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Article

Lactic Acid Bacteria and Formic Acid Improve Fermentation Quality and Beneficial Predicted Functional Characteristics in Mixed Silage Consisting of Alfalfa and Perennial Ryegrass

by
Yao Lei
1,
Maoya Li
1,
Yinghao Liu
2,
Jiachuhan Wang
1,
Xiangjiang He
1,
Yuanyuan Zhao
1,
Yulian Chen
1,
Qiming Cheng
1,3,* and
Chao Chen
1,*
1
College of Animal Science, Guizhou University, Guiyang 550025, China
2
Institute of Grassland Research of Chinese Academy of Agricultural Sciences, Hohhot 010010, China
3
College of Grassland, Resources and Environment, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization of Ministry of Agriculture, Key Laboratory of Grassland Resources, Inner Mongolia Agricultural University, Ministry of Education, Hohhot 010010, China
*
Authors to whom correspondence should be addressed.
Fermentation 2024, 10(1), 43; https://doi.org/10.3390/fermentation10010043
Submission received: 7 November 2023 / Revised: 22 December 2023 / Accepted: 2 January 2024 / Published: 5 January 2024
(This article belongs to the Special Issue The Use of Lactobacillus in Forage Storage and Processing)
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Abstract

:
The purpose of the present study was to investigate the effect of additives on the fermentation properties of ensiled mixed alfalfa and perennial ryegrass silage in the karst terrain of Southwest China. A mixture of alfalfa and perennial ryegrass was ensiled at a ratio of 3:7 using three experimental treatments: (1) CK (without additives) and distilled water (5 mL kg−1 fresh weight (FW)); (2) FA and formic acid (88%) (5 mL kg−1 FW); and (3) LAB combined with the application of Lactiplantibacillus plantarum and Lentilactobacillus buchneri (2 × 107 cfu/g FW). All samples were packed manually into polyethylene bags, and three polyethylene bags from each treatment were sampled on days 7, 15, and 45. The findings demonstrated that the pH values of all the mixed silages gradually decreased during ensiling. The lactic acid (LA) and acetic acid (AA) contents increased gradually with ensiling time and peaked after 45 days of ensiling. After 45 days of ensiling, the FA and LAB groups effectively preserved the nutrient content of the mixed silage, which presented a reduced neutral detergent fiber and acid detergent fiber content (p < 0.05) and higher water-soluble carbohydrate content (p < 0.05) than the CK group. The fermentation quality of the mixed silages in the FA and LAB groups improved, as indicated by higher (p < 0.05) LA contents and lower (p < 0.05) pH and ammoniacal nitrogen contents after 45 days of ensiling compared to those in the CK group. As fermentation progressed, the abundance of harmful microorganisms (Hafnia obesumbacterium, Enterobacteriaceae, and Sphingomonas) and beneficial microorganisms (Lactiplantibacillus and Lentilactobacillus) decreased and increased, respectively. In addition, compared to those in the CK group, the FA group had higher abundances of “lipid metabolism” and “biosynthesis of antibiotics” and lower abundances of “membrane transport”. Briefly, the results of this study suggest that the incorporation of FA and LAB additives could improve the quality of fermented mixed silage, and that FA is better than LAB. This information is useful for combining forage resources to satisfy the requirements for high-protein feed and for manufacturing ruminant feed annually.

1. Introduction

In the karst terrain in Southwest China, where precipitation is plentiful and concentrated, a vast area of dangerously exposed soil and rock has developed, a phenomenon known as rocky desertification [1]. Rocky desertification has a significant negative impact on regional sustainability and national ecological security [2]. A shortage of food supplies for herbivores, resulting from severe rock desertification, has also destroyed the original intact flora, which has a significant negative impact on the development of animal husbandry [3]. Forage for feeding animals is usually collected as hay or made into silage by anaerobic fermentation. However, karst regions are characterized by both high temperatures and precipitation; therefore, silage may be more suitable for preserving fodder in these environments [4].
Alfalfa (Medicago sativa L., AL), often called “the queen of forage crops”, has become a significant pasture for livestock due to its high yield potential and high levels of digestible protein, minerals, and vitamins [5,6]. Recent research has shown that ensiling AL alone usually results in poor fermentation, principally due to low epiphytic lactic acid bacteria populations, low contents of water-soluble carbohydrate (WSC) and dry matter (DM), and a high buffering capacity (BC) in fresh AL [7,8]. Numerous studies have shown that adding gramineous forage with a high WSC content, such as whole-plant maize or sweet sorghum, to AL silage can enhance fermentation [4,8]. Perennial ryegrass (Lolium perenne L., PR) is widely distributed in Asia, Europe, and North Africa and has a long growing season, high adaptability, and high forage yield [9]. PR is a typical cultivated grass in karst areas which is not only rich in nutrients and can be used as a source of livestock forage; at the same time, it has a strong resistance to harsh environments, and is also an important resource for promoting ecological recovery [10]. PR can be stored as feed for ruminants, but the crude protein (CP) content of fresh and stored PR (100 g CP per kg DM) is not sufficient to meet nutritional requirements [11]. Previous studies have shown that the nutritional quality of PR can be improved (>200 g CP/kg DM) by adding high-CP-content forage [12]. Moreover, adding PR with a high WSC content (12.14% DM-14.02% DM) to high-protein forage is an effective solution for improving the fermentation quality of silage [12,13]. Consequently, ensiling AL with PR might be a useful technique for enhancing the nutritional value and fermentation quality of silage. However, even though our previous studies showed that the nutritional quality of mixed silage ensiled with AL and PR improved, the fermentation quality was not satisfactory (pH 4.63–5.94) [14]. Hence, according to previous studies, silage additives are required to further improve the fermentation quality of mixed silage ensiled with AL and PR.
The proportion of dominant bacteria in the bacterial community largely determines the success of the anaerobic fermentation process [15]. One of the most common ways to improve lactic acid bacteria populations in silage is to increase the amount of exogenous lactic acid bacteria, and the other is to suppress harmful bacteria in silage. Lactic acid bacteria additives improve silage quality by increasing the lactic acid (LA) content, reducing the pH, and inhibiting the growth of harmful microorganisms [16]. Generally, heterofermentative lactic acid bacterial strains have been recognized for their antifungal ability to enhance silage aerobic stability, while homofermentative lactic acid bacterial strains are typically the primary contributors to lactic acid fermentation [17,18]. It is well known that formic acid (FA) prevents the growth of mold and yeast, which are typically thought to be the causes of aerobic degradation [19]. However, homologous and allogenic Lactobacillus species, FA succession, and functional changes in these bacteria during mixed silage have not been determined, and are essential for further fermentation regulation. Next-generation 16S rRNA sequencing technology has recently been used to track changes in microbial communities in silage and identify dominant species, since it can reveal the bacterial profile of target samples at the species level [20]. Recent studies have focused on microbial interactions and predictive functional characteristics in addition to changes in microbial diversity. During fermentation, lactic acid bacteria produce many metabolites that are essential for the establishment and maintenance of silage [21]. Therefore, a deeper comprehension of the predicted functional characteristics involved in silage fermentation may offer crucial biological insights into the regulation of silage fermentation. To date, there have been studies on mixed silage of alfalfa with maize and rapeseed [22,23]. However, rather than examining intricate microbial community succession, microbial interactions, and functional predictions, the majority of these studies have focused on changes in silage fermentation characteristics and chemical composition. Consequently, the present study aimed to investigate the effects of FA and lactic acid bacteria on fermentation characteristics, bacterial community succession, and predicted microbial functions in mixed alfalfa and perennial ryegrass silage. We hypothesize that FA and lactic acid bacteria can enhance the nutritional value and fermentation quality of mixed AL and PR silage by restricting the growth of harmful bacteria. In addition, we predicted that mixed silage of AL and PR could provide more balanced nutrients for livestock in karst areas, which could lead to the development of a scientific reference program to address the problem of livestock winter forage shortages in karst areas.

2. Materials and Methods

2.1. Silage Preparation

AL and PR plants were harvested as whole plants and cut to a length of 1–2 cm on 11 April 2021 in Guanling County, Anshun City, Guizhou Province. The DM content in the AL treatment was 29.25% fresh matter (FM), and the CP, neutral detergent fiber (NDF), acid detergent fiber (ADF), and WSC contents were 24.30, 47.43, 24.71, and 6.89%, respectively. The DM content of PR was 34.62% FM, and its CP, NDF, ADF, and WSC contents were 7.54, 45.39, 23.72, and 10.89%, respectively. The epiphytic lactic acid bacteria, yeasts, and coliform bacteria counts on fresh AL were 2.81, 4.23, and <2.00 log10 CFU/g FW, respectively. The numbers of epiphytic lactic acid bacteria, yeasts, and coliform bacteria on the fresh PR were 4.47, 2.63, and <2.00 log10 CFU/g FW, respectively. AL was mixed with PR at a ratio of 3:7 on an FM basis, as this ratio resulted in relatively favorable fermentation quality in the mixed silage in our previous experiments [14]. Approximately 300 g of chopped forage was evenly mixed at a previous ratio of 3:7, and manually loaded into polyethylene bags (25 cm × 30 cm); then, a vacuum-packaging machine (SJ-400, Shanghai Precision Machinery Manufacturing Co., Ltd., Shanghai, China) was used for vacuum-packaging. The following three treatments were used: (1) CK (without additives); distilled water (5 mL kg−1 fresh weight (FW)); (2) FA, formic acid (88%, Zhongke Jiayi Biological Engineering Co., Ltd., Qingzhou, China) (5 mL kg−1 FW); and (3) LAB, a combined application of Lactiplantibacillus plantarum and Lentilactobacillus buchneri (Lactobacillus plantarum and Lactobacillus buchneri were produced by Xi’an Polysource Biotechnology Co., Ltd. (Xi’an, China) and preserved in the China General Microbiological Culture Collection Center under accession numbers HH-LP56 (Lactobacillus plantarum) and JY-16LE8 (Lactobacillus buchneri)) (2 × 107 cfu/g FW). Three polyethylene bags of silage from identical treatments were kept at ambient temperature (22–25 °C), and three bags from each treatment were inspected to analyze microbial diversity after 7 and 45 days of silage, as well as chemical composition and fermentation quality after 7, 15, and 45 days.

2.2. Analysis of Microbial Populations, Chemical Compositions, and Fermentation Quality

The plate count method was used to ascertain the microbial populations of fresh materials, as carried out by Cai et al. [24]. The number of microorganisms was determined by determining the number of colony-forming units (cfu), which were subsequently transformed into logarithmic form and expressed on an FM basis. The samples were dried at 65 °C to estimate the DM content. The dried sample was crushed and stored after being passed through a 1 mm sieve for determination of other indices. The WSC content was determined by the anthrone method [25]. The CP content was measured in accordance with the official procedures of the AOAC [26]. The ADF and NDF contents were detected according to the methods of Van Soest et al. [27].
A total of 20 g of each sample was mixed with 180 mL of sterile water, stored at 4 °C for 24 h, and subsequently filtered through 4 layers of cheesecloth. Organic acids, ammoniacal nitrogen (NH3-N), and pH were measured in the filtrate. The pH was measured with a pH meter [28]. The content of ammonia nitrogen (NH3-N) was determined according to the methods of Broderick et al. [28]. Organic acids (LA, acetic acid (AA), propionic acid (PA), and butyric acid (BA)) were determined by liquid chromatography [29].

2.3. Bacterial Community Analysis

Using the procedure outlined by Li et al. [30], microbial DNA was isolated from the silage sample. In summary, whole DNA was extracted from each sample using a Power Soil DNA Isolation Kit (MO BIO Laboratories) in accordance with the manufacturer’s instructions. Following purification, sterile water was used to dilute the DNA to 1 ng/mL. For PCR amplification and bioinformatic analysis, all microbial DNA samples were promptly transferred to Biomarker Technologies Corporation (Beijing, China). Using particular barcode sequences in combination with a forward primer (50-ACTCCTACGGGAGGCAGCA-30) and reverse primer (50-GGACTACHVGGGTWTCTAAT-30), the 16S rDNA V3–V4 regions were amplified. QIIME (v1.8.0) was used to cluster the operational taxonomic units (OTUs) at a 97% similarity threshold. Alpha diversity (Mothur, v1.41.1) and rarefaction (R, v. 22) were computed using the OTU file. R software (version 4.0.5) was used to graphically present the results of principal coordinate analysis (PCoA), linear discriminant analysis effect size (LefSe) analysis, and canonical correspondence analysis (CCA) in accordance with the package protocols.

2.4. Predicted Metabolic Function Analysis

After using Phylogenetic Investigation of Communities by Reconstruction of Tax4Fun (version 0.3.1), the bacterial function was inferred from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, as reported by Asshauer et al. [31]. IBM, Armonk, NY, USA, and version 8 of GraphPad Prism were used to graphically visualize the differences in the KEGG pathways.

2.5. Statistical Analysis

The effects of treatment (T), ensiling period (D), and their interaction (T × D) on the chemical composition, fermentation quality, and bacterial community data were assessed using two-way analysis of variance. Afterward, the means were compared using Duncan’s multiple range test to ascertain significance. The general linear model approach was used for all the statistical analyses, and SPSS 26 software (IBM Crop., Armonk, NY, USA) was used. Unless specified otherwise, p < 0.05 was used to determine significance.

3. Results

3.1. Chemical Composition of Mixed Silage with Alfalfa and Perennial Ryegrass during the Ensiling Process

The chemical composition of the mixed silage containing AL and PR is listed in Table 1. The DM, CP, NDF, ADF, and WSC contents of the mixed silage were significantly impacted by the treatment and ensiling duration (p < 0.001). The interactions between treatment and ensiling period were significant (p < 0.05) for the CP, NDF, and WSC contents. The contents of DM, CP, NDF, ADF, and WSC in the mixed silage gradually decreased as the fermentation process progressed. The DM and WSC contents in the FA group were higher than those in the CK group after 45 days of ensiling (p < 0.05). Similarly, after 45 days of ensiling, the NDF and ADF contents in the FA and LAB groups were lower than (p < 0.05) those in the CK group. However, the effect of the additives after 45 days of ensiling on the CP content in the mixed silage was small. In this study, the FA treatment had more WSC (4.07% DM) and less ADF (34.13% DM) than the other treatments, indicating that it is the most effective additive for preserving the chemical properties of mixed silage.

3.2. Fermentation Characteristics of Mixed Alfalfa and Perennial Ryegrass Silage during the Ensiling Process

The fermentation characteristics of the mixed silage containing AL and PR are listed in Table 2. The LA, AA, and NH3-N contents of the mixed silage were significantly impacted by the treatment (p < 0.001) and ensiling duration (p < 0.001). The contents of LA, AA, and NH3-N in the mixed silage increased gradually, while the pH decreased gradually during fermentation. During the ensiling period, the pH of the FA group was significantly lower than that of the CK group (p < 0.05). Specifically, the FA group experienced rapid acidification at the preliminary stage of fermentation, and maintained a comparatively steady pH value (<4.2) until fermentation anaphase. However, at the end of ensiling, the pH of the CK group remained above 4.2. After 45 days of ensiling, the pH and NH3-N content were lower, and the LA and AA contents were higher in the FA and LAB groups than in the CK group (p < 0.05). Moreover, the FA group had the lowest pH (4.06) and NH3-N content (2.58% DM), the highest LA content (6.22% DM), and the highest AA content (2.91% DM) among the LAB groups. After 45 days of ensiling, PA and BA were not detected. Overall, additives may enhance the fermentation quality of AL and PR mixed silage, with FA treatment producing the highest-quality silage; this improvement was primarily observed at a low pH (4.06), low NH3-N content (2.58% DM), and high LA content (6.22% DM); PA and BA were not detected.

3.3. Dynamic Changes in the Microbial Community of Mixed Alfalfa and Perennial Ryegrass Silage during the Ensiling Process

The bacterial diversity of the mixed AL and PR silage samples is shown in Table 3. The majority of the samples had good coverage, with values above 0.99, indicating that high-throughput sequencing technology was able to identify the majority of the bacteria. In our study, the OTU, Ace, Chao, Shannon, and Simpson indices of the mixed silage samples decreased as fermentation progressed. Figure 1A shows the bacterial community at the phylum level in the mixed silage samples. Proteobacteria and Firmicutes were the majority phyla in the mixed silages containing AL and PR. Notably, the phylum Firmicutes gradually became the dominant genus during the ensiling process. After 45 days of ensiling, the relative abundance of Firmicutes in the FA and LAB groups was higher than that in the CK group. The graph indicates that the additions had an impact on how the bacterial populations in the mixed silages successively developed. The bacterial community at the genus level in the mixed silage samples is described in Figure 1B. Hafnia obesumbacterium was the most common bacterial genus, followed by the genera Lentilactobacillus and unclassified Enterobacteriaceae, in the AL and PR mixed silage after 7 days of ensiling. Lactiplantibacillus was the most common bacterial genus, followed by the genus Lentilactobacillus, in the AL and PR mixed silage after 45 days of ensiling. The relative abundance of Hafnia obesumbacterium decreased with prolonged ensiling periods, and similarly, the relative abundances of other undesirable microbes, such as unclassified Enterobacteriaceae and Sphingomonas, also decreased. As expected, Lactiplantibacillus, Lentilactobacillus, and Levilactobacillus became the dominant genera, with an average relative abundance of more than 20% in all the treatments. After 45 days of ensiling, compared with those in the CK group, the relative abundances of Lactiplantibacillus, Lentilactobacillus, and Levilactobacillus in the FA and LAB groups was higher, and the relative abundances of Hafnia obesumbacterium, unclassified Enterobacteria, and Sphingomonas were lower. Moreover, the relative abundance of Lactiplantibacillus was the highest in the FA treatment, and the relative abundances of Hafnia obesumberium and Sphingomonas were the lowest. Notably, the LAB group contained a higher relative abundance of Secundilactobacillus. Overall, changes in the bacterial community were observed in the AL and PR mixed silage during the ensiling process. Furthermore, heterofermentative lactic acid bacteria species dominated in the initial stage of fermentation (7 days), while homofermentative lactic acid bacteria species dominated in the subsequent stage of fermentation (45 days).
To explain the influence of bacterial taxa differing in the fermentation process of silages treated with or without inoculants, a latent Dirichlet allocation (LDA) effect size (LEfSe) analysis was performed (Figure 2A). After 7 days of fermentation, the CK group had more Lactococcus and unclassified Enterobacteriaceae, the LAB group had more Lactiplantibacillus and Hafnia obesumberium, and the FA group had more Sphingomonas. After 45 days of fermentation, the CK group had more Paucilactobacillus and Levilactobacillus, the LAB group had more Lentilactobacillus and Secundilactobacillus, and the FA group had more Companilactobacillus.
The PCoA plot at the OTU level demonstrated variance in the bacterial community, as shown in Figure 2B. Components 1 and 2 explained 42.30 and 35.54%, respectively, of the total variance. Silage samples from different treatments and different fermentation periods were well separated and divided into different quadrants in the PCoA plots. The distinct separation of silage samples at 7 and 45 days of fermentation suggested that different microbial communities are present in the silage at various stages of fermentation. The discreteness of the mixed silage samples treated with additives and the CK group suggested that different microbial communities existed in the mixed silage treated with different additives. These findings indicate that the silage additive and fermentation time had greater effects on the microbial community of the mixed silage with AL and PR.

3.4. Predicted Metabolic Functions of the Bacterial Community of Mixed Silage with Alfalfa and Perennial Ryegrass during the Ensiling Process

The functional prediction results for the microbial community of mixed silages during ensiling are shown in Figure 3. In all mixed silages, chemoheterotrophy was the most important functional component of the microbial community after fermentation, aerobic chemoheterotrophy, and ureolysis. The relative abundance of bacteria during fermentation increased with prolonged ensiling, and similarly, the relative abundance of bacteria undergoing aerobic chemoheterotrophy also decreased. Moreover, the relative abundance of fermentation products in the FA treatment was higher than that in the other groups, and the relative abundance of aerobic chemoheterotrophy products was lower than that in the other groups after 45 days of fermentation. Interestingly, at 45 d of fermentation, the FA group had less nitrate fixation ability than the other groups.
The first-level changes in the predicted metabolic pathways after 7 and 45 days of fermentation in mixed silage are depicted in Figure 4A. After 7 days of fermentation, the FA and LAB groups had significantly (p < 0.05) lower abundances of “Cellular Process” and “Human Diseases” and higher abundances of “Genetic Information Processing” and “Metabolism” than did the CK group. After 7 days of fermentation, the FA group had significantly (p < 0.05) lower abundances of “Environmental Information Processing” and higher abundances of “Organismal Systems” than did the CK group. After 45 days of ensiling, the FA and LAB groups had significantly (p < 0.05) lower abundances of “Cellular Process” and “Human Diseases”. The FA group had significantly (p < 0.05) higher abundances of “Organismal Systems” than did the CK group.
The second-level changes in predicted metabolic pathways after 7 and 45 days of fermentation in mixed silage are depicted in Figure 4B. After 7 d of fermentation, the FA group had markedly (p < 0.05) higher abundances of “carbohydrate metabolism”, “amino acid metabolism”, “signal transduction”, and “lipid metabolism” than the CK group. The relative abundances of “cellular community-prokaryotes” and “signal transduction” in the LAB group were significantly (p < 0.05) lower than those in the CK group. The LAB group had markedly (p < 0.05) higher abundances of “carbohydrate metabolism”, and the FA group had markedly (p < 0.05) lower abundances of “membrane transport” than did the CK group. After 45 d of fermentation, the FA and LAB groups had markedly (p < 0.05) higher abundances of “carbohydrate metabolism” than the CK group.
The third-level changes in the predicted metabolic pathways after 7 and 45 days of fermentation of mixed silage are depicted in Figure 4C. After 7 d of fermentation, the FA group had significantly (p < 0.05) lower abundances of “ABC transporters”, “Quorum sensing”, “Biosynthesis of amino acids”, and “Phosphotransferase system (PTS)” and higher abundances of “Biosynthesis of secondary metabolites”, “Microbial metabolism in diverse environments”, “Biosynthesis of antibiotics”, and “Two component system” than did the CK group. After 7 d of fermentation, the LAB group had significantly (p < 0.05) lower abundances of “two component system” and “quorum sensing” and higher abundances of “biosynthesis of secondary metabolites”, “biosynthesis of antibiotics”, “biosynthesis of amino acids”, and “PTS” than the CK group. After 45 d of fermentation, the FA group had significantly (p < 0.05) lower abundances of “ABC transporters”, “Quorum sensing”, and “PTS” and higher abundances of “Biosynthesis of secondary metabolites”, “Biosynthesis of antibiotics”, and “Biosynthesis of amino acids” than did the CK group. After 45 d of fermentation, the LAB group had significantly (p < 0.05) lower abundances of “ABC transporters”, and “PTS” and higher abundances of “biosynthesis of secondary metabolites”, “biosynthesis of antibiotics”, and “biosynthesis of amino acids” than did the CK group.
BugBase analysis identified seven potential microbial phenotypes, including aerobic, anaerobic, facultatively anaerobic, Gram-negative, Gram-positive, potentially pathogenic, and stress-tolerant (Figure 5). As fermentation progressed, the abundances of aerobic, facultative anaerobic, Gram-negative, potentially pathogenic, and stress-tolerant bacteria decreased, and the abundances of anaerobic and Gram-positive bacteria increased in the mixed silage. In this trial, after 7 and 45 days of fermentation, the abundances of aerobic, facultative anaerobic, Gram-negative, potentially pathogenic, and stress-tolerant bacteria decreased, and the abundances of anaerobic and Gram-positive bacteria increased in the FA and LAB groups compared to those in the CK group.

4. Discussion

The chemical compositions of the mixed silages were significantly altered after fermentation. The contents of DM and WSC decreased as the fermentation process advanced, indicating that lactic acid bacteria consumed WSC and generated organic acids during the ensiling process [16]. The FA group contained higher DM and WSC contents than did the CK group. A similar result was obtained by Yuan et al. [32], who reported that the addition of FA at ensiling increased the DM and WSC contents of AL silage compared to those of the CK group. Importantly, the CP content was lower in the mixed silage, which may be associated with extensive protein hydrolysis caused by clostridial fermentation or phytase activity [33]. NH3-N in silage is produced by the decomposition of CP during fermentation [34]. In our study, the increase in NH3-N content was consistent with the decrease in CP content. Moreover, the content of NH3-N in the FA group was significantly lower than that in the CK group, indicating that the bacteria that are able to decompose CP in the FA group were effectively inhibited. Our NDF and ADF results are consistent with those of Dong et al. [12], who reported that the NDF and ADF contents decreased as fermentation progressed in mixed silage of Broussonetia papyrifera and PR.
The pH decreased gradually as the fermentation process advanced, probably because the mixed silage provided enough substrate for the fermentation of lactic acid bacteria. Furthermore, a low pH value of 4.2 was considered to indicate well-preserved silage, and after 45 days of ensiling, only the FA group had a pH value less than 4.2. The addition of FA as an acid to silage directly lowers the pH of the mixed silage, which reduces the pH. This finding has been demonstrated in other studies [35]. The increase in LA content in mixed silage from day 7 to day 45 was due to the increase in the relative abundance of homofermentative lactic acid bacteria (Lactiplantibacillus). The LA concentration in the LAB group increased compared with that in the CK group. This may be because the addition of exogenous lactic acid bacteria promoted lactic acid acidification in mixed silage, which facilitated the homofermentation of mixed silage and thus increased LA production [36]. The LA concentration in the FA group increased compared to that in the CK group. Since FA can quickly produce an acidic environment and inhibit the activity of other microorganisms, lactic acid bacteria can quickly become the dominant bacteria, thus producing more LA. In addition, FA can directly penetrate into bacterial cells and alter their physiological homeostasis (Figure 4), thereby affecting the microbial community structure and influencing LA production. This result was verified by our microbial results (Figure 1B). It was previously reported that AA can be produced by heterofermentative lactic acid bacteria [16]. The content of AA in the mixed silage increased gradually during fermentation, and at the same time, the relative abundance of heterofermentative lactic acid bacteria (Lentilactobacillus and Levilactobacillus) also increased, which was consistent with the findings of a previous study [20]. As shown in Table 2, FA utilization can increase the content of LA in mixed silage. This result suggested that the addition of FA can stimulate the growth of heterotrophic lactic acid bacteria, thereby increasing acetic acid production [36]. However, the LAB group contained more AAs because the LAB group was supplemented with Lentilactobacillus buchneri, which is a heterotrophically fermented lactic acid bacterium that converts LA to AA [37]. The PA and BA contents of silage have been determined to be insufficient [38]. In the present study, PA was detected only in the CK and LAB groups before 15 days of ensiling, which could be related to the higher pH in the mixed silage.
In our study, the OTU, Ace, Chao1, Shannon, and Simpson indices of mixed silage decreased with fermentation, which is consistent with the results of Dong et al. [39]. This may be because with the development of anaerobic and acidic environments in the late ensiling stage, some aerobic, acid-intolerant microorganisms were inhibited, leading to a decrease in bacterial diversity. The composition and structure of the bacterial community in silage strongly affect its fermentation quality [16]. Proteobacteria and Firmicutes were the two phyla that predominated in the mixed silage in the present study. In mixed silage, the relative abundance of Firmicutes increased, while the relative abundance of Proteobacteria decreased after 45 days of ensiling. This is because Firmicutes can grow and reproduce more easily under anaerobic and acidic fermentation conditions [40]. The FA and LAB groups contained a higher relative abundance of Firmicutes because they had lower pH values and more acidic environments, and were more conducive to the growth of Firmicutes. Hafnia obesumbacterium was the prevalent bacterial genus in the AL and PR mixed silage after 7 days of ensiling. Reportedly, Hafnia obesumbacterium belongs to the genus Enterobacter and has the ability to promote protein breakdown in silage [41]. Previous studies have shown that commercial lactic acid bacteria inoculants can reduce pH but cannot inhibit the growth of Hafnia obesumbacterium in AL silage [42]. In our study, we found that the addition of lactic acid bacteria and FA not only reduced the pH but also inhibited the growth of Hafnia obesumbacterium in AL and PR mixed silage. Therefore, we speculate that the lactic acid bacteria and FA mixture we used can effectively inhibit the growth of Hafnia obesumbacterium. Lactococcus species have been shown to produce LA during the early phase of ensiling, and LA is usually replaced by rod-shaped lactic acid bacteria (such as Lactiplantibacillus) during the later phase of ensiling [43]. Therefore, Lactiplantibacillus and Lentilactobacillus were the prevalent bacterial genera in the AL and PR mixed silage after 45 days of ensiling. After 45 days of ensiling, compared with those in the CK group, the relative abundances of Lactiplantibacillus and Lentilactobacillus in the FA and LAB groups were greater. This could explain the better fermentation quality, which was manifested by higher LA and AA contents and lower pH values. The harmful bacterium Sphingomonas can thrive in moist and cool environments [44]. The relative abundances of Hafnia Obesumberium and Sphingomonas were lower in the FA and LAB groups compared with those in the CK group. This result is attributed to the fact that the FA and LAB groups have lower pH values and can inhibit the growth of Hafnia obesumberium and Sphingomonas. The LAB Secundilactobacillus was reported in red radish paocai by Jiang et al. [45]; this genus is a facultatively anaerobic species that produces AA heterofermentatively. This difference might explain why the LAB group had the highest AA content. This study identified the keystone species in mixed silage. The largest contribution to the CK group at 45 d of ensiling was Paucilactobacillus (at the genus level), which was predominantly isolated from fermented plant material, including silage, pickles, and fruit purees [46]. The largest contributor to the FA group at 45 d of ensiling was Companilactobacillus (at the genus level), which was isolated from fermented vegetables, particularly fermented mustard or onion greens, and fruits, sourdough, or related cereal fermentations [46]. The largest contributor to the LAB group at 45 d of ensiling was Lentilactobacillus (at the genus level), which was isolated from silage, fermented vegetables, wine, and cereal mashes [46]. The results showed that additives altered the microbiota in the silage, and the key species identified were completely different among the three treatment groups and the two fermentation periods, which further demonstrated how the regulation of microbial dynamics in mixed silage varies according to the two additives. All the mixed silages were clearly isolated in PCoA plots and partitioned into different quadrants, demonstrating that the ensiling period and additive had the greatest effect on the bacterial community composition. The FA group was far from the CK and LAB groups at 7 d of fermentation, which can be attributed to the higher number of keystone species in the FA group. Furthermore, the FA and LAB groups at 45 d of fermentation were adjacent in the PCoA plot, which can be attributed to the higher abundance of lactic acid bacteria in the FA and LAB groups.
Predicting the functional properties and metabolic pathways of microbial populations enables the evaluation of microbial influences on silage quality [21]. Our results are comparable to those of Li et al., who discovered that chemoheterotrophy is the primary functional component of the bacterial population in paper mulberry silage, followed by fermentation. As fermentation proceeds, the increase in lactic acid bacteria in mixed silage accelerates fermentation, whereas the decrease in aerobic microorganisms and the dominance of lactic acid bacteria leads to less aerobic chemoheterotrophy [47]. The presence of more homofermentative lactic acid bacteria in the FA group resulted in a higher relative abundance of fermentation and a lower relative abundance of aerobic chemoheterotrophy than in the other groups. Previous studies have shown that nitrogen fixation refers to the process by which certain bacteria convert N2 to its more active components (nitrate, nitrite, or ammonia). Therefore, the finding that the FA group at 45 d of fermentation contained a lower AN content was mutually supported by its lower nitrogen-fixing capacity than that of the other treatment groups [48].
KEGG is a bioinformatics resource for obtaining a genomic-level understanding of the function and utility of cells and organisms. Therefore, Tax4Fun-based KEGG analysis was performed to assess the effect of the various additives on the metabolic characteristics of the AL and PR mixed silages. In this study, the “genetic information processing”, “organismal systems” and “metabolism” pathways were promoted, and the “cellular process”, “environmental information processing” and “human disease” pathways were inhibited after 7 days of fermentation in the FA group compared to those in the CK group. These findings indicate that modified cellular characteristics in the FA and LAB groups hinder membrane transport and signal transduction in harmful microorganisms while increasing the rate of proliferation and metabolism of beneficial bacteria (e.g., lactic acid bacteria) [20]. The metabolic pathways at level 1 were altered less between treatments after 45 days of ensiling than after 7 days of ensiling. This could be because during the final stage of fermentation, a stable internal environment developed. In this study, the “carbohydrate metabolism” pathways in the FA and LAB groups were strengthened after 7 and 45 days of ensiling. This demonstrated that the bacteria from the FA and LAB groups had higher WSC metabolism and produced more fermentation compounds, such as LA. In addition, the relative abundance of “membrane transport” in the FA group was less than that in the CK group after 7 days of ensiling. During the fermentation process, some harmful microbes can use transfer proteins to acquire resources for growth and energy, so the FA group contains a limited number of dangerous germs. The building blocks of proteins are known as amino acids, and plants use them extensively for metabolism. The relative abundance of “amino acid metabolism” in the FA group was greater than that in the CK group after 7 days of ensiling. Du et al. demonstrated that amino acid metabolism includes both the generation of bioactive peptides such as antimicrobial peptides and the breakdown of proteins into amino acids and peptides. Therefore, the FA group contained fewer harmful microorganisms [49]. Interestingly, after 7 days of ensiling, the relative abundance of “Signal Transduction” in the FA group was greater than that in the CK group and lower than that in the CK group. Lactic acid bacteria were inoculated in the LAB group, so less “signal transduction” was required during the prefermentation period. In contrast, FA contains more “Signal transduction”, which might be attributable to the high number of key species in the FA group and the symbiotic relationships in the bacterial community [50]. The relative abundance of “lipid metabolism” in the FA group was greater than that in the CK group because lipid metabolites participate in the glycolytic pathway and glyoxalate cycle (acetyl coenzyme A) (dihydroxyacetone phosphate). As a result, increased lipid metabolism supports an increase in silage organic acid levels [51]. The metabolic pathways were specifically analyzed at the third level. The relative abundances of “biosynthesis of secondary metabolites” and “biosynthesis of antibiotics” in the FA and LAB groups were greater than those in the CK group after 7 and 45 days of ensiling, and similar results were found in the study of Fan et al. [14]. The relative abundances of “ABC transporters” in the FA group after 7 days of ensiling and in the FA and LAB groups after 45 days of ensiling were lower than those in the CK group. Because the pH of the FA and LAB groups was lower than that of the CK group, the acidic environment was able to restrict the transit of pathogenic bacteria through the membrane system [52]. Importantly, the relative abundance of “Microbial metabolism in diverse environments” in the FA group after 7 days of ensiling was greater than that in the CK group. We speculate that this difference is due to the enrichment of microorganisms in the FA group after 7 days of ensiling (Figure 2A). Quorum sensing is one of the major signaling mechanisms that directly aids in biofilm formation and regulates the pathogenesis of some plant pathogens [53]. The “two-component system” reacts to a wide range of environmental stimuli and serves as a crucial way for pathogens to communicate in the production of virulent components [54]. The relative abundances of “Quorum Sensing” and “Two-component system” bacteria in the FA and LAB groups, after 7 days of ensiling, were less than those in the CK group, which was attributed to the presence of fewer harmful bacteria in the FA and LAB groups. The lowest relative abundance of “Quorum Sensing” bacteria was found in the FA group after 45 days of ensiling, which was consistent with the bacterial composition of the FA group, where the least harmful bacteria were present (Figure 1B). The relative abundances of “Biosynthesis of amino acids” in the FA and LAB groups after 7 and 45 days of ensiling were greater than those in the CK group. These findings demonstrated that amino acid synthesis was inhibited in the CK groups and that an acidic environment promoted amino acid synthesis by bacteria. The “PTS” is a major mechanism used by bacteria for the uptake of carbohydrates [55]. The relative abundance of “PTS” was higher in the FA and LAB groups than in the CK group after 7 days of ensiling, while the relative abundance of “PTS” was lower in the FA and LAB groups than in the CK group after 45 days of ensiling. We hypothesize that in the early stage of ensiling, diverse bacterial activities are prevalent, resulting in high carbohydrate consumption and a high relative abundance of bacteria in the PTS, whereas in the late stage of ensiling, some bacteria cannot survive in an acidic environment, resulting in lower carbohydrate consumption and a low relative abundance of bacteria in the PTS.
BugBase analysis demonstrated that aerobic and facultatively anaerobic bacteria were gradually substituted by anaerobic bacteria, and Gram-negative bacteria were gradually substituted by Gram-positive bacteria in mixed silage, which is compatible with the silage fermentation process. The FA and LAB groups contained more anaerobic and Gram-positive bacteria and fewer aerobic, facultatively anaerobic, and Gram-negative bacteria than did the CK group, indicating that the FA and LAB groups had better fermentation quality. As fermentation proceeds, an acidic environment gradually forms, and lactic acid bacteria dominate, causing potential pathogenic bacteria to slowly disappear and leading to a reduction in the abundance of potentially pathogenic microorganisms. Notably, the lowest relative abundance of potentially pathogenic phenotypes was measured in the FA group, suggesting that the addition of FA is beneficial for reducing some of the pathogenic diseases that can occur in animals fed silage. The relative abundance of stress tolerance is associated with oxygen stress, and oxidative stress is thought to be an important factor in aging and disease [56]. With less air in the silage and the formation of an acidic environment, the oxidation capacity decreases; thus, the incidence of disease is reduced in animals.

5. Conclusions

According to our research findings, the addition of FA and LAB improved the silage quality of mixed alfalfa and perennial ryegrass silage, and FA addition contributed the most to fermentation quality and nutrient conservation. FA provided a sufficiently acidic environment that increased the relative abundance of lactic acid bacteria while restricting the growth of uncultured bacteria such as Hafnia obesumberium, Enterobacteria, and Sphingomonas. In addition, compared to those in the CK group, the FA group had higher abundances of “lipid metabolism” and “biosynthesis of antibiotics”, and lower abundances of “membrane transport”. This information is useful for combining forage resources to meet demand for high-protein feed in karst regions, and for producing ruminant feed annually.

Author Contributions

Y.L. (Yao Lei): Data curation, Visualization, Writing—original draft. M.L.: Formal analysis, Investigation, Y.L. (Yinghao Liu) and Y.C.: Investigation. J.W., X.H. and Y.Z. contributed to the grammar and language revision of the paper and contributed to this experiment. Q.C.: Conceptualization, Methodology, Validation, Writing—review and editing, Supervision, Funding acquisition. C.C.: Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by the National Key Research and Development Subject (2021YFD1300302), the Guizhou Provincial Science and Technology Project (Qiankehe Chengguo [2022]005), the Guizhou Provincial Science and Technology Project (Qiankehe Jichu-ZK [2023]120), the Guizhou Provincial Science and Technology Project (QKHPTRC-CXTD [2022]011), and the Guizhou University Cultivation Project (Guida Peiyu [2020]9).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Our raw sequence data are available under NCBI project PRJNA778048 with accession number SRP344966.

Conflicts of Interest

The authors declare no conflicts of interest. The authors declare that they have no competing interests or personal relationships which might have potentially influenced the results discussed in this paper.

Abbreviations

AL: Alfalfa; PR: Perennial ryegrass; LA: Lactic acid; AA: Acetic acid; BA: Butyric acid; DM: Dry matter; LAB: Combined application of Lactiplantibacillus plantarum and Lentilactobacillus buchneri; FA: Formic acid; CP: Crude protein; WSC: Water-soluble carbohydrate; FM: Fresh matter; FW: Fresh weight; NH3-N: Ammonia–nitrogen; PA: Propionic acid; ANOVA: Analysis of variance; OTUs: Operational classification units; cfu: Colony-forming units

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Fermentation 10 00043 g001
Figure 1. The relative abundance of bacteria at the phylum (A) and genus (B) levels in mixed silage of alfalfa and perennial ryegrass treated with different additives. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
Figure 1. The relative abundance of bacteria at the phylum (A) and genus (B) levels in mixed silage of alfalfa and perennial ryegrass treated with different additives. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
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Fermentation 10 00043 g002
Figure 2. The LDA value (A) distribution and evolutionary branch of different species in mixed silage consisting of alfalfa and perennial ryegrass treated with different additives. The principal coordinate analysis (PCoA) plot at the operational taxonomic unit (OTU) level (B) in mixed silage of alfalfa and perennial ryegrass. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
Figure 2. The LDA value (A) distribution and evolutionary branch of different species in mixed silage consisting of alfalfa and perennial ryegrass treated with different additives. The principal coordinate analysis (PCoA) plot at the operational taxonomic unit (OTU) level (B) in mixed silage of alfalfa and perennial ryegrass. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
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Fermentation 10 00043 g003
Figure 3. The dynamics of predicted microbial functional profiles of the relative abundances of the top 10 functions in mixed silage containing alfalfa and perennial ryegrass treated with different additives. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
Figure 3. The dynamics of predicted microbial functional profiles of the relative abundances of the top 10 functions in mixed silage containing alfalfa and perennial ryegrass treated with different additives. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
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Fermentation 10 00043 g004aFermentation 10 00043 g004b
Figure 4. “*” indicates significant differences between treatments. Bar graphs showing 16S rRNA gene-predicted functional profiles on the level 1 metabolic pathways (A), level 2 metabolic pathways (B), and level 3 metabolic pathways (C) of mixed silage consisting of alfalfa and perennial ryegrass treated with different additives for 7 and 45 days obtained with Tax4Fun. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
Figure 4. “*” indicates significant differences between treatments. Bar graphs showing 16S rRNA gene-predicted functional profiles on the level 1 metabolic pathways (A), level 2 metabolic pathways (B), and level 3 metabolic pathways (C) of mixed silage consisting of alfalfa and perennial ryegrass treated with different additives for 7 and 45 days obtained with Tax4Fun. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri.
Fermentation 10 00043 g004aFermentation 10 00043 g004b
Fermentation 10 00043 g005
Figure 5. BugBase feature prediction included the aerobic, anaerobic facultative anaerobic, mobile element content, potential pathogenicity, stress tolerance, biofilm formation, and Gram-negative features of mixed silage consisting of alfalfa and perennial ryegrass treated with different additives for 7 and 45 days. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri. "*" means significant difference between different treatments, and "**" means very significant difference between different treatments.
Figure 5. BugBase feature prediction included the aerobic, anaerobic facultative anaerobic, mobile element content, potential pathogenicity, stress tolerance, biofilm formation, and Gram-negative features of mixed silage consisting of alfalfa and perennial ryegrass treated with different additives for 7 and 45 days. CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri. "*" means significant difference between different treatments, and "**" means very significant difference between different treatments.
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Table 1. Effects of additives on the dynamics of chemical compositions in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
Table 1. Effects of additives on the dynamics of chemical compositions in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
ItemsTreatmentEnsiling Period (D)SEMp-Value
(T)Day 7Day 15Day 45TDT × D
DMCK21.09 Ab17.78 Bb17.29 Bb
%FMLAB20.71 Ab17.41 Bb17.27 Bb0.100 <0.001<0.0010.827
FA22.36 Aa18.90 Ba18.70 Ba
CPCK16.62 Ac16.48 Ac15.70 Ba
%DMLAB18.13 Ab17.29 Ab16.05 Ba0.082 <0.001<0.0010.002
FA19.39 Aa18.75 Aa16.12 Ba
NDFCK56.76 Aa51.87 Ba51.62 Ba
%DMLAB51.63 Ab49.48 Bb46.53 Cb0.244 <0.001<0.0010.041
FA48.84 Ab48.57 Ab46.63 Bb
ADFCK46.79 Aa42.84 Ba37.96 Ca
%DMLAB42.13 Ab35.78 Bb34.59 Cb0.343 <0.001 <0.0010.222
FA43.40 Ab34.40 Bb34.13 Cb
WSCCK3.42 Ab2.70 Ab1.69 Bc
%DMLAB3.50 Ab2.46 Bb2.46 Bb0.056 <0.001<0.0010.034
FA5.04 Aa4.07 Ba4.04 Ba
A–C Means of ensiling days within a column with different superscripts differ in the same additive treatment (p < 0.05); a–c Means of additive treatment within a row with different superscripts differ in the same ensiling day (p < 0.05); DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri; SEM, standard error of the mean; T, treatment; D, ensiling days; D × T, ensiling days and treatment interaction.
Table 2. Effects of additives on the dynamics of fermentation quality in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
Table 2. Effects of additives on the dynamics of fermentation quality in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
ItemsTreatmentEnsiling Period (D)SEMp-Value
(T)Day 7Day 15Day 45TDT × D
pHCK4.96 Aa4.83 ABa4.67 Ba
LAB4.59 Ab4.58 Abb4.47 Bb0.022<0.0010.0480.232
FA4.14 Ac4.07 ABc4.06 Bc
LACK0.24 Cc3.62 Bc4.35 Ac
%DMLAB1.81 Cb4.53 Bb5.71 Ab0.103 <0.001<0.0010.288
FA2.03 Ca5.48 Ba6.22 Aa
AACK0.14 Bc1.52 Ab1.54 Ab
%DMLAB2.15 Aa2.20 Aab2.91 Aa0.091 <0.001<0.0010.099
FA1.22 Bb2.75 Aa2.47 Aa
PACK0.240.21-
%DMLAB0.18------
FA---
BACK---
%DMLAB-------
FA---
NH3-NCK2.82 Ca3.56 ABa4.36 Aa
%DMLAB1.86 Cb2.12 Bb3.17 Ab0.109<0.001 0.0010.646
FA1.57 Cb2.45 Bb2.58 Ab
A–C Means of ensiling days within a column with different superscripts differ in the same additive treatment (p < 0.05); a–c Means of additive treatment within a row with different superscripts differ in the same ensiling day (p < 0.05); CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri; T, treatment; D, ensiling days; D × T, ensiling days and treatment interaction; SEM, standard error of the mean; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; NH3-N, ammonia nitrogen.
Table 3. Effects of additives on the diversity and richness of the bacterial microbiota in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
Table 3. Effects of additives on the diversity and richness of the bacterial microbiota in mixed silage consisting of alfalfa and perennial ryegrass during ensiling.
ItemsTreatmentEnsiling Period (D)SEMp-Value
(T)Day 7Day 45TDT × D
Observed speciesCK529 a465 a
LAB470 b445 a2.7900.003<0.001<0.001
FA465 b446 a
ACECK559.06 a476.53 a
LAB486.48 c479.33 a2.059<0.001<0.001<0.001
FA517.94 b490.50 a
Chao1CK564.95 a491.34 a
LAB514.31 b497.33 a2.1840.003 <0.0010.001
FA532.39 b488.77 a
SimpsonCK0.92 ab 0.90 a
LAB0.86 b0.75 b0.008<0.001 <0.001<0.001
FA0.96 a0.67 c
ShannonCK5.12 b4.51 a
LAB4.48 b3.44 a0.01410.040<0.0010.019
FA6.24 a3.44 a
CoverageCK0.9980.997
LAB0.9980.999
FA0.9970.998
a–c Means of additive treatment within a row with different superscripts differ in the same ensiling day (p < 0.05); CK, without additives; FA, formic acid; LAB, Lactiplantibacillus plantarum combined with Lentilactobacillus buchneri; T, treatment; D, ensiling days; D × T, ensiling days and treatment interaction; SEM, standard error of the mean; OTU, operational taxonomic uni.
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Lei, Y.; Li, M.; Liu, Y.; Wang, J.; He, X.; Zhao, Y.; Chen, Y.; Cheng, Q.; Chen, C. Lactic Acid Bacteria and Formic Acid Improve Fermentation Quality and Beneficial Predicted Functional Characteristics in Mixed Silage Consisting of Alfalfa and Perennial Ryegrass. Fermentation 2024, 10, 43. https://doi.org/10.3390/fermentation10010043

AMA Style

Lei Y, Li M, Liu Y, Wang J, He X, Zhao Y, Chen Y, Cheng Q, Chen C. Lactic Acid Bacteria and Formic Acid Improve Fermentation Quality and Beneficial Predicted Functional Characteristics in Mixed Silage Consisting of Alfalfa and Perennial Ryegrass. Fermentation. 2024; 10(1):43. https://doi.org/10.3390/fermentation10010043

Chicago/Turabian Style

Lei, Yao, Maoya Li, Yinghao Liu, Jiachuhan Wang, Xiangjiang He, Yuanyuan Zhao, Yulian Chen, Qiming Cheng, and Chao Chen. 2024. "Lactic Acid Bacteria and Formic Acid Improve Fermentation Quality and Beneficial Predicted Functional Characteristics in Mixed Silage Consisting of Alfalfa and Perennial Ryegrass" Fermentation 10, no. 1: 43. https://doi.org/10.3390/fermentation10010043

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