Variation in the chemical composition of triticale silage
Triticale, rich in starch, protein, and fiber, offers high yields and shows rapid growth and drought resistance. However, its hollow stem makes it susceptible to aerobic deterioration [25]. Appropriate additives can improve the triticale silage quality and enhance livestock production.
Cereal crop silage is widely used as a livestock feed because it is rich in proteins and starch. To the best of our knowledge, LAB do not utilize starch during silage fermentation. However, the starch content in the S. bovis-treated group was lower than in the L. plantarum-treated and CON groups at an early stage. This difference might be because the α-amylase secreted by S. bovis acts on starch molecules, cutting the glycosidic bonds through hydrolysis, breaking starch into smaller sugar molecules, and converting insoluble starch into soluble sugar [26, 27]. In contrast, Jones et al. demonstrated that starch in alfalfa silage was not hydrolyzed by S. bovis as expected [10]. The reason for this difference may be that the low starch content in alfalfa (1–3%) insufficiently supports the results presented, whereas the higher starch content in triticale (12–15%) was more likely to be significant during statistical analysis.
In addition, S. bovis and L. plantarum had a synergistic effect that enabled them to utilize WSC and rapidly reduce the pH value. In line with this, several studies have demonstrated that compared with LAB-based silage, the use of S. bovis results in a reduction of 30% or more in the doubling time [27, 28], leading to faster pH reduction in silage, similar to Enterococcus faecium [10]. Previous studies have shown that LAB additives reduce DM and CP loss [29, 30]. In the present study, LAB treatment resulted in less DM loss than in the CON group. This difference may be attributed to swift fermentation leading to rapid pH reduction, inhibiting the growth of detrimental bacteria such as Enterobacterium and Clostridium. Furthermore, LAB produce antimicrobial substances such as organic acids, hydrogen peroxide, and bacteriocins, which inhibit the growth of harmful bacteria [31].
Variation in the fermentation characteristics of triticale silage
LAB produce a significant amount of LA during ensiling, thus reducing the pH [32]. Consequently, pH is a critical parameter for evaluating silage fermentation efficiency [33]. In this study, the pH values of all groups decreased below 4.0, suggesting that fresh triticale could be effectively fermented and preserved with or without the inoculant. This may be due to the high WSC content, low buffering capacity, and sufficient epiphytic LAB content of the raw materials [34]. Nevertheless, the ST and LS groups inoculated with S. bovis exhibited higher LA production and lower pH during the early stages of ensiling. Generally, for silage, lower pH values result in higher LA production [35, 36]. This result is verified in the present study. The higher LA concentration in the inoculated group could be due to the shorter time and greater number of LAB required to produce organic acids during the early stages. All groups underwent homofermentative fermentation (LA/AA > 3.0). On days 3 and 7, the LA and AA concentrations were higher in the ST and LS groups, but their LA/AA ratios were significantly lower than those in the CON and LP groups. In contrast, Zhao et al.[30] reported that Leuconostoc lactis and Weissella confusa had relatively high LA/AA ratios during ensiling. The reason for this discrepancy could be that S. bovis functions as a quick starter during fermentation, encouraging the growth of homofermentative LAB(producing LA) at the beginning of fermentation, while accelerating the growth of heterofermentative LAB (producing AA). However, with decreasing pH, the growth of S. bovis was inhibited. This also explains why AA production and LA/AA ratio converged at a later stage. Typically, propionic acid and BA are undesirable products in silage because their production results in energy wastage, and the PA content of high-quality silage should be between 1 and 10 g/kg DM [37, 38]. In the present study, PA concentrations were low across all groups (1.06–7.50 g/kg DM), and BA was undetected. This also indicated no secondary fermentation during ensiling, indicating the excellent quality of silage fermentation. Notably, the PA concentrations in the ST and LS groups with S. bovis were lower than those in the CON and LP groups. This may be attributed to the involvement of S. bovis, which inhibits the growth of some PA-producing bacteria, and its low pH, which restricts the conversion of lactate to propionate. Typically, clostridia decompose proteins to produce AN, which results in poor fermentation quality. However, our study did not detect BA, indicating that harmful bacteria such as clostridia were inhibited. Some LAB produce proteases during ensiling, which further generate AN via protein degradation [39, 40]. This may explain why the LS group had the highest LA concentration and lowest pH but still had a higher AN content in the early stage.
Variation in the bacterial community of triticale silage
Previous studies have elucidated the role of S. bovis in silage by analyzing its chemical properties and fermentation characteristics [10, 11, 15]. The 16S rRNA sequencing technology can reveal microbial diversity and dynamic variations more accurately and comprehensively than traditional methods. To the best of our knowledge, this is the first study to use NGS technology to assess the effects of S. bovis in silage on microbial communities and to predict variations in metabolic function. The coverage of fresh triticale and silage was more than 0.99, indicating that most of the target gene regions for sequencing were read and covered, thereby ensuring data integrity and accuracy [41]. Bacterial diversity is generally lower in low pH value silage, and pH largely determines the bacterial α-diversity [42]. Similar results were observed in our study, where inoculation with LAB reduced α-diversity compared to CON, and the combined addition of L. plantarum and S. bovis further intensified this effect. This may be attributed to the rapid proliferation of S. bovis and the antibacterial properties of L. plantarum, which inhibit undesirable bacterial growth [10, 43].
Additionally, combining the results of the PCoA and Venn plots further explained the impact of microbial community differences between the treatments on ecological functionality and environmental adaptability. The PCoA results revealed that anaerobic fermentation significantly influenced the microbial composition of triticale, leading to a distinct separation among all treatments during the early ensiling stage (Fig. 1). When compared to CON, the Venn plots indicated that the unique ASVs were reduced in all treatments on day 7 but increased on day 30; conversely, common ASVs decreased during ensiling (Fig. 2). This could be attributed to the promotion of LA production by inoculation in the early stages, creating an acidic environment that inhibits the growth of aerobic and other undesirable bacteria, further facilitating the proliferation of LAB in the later stages [25].
Microbial communities play a crucial role in the silage fermentation quality [25]. In this study, we observed a clear succession of Proteobacteria to Firmicutes before and after fermentation, which is consistent with the literature [25, 29, 30]. This can be mainly attributed to the acidic environment, which suppresses the growth of acid-sensitive bacteria (Enterococcus, Staphylococcus, Yeast, Mold, etc.) while promoting the proliferation of LAB. Moreover, Firmicutes can degrade complex organic compounds found in silage, including starch, proteins, and cellulose [44]. In the present study, the ST group(day 7) showed the highest abundance of Firmicutes. In addition, S. bovis increased starch hydrolysis, resulting in more WSC for Firmicute utilization and further increasing its abundance.
At the genus level, Rosenbergiella, Enterobacter, and Pantoea were the predominant genera in fresh triticale, which is consistent with the results of previous studies [45, 46]. Rosenbergiella and Pantoea compete with LAB for sugars, whereas Enterobacter produces ammonia and other putrefaction products that are detrimental to fermentation. After ensiling, the abundance of Rosenbergiella, Enterobacter, and Pantoea significantly decreased and were replaced by LAB and Weissella. This can be attributed to the anaerobic and acidic environment, which limited the growth of these undesirable bacteria. LAB remain unaffected because of their vital acid tolerance, and organic acids produced by LAB proliferation further reduce the pH and inhibit undesirable bacteria [47]. Moreover, the abundances of Rosenbergiella, Enterobacter, and Pantoea in the ST and LS groups were significantly lower than those in the CON and LP groups after 7 days. This might be because S. bovis can quickly oxidize oxygen, which helps acid-resistant L. plantarum to lower the pH and inhibit harmful bacteria. Additionally, S. bovis produces antibacterial substances, such as organic acids and bovicin HC5 [10, 13], which suppress undesirable bacteria in silage.
Weissella, Lactococcus, and Pediococcus are commonly encountered during the initial stages of silage fermentation [48]. Yang et al. [49] reported that with pH reduction, acid-tolerant Lactobacilli replaced Weissella, Pediococcus, and Lactococcus during ensiling, which is similar to the results of our study. In this study, inoculation with S. bovis and L. plantarum increased the proportion of Pediococcus and Lactococcus in silage on day 7 compared to that in the CON group. On day 30, the CON group had significantly higher Weissella and Lactococcus abundance than the other groups. This may be because the inoculant promotes LAB growth during the early stages, whereas the CON group without additives exhibits slower fermentation. Additionally, the abundance of S. bovis decreased significantly over time, which was consistent with the changes in Lactococcus and Pediococcus. This suggests that S. bovis rapidly initiates fermentation during the early stages to create favorable conditions for other LAB; however, its growth is gradually inhibited as the pH decreases.
Variation in bacterial metabolism of triticale silage
Microorganisms convert substrates into various metabolites via complex metabolic pathways that affect silage fermentation quality [37]. KEGG analysis showed that most of the metabolic pathways were involved in metabolism (Fig. S1), indicating that metabolism may be the most critical factor influencing triticale silage fermentation. At level 2, carbohydrate, amino acid, energy, and other amino acid metabolic pathways were upregulated after 30 days. A previous study showed a significant positive correlation between the metabolism of amino acids and carbohydrates and the main products of high-quality silage [50]. Amino acid decarboxylation, malate decarboxylation, and arginine deamination are the three main energy metabolic pathways involved in LA accumulation during LAB fermentation [51]. These upregulated metabolic pathways indicated that bacteria (presumably lactobacilli) remained active, breaking down polysaccharides into monosaccharides and LA. Correspondingly, the upregulated groups exhibited a higher levels of lactobacilli abundance and LA production in this study. Nutrient loss can occur owing to respiration, proteolysis, and lipolysis during the initial stages of ensilage. Proper ensilage techniques help maintain the nutrient content by minimizing losses due to spoilage and respiration [14]. In the present study, lipid metabolism pathways were downregulated on day 30 compared to day 7. This may be because plant matter is primarily fermented by LAB, which convert sugars into LA, thus lowering pH, inhibiting undesirable bacteria, and preserving nutrients, including lipids, in the silage [52]. Furthermore, our results indicated that the CON group showed significantly lower levels of upregulated pathways and significantly higher levels of downregulated pathways than the inoculant-treated group. This was consistent with the higher pH, lower lactate production, and lower LAB abundance observed in the CON group. The lack of additional LAB may explain the delayed fermentation, lower LA production, and higher pH values in the CON group.
In order to investigate the utilization of sugars in silage using additives, we analyzed carbohydrate metabolism. During ensiling, starch and sucrose provide energy, and their metabolism produces organic acids such as LA, which are crucial for lowering silage pH and preventing the accumulation of spoilage organisms [53]. Amino sugar and nucleotide sugar metabolism produces intermediates for synthesizing cellular components, including those that improve silage quality, such as 3-phosphoglycerate, amino acids, and coenzyme A [14]. Interestingly, on day 7, the group inoculated with S. bovis showed significant upregulation of these two metabolic pathways compared to the non-inoculated group. Similar results were obtained for glycolysis/gluconeogenesis and galactose metabolism. Upregulation of these metabolic pathways indicates that microorganisms, particularly S. bovis, actively ferment sugars to produce LA and other organic acids. This is consistent with the fermentation and microbial composition results.
Correlation between carbohydrate content, fermentation characteristics, and microflora
Correlations between microbial composition and various chemical indicators provide a deeper understanding of the fermentation mechanisms of silage and the microbiological basis of its quality. During ensiling, the interaction between the chemical composition and microorganisms affects the metabolic products of the final silage. These parameters are positively correlated with beneficial bacteria and negatively correlated with harmful bacteria [54]. In this study, Streptococcus and Lactococcus positively affected pH, LA, AA, and LA/AA but negatively affected PA. Our findings suggest that their involvement in early fermentation leads to organic acid production and decreased pH, consistent with previous studies [55, 56]. PA is undesirable for fermentation because of its potential energy wastage [37].
Typically, Lactobacillus dominates silage fermentation and positively affects pH and organic acids [5, 14]. However, this study found no significant correlations between Lactobacillus and WSC, pH, LA, or AA levels. This may be because of the ecological niche overlap between Lactobacillus and Streptococcus during the early stages of silage production, resulting in no statistically significant correlation with Lactobacillus. In addition, we found a significant negative correlation between Pantoea and AN, consistent with the findings of Ogenade et al., who reported that an increased Pantoea abundance could reduce AN concentrations in silage [57]. However, the specific mechanism through which Pantoea degrades AN remains unclear and requires further investigation.