1. Introduction
The shortage of feed resources and the rising prices of feed lead to the high production cost of the breeding industry, which is one of the most important factors restricting the development of the modern breeding industry [
1]. To solve this problem, research and exploration have been carried out in the hope of finding new sources of cheap feed. Hybrid
Pennisetum (HP) is a triploid hybrid produced by the
Pennisetum americanum and
P. purpureum [
2]. It has the characteristics of strong adaptability, fast growth rate, high yield, good palatability, high crude protein content, more balanced amino acid content, and rich nutrition; as such, it is a high-quality feed for feeding poultry [
3]. Through silage processing, not only can the nutritional value of HP be preserved to the greatest extent, but also the palatability can be improved to a certain extent. However, due to the high buffering capacity (BC), low water-soluble carbohydrate (WSC), and the lack of lactic acid bacteria, HP was difficult to silage [
4]. Therefore, foreign additives such as inorganic additives, organic additives, and microbial agents are usually added in the ensilage process to improve the success rate of ensilage.
Lactobacillus plantarum (LP) belongs to homogenous fermentation lactic acid bacteria, which is currently one of the most commonly used microbial preparations in silage production. Studies have shown that using LP in the silage process can inhibit the growth of yeast and mold, avoid nutrient loss caused by aerobic degradation of silage, and improve the fermentation quality of silage with the increase of the concentration of LP [
5,
6,
7], probably because it can quickly generate a large amount of lactic acid, reduce environmental pH, inhibit harmful microbial activity, reduce the decomposition of protein and WSC, reduce the loss of nutrients in feed, and significantly improve the success rate of silage and the nutritional value [
8,
9].
Bacillus licheniformis (BL) is a Gram-positive thermophilic bacterium commonly found in soil [
10]. It not only has a unique biological oxygen capture mechanism to rapidly create an anaerobic environment but also can produce antibacterial active substances to inhibit the growth and reproduction of pathogenic bacteria [
11]. Studies have shown that BL is deemed safe for the intended species, humans who consume products made from animals that were fed treated silage, and the environment [
12]. BL has strong activities of protease, lipase, and amylase. It can promote the degradation of nutrients in feed so that animals can absorb and utilize feed more fully. Mara G. et al. found that BL had a strong ability to degrade fiber [
13]. Similarly, C. A. Oliveira et al. used BL to treat animal feed, which could significantly improve the digestibility of neutral detergent fiber (NDF) and starch in feed and then improve the feed utilization rate [
14]. At present, there are few studies on the application of BL in silage, and its influence on silage needs further study.
The HP was used as the silage material, and silage was prepared by using different concentrations of LP and BL. The study aimed to study the effects of LP and BL on the quality of HP silage and their optimal dosage and to provide a reference for their application in the silage processing industry.
4. Discussions
Silage is one of the most common methods used in animal husbandry to preserve green fodder. The production principle of silage is to make lactic acid bacteria proliferate in an anaerobic environment, consume oxygen to quickly produce a large amount of lactic acid, and reduce the pH value of the environment, thereby inhibiting the activity of harmful microorganisms, so that feed nutrients can be preserved [
22].
During the ensilage process, with the vigorous activities of plant respiration and aerobic microbes, nutrients in the feed are constantly consumed, resulting in the production of water, carbon dioxide, and free ammonia, which leads to the decrease of DM content in silage [
23]. LP is a homogenous fermentative lactic acid bacterium that can quickly produce a large amount of lactic acid during storage, reduce the environmental pH, inhibit the activities of harmful microorganisms such as yeast and mold, avoid nutrient loss caused by aerobic degradation of silage, and significantly improve the success rate and nutritional value of silage [
6]. This was consistent with the results of this study, and DM content in the LP group was higher than that in the control group. BL can produce anti-active substances that have a unique mechanism of biological oxygen uptake and can inhibit the growth and reproduction of pathogenic bacteria [
9]. This may be the reason for the higher DM content in the BL5 group than in the control group. However, the BL has strong protease, lipase, and amylase activity, which can promote the degradation of nutrients in the feed [
24,
25]. Therefore, the DM in the BL7 group was significantly lower than that in the BL5 group. CP is one of the most important nutrient elements in the feed, the content of which is an important indicator of evaluating the quality of silage. In this experiment, the addition of LP had no significant effect on the CP content of HP silage. Furthermore, the BL5 group was significantly higher than the LP5 group and the control group, which may be because BL can effectively inhibit the degradation of CP by microorganisms in silage. However, with the increase of BL addition, the CP content gradually decreased, which may be caused by the protease contained in BL, and it also explains that high concentrations of BL will deplete nutrients in silage and reduce DM content [
13]. The high content of NDF and ADF in the feed will affect the feed intake and digestibility of animals [
26]. In this experiment, the use of LP had no significant effect on the NDF content of the HP silage but decreased the ADF content of the silage, which is consistent with the findings of Wang et al. and Zhang et al. [
27,
28]. Moreover, Mara G et al. also found that
Bacillus licheniformis had a strong ability to degrade fiber, and its main effect may be to promote the decomposition of ADF [
13]. WSC is an energy source for microbial activity in silage, and rapid depletion of WSC is often accompanied by strong microbial activity [
29]. Compared with the control group, the WSC content of the LP group was significantly increased, and the LP7 group was significantly higher than the control group, indicating that
Lactobacillus plantarum can effectively inhibit the colonization of harmful microorganisms, reduce the consumption of WSC, and improve the nutritional value of feed, and the result showed a dose-dependent effect. There was no significant difference in WSC between the BL group and the control group.
pH is one of the important indicators to evaluate the quality of silage. Most harmful bacteria will not survive in acidic environments, so the pH below 4.2 is considered one of the criteria for quality silage [
30]. In this experiment, the pH values of the treatment groups were all lower than 4.2, which met the standard of high-quality silage. AN is an index reflecting the decomposition degree of amino acids and proteins in silage. The higher the value is, the greater the decomposition degree of protein is, and the worse the fermentation quality is [
31]. Moreover, the absorption and utilization capacity of AN in an animal is far lower than that of true protein, so the higher the content of AN, the lower the nutritional value of the feed. The AN content in experimental groups was much lower than that in the control group, showing that LP and BL can effectively inhibit the degradation of protein feed and improve the feed nutrition value. This may be because they can inhibit the activities of harmful microorganisms such as mold and clostridium in silage and avoid the complete decomposition of feed protein into AN. LP is a homofermentative bacteria that can promote the production of LA, which is consistent with the results of this experiment [
32]. The AA content of the LP5 group and BL5 group was significantly higher than that of the control group, which may be caused by insufficient sugar. Lactic acid bacteria (LAB) are generally homofermentative bacteria capable of producing LA. However, when the sugar content of the silage is too low, part of the LAB will turn to heterofermentation, producing LA and an equivalent amount of AA [
33]. With the increase of LP and BL supplemental, the homofermentative LAB competitively inhibited the activity of heterofermentative LAB, thereby reducing the AA content.
The silage process is often accompanied by intense microbial activities, and the use of silage additives often has a great influence on the microbial community of silage [
34]. Through 16S rRNA high throughput sequencing technology, we can have a deeper understanding of how silage additives improve the quality of silage by affecting the microbial community. Goods refers to the coverage of the sample library, and the higher the value of goods, the higher the reliability of the result. The larger the Chao1 and observed species are, the higher the community richness is. The Simpson index and the Shannon index are used in evaluating species diversity. Pielou’s evenness index characterizes sample evenness [
35,
36]. Goods’ coverage in each group was higher than 0.99, indicating that the sequencing data coverage was good enough to reflect the silage flora of each treatment group. A large number of studies have shown that
Bacillus licheniformis has anti-pathogen activity and its fermentation products can inhibit the growth of harmful bacteria such as
Clostridium perfringens and
Staphylococcus aureus in vitro [
37,
38]. This is consistent with the results of this experiment.
Bacillus licheniformis may inhibit the growth of harmful strains and reduce the abundance of silage colonies through its fermentation products. The β-diversity reflects the differences in bacterial communities among samples. From the PCOA figure, we can see that there is an obvious separation between the experimental groups and the control group, indicating that the addition of different strains has a great impact on the microbial community of HP silage.
The application of bacterial fertilizer can not only change the richness and diversity of bacteria but also change the composition and structure of the bacterial community. At the phylum level, the main phyla in each treatment group were
Firmicutes and
Proteobacteria, which was consistent with the results of Chen and Zi et al. [
30,
39].
Firmicutes are Gram-positive bacteria, mainly including spore-producing, non-spore-producing and mycoplasma bacteria, which can degrade macromolecular substances, such as cellulose, starch, protein, etc., and increase the abundance of
Firmicutes will improve the acid production capacity, resulting in the reduction of pH, and further decompose the ADF [
40]. This is consistent with the results of this experiment,
Firmicutes in BL groups were significantly increased compared with the control group, and pH and ADF were decreased compared with the control group.
Proteobacteria are Gram-negative bacteria, some of which can undergo deamination reactions to generate ammonia, which slows down the decrease of pH [
41]. This may be the reason why the pH of the LP groups is difficult to decrease.
The dominant bacteria genus in each treatment group was
Lactobacillus, which was consistent with the results of Shah et al. [
42]. The dominant bacteria genus on the surface of HP was
Lactobacillus.
Lactobacillus is the main bacteria of silage fermentation, belonging to the fermentative bacteria homotype. Its fermentation product lactic acid can quickly reduce the pH of silage, inhibit the growth of fungi, clostridium, and other bacteria, and improve the success rate of silage and fermentation quality.
Weissella is one of the most common heteromorphic fermentation bacteria in silage, which can produce the same amount of AA as LA. With the accumulation of organic acids and the decrease of pH value, its growth will be inhibited [
43]. Studies have shown that LA is positively correlated with lactic acid bacteria and negatively correlated with
Weissella [
44]. It can be seen from the results of this experiment that LP can increase the abundance of
Lactobacillus and reduce the abundance of
Weissella, thereby increasing the content of LA, reducing the content of AA, and improving the quality of feed silage. Furthermore, the degree of its effect was positively correlated with the concentration of LP addition. The relative abundance of
Lactobacillus in the BL group was also higher than that in the control group, and the relative abundance of
Weissella was lower than that in the control group, but the mechanism of action was different from LP. Adding LP can directly increase the abundance of
Lactobacillus, while BL can quickly create an anaerobic environment to promote the rapid growth and reproduction of
Lactobacillus through its unique biological oxygen capture mechanism [
45]. At the same time, the antibacterial substances produced by its fermentation can effectively inhibit the growth of mold, clostridium, and other harmful microorganisms in silage, so as to increase the relative abundance of some bacteria in forage silage [
46].
Pediococcus is one of the microorganisms involved in the early fermentation of silage. The content of
Pediococcus decreases because LP directly increases the relative abundance of
Lactobacilli. BL has no significant effect on
Pediococcus. Studies have shown that
Flavobacterium belongs to
Bacteroidetes and can decompose complex polysaccharides such as starch, chitin, pectin, and carboxymethyl cellulose [
47]. The content of
Flavobacterium in the LP groups was significantly higher than that in the control group and BL group, which may be the reason for the higher WSC content in LP groups.
The function and pathway of HP silage were predicted using PICRUSt2. The results showed that among the top five functions, the addition of LP reduced the relative abundance of ABC-2 type transport system ATP-binding protein, sucrose-6-phosphatase [EC:3.1.3.24], ABC-2 type transport system permease protein, and LacI family transcriptional regulator, while the use of BL increased the relative abundance of ABC-2 type transport system ATP-binding protein, sucrose-6-phosphatase [EC:3.1.3.24], ABC-2 type transport system permease protein, LacI family transcriptional regulator and probable phosphoglycerate mutase [EC:5.4.2.12]. Similarly, the performance of LP and BL in the top five pathways is also very different. The use of LP decreased the relative abundance of secondary bile acid biosynthesis, fatty acid biosynthesis, D-glutamine and D-glutamate metabolism, and lysine biosynthesis, whereas the use of BL increased the relative abundance of the top five pathways. These results indicate that both LP and BL have a great influence on the function and pathway of the bacterial community in silage, but their modes of action are quite different, which may be related to their mechanism of action in silage, and further research is needed.
In vitro fermentation gas production is an important indicator for predicting the degree of feed fermentation, which can reflect the digestion and degradation characteristics of ruminants to feed [
48]. The amount of gas production is related to the nutritional value of feed, rumen microbial activity, and microbial utilization of feed. The higher the nutritional value of the feed, the stronger the fermentability and the higher the gas production [
21]. In this experiment, the gas production of each treatment group at each time point was higher than that of the control group, indicating that both LP and BL can improve the fermentability of silage and improve the nutritional value of feed, which is also consistent with the results of the above silage nutritional indicators. The amount of gas production in the LP group is positively correlated with the amount of LP added, while the trend of gas production in the BL group is just the opposite. In the BL group, only the gas production of the BL5 group was significantly higher than that of the control group in the first 24 h. When the time was increased to 48 h, there was no significant difference in the gas production of the BL group. This may be because the use of BL changed the composition of chemical components in the silage, making it easier to use and ferment, but the impact on total gas production is limited. The gas production in the LP7 group was significantly higher than that in the control group from beginning to end, and the gas production in the LP7 group was the highest among the treatment groups at 48 h, indicating that the effect of LP7 was the best and better than that of the BL5 group.
The pH value in the rumen is one of the important indicators to measure the rumen environment. Calsamiglia et al. [
49] indicated that the range of rumen pH should be controlled between 5.5 and 7.5. All treatments in this experiment were between 6.60 and 6.82, which is within the normal range. The VFA content of rumen fluid depends on the source of the fermentation substrate, microbial population, and rumen environment, and is an important indicator for evaluating rumen health and fermentation status [
50]. There were no significant differences in the VFA indexes among the test groups, indicating that the use of LP and BL would not affect the growth activity of rumen microorganisms and the health of the rumen.