Characteristics of triticale before and after ensiling
The characteristics of silage raw materials are important factors affecting the fermentation quality of silage. In this research, raw triticale has high nutritional value to ensilage. Triticale silage, however, is susceptible to aerobic deterioration due to its hollow stem. The present surroundings provide a suitable atmosphere for the proliferation and development of deleterious microorganisms. Therefore, the finding of suitable additives is of great significance for inhibiting harmful bacteria and protecting silage. Bacterial inoculants have been developed to shorten the primary fermentation process and achieve a good fermentation quality. Bacillus spp, a facultative anaerobic bacterium, can consume oxygen sources to create an anaerobic environment, which stimulates lactic acid bacteria growth [12] and the acidification of silages, consequently decreasing the nutrient loss by inhibiting the growth of spoilage microbes. The degree of protein degradation in silage is frequently determined by the accumulation of NH3-N (typically less than 10–15% of total nitrogen) during the ensiling process [22]. In general, ruminants absorb NH3-N less efficiently than true protein. In this research, the inoculants had decreased the concentration of NH3-N. Proteolysis of ensiled triticale inhibited most by the combination of LP and BC [23]. This may be due to the rapid drop in pH, which inhibits the activity of plant proteases and decomposable protein strains [24], as well as the bacteriostatic effect of B. coagulans in suppressing mold activity and reducing the conversion efficiency of protein substances to NH3-N. The decrease of NDF and ADF contents suggested that plant structural carbohydrate was degraded, which may be attributed to the Bacillus' production of fibrinolytic enzymes during the silage fermentation process [25]. The results suggested an improved digestibility. Gao et al. reported that the WSC concentration of the LP treated straws was lower than that of the control silage [26]. This is in accordance with our study where silages with LP or BC inoculants had fewer WSC residues. Furthermore, the synergistic effect of the inoculants was observed to reduce the concentrations of WSC, as anaerobic bacteria decompose the soluble sugar into lactic acid during the second stage of fermentation [27].
A rapid pH drop of ensiling plays a crucial role inhibiting undesirable microorganism and decreasing nutrients losses during ensiling [28]. The pH of the LP groups was all below 4.2, which is the criteria for high-quality silage. L. plantarum, a homofermentative bacterium, can encourage the formation of lactic acid, which is compatible with the results of this study [29]. The highest concentration of LA was observed in the LP + BC-treated silage, likely due to the accelerating of the growth of homofermentative LAB rapidly and effectively metabolizing WSC to LA during fermentation by BC. Lactic acid is capable of inhibiting the growth of deleterious organisms and reducing the formation of BA. However, the decrease in AA observed in ensiled triticale after LP inoculation is likely attributable to the suppressive effect of homofermentative LAB on heterofermentative LAB. The growth and reproduction of molds and fungi can be effectively inhibited by propionic acid, which also has the capability to curtail the occurrence of secondary fermentation. This acid can promote the growth of LAB to a certain extent, thereby regulating the silage process and minimizing the decomposition of proteins [30, 31]. Butyric acid is usually present in the fermentation by Clostridium and represents poor fermentation [8]. The combination of LP and BC was better for enhancing the silage quality of triticale silage in terms of nutritional content and fermentation characteristics.
Opening the silage bag exposes the aerobic microorganisms in the silage to oxygen, allowing them to multiply rapidly using the nutrients present. Inoculation with either LP or BC alone, or a combination of both, has been proven to prolong the aerobic stabilization time of triticale silage. This is likely due to LP's ability to establish a stable acidic environment, which impedes the growth of spoilage bacteria and prevents yeast from metabolizing lactic acid and WSC [32]. Additionally, B. coagulans has been observed to produce antibiotics, which inhibit the proliferation of harmful microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella and Candida albicans [33]. The LP + BC-treated silage exhibited the greatest aerobic stability, indicating a synergistic effect between the two.
Microbial communities of triticale silage
The microbial community and their metabolites play an essential role in the fermentation quality of silage. The application of inoculants has a significant effect on the competition among microbes. Based on our findings, it can be inferred that the sequencing data was deemed satisfactory for precise analysis of the bacterial population, given that the coverage value for each treatment was 0.99. The Shannon and Chao1 indices serve as indicators of bacterial community diversity and richness, respectively [34]. The bacterial diversity was lower and the richness was higher in the silages treated with LP or BC alone, which is in line with the findings of Liu et al. [6]. This may be due to the antibacterial activity of BC and lower pH [33].
The β-diversity analysis was utilized to assess the disparity of bacterial communities across samples, and the microbial composition of triticale silage was significantly impacted by ensiling in this study. The PCoA results indicated that a single additive could significantly modify the bacterial composition of triticale silage. Moreover, the inoculation with LP was found to be the primary factor of alterations during anaerobic fermentation. The Venn diagram revealed that BC augmented the total and unique OTU counts in comparison to the control silage. This could be attributed to the inhibited activity of BC and reduced production of bacteriocin [14]. The interaction between LP and BC prevented the growth of certain microorganisms in triticale silage by enhancing the production of lactic acid, thus decreasing the pH of silage and creating an acidic environment, further inhibiting the proliferation of Clostridium and other undesirable bacteria.
The ensiling process is primarily driven by bacterial fermentation, whereby the microflora present on the plant material plays a crucial role in determining the quality of silage produced. In the current investigation, it was observed that Cyanobacteria dominated the microbial community on triticale prior to ensiling, with a subsequent shift towards Firmicutes during the ensiling process. This finding is consistent with the results of a previous study conducted by Yuan et al., which reported a marked increase in the abundance of Firmicutes in Napier grass silage with prolonged storage time [35]. Firmicutes are gram-positive bacteria that could break down a variety of macromolecules, including cellulose, protein, and starch. After silage fermentation, there is an increase in Firmicutes species such Lactobacillus and Enterococcus. This may result in the reduction of NDF, ADF, and NH3-N in silage
We assessed bacterial communities at the genus and species levels in order to future reveal the compositions of the bacterial communities in triticale silage that had been inoculated with the strains of LP and BC. Apparently, Enterococcus, Pediococcus and Weissella have been observed by many researchers in the untreated barley silage [36], oat silage [37] and rice straw silage [38]. To date, it has been identified that Pediococcus and Weissella are regarded as unwanted in silage and encourage the development of microbes that cause spoilage. The addition of LP or BC resulted in a decrease in the relative abundance of Pediococcus and Weissella compared to a control group. In addition, the most abundant genus was Lactobacillus, which is essential for the decrease in pH during the later stages of ensiling and provides a stable environment [39]. These results indicated that exogenous microorganism could change the composition of bacterial community in triticale silage. In the present study, the dominant bacteria composition in the treatment group was similar, but there was some difference degree of relative abundance. The LP + BC-treated silage had a lower abundance of Lactobacillus than LP-treated silage, yet the fermentation quality was higher due to the addition of BC, which rapidly consumes oxygen and initiates fermentation, and acid-resistant LP, which helps to further reduce the pH. Additionally, higher abundance of Enterococcus and Serratia was found in the LP + BC- treated silage, which might suggest that the synergy of the two additions is good for the growth of Enterococcus and Serratia. Enterococcus is a kind of cumulative anaerobic bacterium that mostly produces L (+)-LA, contributes to enhancing the fermentation quality of silage. Prodigiosin, which are secondary metabolites produced by Serratia, may inhibit the growth of the fungus [40]. The greater aerobic steadiness in the LP + BC- treated silage could be attributable to this.
Ensiling promoted Lactobacillus, Enterococcus, and Bacillus proportion, which may have aided the fermentation process in creating an acidic environment that inhibited most microorganisms, as suggested by Cai et al [41]. Acetobacter microorganisms produce AA, which can cause aerobic deterioration of silage [42]. Acetobacter aceti is suitable for growing in an environment with a pH of about 7.0, inhibiting the growth of LAB and leading to silage fermentation, which may be one of the reasons for the high pH and AA of the BC-treated silage.
The correlation between fermentation characteristics and microflora
A strong association between the top 8 known bacterial genera and 10 environmental parameters was found using Spearman analysis. Lactobacillus displayed a beneficial impact on LA and LA/AA, while also exhibiting a detrimental effect on NH3-N, as reported by Yang et al [43]. Additionally, propionic acid was found to be highly soluble and permeable, thus altering the microbial composition of silage during early stages of fermentation, resulting in an increase in lactic acid bacteria content and promoting silage fermentation [44, 45]. In this study, propionic acid created conditions conducive to the survival of LP by inhibiting the growth of Weissella and Pediococcus, so that LP rapidly proliferate, thus increasing the content of lactic acid bacteria and promoting silage fermentation. The results showed that propionic acid could effectively inhibit the spoilage and growth of undesirable microorganisms during fermentation. Bacillus had negative effects on neutral detergent fiber and acetic acid. This could be due to the fact that Bacillus bacteria can synthesize a range of digestive enzymes, such as galase, glucanase and cellulase, which assist in the degradation of complex carbohydrates in feed [46]. Additionally, Bacillus could also produce a variety of metabolites, including LA, antibiotics, and coagulin, which inhibit the growth of pathogenic and spoilage bacteria.