Insights into the phage community structure and potential function in silage fermentation.

The virus that infects bacteria known as phage, plays a crucial role in the biogeochemical cycling of nutrients. However, the community structure and potential functions of phages in silage fermentation remain largely unexplored. In this study, we utilized viral metagenomics (viromics) to investigate the types, lifestyles, functions, and nutrient utilization patterns of phages in silage. Our findings indicated a high prevalence of annotated phages belonging to Caudovirales and Geplafuvirales, as well as unclassified phages in silage. The predominant host types for these phages were Campylobacterales and Enterobacterales. Virulent phages dominated the silage environment due to their broader range of hosts and enhanced survival capabilities. All identified phages present in silage were found to be non-pathogenic. Although temperate and virulent phages carried distinct genes associated with nutrient cycling processes, the shared genes (prsA) involved in carbon metabolism underscore the potential significance of phages in regulating carbon metabolism in silage. Overall, our findings provide a valuable foundation for further exploring the complex interactions between phages and microorganisms in regulating silage fermentation quality.

Dietary Paper Mulberry Silage Supplementation Improves the Growth Performance, Carcass Characteristics, and Meat Quality of Yangzhou Goose.

There have been few investigations into the health benefits and meat quality of supplementing Yangzhou geese with paper mulberry silage. One hundred and twenty 28-day-old Yangzhou geese were selected for the experiment and randomly divided into two groups: a control group (CON) and a paper mulberry silage group (PM), with six replicates in each group. The experiment lasted for a total of 6 weeks. The experiment found that compared with CON, PM had a promoting effect on the average daily weight gain of Yangzhou geese (p = 0.056). Sensory and nutritional analysis of breast muscles revealed a decrease in a* value (p < 0.05) and an increase in protein content (p < 0.05) following PM treatment. Through untargeted metabolomics analysis of breast muscle samples, it was found that 11 different metabolites, including guanidinoacetic acid and other substances, had a positive effect on amino acid metabolism and lipid antioxidant pathways of PM treatment. Overall, the strategy of feeding Yangzhou geese with paper mulberry silage is feasible, which can improve the sensory quality and nutritional value of goose meat. The experiment provides basic data for the application form of goose breeding, so exploring the impact of substances within paper mulberry on goose meat should be focused on in the future.

Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality.

The naturally attached phyllosphere microbiota play a crucial role in plant-derived fermentation, but the structure and function of phyllosphere endophytes remain largely unidentified. Here, we reveal the diversity, specificity, and functionality of phyllosphere endophytes in alfalfa (Medicago sativa L.) through combining typical microbial culture, high-throughput sequencing, and genomic comparative analysis. In comparison to phyllosphere bacteria (PB), the fermentation of alfalfa solely with endophytes (EN) enhances the fermentation characteristics, primarily due to the dominance of specific lactic acid bacteria (LAB) such as Lactiplantibacillus, Weissella, and Pediococcus. The inoculant with selected endophytic LAB strains also enhances the fermentation quality compared to epiphytic LAB treatment. Especially, one key endophytic LAB named Pediococcus pentosaceus EN5 shows enrichment of genes related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes and higher utilization of carbohydrates. Representing phyllosphere, endophytic LAB shows great potential of promoting ensiling and provides a novel direction for developing microbial inoculant.

Effects of Oat Bran Addition on the Growth Performance and Intestinal Health of Nile Tilapia (Oreochromis niloticus) Exposed to Copper Ions.

This study investigated the effects of the oat bran addition on the growth performance and intestinal health of Nile tilapia (Oreochromis niloticus) exposed to copper ions. Four groups of diets containing 0%, 5%, 10%, and 20% oat bran were fed to Nile tilapia for four weeks. The results showed that oat bran had a dose-dependent effect on the growth performance of Nile tilapia. The addition of oat bran can increase the relative abundance of Delftia, which is capable of degrading heavy metals in the intestinal tract and alleviating the intestinal damage caused by copper ion stress. Compared to the control group, the 5% oat bran group had an increased intestinal antioxidant capacity. The relative gene expression of proinflammatory factors (NF-κB, IL-1ß) was significantly downregulated in the 5% oat bran group (P < 0.05), and the relative gene expression of anti-inflammatory factors (TGF-ß), HIF-1α, occludin, and claudin was significantly upregulated (P < 0.05). In conclusion, we suggest that 5% oat bran should be added to the diet to improve the growth performance of Nile tilapia and alleviate the negative effects of copper ion stress on intestinal health.

Exploring the rumen microbiota of Hu lambs in response to diet with paper mulberry.

Paper mulberry (Broussonetia papyrifera), as a new woody forage with high-protein characteristic, is being widely used in ruminant feeding. However, little is known about the comprehensive microbiota picture of whole ruminal niches (liquid, solid, and epithelium) under paper mulberry diet. To gain a better understanding of feeding paper mulberry on the rumen microbiota, the effects of fresh paper mulberry, paper mulberry silage, or a conventional high-protein alfalfa silage on rumen fermentation products and microbiota in rumen niches of Hu lambs were studied. Forty-five Hu lambs were randomly divided into 3 treatments with 15 replicates in each treatment. No significant difference was observed among treatments in the average daily gain (ADG). The fresh paper mulberry treatment had lower (P < 0.05) pH and higher (P < 0.05) total volatile fatty acids (TVFA) compared with silage treatments, but the fermentation parameters did not show significant differences between paper mulberry silage and alfalfa silage treatments. The Shannon index did not show a significant difference (P < 0.05) among treatments except between fresh paper mulberry and alfalfa silage treatment in rumen epithelial niches. Butyrivibrio and Treponema were the predominant genera in the rumen epithelial fraction, while Prevotella and Rikenellaceae_RC9 dominated in both rumen liquid and solid fractions. These results indicated the paper mulberry supplement did not have distinct impact on the microbial diversity and growth performance compared with alfalfa silage, especially for paper mulberry silage, which might help us develop an alternative animal feeding strategy of replacing alfalfa with paper mulberry. KEY POINTS: • Feeding paper mulberry silage did not show significant impact on the growth performance compared with alfalfa silage treatment. • Feeding fresh paper mulberry reduced rumen pH value and increased total volatile fatty acid. • The microbial diversity did not show significant difference among treatments.

Innovative utilization of herbal residues: Exploring the diversity of mechanisms beneficial to regulate anaerobic fermentation of alfalfa.

In order to increase the utilization of herbal residues, realize efficient utilization of resources, the bacterial community and anaerobic fermentation characteristics of alfalfa ensiling treated with 36 kinds of herbal residues were studied. All the herbal residues improved the anaerobic fermentation quality in different degrees, indicated by lower pH, NH3-N and butyric acid concentrations. However, the contents of lactic and acetic acids varied widely in silage with different herbal residues. Pearson's correlation analysis showed that the improved fermentation quality was closely associated with the variation of lactic acid bacteria community. Consequently, the herbal residues could improve anaerobic fermentation quality by stimulating desirable Lactobacillus species and inhibiting undesirable microbes. This study provides new insights for efficient utilization of herbal residues.

Exploring the Effects of Different Bacteria Additives on Fermentation Quality, Microbial Community and In Vitro Gas Production of Forage Oat Silage.

Bacterial inoculants are considered as a good choice for successful ensiling, playing a key role in improving the silage quality. However, the potential of different bacteria, especially the propionic acid bacteria, in forage oat ensiling is yet to be explored. Therefore, the purpose of this study was to investigate the regulation effects of different bacterial additives on the fermentation quality of forage oat silage. Four additives (Lactiplantibacillus plantarum F1, LP; Lacticaseibacillus 0rhamnosus XJJ01, LR; Lacticaseibacillus paracasei XJJ02, LC; and Propionibacterium acidipropionici 1.1161, PP; without additives, CK) were inoculated in forage oat silage, and the fermentation quality and organic compounds were determined after 60 days of ensiling. Notably, LR showed higher dry matter preservation compared to other additives and CK. In addition, LP and LR showed strong lactic acid synthesis capacity, resulting in lower pH compared to other additives and CK. The treatments of PP and LC increased the bacterial diversity in silage, while the bacterial community in the LR group was different from that in other groups. In addition, the PP- and LC-treated oat silage showed significantly lower total in vitro gas production and a lower methane content. These results suggest that LP is more favorable for producing high-quality oat silage than LR, LC, or PP. Both the PP- and LC- treated oat silage may reduce rumen greenhouse gas emissions.

Perilla frutescens as potential antimicrobial modifier to against forage oat silage spoilage.

The aim of this study was to investigate the influence of Perilla frutescens, alone or in combination with Lactobacillus plantarum a214 or citric acid, on forage oat silage quality, bacterial and fungal microbiological profile during ensiling and aerobic exposure. With the exception of Perilla frutescens, all additives could improve silage quality of forage oat based on lower ammonia-nitrogen content and higher residual of water soluble carbohydrates during anaerobic fermentation compared to control silage, especially in Perilla frutescens combined with citric acid (CAPF). Lactobacillus was the dominant bacteria in all silages, while CAPF group increased the relative abundance of Lactobacillus lindneri and Lactobacillus brevis compared to control silage. The application of Perilla frutescens suppressed the relative abundance of yeasts such as Pichia fermentans and Wickerhamomyces anomalus in response to aerobic exposure, especially in CAPF treatment, leading to high acetic acids and lower dry matter loss, as well as good aerobic stability. Therefore, Perilla frutescens, alone or in combination with citric acid, has potential to improve aerobic stability of forage oat silage by shifting bacterial and fungal community composition, and can be used as new additive to prepare high-quality silage for animal production.

Effects of Lactic Acid Bacteria on Microbial Metabolic Functions of Paper Mulberry Silage: A BIOLOG ECO Microplates Approach.

Lactic acid bacteria occupy an important position in silage microorganisms, and the effects of exogenous lactic acid bacteria on silage quality have been widely studied. Microbial metabolism has been proved as an indicator of substrate utilization by microorganisms. Paper mulberry is rich in free carbohydrate, amino acids, and other components, with the potential to be decomposed and utilized. In this study, changes in the microbial metabolism characteristics of paper mulberry silage with Lactiplantibacillus plantarum (LP) and Lentilactobacillus buchneri (LB) were studied along with a control (CK) using BIOLOG ECO microplates. The results showed that average well-color development (AWCD), Shannon diversity, Shannon evenness, and Simpson diversity exhibited significant temporal trends. LB and LP responded differently in the early ensiling phase, and the AWCD of LB was higher than LP at 7 days. Principal component analysis revealed that CK, LB, and LP samples initially clustered at 3 days and then moved into another similar cluster after 15 days. Overall, the microplates methodology applied in this study offers important advantages, not least in terms of accuracy.

Screening of Lactic Acid Bacteria with Inhibitory Activity against ETEC K88 as Feed Additive and the Effects on Sows and Piglets.

Enterotoxigenic Escherichia coli (ETEC), which expresses K88 is the principal microorganism responsible for bacterial diarrhea in pig husbandry, and the indiscriminate use of antibiotics has caused many problems; therefore, antibiotics need to be replaced in order to prevent diarrhea caused by ETEC K88. The objective of this study was to screen excellent lactic acid bacteria (LAB) strains that inhibit ETEC K88 and explore their effects as probiotic supplementation on reproduction, growth performance, diarrheal incidence, and antioxidant capacity of serum in sows and weaned piglets. Three LAB strains, P7, P8, and P15, screened from 295 LAB strains and assigned to Lactobacillus (L.) reuteri, L. amylovorus, and L. johnsonii with high inhibitory activity against ETEC K88 were selected for a study on feeding of sows and weaned piglets. These strains were chosen for their good physiological and biochemical characteristics, excellent exopolysaccharide (EPS) production capacity, hydrophobicity, auto-aggregation ability, survival in gastrointestinal (GI) fluids, lack of hemolytic activity, and broad-spectrum activity against a wide range of microorganisms. The results indicate that LAB strains P7, P8, and P15 had significant effects on improving the reproductive performance of sows and the growth performance of weaned piglets, increasing the activity of antioxidant enzymes and immune indexes in both.

Impacts of Low Temperature and Ensiling Period on the Bacterial Community of Oat Silage by SMRT.

The objective of this study was to investigate how storage temperatures influence the bacterial community of oat silage during the ensiling process via PacBio single molecule, real-time sequencing technology (SMRT). Forage oat was ensiled at four different temperatures (5 °C, 10 °C, 15 °C, and 25 °C) and ensiling days (7, 14, 30, and 60 days). With the rise in storage temperature, the lactic acid content showed an increased trend. Acetic acid production was observed highest in silage fermented at 5 °C compared with other treatments, and Enterococcus mundtii was also the dominant bacterial species. Lactiplantibacillus pentosus and Loigolactobacillus rennini were exclusively detected in silages at 10 °C, 15 °C, and 25 °C, and dominated the fermentation after 60 days of ensiling at 10 °C and 25 °C, respectively. In addition, L. pentosus, L. rennini, and E. mundtii may be related to changes in the fermentation products due to the differences in ensiling temperature. In conclusion, results of this study improve our understanding of the complicated microbial composition underlying silage fermentation at low temperatures, which might contribute to target-based regulation methods for enhancing silage quality and developing new inoculants.

Exploring the Addition of Herbal Residues on Fermentation Quality, Bacterial Communities, and Ruminal Greenhouse Gas Emissions of Paper Mulberry Silage.

This study aimed to investigate the influence of herbal residues on the fermentation quality and ruminal fermentation of paper mulberry silage. Clove, mint, and purple perilla residues were used as additives. Silage treatments were designed as control (no additives), 5% of clove, 5% of mint, and 5% of purple perilla. After 21 and 75 days of fermentation, the fermentation characteristics, bacterial communities, and ruminal greenhouse gas emissions in vitro incubation of paper mulberry were analyzed. The results showed that the used herbal residues could reduce the protein losses in paper mulberry silage based on the lower contents of ammoniacal nitrogen and nonprotein nitrogen. Compared with control, higher lactic acid and propionic acid contents were observed in the silages treated with mint and purple perilla but with a higher acetic acid content in clove treatment. Real-time sequencing technology (single-molecule real-time) revealed that Lactobacillus was the dominant bacteria in all silages at the genus level, whereas the bacterial abundance in the treated silages differed greatly from control at the species level. Lactobacillus hammesii abundance was the highest in control, whereas Lactobacillus acetotolerans was the first predominant in the treated silages. All the additives enhanced the digestibility of in vitro dry matter significantly. However, purple perilla decreased the production of total gas, methane, and carbon dioxide. The findings discussed earlier suggested that herbal residues have potential effects in improving fermentation quality, reducing protein loss, and modulating greenhouse gas emissions in the rumen of paper mulberry silage by shifting bacterial community composition.

Exploring the silage quality of alfalfa ensiled with the residues of astragalus and hawthorn.

This study evaluated the effect of astragalus (AS) and hawthorn (HN) residues on the silage quality of alfalfa. Alfalfa was ensiled with additives (AS, HN and AS + HN) or without additives for 60 days. Results showed that the silage treated with AS and HN had lower contents of butyric acid and NH3-N compared with control, whereas no significant differences of pH and lactic or acetic acid content were found. Additionally, the treated silages also exhibited lower copy numbers of Clostridium, Enterobacter and fungi during ensiling process established by qPCR. In conclusion, AS and HN could be used as additives to control the growth of unfavorable microorganism and enhance the silage quality.

Characterization of Ligninolytic Bacteria and Analysis of Alkali-Lignin Biodegradation Products.

Ligninolytic bacteria degrading lignin were isolates and identified, and their biodegradation mechanism of alkaline-lignin was investigated. Four strains with lignin degradation capability were screened and identified from the soil, straw, and silage based on their decolorizing capacity of aniline blue and colony size on alkaline-lignin medium. The degradation ratio of Bacillus aryabhattai BY5, Acinetobacter johnsonii LN2, Acinetobacter lwoffii LN4, and Micrococcus yunnanensis CL32 have been assayed using alkaline-lignin as the unique carbon source. Further, the Lip (lignin peroxidase) and Mnp (manganese peroxidase) activities of strains were investigated. Lip activity of A. lwoffii LN4 was highest after 72 h of incubation and reached 7151.7 U · l-1. Mnp activity of M. yunnanensis CL32 was highest after 48 h and reached 12533 U · l-1. The analysis of alkaline-lignin degradation products by GC-MS revealed that the strains screened could utilize aromatic esters compounds such as dibutyl phthalate (DBP), and decomposite monocyclic aromatic compounds through the DBP aerobic metabolic pathway. The results indicate that B. aryabhattai BY5, A. johnsonii LN2, A. lwoffii LN4, and M. yunnanensis CL32 have high potential to degrade alkaline-lignin, and might utilize aromatic compounds by DBP aerobic metabolic pathway in the process of lignin degradation.

Effects of ferulic acid esterase-producing Lactobacillus fermentum and cellulase additives on the fermentation quality and microbial community of alfalfa silage.

BACKGROUND: Alfalfa (Medicago sativa) is an important forage material widely used for animal feed production. Ensiling is an effective method for preserving alfalfa, but it has shown some limitations in the production of high-quality alfalfa silage due to its low water soluble carbohydrates (WSC) content and high buffering capacity. Lactic acid bacteria (LAB) and cellulase are often used as silage additives to promote the ensiling process and enhance fermentation quality. METHODS: Experiments were conducted to investigate the effects of ferulic acid esterase (FAE)-producing Lactobacillus fermentum 17SD-2 (LF) and cellulase (CE) on the fermentation quality and microbial community of alfalfa silage. After 60 days of ensiling, analysis of fermentation quality and bacterial diversity in alfalfa silages were conducted using high-performance liquid chromatography and high-throughput sequencing methods. RESULTS: Alfalfa was ensiled with additives (LF, CE, and LF+CE) or without additives for 60 days. All additives increased lactic acid and decreased pH values and ammonia-N contents compared to control. Among all treatments, the combined addition of LF and CE showed lowest pH (4.66) and ammonia-N (NH3-N, 0.57% DM) content, highest contents of lactic acid (LA, 10.51% DM), dry matter (DM, 22.54%) and crude protein (CP, 24.60% DM). Combined addition of LF and CE performed better in reducing neutral detergent fiber (NDF, 29.76% DM) and acid detergent fiber (ADF, 22.86% DM) contents than the addition of LF (33.71, 27.39% DM) or CE (32.07, 25.45% DM) alone. Moreover, the microbial analysis indicated that LF+CE treatments increased the abundance of desirable Lactobacillus and inhibited the growth of detrimental Enterobacter and Clostridia in alfalfa silage. DISCUSSION: Combined addition of FAE-producing LF and CE is more effective than treatments of LF or CE alone in improving fermentation quality and nutrition values of alfalfa silage. This is likely due to a synergistic effect of CE and FAE produced by LF on plant cell wall degradation, indicating that these additives promote each other to improve fiber degradation and silage fermentation. In conclusion, combined addition of FAE-producing LF and CE could be a feasible way to improve alfalfa silage quality.

Effects of Lactic Acid Bacteria Isolated From Rumen Fluid and Feces of Dairy Cows on Fermentation Quality, Microbial Community, and in vitro Digestibility of Alfalfa Silage.

The objective of this study was to select lactic acid bacteria (LAB) isolated from the rumen fluid and feces of dairy cows, and evaluate their effects on silage quality of alfalfa after 30 or 60 days of ensiling. One hundred and four LAB strains were isolated from rumen fluid and feces of six dairy cows, of which four strains (Lactobacillus plantarum F1, L. plantarum F50, Lactobacillus salivarius L100, and Lactobacillus fermentum L120) and one commercial inoculant (GFG) isolated from forage were employed for further study. The silages treated with F1 had the lowest (P < 0.05) pH value and the highest (P < 0.05) lactic acid (LA) content in all treatments. Besides, higher (P < 0.05) in vitro digestibility was also observed in F1-treated silage after 60 days of ensiling. The microbial analysis showed that the Lactobacillus abundance in the F1-treated silages increased to 60.32%, higher than other treatments (5.12-47.64%). Our research indicated that strain F1 could be an alternative silage inoculant, and dairy cows could be a source for obtaining excellent LAB for ensiling.

Fermentation characteristics, chemical composition and microbial community of tropical forage silage under different temperatures.

OBJECTIVE: In tropical regions, as in temperate regions where seasonality of forage production occurs, well-preserved forage is necessary for animal production during periods of forage shortage. However, the unique climate conditions (hot and humid) and forage characteristics (high moisture content and low soluble carbohydrate) in the tropics make forage preservation more difficult. The current study used natural ensiling of tropical forage as a model to evaluate silage characteristics under different temperatures (28°C and 40°C). METHODS: Four tropical forages (king grass, paspalum, white popinac, and stylo) were ensiled under different temperatures (28°C and 40°C). After ensiling for 30 and 60 days, samples were collected to examine the fermentation quality, chemical composition and microbial community. RESULTS: High concentrations of acetic acid (ranging from 7.8 to 38.5 g/kg dry matter [DM]) were detected in silages of king grass, paspalum and stylo with relatively low DM (ranging from 23.9% to 30.8% fresh material [FM]) content, acetic acid production was promoted with increased temperature and prolonged ensiling. Small concentrations of organic acid (ranging from 0.3 to 3.1 g/kg DM) were detected in silage of white popinac with high DM content (50.8% FM). The microbial diversity analysis indicated that Cyanobacteria originally dominated the bacterial community for these four tropical forages and was replaced by Lactobacillus and Enterobacter after ensiling. CONCLUSION: The results suggested that forage silages under tropical climate conditions showed enhanced acetate fermentation, while high DM materials showed limited fermentation. Lactobacillus and Enterobacter were the most probable genera responsible for tropical silage fermentation.

Assessing the fermentation quality and microbial community of the mixed silage of forage soybean with crop corn or sorghum.

The silage quality of forage soybean (FS) rich in protein with crop corn (CN) or sorghum (SG) rich in water soluble carbohydrate was investigated, and microbial community after ensiling was analyzed. Results showed that pH in mixed silages dropped to 3.5-3.8 lower than 100%FS silage (4.5). Microbial analysis indicated that mixed ensiling could influence the microbial community. Although Lactobacillus and Weissella were the dominant genera in all silage samples, Lactobacillus abundance in mixed silages (33-76%) was higher compared with 100%FS silage (27%). In conclusion, FS ensiled with CN or SG could be an alternative approach to improve FS silage quality.

Effects of lactic acid bacteria and molasses additives on the microbial community and fermentation quality of soybean silage.

The objective was to study effects of lactic acid bacteria (L) and molasses (M) on the microbial community and fermentation quality of soybean silage. Soybean was ensiled with no additive control (C), 0.5% molasses (0.5%M), 0.5%M+L (0.5%ML), 2%M, 2%M+L (2%ML) for 7, 14, 30 and 60days. The M-treated silages could increase the content of lactic acid and decrease butyric acid than control. Besides, higher crude protein was also observed in M-treated silages. With prolonged ensiling time, there was a reduction of the ratio of lactic acid/acetic acid in the 2%M-treated and 2%ML-treated silages. The combined addition of L and 2%M could enhance the account of desirable Lactobacillus and inhibit the growth of undesirable microorganism such as Clostridia and Enterobacter. In summary, the silage quality of soybean was improved with the addition of L and M.

Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn, and wilted alfalfa silage using denaturing gradient gel electrophoresis and next-generation sequencing.

The microbiota of pre-ensiled crop and silage were examined using denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Wilted Italian ryegrass (IR), whole crop corn (WC), and wilted alfalfa (AL) silages stored for 2 months were examined. All silages contained lactic acid as a predominant fermentation product. Across the three crop species, DGGE detected 36 and 28 bands, and NGS identified 253 and 259 genera in the pre-ensiled crops and silages, respectively. The NGS demonstrated that, although lactic acid bacteria (LAB) became prevalent in all silages after 2 months of storage, the major groups were different between crops: Leuconostoc spp. and Pediococcus spp. for IR silage, Lactobacillus spp. for WC silage, and Enterococcus spp. for AL silage. The predominant silage LAB genera were also detected by DGGE, but the presence of diverse non-LAB species in pre-ensiled crops was far better detected by NGS. Likewise, good survival of Agrobacterium spp., Methylobacterium spp., and Sphingomonas spp. in IR and AL silages was demonstrated by NGS. The diversity of the microbiota described by principal coordinate analysis was similar between DGGE and NGS. Our finding that analysis of pre-ensiled crop microbiota did not help predict silage microbiota was true for both DGGE and NGS.