial counting, LAB and yeast were screened in sugarcane top silage of CK and AF groups based on colony size, color, and shape respectively. Continuous subculturing was performed on MRS and PDA plates until pure colonies were established. Then, Gram staining and methylene blue staining were used to examine the macroscopic colony morphology and microscopic cellular characteristics of the obtained pure LAB and yeast, respectively. For molecular identification, LAB and yeast DNA extraction was performed using bacterial DNA kit (Tiangen Biotech Co. Ltd., Beijing, China) and yeast genome extraction kit (Solarbio Lifesciences, Beijing, China), according to the manufacturer’s instructions, respectively. Subsequently, the 16S rDNA of LAB was amplified by the PCR with primers were 27 F (5’AGAGTTTGATCCTGGCTCAG3′) and 1492R (5’GTTTACCTTGTTACGACTT 3′). The ITS region of yeast was amplified using the primer pair ITS1-(5′ TCCGTAGGTGAACCTGCGG 3′) and ITS4-(5′ TCCTCCGCTTATTGATATGC 3′). The 16S rRNA and ITS sequences were aligned with those in the GenBank database for species identification via the NCBI BLAST analysis [ 36 ]. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 12 software (V12.1.1) to perform molecular evolutionary analysis on the identified LAB and yeast strains.

Haemolytic activity of the LAB and yeast isolates was evaluated according to Mangia et al. [ 37 ] and made some modification. Specifically, the isolated bacteria and yeast were passaged three times in MRS (Beijing Land Bridge Technology, Beijing, China) and PDB (Potato Dextrose Broth, Beijing Land Bridge Technology, Beijing, China) liquid media, respectively. The third-generation cultures were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, while the cells precipitation was washed three times with sterile physiological saline, and centrifuge again. Then, the LAB and yeast suspension were prepared by adding sterile physiological saline and normalized to 1.0 McFarland (570 nm; 3 × 10 8 CFU/mL), respectively. 10 μL of the LAB and yeast suspension was spotted onto blood agar plates containing 5% (v/v) sheep blood (Solarbio Lifesciences, Beijing, China). The LAB were anaerobically cultured at 37 °C for 48 h, and the yeast was cultured at 30 °C for 72 h. Hemolysis was then observed on the plates to determine whether complete hemolysis, β-hemolysis (a clear halo around each colony), α-hemolysis (a green halo around each colony), or γ-hemolysis (no halo around each colony) occurred. The utilization of carbon sources such as glucose, lactose, maltose, mannitol, sucrose, arabinose, xylose, sorbitol, rhamnose, salicin, aescin, muscarin, raffinose, fructose, melibiose, cellobiose, starch, and galactose by the yeast and LAB strains was determined using microbial chemical reaction tubes (Hangzhou Binhe Microbial Reagent Co., Ltd., Hangzhou, China).

To evaluate the organic acid‑producing ability and metabolic flexibility of the isolated LAB ( Lactiplantibacillus ) and yeast ( Wickerhamomyces ) strains on diverse carbon sources, a co‑fermentation experiment was conducted using three fruit peels (orange, dragon fruit, mango). For each substrate, 36 g of crushed peel waste was placed in a 250 mL Erlenmeyer flask with 12 mL molasses and 120 mL sterile water. Then, 1% sterile saline and a 1:1 compound microbial agent consisting of the isolated LAB Lactiplantibacillus and the yeast Wickerhamomyces (based on fresh weight) was added. The mixture was incubated anaerobically at 25 °C for 7 days (four replicates per treatment), after which 2 mL samples were taken to measure LA and AA content. Efficient LA/AA production from these three distinct carbon sources confirmed the robustness of the strains, directly supporting their application in sugarcane top silage for improved fermentation quality.

Statistical analysis

Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, Illinois, USA). A general linear model (GLM) was used for two-way analysis of variance on ensiling times, additives, and their interaction on microbial counts and fermentation quality of the sugarcane tops silage. One-way analysis of variance (ANOVA) was performed on the microbial population and fermentation quality of the sugarcane tops silage after aerobic exposure and the LA and AA content in fruit peel waste fermentation substrate.

Due to the small sample size ( n = 3 per treatment) and the non-normal distribution of microbial relative abundance data, non-parametric tests were used: the Wilcoxon rank-sum test for pairwise comparisons, and LEfSe (Linear discriminant analysis Effect Size) with the Kruskal‑Wallis test followed by Linear Discriminant Analysis (LDA) for differential abundance analysis. LEfSe analyses were performed using LEfSe software (version 1.0). The Spearman’s correlation coefficients were assessed to determine the relationships between biomarker microorganisms and fermentation parameters and predictive function in silage in the R (v4.0.3). Statistical significance was set at P < 0.05.

Results and discussion

Microbial counts and fermentation quality of sugarcane tops silage

The dynamic changes of countable microorganisms during sugarcane tops silage fermentation are shown in Fig. 1 A-C (Table S3). LAB produce LA under anaerobic conditions by using WSC, which lowers the pH and thus effectively inhibits the growth of harmful microorganisms [ 8 ]. In AB group, the LAB counts were lower than 5 log CFU/g of FM during 3–60 days of ensiling (Fig. 1 A). However, when LAB counts were below 5 log CFU/g of FM, silage is likely to fail (e.g., the proliferation of harmful microorganisms) [ 38 , 39 ]. So, during the 3–60 days of ensiling, the yeasts and molds counts remained the highest in AB group ( P < 0.05, Fig. 1 B, C). Moreover, during 3–60 days of ensiling, the LAB counts in both the CK and AF groups reached its maximum on day 30 (Fig. 1 A). Based on this observation, we hypothesize that inhibiting yeasts in the epiphytic microbiota may not delay the time required for LAB to reach a stable growth stage in sugarcane top silage under the conditions tested. Further experiments are needed to test this hypothesis. But, on 30 and 60 days of ensiling, the LAB counts were significantly highest in AF group ( P ≤ 0.01, Fig. 1 A). A possible reason is that in the later stages of silage, the number of microorganisms competing with LAB for sugar substrates decreases. Our study confirmed this result, the fungi (yeasts and mold) counts were significantly lowest in AF group during 30–60 days of ensiling ( P < 0.05, Fig. 1 C, D). Fig. 1 Fermentation quality of sugarcane tops silage. Typical plate count method for counting LAB ( A ), yeast ( B ), and molds ( C ); The content of pH ( D ), LA ( E ), AN ( F ), AA ( G ), BA ( H ), and ethanol ( I ) in silage during ensiling; Temperature in 60-days silage was continuously recorded for 192 h during 8 days of aerobic exposure ( J ); The counts of LAB, EB, yeast, and molds in silage on day 8 of aerobic exposure ( K ); The fermentation parameters (including pH, LA and AN) of silage on day 8 of aerobic exposure ( L ); The contents of volatile organic compounds (including AA, PA, and BA) and ethanol in silage on day 8 of aerobic exposure ( M ). LAB, lactic acid bacteria; LA, lactic acid; AA, acetic acid; BA, butyric acid; AN ammonia nitrogen. CK, untreated silage; AB, silage with epiphytic microorganisms after addition of antibacterial agent; AF, silage with epiphytic microorganisms after addition of antifungal agent; NA, silage withγ-sterilized. Statistical significance ( P < 0.05) among the treatments according to two-way ANOVA and HSD test at the 5% level ( n = 3, biologically independent samples). a−c Different lowercase letters between groups indicate significant differences

The dynamic changes of fermentation parameter during sugarcane tops silage fermentation are shown in the Fig. 1 D-I (Table S4). Silage pH is a key indicator of silage quality, and when the pH value reaches 4.5 or below, the fermentation quality of silage is considered good [ 40 ]. In this study, only found in AF and CK groups, the pH was significantly decreased to below 4.5 during 3–60 days of ensiling ( P < 0.05, Fig. 1 D). This result is also reported in previous study on sugarcane top silage [ 41 ]. This is because abundant LAB present in silage dominate the fermentation process (Fig. 1 A). Meanwhile, the LA content was significantly increased in AF and CK groups during 3–60 days of ensiling ( P < 0.05, Fig. 1 E). This is mainly related to the continuous reproduction of LAB during ensiling (Fig. 1 A). Similarly, the AF group had a higher number of LAB after 30 and 60 days of ensiling (Fig. 1 A), resulting in the highest LA content at these time points ( P < 0.05, Fig. 1 E). AN reflects protein decomposition during ensiling, driven by plant proteases (early) and proteolytic microorganisms (e.g., clostridia) under suboptimal conditions [ 42 ]. LAB can indirectly affect AN by influencing pH decline and suppressing proteolytic bacteria [ 43 ]. In the AF group, slower pH decline due to yeast inhibition prolonged plant protease activity and allowed limited clostridial activity, leading to the highest AN content ( P < 0.05, Fig. 1 F). This is mainly due to that some LAB strain can use AA as the main fermentation product and overcome the inhibitory effect of low pH by producing AN through the arginine deiminase pathway [ 44 ]. Exactly, the AA content was highest in AF group during 3–60 days of ensiling ( P < 0.05, Fig. 1 G). The increase in AA content over ensiling days may be attributed to the proliferation of heterofermentative LAB (e.g., Lactobacillus buchneri ), which convert LA and/or sugars into AA [ 45 , 46 ]. In addition, BA content is only affected by ensiling time, and BA content decreases significantly over time ( P < 0.05, Fig. 1 H). This is mainly attributed to the fact that BA-producing bacteria have difficulty adapting to the acidic environment as silage is processed [ 47 ]. Furthermore, ethanol content was highest in AB group during 3–60 days of ensiling ( P < 0.05, Fig. 1 I). This is mainly due to that yeast becomes most dominant in silage by inhabiting epiphytic LAB (Fig. 1 B), and produces a large amount of ethanol by decomposing sugar substrates [ 40 ].

The temperature changes during 8 days of aerobic exposure of 60-day silage are shown in Fig. 1 J. Previous study has point out that when the temperature of silage is more than 2 °C higher than the ambient temperature, it is considered to be spoiled [ 48 ]. In this study, the AB and NA groups exceeded the ambient temperature by 2 °C after 4 h and 36 h, respectively. This is mainly attributed to the highest pH and lowest LA content in silage (Fig. 1 D, E), which makes it difficult to effectively inhibit the growth of microorganisms that cause aerobic deterioration. This is likely because even after aerobic exposure, the LA still accumulates in the silage, which reduces energy conversion efficiency by inhibiting the metabolic activity of other aerobic microorganisms, thereby reducing heat production [ 49 ]. Therefore, the CK and AF groups have better aerobic stability. The lowest mold count in AF groups was confirmed this result ( P < 0.05, Fig. 1 K). Moreover, the LAB and EB counts were highest in CK and AF groups ( P < 0.05, Fig. 1 K). The presence of LAB is associated with the acidic environment created by LA, while pH > 5.0 is a suitable environment for the reproduction of EB. However, in CK and AF groups, the yeasts count was also highest ( P < 0.05, Fig. 1 K). The reason may be that residual LA can serve as a nutrient substrate for yeast growth and reproduction under air condition [ 50 ]. This may be also due to the higher residual LA content in AF and CK groups (Fig. 1 L), the silage pH in the AF and CK groups was significantly lower than that in the other two groups after aerobic exposure (Fig. 1 L). Additionally, in our study, the AN content was no more than 0.50 g/kg DM in all sugarcane tops silage on 8 days of aerobic exposure (Fig. 1 L). This is mainly related to sugarcane tops, which are low-protein forages. Notably, the AA content was highest in AF group ( P < 0.05, Fig. 2 F). This is may be due to the yeast can convert organic matter into ethanol, while acetic acid bacteria can oxidize ethanol into AA under air condition [ 51 ]. This may also explain why the aerobic retention time was longer in the AF group (Fig. 1 J). In contrast, due to the highest yeasts and mold counts under air condition, the highest ethanol content was only found in CK group ( P < 0.05, Fig. 1 M). A study has shown that under aerobic conditions, molds convert macromolecules such as starch and protein into glucose and amino acids, providing substrates for subsequent alcoholic fermentation by yeast [ 52 ]. Fig. 2 Bacterial community succession with different treatments. Silage bacterial community composition in phylum ( A ) and genus ( B ) level; Biomarker bacteria in CK and AB groups after 3 ( C ), 7 ( D ), 12 ( E ), 30 ( F ), and 60 ( G ) days of ensiling and 8-day of aerobic exposure ( H ); Biomarker bacteria in AF and AB groups after 3 ( C ), 7 ( D ), 12 ( E ), 30 ( F ), and 60 ( G ) days of ensiling and 8-day of aerobic exposure ( H ). CK, untreated silage; AB, silage with epiphytic microorganisms after addition of antibacterial agent; AF, silage with epiphytic microorganisms after addition of antifungal agent; NA, silage withγ-sterilized

Bacterial community of sugarcane tops silage

Changes in bacterial communities at the phyla level in sugarcane tops silage, before and after aerobic exposure, are shown in Fig. 2 A. In this study, Bacillota and Pseudomonadota were the top 2 bacterial phyla in all sugarcane tops silage whether before or after aerobic exposure. Gu et al. [ 19 ] has also reported that Bacillota and Pseudomonadota were the dominant bacterial phyla in sugarcane top silage. Bacillota (especially Lactobacillus ) are ideal for dominance in silage because they are major producers of LA; however, Pseudomonadota are a main source of putrefaction-related pathogens and opportunistic pathogens [ 53 ]. In this study, Bacillota was first dominant in CK and AF groups, Pseudomonadota was first dominant in AB group. This result also indicates that inhibiting epiphytic LAB, numerous yeasts cannot exert a strong inhibitory effect on the reproduction of harmful bacteria [ 13 – 15 ]. In addition, Bacteroidota primarily function to hydrolyze complex macromolecules (such as plant cell walls) and release enzymes that degrade polysaccharides, thereby improving the digestibility of silage. But, Bacteroidota were the top 3 bacterial phyla in all sugarcane tops silage whether before or after aerobic exposure.

Changes in bacterial communities at the genus level in sugarcane tops silage, before and after aerobic exposure, are shown in Fig. 2 B. In this study, Weissella was the first dominant bacteria in CK and AF groups on 3 days and 7 days of ensiling. Weissella was the first dominant bacteria also reported in Italian ryegrass silage after 3 days of ensiling [ 54 ]. A species from Weissella (e.g., Weissella cibaria ) can undergo heterofermentation to produce AA [ 55 ]. In the early stages of ensiling, the high concentration of AA produced in CK and AF groups also confirms this result (Fig. 1 G). Same as the results of CK and AF groups in this study, Niu et al. [ 56 ] also reported that with pH reduction, acid-tolerant Lactiplantibacillus replaced Weissella during ensiling. It is possible that the inhibition of some epiphytic yeasts reduced substrate competition, thus leading to a faster occurrence of this phenomenon in the AF group compared to the CK group. However, Vibrionimonas was dominant in AB and NA group whether before or after aerobic exposure. Previous study has shown that some strain of Vibrionimonas was isolated during the aerobic spoilage process of tilapia fillets [ 57 ]. This might explain why aerobic deterioration was more likely to occur in AB and NA groups (Fig. 1 J). In addition, in this study, unlike the CK group where Lactiplantibacillus was the first dominant bacteria, in AF group, Lactiplantibacillus was the second dominant bacteria, accompanied by high abundance of Enterobacter (> 50%) after aerobic exposure. Enterobacter is considered a commonly undesirable bacteria that easily causes spoilage of silage. While, previous study has shown that under aerobic conditions, when high numbers of Enterobacter in silage, it will ferment sugars to produce AA, which inhibits the growth of other aerobic and harmful microorganisms (e.g., mold), thereby improving the aerobic stability of silage [ 39 , 58 ]. Another possibly reason is that Enterobacter can directly converted LA produced by Lactiplantibacillus to AA [ 59 ]. The fact that the AF group had better aerobic stability, higher AA content, and the lowest mold count after aerobic exposure also confirms this result (Fig. 1 J, K and M). Enterobacter is traditionally classified as opportunistic pathogens, which can cause infection in animals under certain conditions. But, most members of the Enterobacter genus are non-pathogenic bacteria [ 60 ].

To identify the key bacteria in well fermented sugarcane top silage, LEfSe was used to analyze the differences in bacterial taxa of AB and CK (Fig. 2 C-H) or AF (Fig. 2 I-N) groups before and after aerobic exposure (linear discrimination analysis [LDA] > 4.0). The many undesirable bacteria belong to Enterobacteriaceae (e.g., Enterobacter ) were significantly enriched in AB group during ensiling and after aerobic exposure (Fig. 2 C-H and I-N). The possible reason is that some strains from the Enterobacteriaceae are not only ethanol-resistant but can also produce high levels of ethanol under certain specific conditions [ 61 ]. However, during the 3–60 days of ensiling, the Lactobacillaceae were significantly enriched in CK and AF group (Fig. 2 C-G and I-M). The Lactobacillaceae typically predominant in well-fermented silage [ 62 ]. The fact that LA fermentation was the dominant process in both CK and AF groups confirms this result during ensiling (Fig. 1 D). Moreover, during the 3–60 days of ensiling, the Lactobacillaceae species Lactiplantibacillus and Weissella were also significantly enriched in CK and AF group. Lactiplantibacillus, as a homofermentative LAB, is considered a key species for LA-producing in the later stages of silage [ 59 ]. In contrast, as a typical heterofermentative LAB, Weissella is the main microorganism that inhibits harmful microorganisms in the early stages of silage by producing LA. Similarly, the Lactiplantibacillus was significantly enriched in CK and AF group after aerobic exposure. Studies have shown that the accumulation of Lactiplantibacillus spp. (e.g., Lactiplantibacillus plantarum ) in silage can effectively extend the aerobic storage time of silage [ 63 , 64 ]. This is mainly related to the LA produced by their metabolism, which can inhibit the growth of aerobic putrefactive microorganisms by creating an acidic environment. The wilcoxon test was used to further analyze the differences in common LAB between the CK and AF groups before and after aerobic exposure (Fig. 3 A-F). Compared with the CK group, Lactobacillaceae and Lactiplantibacillus abundance were significantly higher in the AF group only at 30 and 60 days of ensiling ( P < 0.05). This result explain why the AF group accumulated more LA after 30 and 60 days of ensiling (Fig. 2 E). However, Lactobacillaceae and Lactiplantibacillus abundance were lower in the AF group compared with CK group after aerobic exposure. This may be the reason for the massive proliferation of its competitor, Enterobacter [ 65 ]. Su et al. [ 59 ] also found that the proliferation of Enterobacter in mulberry silage during ensiling under aerobic conditions led to a reduction in Lactiplantibacillus . This may because Enterobacter converted the LA produced by Lactiplantibacillus into AA, which increased the silage pH and thus disrupted the acidic growth environment preferred by Lactiplantibacillus [ 63 ]. Fig. 3 Differences in relative abundance of LABs in normally fermented sugarcane top silage. The differences in common LABs between the CK and AF groups at 3 ( A ), 7 ( B ), 12 ( C ), 30 ( D ), and 60 ( E ) days of ensiling and 8-day of aerobic exposure ( F ) were compared. CK, untreated silage; AF, silage with epiphytic microorganisms after addition of antifungal agent. Statistical significance ( P < 0.05) among the treatments according to wilcoxon test at the 5% level ( n = 3, biologically independent samples). NS, not significantly; *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01

Fungal community of sugarcane tops silage

Changes in fungal communities at the phyla level in sugarcane tops silage, before and after aerobic exposure, are shown in Fig. 4 A. In this study, Ascomycota and Basidiomycota were the top 2 dominant fungal phyla in all groups before aerobic exposure. Zhang et al. [ 20 ] also reported that Ascomycota and Basidiomycota were the most abundant phyla in sugarcane tops silage during 2–60 days of ensiling. While, after aerobic exposure, Ascomycota were the most dominant fungal phyla in all groups. This result also reported by Gu et al. [ 21 ], who found that the Ascomycota was the first dominant fungal phyla after 2–14 days of aerobic exposure. Ascomycetes and Basidiomycetes are the main groups involved in cellulolysis, and both are most diverse and species-rich group in the fungal kingdom. These results indicated that inhibiting either epiphytic LAB or yeast does not affect the high abundance of fungi in silage at phyla level. Fig. 4 Fungal community succession with different treatments. Silage bacterial community composition in phylum ( A ) and genus ( B ) level; Biomarker bacteria in CK and AB groups after 3 ( C ), 7 ( D ), 12 ( E ), 30 ( F ), and 60 ( G ) days of ensiling and 8-day of aerobic exposure ( H ); Biomarker fungi in CK and AF groups after 3 ( C ), 7 ( D ), 12 ( E ), 30 ( F ), and 60 ( G ) days of ensiling and 8-day of aerobic exposure ( H ). CK, untreated silage; AB, silage with epiphytic microorganisms after addition of antibacterial agent; AF, silage with epiphytic microorganisms after addition of antifungal agent; NA, silage withγ-sterilized

Changes in fungal communities at the genera level in sugarcane tops silage, before and after aerobic exposure, are shown in Fig. 4 B. In this study, Fusarium was the most dominant fungal genera in both CK and AF groups during 3–60 days of ensiling. Fusarium is common mold types in corn silage, bean silage and forage silage [ 66 ], and produces mycotoxins (e.g., zearalenone, deoxynivalenol, fumonisins) that pose health risks to livestock, including reduced feed intake, immune suppression, and reproductive disorders [ 4 , 67 ]. However, under the low‑pH, anaerobic conditions of well‑fermented silage, Fusarium activity and mycotoxin production are minimal [ 68 ]; thus, DNA detection does not confirm toxin accumulation. Direct quantification of these mycotoxins is needed in future studies. Furthermore, in this study, Wickerhamomyces was the most dominant fungal genera in AB group during 3–60 days of ensiling. Wickerhamomyces was the dominant genera also reported in oat silages [ 69 ]. Our data revealed that the AB group had the lowest LA content and highest pH (Fig. 1 D, E), conditions that are less inhibitory to Wickerhamomyces . This observation may directly explain the dominance of this genus in the AB group. Additionally, it has been reported that Wickerhamomyces species can secrete mycotoxins to inhibit competing fungi such as Fusarium [ 9 , 70 ], suppress other fungi (e.g., Saccharomymycopsis ) by resource competition [ 71 ], and tolerate high ethanol concentrations [ 59 , 72 ]. Furthermore, Shimizu et al. [ 72 ] have reported that Wickerhamomyces has a strong tolerance to low pH. This may explain why it persists during the 3–60 days of ensiling in both the CK and AF groups, especially reaching its highest abundance at 30 days of ensiling under conditions of high LA concentrations. While, after aerobic exposure, the Wickerhamomyces was the first dominant genera in AB (70.33%) and AF group (48.87%) and that was the second dominant genera (17.50%) in CK group. The similar result was also reported in wilted king grass silage, untreated sainfoin silage, whole crop corn silage, and mulberry silage after aerobic exposure [ 59 , 73 – 75 ]. However, Wickerhamomyces enrichment in the poorly-fermented silages are more triggered aerobic putrefaction, while in well-fermented silage, it promotes aerobic stability [ 59 , 73 , 74 ]. The high abundance of this Wickerhamomyces in AB group may be because have the ability of antimicrobial and the significant competitive edge of Wickerhamomyces after aerobic exposure. While, the high abundance of this yeast in the AF and CK group is most likely due to that the abundant LA and AA in AF and CK groups can serve as a nutrient substrate for Wickerhamomyces to enhances ester production at low ethanol concentrations [ 59 , 76 ]. Wickerhamomyces can also metabolize AA through glycolysis and the tricarboxylic acid cycle [ 71 , 75 ], a product that ensures the stability of silage. However, in the presence of abundant Wickerhamomyces , the aerobic stability in AB group was inferior to that in CK and AF groups (Fig. 1 J). This indicates that only Wickerhamomyces enriched in well fermented silage will have AA as its main metabolite, thus extending the aerobic exposure time of silage. The differences in fungal taxa between the CK and AF groups during 3–60 days of ensiling and after 8 days of aerobic exposure were analyzed using LEfSe to identify the key fungi that coexist with LAB in well fermented silage (Fig. 4 C-H, LDA > 4.0). Wickerhamomyces was consistently significantly enriched in the AF group during ensiling and after aerobic exposure. This indicates that Wickerhamomyces plays a positive role in acidic anaerobic environments, especially in the later stages of ensiling when its abundance increases and it coexists with high abundance of Lactiplantibacillus to achieve high production of organic acids (LA and AA). While, after aerobic exposure, its abundance increases dramatically and increases the yield of AA in silage.

Changes in bacterial phenotypes

To identify the sources of bacterial contamination risk in sugarcane top silage, we used the bugbase algorithm to predict bacterial potential pathogenic phenotypes in the silage (Fig. 5 A). The BugBase-predicted potential pathogenic abundance were always down-regulated only in AF group during ensiling and after aerobic exposure. This is mainly related to the downregulation of Gram-negative bacteria in sugarcane top silage (Fig. 5 A). Gram-negative bacteria can cause serious systemic infections, so their reduction is beneficial to fermentation quality [ 77 ]. The reason may be the decrease in the abundance of Gram-negative bacteria Raoultella belong to Enterobacteriaceae [ 78 ]. While, the BugBase-predicted potential pathogenic abundance in CK group changed from up-regulated to down-regulated as the ensiling time prolonged. Also, the BugBase-predicted potential pathogenic abundance were down-regulated in CK group after aerobic exposure. This change in CK group was mainly related to the downregulation of Gram-positive bacteria. Weissella , as gram-positive bacteria, is usually classified as opportunistic pathogens [ 79 ]. Therefore, the downregulation of BugBase-predicted potential pathogenic abundance in CK group may be related to the decline in the abundance of Weissella during ensiling and after aerobic exposure. Similar result also found in AB group during ensiling. This change in AB group was mainly related to the downregulation of Gram-negative bacteria. This also possibly due to the decline in the relative abundance of Gram-negative bacteria Enterobacter in sugarcane top silage during ensiling. However, the BugBase-predicted potential pathogenic abundance were up-regulated in AB group after aerobic exposure. This change in AB group was mainly related to the upregulation of Gram-negative bacteria. This could be attributed to high abundance of the Gram-negative bacteria Vibrionimonas in sugarcane top silage after aerobic exposure. Studies have shown that Vibrionimonas is a Gram-negative bacterium with potential pathogenicity [ 80 , 81 ]. These results indicates that in well-preserved sugarcane top silage with abundant coexistence of Wickerhamomyces and Lactiplantibacillus , can reduce the source of potential pathogenic bacteria whether before or after aerobic exposure. Fig. 5 Bugbase bacterial phenotype prediction. CK, untreated silage; AB, silage with epiphytic microorganisms after addition of antibacterial agent; AF, silage with epiphytic microorganisms after addition of antifungal agent; NA, silage withγ-sterilized

Correlation analysis between the microbiome, environmental factors and predictive function

Spearman’s rank correlation coefficient was used to reveal the correlation between fermentation parameters, predictive function, and key LAB and yeast from the CK and AF groups (Fig. 6 ). Positive correlations were observed between LA and AA content and Lactiplantibacillus ( P < 0.05). Lactiplantibacillus is an important source of organic acids such as LA and AA in silage due to its metabolic flexibility in silage, which allows it to adapt to changes in the fermentation process. Positive correlations were also found between LA and AA content and Wickerhamomyces ( P < 0.05). Some studies have indicated that certain strains from the non- Saccharomyces genus Wickerhamomyces (e.g., Wickerhamomyces anomalus ) can directly produce LA [ 82 ] and AA [ 71 , 75 ] by utilizing sugar substrates. Moreover, a positive correlation was detected between Lactiplantibacillus and Wickerhamomyces ( P < 0.05). Sudun et al. [ 83 ] have pointed that some yeasts can provide a nutrient substrate (e.g., vitamins and growth factors) for LAB growth. LA and AA produced by LAB can serve as substrates for yeast esterification [ 59 , 76 ]. In contrast, the LA, AA content, Wickerhamomyces and Lactiplantibacillus were all showed negative correlations with BugBase-predicted potential pathogenic abundance ( P < 0.05). One possible explanation is that the coexistence of these two microbes may promote LA or AA production, which inhibits potentially pathogenic bacteria both during ensiling and after aerobic exposure, as evidenced by the decrease in Enterobacter and Vibrionimonas , respectively. The reduction in potentially pathogenic aerobic deteriorating bacteria such as Vibrionimonas may explain the better aerobic stability in CK and AF groups. Fig. 6 Correlation between biomarker microorganisms and fermentation parameters (including LA and AA) and bacterial phenotypes in well-preserved sugarcane top silage. LA, lactic acid; AA, acetic acid. *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, P < 0.001

Isolation and identification of LAB and yeast from the sugarcane tops silage

Based on the above results, and to further clarify the impact of the coexistence of Lactiplantibacillus and Wickerhamomyces on fermentation quality, this study used isolation and culture methods to isolate common Lactiplantibacillus and Wickerhamomyces 's yeast strains from well-preserved sugarcane top silage (CK and AF groups) and carried out co-fermentation in fruit pomace. The colony morphology (color, surface, edge, and elevation) of the isolated yeast and LAB was macroscopically observed by staining (Fig. 7 A, B). A phylogenetic tree was then constructed to show the phylogenetic relationships between the isolated LAB (G35) and yeast (G32-15) strains and various yeast and LAB groups in the GenBank database, respectively, and the G35 was identified as Lactiplantibacillus plantarum with 100% similarity (Fig. 7 C), the G32-15 was identified Wickerhamomyces anomalus with 98% similarity (Fig. 7 D). Hemolysis tests revealed that both the isolated Lactiplantibacillus plantarum G35 and Wickerhamomyces anomalus G32-15 exhibited γ-hemolysis (Fig. 7 E, F), indicating no hemolytic activity on blood agar. As shown in Table S5, among the tested carbon sources, Lactiplantibacillus plantarum G35 could not utilize mannitol, rhamnose, raffinose, or starch, while Wickerhamomyces anomalus G32-15 failed to utilize only aescin and starch. To evaluate the organic acid‑producing ability and metabolic flexibility of the isolated Lactiplantibacillus plantarum G35 and Wickerhamomyces anomalus G32-15 on diverse carbon sources, a co‑fermentation experiment was conducted using three fruit peels (orange, dragon fruit, mango). After 7 days of fermentation with the compound inoculant (G35 and G32-15), the content of both LA (Fig. 7 G) and AA (Fig. 7 H) increased, especially in mango peel and dragon fruit peel. Fig. 7 Microscopic examination and morphological observation of isolated Lactiplantibacillus ( A ) and Wickerhamomyces ( B ). Phylogenetic trees of isolated Lactiplantibacillus ( C ) and Wickerhamomyces ( D ). Hemolysis test for isolating Lactiplantibacillus ( E ) and Wickerhamomyces ( F ). Screened Lactiplantibacillus and Wickerhamomyces co-fermented fruit peel waste to produce LA ( G ) and AA ( H ). OPWCK, orange peel waste treated with 1% sterile saline; OPWT, orange peel waste treated with 1% compound microbial agent; DFPWCK, dragon fruit peel waste treated with 1% sterile saline; DFPWT, dragon fruit peel waste treated with 1% compound microbial agent; MPWCK, mango peel waste treated with 1% sterile saline; MPWT, mango peel waste treated with 1% compound microbial agent. The compound microbial agent consisted of the isolated Lactiplantibacillus spp. and Wickerhamomyces spp. in a 1:1 ratio

Conclusion

The results of the present study revealed that in well-preserved sugarcane top silage, the coexistence of Lactiplantibacillus , the core bacterium of silage, and Wickerhamomyces , a key fungus, can lead to the accumulation of LA and AA in the silage, inhibiting aerobic spoilage and reducing the occurrence of pathogens. However, the absence of Lactiplantibacillus in sugarcane top silage, where only Wickerhamomyces is abundant, not only leads to excessive ethanol production and increases BugBase-predicted potential pathogenic abundance after aerobic exposure, but makes it more susceptible to aerobic spoilage. Accordingly, Lactiplantibacillus plantarum G35 and Wickerhamomyces anomalus G32-15 with both safety and broad carbon source assimilation were isolated from the well-preserved silage. Moreover, The LA and AA contents increases after co-fermentation of fruit peel waste by Wickerhamomyces anomalus G32-15 and Lactiplantibacillus plantarum G35. This study identified and isolated the key coexisting microorganisms in sugarcane top silage that correlate with fermentation quality and safety, and provided an innovative strategy for developing microbial agents to target and regulate silage quality. Nevertheless, the observed associations are based on correlational analyses; further mechanistic investigations are required to establish causal relationships.

Supplementary Information

Supplementary Material 1.

Abbreviations

Dry matter

Fresh matter

Colony forming units

Lactic acid bacteria

Enterobacteria

Lactic acid

Acetic acid

Propionic acid

Butyric acid

Deoxyribonucleic acid

Ribonucleic acid

Operational taxonomic units

Linear discriminant analysis effect size

Linear discriminant analysis

Standard error of the mean

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Qichao Gu and Qiuxiang Ye contributed equally to this work.

Authors’ contributions

Qichao Gu: Writing – original draft, Writing – review & editing, Methodology, Investigation, Data curation, Conceptualization. Qiuxiang Ye: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Jia Wang: Methodology, Investigation. Zhilin Yan: Methodology, Investigation. Xiaohua Huang: Investigation. Chenghuan Qin: Investigation. Caixiang Wei: Investigation. Qi Yan: Investigation. Xing Gao: Investigation. Yongqi Tan: Investigation. Xinghua Cai: Investigation. Bo Lin: Investigation. Caixia Zou: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization.

Funding

This work was supported by the National Key Research and Development Program of China [grant number 2022YFD1300602], the National Natural Science Foundation of China [grant numbers 312360851], and the Innovation Project of Guangxi Graduate Education [grant number YCBZ2023050].

Data availability

The datasets generated or analyzed during the current study are available from the corresponding author upon reasonable request (zou.cx@gxu.edu.cn). Bacterial and fungal raw sequence data were deposited in the sequence read archive at NCBI ( https://www.ncbi.nlm.nih.gov/ ) under the accession numbers PRJNA1290004 and PRJNA1290002, respectively.

Declarations

Ethics approval and consent to participate

The collection of sugarcane tops was authorized by the College of Life Sciences and Technology, Guangxi University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

References

1. Ávila CLS Carvalho BF Silage fermentation—updates focusing on the performance of micro-organisms J Appl Microbiol 2020 128 4 966 984 10.1111/jam.14450 31519038 Ávila CLS, Carvalho BF. Silage fermentation—updates focusing on the performance of micro-organisms. J Appl Microbiol. 2020;128(4):966–84. 10.1111/jam.14450. 31519038 10.1111/jam.14450

2. Oude Elferink S, Driehuis F, Gottschal JC, Spoelstra SF. Silage fermentation processes and their manipulation. In: FAO electronic conference on tropical silage. 2000. p. 1–28.

3. Kung JL Shaver RD Grant RJ Schmidt RJ Silage review: interpretation of chemical, microbial, and organoleptic components of silages J Dairy Sci 2018 101 5 4020 4033 10.3168/jds.2017-13909 29685275 Kung JL, Shaver RD, Grant RJ, Schmidt RJ. Silage review: interpretation of chemical, microbial, and organoleptic components of silages. J Dairy Sci. 2018;101(5):4020–33. 10.3168/jds.2017-13909. 29685275 10.3168/jds.2017-13909

4. Jiang H Okoye CO Wu Y Gao L Li X Wang Y Silage pathogens and biological control agents: effects, action mechanisms, challenges and prospects Biocontrol 2024 69 1 1 17 10.1007/s10526-023-10236-z Jiang H, Okoye CO, Wu Y, Gao L, Li X, Wang Y, et al. Silage pathogens and biological control agents: effects, action mechanisms, challenges and prospects. Biocontrol. 2024;69(1):1–17. 10.1007/s10526-023-10236-z.

5. Gou W Tang Y Zhang M Mao N Lu G Li L Ensuring the safety and quality of Phalaris arundinacea silage: the role of wilting and lactic acid bacteria Ind Crops Prod 2025 234 121589 10.1016/j.indcrop.2025.121589 Gou W, Tang Y, Zhang M, Mao N, Lu G, Li L, et al. Ensuring the safety and quality of Phalaris arundinacea silage: the role of wilting and lactic acid bacteria. Ind Crops Prod. 2025;234:121589. 10.1016/j.indcrop.2025.121589.

6. Tuovinen OH Niemelä SI Rajala-Schultz PJ The role of microbes in ensiling Microorganisms 2025 13 10 2237 10.3390/microorganisms13102237 41156697 PMC12565874 Tuovinen OH, Niemelä SI, Rajala-Schultz PJ. The role of microbes in ensiling. Microorganisms. 2025;13(10):2237. 10.3390/microorganisms13102237. 41156697 10.3390/microorganisms13102237 PMC12565874

7. Guan H Ke W Yan Y Shuai Y Li X Ran Q Screening of natural lactic acid bacteria with potential effect on silage fermentation, aerobic stability and aflatoxin B1 in hot and humid area J Appl Microbiol 2020 128 5 1301 1311 10.1111/jam.14570 31898381 Guan H, Ke W, Yan Y, Shuai Y, Li X, Ran Q, et al. Screening of natural lactic acid bacteria with potential effect on silage fermentation, aerobic stability and aflatoxin B1 in hot and humid area. J Appl Microbiol. 2020;128(5):1301–11. 10.1111/jam.14570. 31898381 10.1111/jam.14570

8. Zhou ZH Jia S Gao Y Li X Lin Y Yang F Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality Commun Biol 2024 7 1 117 10.1038/s42003-024-05816-3 38253824 PMC10803313 Zhou ZH, Jia S, Gao Y, Li X, Lin Y, Yang F, et al. Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality. Commun Biol. 2024;7(1):117. 10.1038/s42003-024-05816-3. 38253824 10.1038/s42003-024-05816-3 PMC10803313

9. Rungchaiwattanakul T Chareonpornwattana S Sirisomboon CD Antagonistic activity of Wickerhamomyces anomalus , Kluyveromyces marxianus and L actobacillus paracasei against growth and aflatoxin production of Aspergillus flavus in corn silage Agric Nat Res 2022 56 4 673 686 Rungchaiwattanakul T, Chareonpornwattana S, Sirisomboon CD. Antagonistic activity of Wickerhamomyces anomalus , Kluyveromyces marxianus and L actobacillus paracasei against growth and aflatoxin production of Aspergillus flavus in corn silage. Agric Nat Res. 2022;56(4):673–86.

10. Javed A Ajmal M Hanif NQ Akram A Effects of inoculation of corn silage with Saccharomyces cerevisiae on silage fermentation characteristics, nutrient digestibility, mycoflora and aflatoxin production Nat Prod Res 2024 38 20 3488 3497 10.1080/14786419.2023.2252154 37647099 Javed A, Ajmal M, Hanif NQ, Akram A. Effects of inoculation of corn silage with Saccharomyces cerevisiae on silage fermentation characteristics, nutrient digestibility, mycoflora and aflatoxin production. Nat Prod Res. 2024;38(20):3488–97. 10.1080/14786419.2023.2252154. 37647099 10.1080/14786419.2023.2252154

11. Hatoum R Labrie S Fliss I Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications Front Microbiol 2012 3 421 10.3389/fmicb.2012.00421 23267352 PMC3525881 Hatoum R, Labrie S, Fliss I. Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications. Front Microbiol. 2012;3:421. 10.3389/fmicb.2012.00421. 23267352 10.3389/fmicb.2012.00421 PMC3525881

12. Menezes AGT Ramos CL Cenzi G Melo DS Dias DR Schwan RF Probiotic potential, antioxidant activity, and phytase production of indigenous yeasts isolated from indigenous fermented foods Probiotics Antimicrob Proteins 2020 12 280 288 10.1007/s12602-019-9518-z 30685824 Menezes AGT, Ramos CL, Cenzi G, Melo DS, Dias DR, Schwan RF. Probiotic potential, antioxidant activity, and phytase production of indigenous yeasts isolated from indigenous fermented foods. Probiotics Antimicrob Proteins. 2020;12:280–8. 10.1007/s12602-019-9518-z. 30685824 10.1007/s12602-019-9518-z

13. Xu S Yang J Qi M Smiley B Rutherford W Wang Y Impact of Saccharomyces cerevisiae and Lactobacillus buchneri on microbial communities during ensiling and aerobic spoilage of corn silage J Anim Sci 2019 97 3 1273 1285 10.1093/jas/skz021 30715344 PMC6396264 Xu S, Yang J, Qi M, Smiley B, Rutherford W, Wang Y, et al. Impact of Saccharomyces cerevisiae and Lactobacillus buchneri on microbial communities during ensiling and aerobic spoilage of corn silage. J Anim Sci. 2019;97(3):1273–85. 10.1093/jas/skz021. 30715344 10.1093/jas/skz021 PMC6396264

14. Zhou X Ouyang Z Zhang X Wei Y Tang S Ma Z Sweet corn stalk treated with Saccharomyces cerevisiae alone or in combination with Lactobacillus plantarum : nutritional composition, fermentation traits and aerobic stability Animals 2019 9 9 598 10.3390/ani9090598 31450836 PMC6770685 Zhou X, Ouyang Z, Zhang X, Wei Y, Tang S, Ma Z, et al. Sweet corn stalk treated with Saccharomyces cerevisiae alone or in combination with Lactobacillus plantarum : nutritional composition, fermentation traits and aerobic stability. Animals. 2019;9(9):598. 10.3390/ani9090598. 31450836 10.3390/ani9090598 PMC6770685

15. Zhou X Ouyang Z Zhang X Wei Y Tang S Tan Z Effects of a high-dose Saccharomyces cerevisiae inoculum alone or in combination with Lactobacillus plantarum on the nutritional composition and fermentation traits of maize silage Anim Prod Sci 2020 60 6 833 842 10.1071/AN18701 Zhou X, Ouyang Z, Zhang X, Wei Y, Tang S, Tan Z, et al. Effects of a high-dose Saccharomyces cerevisiae inoculum alone or in combination with Lactobacillus plantarum on the nutritional composition and fermentation traits of maize silage. Anim Prod Sci. 2020;60(6):833–42. 10.1071/AN18701. 10.3390/ani9090598 PMC6770685 31450836

16. Wang Y Li Y Liu T Guo X Ren H Jing W Pichia anomala enhances the growth and lactic acid production of Leuconostoc citreum : synergistic improvement for sweet sorghum silage quality Bioresour Technol 2026 10.1016/j.biortech.2026.134128 41638590 Wang Y, Li Y, Liu T, Guo X, Ren H, Jing W, et al. Pichia anomala enhances the growth and lactic acid production of Leuconostoc citreum : synergistic improvement for sweet sorghum silage quality. Bioresour Technol. 2026. 10.1016/j.biortech.2026.134128. 41638590 10.1016/j.biortech.2026.134128

17. Duniere L Jin L Smiley B Qi M Rutherford W Wang Y Impact of adding Saccharomyces strains on fermentation, aerobic stability, nutritive value, and select Lactobacilli populations in corn silage J Anim Sci 2015 93 5 2322 2335 10.2527/jas.2014-8287 26020328 Duniere L, Jin L, Smiley B, Qi M, Rutherford W, Wang Y, et al. Impact of adding Saccharomyces strains on fermentation, aerobic stability, nutritive value, and select Lactobacilli populations in corn silage. J Anim Sci. 2015;93(5):2322–35. 10.2527/jas.2014-8287. 26020328 10.2527/jas.2014-8287

18. Hu J Ma S Wang F Xing H Bai J Wei Y Design and development of sugarcane top chopper and its field performance Sugar Tech 2021 23 5 1192 1198 10.1007/s12355-021-00990-8 Hu J, Ma S, Wang F, Xing H, Bai J, Wei Y, et al. Design and development of sugarcane top chopper and its field performance. Sugar Tech. 2021;23(5):1192–8. 10.1007/s12355-021-00990-8.

19. Gu Q Zhang J Lin B Ding H Yan Q Wei C Effects and function of citric acid on fermentation quality and microbial community in sugarcane tops silage with high and low water-soluble carbohydrate content BMC Plant Biol 2025 25 1 99 10.1186/s12870-025-06063-2 39849361 PMC11755813 Gu Q, Zhang J, Lin B, Ding H, Yan Q, Wei C, et al. Effects and function of citric acid on fermentation quality and microbial community in sugarcane tops silage with high and low water-soluble carbohydrate content. BMC Plant Biol. 2025;25(1):99. 10.1186/s12870-025-06063-2. 39849361 10.1186/s12870-025-06063-2 PMC11755813

20. Zhang L Zhou X Gu Q Liang M Mu S Zhou B Analysis of the correlation between bacteria and fungi in sugarcane tops silage prior to and after aerobic exposure Bioresour Technol 2019 291 121835 10.1016/j.biortech.2019.121835 31352166 Zhang L, Zhou X, Gu Q, Liang M, Mu S, Zhou B, et al. Analysis of the correlation between bacteria and fungi in sugarcane tops silage prior to and after aerobic exposure. Bioresour Technol. 2019;291:121835. 10.1016/j.biortech.2019.121835. 31352166 10.1016/j.biortech.2019.121835

21. Gu Q Zhang L Zhou X Lin B Zou C Effects of sugarcane variety and nitrogen application level on the quality and aerobic stability of sugarcane tops silage Front Plant Sci 2023 14 1148884 10.3389/fpls.2023.1148884 37324669 PMC10262686 Gu Q, Zhang L, Zhou X, Lin B, Zou C. Effects of sugarcane variety and nitrogen application level on the quality and aerobic stability of sugarcane tops silage. Front Plant Sci. 2023;14:1148884. 10.3389/fpls.2023.1148884. 37324669 10.3389/fpls.2023.1148884 PMC10262686

22. Mogodiniyai Kasmaei K Passoth V Spörndly R Udén P A new sterilization and inoculation method in silage research Grass Forage Sci 2015 70 4 668 673 10.1111/gfs.12153 Mogodiniyai Kasmaei K, Passoth V, Spörndly R, Udén P. A new sterilization and inoculation method in silage research. Grass Forage Sci. 2015;70(4):668–73. 10.1111/gfs.12153.

23. Malek MA Chowdhury NA Matsuhashi S Hashimoto S Kume T Radiation and fermentation treatment of cellulosic wastes Mycoscience 1994 35 95 98 10.1007/BF02268535 Malek MA, Chowdhury NA, Matsuhashi S, Hashimoto S, Kume T. Radiation and fermentation treatment of cellulosic wastes. Mycoscience. 1994;35:95–8. 10.1007/BF02268535.

24. Klein M Kimmelman LJ The correlation between the inhibition of drug resistance and synergism in streptomycin and penicillin J Bacteriol 1947 54 3 363 370 10.1128/jb.54.3.363-370.1947 16561369 PMC526561 Klein M, Kimmelman LJ. The correlation between the inhibition of drug resistance and synergism in streptomycin and penicillin. J Bacteriol. 1947;54(3):363–70. 10.1128/jb.54.3.363-370.1947. 16561369 10.1128/jb.54.3.363-370.1947 PMC526561

25. Yao JDC, Moellering RC. Antibacterial Agents. In: Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, Warnock DW, editors. Manual of Clinical Microbiology. 1st edition. Wiley; 2011. p. 1041–81. 10.1128/9781555816728.ch65.

26. Te Welscher YM Van Leeuwen MR De Kruijff B Dijksterhuis J Breukink E Polyene antibiotic that inhibits membrane transport proteins Proc Natl Acad Sci U S A 2012 109 11156 11159 10.1073/pnas.1203375109 22733749 PMC3396478 Te Welscher YM, Van Leeuwen MR, De Kruijff B, Dijksterhuis J, Breukink E. Polyene antibiotic that inhibits membrane transport proteins. Proc Natl Acad Sci U S A. 2012;109:11156–9. 10.1073/pnas.1203375109. 22733749 10.1073/pnas.1203375109 PMC3396478

27. Nocua-Báez LC Uribe-Jerez P Tarazona-Guaranga L Robles R Cortés JA Azoles of then and now: a review Rev Chil Infectol 2020 37 219 230 10.4067/s0716-10182020000300219 32853312 Nocua-Báez LC, Uribe-Jerez P, Tarazona-Guaranga L, Robles R, Cortés JA. Azoles of then and now: a review. Rev Chil Infectol. 2020;37:219–30. 10.4067/s0716-10182020000300219. 10.4067/s0716-10182020000300219 32853312

28. Nishino N Touno E Ensiling characteristics and aerobic stability of direct‐cut and wilted grass silages inoculated with Lactobacillus casei or Lactobacillus buchneri J Sci Food Agric 2005 85 1882 1888 10.1002/jsfa.2189 Nishino N, Touno E. Ensiling characteristics and aerobic stability of direct‐cut and wilted grass silages inoculated with Lactobacillus casei or Lactobacillus buchneri . J Sci Food Agric. 2005;85:1882–8. 10.1002/jsfa.2189.

29. Erwin ES Marco GJ Emery EM Volatile fatty acid analyses of blood and rumen fluid by gas chromatography J Dairy Sci 1961 44 9 1768 1771 10.3168/jds.S0022-0302(61)89956-6 Erwin ES, Marco GJ, Emery EM. Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J Dairy Sci. 1961;44(9):1768–71. 10.3168/jds.S0022-0302(61)89956-6.

30. Bolger AM Lohse M Usadel B Trimmomatic: a flexible trimmer for Illumina sequence data Bioinformatics 2014 30 2114 2120 10.1093/bioinformatics/btu170 24695404 PMC4103590 Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. 10.1093/bioinformatics/btu170. 24695404 10.1093/bioinformatics/btu170 PMC4103590

31. Edgar RC UPARSE: highly accurate OTU sequences from microbial amplicon reads Nat Methods 2013 10 10 996 998 10.1038/nmeth.2604 23955772 Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10(10):996–8. 10.1038/nmeth.2604. 23955772 10.1038/nmeth.2604

32. Caruso V Song X Asquith M Karstens L Performance of microbiome sequence inference methods in environments with varying biomass mSystems 2019 4 1 1110 1128 10.1128/msystems.00163-18 PMC6381225 30801029 Caruso V, Song X, Asquith M, Karstens L. Performance of microbiome sequence inference methods in environments with varying biomass. mSystems. 2019;4(1):1110–28. 10.1128/msystems.00163-18. 10.1128/mSystems.00163-18 PMC6381225 30801029

33. Chiarello M McCauley M Villéger S Jackson CR Ranking the biases: the choice of OTUs vs . ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold PLoS ONE 2022 17 e0264443 10.1371/journal.pone.0264443 35202411 PMC8870492 Chiarello M, McCauley M, Villéger S, Jackson CR. Ranking the biases: the choice of OTUs vs . ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold. PLoS ONE. 2022;17:e0264443. 10.1371/journal.pone.0264443. 35202411 10.1371/journal.pone.0264443 PMC8870492

34. Jeske JT Gallert C Microbiome analysis via OTU and ASV-based pipelines—a comparative interpretation of ecological data in WWTP systems Bioengineering 2022 9 146 10.3390/bioengineering9040146 35447706 PMC9029325 Jeske JT, Gallert C. Microbiome analysis via OTU and ASV-based pipelines—a comparative interpretation of ecological data in WWTP systems. Bioengineering. 2022;9:146. 10.3390/bioengineering9040146. 35447706 10.3390/bioengineering9040146 PMC9029325

35. Kim M Chun J 16S rRNA gene-based identification of bacteria and archaea using the EzTaxon server Methods in microbiology 2014 Elsevier 61 74 Kim M, Chun J. 16S rRNA gene-based identification of bacteria and archaea using the EzTaxon server. In: Methods in microbiology. Elsevier; 2014. p. 61–74.

36. Ennahar S Cai Y Fujita Y Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis Appl Environ Microbiol 2003 69 1 444 451 10.1128/AEM.69.1.444-451.2003 12514026 PMC152408 Ennahar S, Cai Y, Fujita Y. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl Environ Microbiol. 2003;69(1):444–51. 10.1128/AEM.69.1.444-451.2003. 12514026 10.1128/AEM.69.1.444-451.2003 PMC152408

37. Mangia NP Saliba L Deiana P Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage Ann Microbiol 2019 69 2 161 170 10.1007/s13213-018-1416-1 Mangia NP, Saliba L, Deiana P. Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage. Ann Microbiol. 2019;69(2):161–70. 10.1007/s13213-018-1416-1.

38. Pahlow G, Muck RE, Driehuis F, Elferink SJWHO, Spoelstra SF. Microbiology of Ensiling. In: Buxton DR, Muck RE, Harrison JH, editors. Agronomy Monographs. 1st edition. Wiley; 2003. p. 31–93. 10.2134/agronmonogr42.c2.

39. McDonald P Henderson N Heron S The biochemistry of silage 1991 Marlow Chalcombe Publications McDonald P, Henderson N, Heron S. The biochemistry of silage. Marlow: Chalcombe Publications; 1991.

40. Pang H Tan Z Wang Y Bourne GH Danielli JF Jeon KW Dominant Factors Affecting Silage Cultural history and modern production technology of silage 2025 London Academic 79 Pang H, Tan Z, Wang Y. Dominant Factors Affecting Silage. In: Bourne GH, Danielli JF, Jeon KW, editors. Cultural history and modern production technology of silage. London: Academic; 2025. p. 79.

41. Ren F He R Zhou X Gu Q Xia Z Liang M Dynamic changes in fermentation profiles and bacterial community composition during sugarcane top silage fermentation: a preliminary study Bioresour Technol 2019 285 121315 10.1016/j.biortech.2019.121315 30965280 Ren F, He R, Zhou X, Gu Q, Xia Z, Liang M, et al. Dynamic changes in fermentation profiles and bacterial community composition during sugarcane top silage fermentation: a preliminary study. Bioresour Technol. 2019;285:121315. 10.1016/j.biortech.2019.121315. 30965280 10.1016/j.biortech.2019.121315

42. Chen Q Yu B Zhu Y Xiong H Guo Y Liu D Effects of different concentrations of Lactiplantibacillus plantarum and Bacillus licheniformis on silage fermentation parameter, chemical composition and microbial community of Pennisetum sinese Front Microbiol 2025 16 1532060 10.3389/fmicb.2025.1532060 40231240 PMC11994720 Chen Q, Yu B, Zhu Y, Xiong H, Guo Y, Liu D, et al. Effects of different concentrations of Lactiplantibacillus plantarum and Bacillus licheniformis on silage fermentation parameter, chemical composition and microbial community of Pennisetum sinese . Front Microbiol. 2025;16:1532060. 10.3389/fmicb.2025.1532060. 40231240 10.3389/fmicb.2025.1532060 PMC11994720

43. Zhang C Shu G Zhu Z Li Y Fang Z Chen L Dynamic effects of exogenous and epiphytic Pediococcus pentosaceus on quality and bacterial community succession of silage mulberry leaves Agriculture Basel 2025 15 16 1726 10.3390/agriculture15161726 Zhang C, Shu G, Zhu Z, Li Y, Fang Z, Chen L, et al. Dynamic effects of exogenous and epiphytic Pediococcus pentosaceus on quality and bacterial community succession of silage mulberry leaves. Agriculture Basel. 2025;15(16):1726. 10.3390/agriculture15161726.

44. Oshiro M Tanaka M Zendo T Nakayama J Impact of pH on succession of sourdough lactic acid bacteria communities and their fermentation properties Biosci Microbiota Food Health 2020 39 3 152 159 10.12938/bmfh.2019-038 32775134 PMC7392915 Oshiro M, Tanaka M, Zendo T, Nakayama J. Impact of pH on succession of sourdough lactic acid bacteria communities and their fermentation properties. Biosci Microbiota Food Health. 2020;39(3):152–9. 10.12938/bmfh.2019-038. 32775134 10.12938/bmfh.2019-038 PMC7392915

45. Oude Elferink SJWH Krooneman J Gottschal JC Spoelstra SF Faber F Driehuis F Anaerobic conversion of lactic acid to acetic acid and 1,2-Propanediol by Lactobacillus buchneri Appl Environ Microbiol 2001 67 125 132 10.1128/AEM.67.1.125-132.2001 11133436 PMC92530 Oude Elferink SJWH, Krooneman J, Gottschal JC, Spoelstra SF, Faber F, Driehuis F. Anaerobic conversion of lactic acid to acetic acid and 1,2-Propanediol by Lactobacillus buchneri . Appl Environ Microbiol. 2001;67:125–32. 10.1128/AEM.67.1.125-132.2001. 11133436 10.1128/AEM.67.1.125-132.2001 PMC92530

46. Gomes ALM Bueno AVI Osmari MP Machado J Nussio LG Jobim CC Effects of obligate heterofermentative lactic acid bacteria alone or in combination on the conservation of sugarcane silage Front Microbiol 2021 12 643879 10.3389/fmicb.2021.643879 34040591 PMC8141596 Gomes ALM, Bueno AVI, Osmari MP, Machado J, Nussio LG, Jobim CC, et al. Effects of obligate heterofermentative lactic acid bacteria alone or in combination on the conservation of sugarcane silage. Front Microbiol. 2021;12:643879. 10.3389/fmicb.2021.643879. 34040591 10.3389/fmicb.2021.643879 PMC8141596

47. Chiou PW-S, Chang S-H, Yu B. The effects of wet sorghum distillers’ grains inclusion on napiergrass silage quality. J Sci Food Agric. 2000; 80: 1199-1205 10.1002/1097-0010(200006)80:8<1199::AID-JSFA620>3.0.CO;2-R

48. Schmidt RJ Kung JL The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations J Dairy Sci 2010 93 4 1616 1624 10.3168/jds.2009-2555 20338439 Schmidt RJ, Kung JL. The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations. J Dairy Sci. 2010;93(4):1616–24. 10.3168/jds.2009-2555. 20338439 10.3168/jds.2009-2555

49. Wilkinson JM Davies DR The aerobic stability of silage: key findings and recent developments Grass Forage Sci 2013 68 1 1 19 10.1111/j.1365-2494.2012.00891.x Wilkinson JM, Davies DR. The aerobic stability of silage: key findings and recent developments. Grass Forage Sci. 2013;68(1):1–19. 10.1111/j.1365-2494.2012.00891.x.

50. Silva LD Pereira OG Silva TC Leandro ES Paula RA Santos SA Effects of Lactobacillus buchneri isolated from tropical maize silage on fermentation and aerobic stability of maize and sugarcane silages Grass Forage Sci 2018 73 3 660 670 10.1111/gfs.12360 Silva LD, Pereira OG, Silva TC, Leandro ES, Paula RA, Santos SA, et al. Effects of Lactobacillus buchneri isolated from tropical maize silage on fermentation and aerobic stability of maize and sugarcane silages. Grass Forage Sci. 2018;73(3):660–70. 10.1111/gfs.12360.

51. Li Y He D Niu D Zhao Y Acetic acid production from food wastes using yeast and acetic acid bacteria micro-aerobic fermentation Bioprocess Biosyst Eng 2015 38 863 869 10.1007/s00449-014-1329-8 25416587 Li Y, He D, Niu D, Zhao Y. Acetic acid production from food wastes using yeast and acetic acid bacteria micro-aerobic fermentation. Bioprocess Biosyst Eng. 2015;38:863–9. 10.1007/s00449-014-1329-8. 25416587 10.1007/s00449-014-1329-8

52. Yang Q Yao H Liu S Mao J Interaction and application of molds and yeasts in Chinese fermented foods Front Microbiol 2022 12 664850 10.3389/fmicb.2021.664850 35496819 PMC9041164 Yang Q, Yao H, Liu S, Mao J. Interaction and application of molds and yeasts in Chinese fermented foods. Front Microbiol. 2022;12:664850. 10.3389/fmicb.2021.664850. 35496819 10.3389/fmicb.2021.664850 PMC9041164

53. Soundharrajan I Min CW Jung JS Choi KC Effects of the novel Lacticaseibacillus paracasei K-68 inoculant on nutrient content, fermentation, and microbial dynamics changes in Dacheongok corn silage Fermentation 2025 11 6 304 10.3390/fermentation11060304 Soundharrajan I, Min CW, Jung JS, Choi KC. Effects of the novel Lacticaseibacillus paracasei K-68 inoculant on nutrient content, fermentation, and microbial dynamics changes in Dacheongok corn silage. Fermentation. 2025;11(6):304. 10.3390/fermentation11060304.

54. Yin X Long J Zhao J Wang S Dong Z Li J Effect of growth stage on Italian ryegrass ( Lolium multiflorum Lam .) silage fermentation from microbiological perspective Chem Biol Technol Agric 2023 10 1 19 10.1186/s40538-023-00394-8 Yin X, Long J, Zhao J, Wang S, Dong Z, Li J, et al. Effect of growth stage on Italian ryegrass ( Lolium multiflorum Lam .) silage fermentation from microbiological perspective. Chem Biol Technol Agric. 2023;10(1):19. 10.1186/s40538-023-00394-8.

55. Björkroth KJ Schillinger U Geisen R Weiss N Hoste B Holzapfel WH Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples Int J Syst Evol Microbiol 2002 52 1 141 148 10.1099/00207713-52-1-141 11837296 Björkroth KJ, Schillinger U, Geisen R, Weiss N, Hoste B, Holzapfel WH, et al. Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples. Int J Syst Evol Microbiol. 2002;52(1):141–8. 10.1099/00207713-52-1-141. 11837296 10.1099/00207713-52-1-141

56. Niu Y Guo Y Huang R Niu J Wang Y Zhang P Fermentative profile and bacterial community structure of whole-plant triticale silage ( Triticosecale Wittmack ) with or without the addition of Streptococcus bovis and Lactiplantibacillus plantarum mSphere 2025 10 2 e00894-24 10.1128/msphere.00894-24 39873502 PMC11852913 Niu Y, Guo Y, Huang R, Niu J, Wang Y, Zhang P, et al. Fermentative profile and bacterial community structure of whole-plant triticale silage ( Triticosecale Wittmack ) with or without the addition of Streptococcus bovis and Lactiplantibacillus plantarum . mSphere. 2025;10(2):e00894-24. 10.1128/msphere.00894-24. 39873502 10.1128/msphere.00894-24 PMC11852913

57. Duan S Zhou X Xiao H Miao J Zhao L Characterization of bacterial microbiota in tilapia fillets under different storage temperatures J Food Sci 2019 84 6 1487 1493 10.1111/1750-3841.14630 31066925 Duan S, Zhou X, Xiao H, Miao J, Zhao L. Characterization of bacterial microbiota in tilapia fillets under different storage temperatures. J Food Sci. 2019;84(6):1487–93. 10.1111/1750-3841.14630. 31066925 10.1111/1750-3841.14630

58. Östling C Lindgren S Influences of enterobacteria on the fermentation and aerobic stability of grass silages Grass Forage Sci 1995 50 1 41 47 10.1111/j.1365-2494.1995.tb02292.x Östling C, Lindgren S. Influences of enterobacteria on the fermentation and aerobic stability of grass silages. Grass Forage Sci. 1995;50(1):41–7. 10.1111/j.1365-2494.1995.tb02292.x.

59. Su Y Yu Q Xi Y Rong Y Long Y Xie Y Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum , on the aerobic stability, microbial composition and in vitro degradability of mulberry silage BMC Microbiol 2025 25 1 439 10.1186/s12866-025-04165-3 40670920 PMC12265255 Su Y, Yu Q, Xi Y, Rong Y, Long Y, Xie Y, et al. Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum , on the aerobic stability, microbial composition and in vitro degradability of mulberry silage. BMC Microbiol. 2025;25(1):439. 10.1186/s12866-025-04165-3. 40670920 10.1186/s12866-025-04165-3 PMC12265255

60. Haq IU Sarwar MK Mohyuddin Z Haq IU Iiaz S Microbial determinants in silage rotting: a challenge in winter fodders Sustainable winter fodder 2021 Boca Raton CRC Press 301 329 Haq IU, Sarwar MK, Mohyuddin Z. Microbial determinants in silage rotting: a challenge in winter fodders. In: Haq IU, Iiaz S, editors. Sustainable winter fodder. Boca Raton: CRC Press; 2021. p. 301–29.

61. Sarkar D Prajapati S Poddar K Sarkar A Production of ethanol by Enterobacter sp. EtK3 during fruit waste biotransformation Int Biodeterior Biodegrad 2019 145 104795 10.1016/j.ibiod.2019.104795 Sarkar D, Prajapati S, Poddar K, Sarkar A. Production of ethanol by Enterobacter sp. EtK3 during fruit waste biotransformation. Int Biodeterior Biodegrad. 2019;145:104795. 10.1016/j.ibiod.2019.104795.

62. Wang Y Sun Y Huang K Gao Y Lin Y Yuan B Multi-omics analysis reveals the core microbiome and biomarker for nutrition degradation in alfalfa silage fermentation mSystems 2024 9 11 e00682-24 10.1128/msystems.00682-24 39440963 PMC11575373 Wang Y, Sun Y, Huang K, Gao Y, Lin Y, Yuan B, et al. Multi-omics analysis reveals the core microbiome and biomarker for nutrition degradation in alfalfa silage fermentation. mSystems. 2024;9(11):e00682-24. 10.1128/msystems.00682-24. 39440963 10.1128/msystems.00682-24 PMC11575373

63. Long Y Li M Su Y Yu Q Rong Y Xi Y Exploring the aerobic stability, antioxidant and microbial community of Broussonetia papyrifera ensiled with ferulic acid esterase-producing Lactiplantibacillus plantarum in combination with cellulase and/or xylanase BMC Microbiol 2025 25 1 533 10.1186/s12866-025-04185-z 40847280 PMC12374340 Long Y, Li M, Su Y, Yu Q, Rong Y, Xi Y, et al. Exploring the aerobic stability, antioxidant and microbial community of Broussonetia papyrifera ensiled with ferulic acid esterase-producing Lactiplantibacillus plantarum in combination with cellulase and/or xylanase. BMC Microbiol. 2025;25(1):533. 10.1186/s12866-025-04185-z. 40847280 10.1186/s12866-025-04185-z PMC12374340

64. Zhang H Wu J Zhao X Yan P Yang R Yan J Improving aerobic stability and methane production of maize stover silage with lactic acid bacteria inoculants: focus on pentose-fermentation Ind Crops Prod 2023 201 116861 10.1016/j.indcrop.2023.116861 Zhang H, Wu J, Zhao X, Yan P, Yang R, Yan J, et al. Improving aerobic stability and methane production of maize stover silage with lactic acid bacteria inoculants: focus on pentose-fermentation. Ind Crops Prod. 2023;201:116861. 10.1016/j.indcrop.2023.116861.

65. Peng S Xie L Cheng Y Wang Q Feng L Li Y Effect of Lactiplantibacillus and sea buckthorn pomace on the fermentation quality and microbial community of paper mulberry silage Front Plant Sci 2024 15 1412759 10.3389/fpls.2024.1412759 39280944 PMC11395498 Peng S, Xie L, Cheng Y, Wang Q, Feng L, Li Y, et al. Effect of Lactiplantibacillus and sea buckthorn pomace on the fermentation quality and microbial community of paper mulberry silage. Front Plant Sci. 2024;15:1412759. 10.3389/fpls.2024.1412759. 39280944 10.3389/fpls.2024.1412759 PMC11395498

66. Ogunade IM Jiang Y Cervantes AP Kim DH Oliveira AS Vyas D Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: effects of Escherichia coli O157: H7 and silage additives J Dairy Sci 2018 101 3 2048 2059 10.1016/j.indcrop.2025.121589 29274960 Ogunade IM, Jiang Y, Cervantes AP, Kim DH, Oliveira AS, Vyas D, et al. Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: effects of Escherichia coli O157: H7 and silage additives. J Dairy Sci. 2018;101(3):2048–59. 10.1016/j.indcrop.2025.121589. 29274960 10.3168/jds.2017-12876

67. Radko L Dudek K Żakowicz P Smulski S Kozdrowski R Health problems with mycotoxins in cattle—a review Molecules 2025 31 43 10.3390/molecules31010043 41515340 PMC12786409 Radko L, Dudek K, Żakowicz P, Smulski S, Kozdrowski R. Health problems with mycotoxins in cattle—a review. Molecules. 2025;31:43. 10.3390/molecules31010043. 41515340 10.3390/molecules31010043 PMC12786409

68. Eckard S Wettstein FE Forrer H-R Vogelgsang S Incidence of Fusarium species and mycotoxins in silage maize Toxins 2011 3 949 967 10.3390/toxins3080949 22069750 PMC3202868 Eckard S, Wettstein FE, Forrer H-R, Vogelgsang S. Incidence of Fusarium species and mycotoxins in silage maize. Toxins. 2011;3:949–67. 10.3390/toxins3080949. 22069750 10.3390/toxins3080949 PMC3202868

69. Bao Y Yangzong Z Yuan Z Shi R Feng K Xin P Song T The microbial communities and natural fermentation quality of ensiling oat ( Avena sativa L. ) harvest from different elevations on the Qinghai-Tibet Plateau Front Microbiol 2023 13 1108890 10.3389/fmicb.2022.1108890 36713149 PMC9873999 Bao Y, Yangzong Z, Yuan Z, Shi R, Feng K, Xin P, et al. The microbial communities and natural fermentation quality of ensiling oat ( Avena sativa L. ) harvest from different elevations on the Qinghai-Tibet Plateau. Front Microbiol. 2023;13:1108890. 10.3389/fmicb.2022.1108890. 36713149 10.3389/fmicb.2022.1108890 PMC9873999

70. de Oliveira AJ Ono MA Suguiura IMS Zucareli C Garcia EB Olchanheski LR Potential of yeasts as biocontrol agents against Fusarium graminearum in vitro and on corn J Appl Microbiol 2023 134 12 lxad296 10.1093/jambio/lxad296 38049375 de Oliveira AJ, Ono MA, Suguiura IMS, Zucareli C, Garcia EB, Olchanheski LR, et al. Potential of yeasts as biocontrol agents against Fusarium graminearum in vitro and on corn. J Appl Microbiol. 2023;134(12):lxad296. 10.1093/jambio/lxad296. 38049375 10.1093/jambio/lxad296

71. Deng J Zeng B Wei C Huang Z Effect of intensified fermentation with Wickerhamomyces anomalus on fungal community structure of fermented grains and flavor compounds of Xiaoqu Baijiu Foods 2025 14 19 3365 10.3390/foods14193365 41097534 PMC12524076 Deng J, Zeng B, Wei C, Huang Z. Effect of intensified fermentation with Wickerhamomyces anomalus on fungal community structure of fermented grains and flavor compounds of Xiaoqu Baijiu. Foods. 2025;14(19):3365. 10.3390/foods14193365. 41097534 10.3390/foods14193365 PMC12524076

72. Shimizu Y Konno Y Tomita Y Wickerhamomyces psychrolipolyticus f.a., sp. nov., a novel yeast species producing two kinds of lipases with activity at different temperatures Int J Syst Evol Microbiol 2020 70 2 1158 1165 10.1099/ijsem.0.003894 31833830 Shimizu Y, Konno Y, Tomita Y. Wickerhamomyces psychrolipolyticus f.a., sp. nov., a novel yeast species producing two kinds of lipases with activity at different temperatures. Int J Syst Evol Microbiol. 2020;70(2):1158–65. 10.1099/ijsem.0.003894. 31833830 10.1099/ijsem.0.003894

73. Chen R Li M Yang J Chen L Zi X Zhou H Exploring the effect of wilting on fermentation profiles and microbial community structure during ensiling and air exposure of king grass silage Front Microbiol 2022 13 971426 10.3389/fmicb.2022.971426 36160258 PMC9493678 Chen R, Li M, Yang J, Chen L, Zi X, Zhou H, et al. Exploring the effect of wilting on fermentation profiles and microbial community structure during ensiling and air exposure of king grass silage. Front Microbiol. 2022;13:971426. 10.3389/fmicb.2022.971426. 36160258 10.3389/fmicb.2022.971426 PMC9493678

74. Pang H Zhou P Yue Z Wang Z Qin G Wang Y Fermentation characteristics, chemical composition, and aerobic stability in whole crop corn silage treated with lactic acid bacteria or Artemisia argyi Agriculture 2024 14 7 1015 10.3390/agriculture14071015 Pang H, Zhou P, Yue Z, Wang Z, Qin G, Wang Y, et al. Fermentation characteristics, chemical composition, and aerobic stability in whole crop corn silage treated with lactic acid bacteria or Artemisia argyi . Agriculture. 2024;14(7):1015. 10.3390/agriculture14071015.

75. Huang Z Zeng B Deng J Ren Z Xie J Wei C Succession of microbial community structure in fermented grains during the fermentation of strong-flavor Baijiu and its impact on the metabolism of acids, alcohols, and esters Food Sci Biotechnol 2024 33 15 3501 3513 10.1007/s10068-024-01591-3 39493398 PMC11525368 Huang Z, Zeng B, Deng J, Ren Z, Xie J, Wei C. Succession of microbial community structure in fermented grains during the fermentation of strong-flavor Baijiu and its impact on the metabolism of acids, alcohols, and esters. Food Sci Biotechnol. 2024;33(15):3501–13. 10.1007/s10068-024-01591-3. 39493398 10.1007/s10068-024-01591-3 PMC11525368

76. Cai W Wan Y Chen Y Fan H Li M Wu S Transcriptomics to evaluate the influence mechanisms of ethanol on the ester production of Wickerhamomyces anomalus with the induction of lactic acid Food Microbiol 2024 122 104556 10.1016/j.fm.2024.104556 38839235 Cai W, Wan Y, Chen Y, Fan H, Li M, Wu S, et al. Transcriptomics to evaluate the influence mechanisms of ethanol on the ester production of Wickerhamomyces anomalus with the induction of lactic acid. Food Microbiol. 2024;122:104556. 10.1016/j.fm.2024.104556. 38839235 10.1016/j.fm.2024.104556

77. Wang Z Li Q Li J Shang L Li J Chou S pH-responsive antimicrobial peptide with selective killing activity for bacterial abscess therapy J Med Chem 2022 65 7 5355 5373 10.1021/acs.jmedchem.1c01485 35294199 Wang Z, Li Q, Li J, Shang L, Li J, Chou S, et al. pH-responsive antimicrobial peptide with selective killing activity for bacterial abscess therapy. J Med Chem. 2022;65(7):5355–73. 10.1021/acs.jmedchem.1c01485. 35294199 10.1021/acs.jmedchem.1c01485

78. Paterson DL Resistance in gram-negative bacteria: Enterobacteriaceae Am J Infect Control 2006 34 5 S20 S28 10.1016/j.ajic.2006.05.238 16813978 Paterson DL. Resistance in gram-negative bacteria: Enterobacteriaceae. Am J Infect Control. 2006;34(5):S20–8. 10.1016/j.ajic.2006.05.238. 16813978 10.1016/j.ajic.2006.05.238

79. Kim E Yang SM Kim HY Weissella and the two Janus faces of the genus Appl Microbiol Biotechnol 2023 107 4 1119 1127 10.1007/s00253-023-12387-6 36680587 Kim E, Yang SM, Kim HY. Weissella and the two Janus faces of the genus. Appl Microbiol Biotechnol. 2023;107(4):1119–27. 10.1007/s00253-023-12387-6. 36680587 10.1007/s00253-023-12387-6

80. Li Z Ke L Huang C Peng S Zhao M Wu H Effects of seawater from different sea areas on abalone gastrointestinal microorganisms and metabolites Microorganisms 2025 13 4 915 10.3390/microorganisms13040915 40284752 PMC12029763 Li Z, Ke L, Huang C, Peng S, Zhao M, Wu H, et al. Effects of seawater from different sea areas on abalone gastrointestinal microorganisms and metabolites. Microorganisms. 2025;13(4):915. 10.3390/microorganisms13040915. 40284752 10.3390/microorganisms13040915 PMC12029763

81. Albert RA Zitomer D Dollhopf M Schauer-Gimenez AE Struble C King M Proposal of Vibrionimonas magnilacihabitans gen. nov., sp. nov., a curved Gram-stain-negative bacterium isolated from lake water Int J Syst Evol Microbiol 2014 64 Pt_2 613 620 10.1099/ijs.0.056663-0 24170777 Albert RA, Zitomer D, Dollhopf M, Schauer-Gimenez AE, Struble C, King M, et al. Proposal of Vibrionimonas magnilacihabitans gen. nov., sp. nov., a curved Gram-stain-negative bacterium isolated from lake water. Int J Syst Evol Microbiol. 2014;64(Pt_2):613–20. 10.1099/ijs.0.056663-0. 24170777 10.1099/ijs.0.056663-0

82. Osburn K Amaral J Metcalf SR Nickens DM Rogers CM Sausen C Primary souring: a novel bacteria-free method for sour beer production Food Microbiol 2018 70 76 84 10.1016/j.fm.2017.09.007 29173643 Osburn K, Amaral J, Metcalf SR, Nickens DM, Rogers CM, Sausen C, et al. Primary souring: a novel bacteria-free method for sour beer production. Food Microbiol. 2018;70:76–84. 10.1016/j.fm.2017.09.007. 29173643 10.1016/j.fm.2017.09.007

83. Sudun W Arakawa K Miyamoto M Miyamoto T Interaction between lactic acid bacteria and yeasts in Airag, an alcoholic fermented milk Anim Sci J 2013 84 1 66 74 10.1111/j.1740-0929.2012.01035.x 23302085 Sudun W, Arakawa K, Miyamoto M, Miyamoto T. Interaction between lactic acid bacteria and yeasts in Airag, an alcoholic fermented milk. Anim Sci J. 2013;84(1):66–74. 10.1111/j.1740-0929.2012.01035.x. 23302085 10.1111/j.1740-0929.2012.01035.x