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This study aimed to evaluate the effectiveness of unprotected and rumen-protected L-carnitine through in vitro tests, rumen degradation tests, and in vivo tests. Twelve rumen-fistulated crossbred rams with similar body weights of 55 ± 3.6 kg and ages of 3 ± 0.2 yr old were divided into three treatment groups in a 3 × 3 Latin square design, G1 (basal diet with no additives), G2 (unprotected L-carnitine), or G3 (rumen-protected L-carnitine). Ruminal fluid and blood samples were collected before morning feeding on the last day of each experimental period (21 d). The percentage of L-carnitine remaining in the simulated rumen and abomasum and rumen increased with the increase in the wall material ratio (P

Bacillus, an excellent organic-degrading agent, can degrade lignocellulose. Notably, some B. velezensis strains encode lignocellulases. However, their ability to degrade lignocellulose in fermented feed is not much appreciated. This study performed a comparative genomic analysis of twenty-three B. velezensis strains to find common carbohydrate-active enzymes (CAZymes) encoding genes and evaluated their potential to degrade lignocellulose. The comparative genomic and CAZyme database-based analyses identified several potential CAZymes genes that degrade cellulose (GH1, GH4, GH5, GH13, GH16, GH32, PL1, and PL9), hemicellulose (GH11, GH26, GH43, GH51, and CE3) and lignin (AA4, AA6, AA7, and AA10). Furthermore, Illumina RNA-seq transcriptome analysis revealed the expression of more than 1794 genes in B. velezensis CL-4 fermented corn germ meal at 48 h (FCGM 48 h). Gene ontology analysis of expressed genes revealed their enrichment in hydrolase activity (breaking the glycosyl bonds during carbohydrate metabolism), indicating the upregulation of CAZymes. In total, 58 differentially upregulated CAZymes-encoding genes were identified in FCGM 48 h compared to FCGM 0 h. The upregulated CAZymes-encoding genes were related to cellulose (6-phospho-β-galactosidase and 6-phospho-α-glucosidase), starch (α-glucosidase and α-amylase), pectin (pectin lyase), and hemicellulose (arabinan endo-1,5-α-L-arabinosidase, xylan 1,4-beta-xylosidase, α-N-arabinofuranosidase, and acetyl xylan esterase). Importantly, arabinoxylan degradation mainly occurred in FCGM 48 h, followed by partial degradation of cellulose, pectin, and starch. This study can support the development of enzymatic cocktails for the solid-state fermented feed (SFF).

Sodium bicarbonate pretreatment and solid-state fermentation (SSF) were used to maximize the nutritional value of corn germ meal (CGM) by inoculating it with Bacillus velezensis CL-4 (isolated from chicken cecal contents and capable of degrading lignocellulose). Based on genome sequencing, B. velezensis CL-4 has a 4,063,558 bp ring chromosome and 46.27% GC content. Furthermore, genes associated with degradation of lignocellulose degradation were detected. Pretreatment of CGM (PCGM) with sodium bicarbonate (optimized to 0.06 g/mL) neutralized low pH. Fermented and pretreated CGM (FPCGM) contained more crude protein (CP), soluble protein of trichloroacetic acid (TCA-SP), and total amino acids (aa) than CGM and PCGM. Degradation rates of cellulose and hemicellulose were reduced by 21.33 and 71.35%, respectively, after 48 h fermentation. Based on electron microscopy, FPCGM destroys the surface structure and adds small debris of the CGM substrate, due to lignocellulose breakdown. Furthermore, 2-oxoadipic acid and dimethyl sulfone were the most important metabolites during pretreatment. Concentrations of adenosine, cytidine, guanosine, S-methyl-5'-thioadenosine, and adenine decreased significantly after 48 h fermentation, whereas concentrations of probiotics, enzymes, and fatty acids (including palmitic, 16-hydroxypalmitic, and linoleic acids) were significantly improved after fermentation. In conclusion, the novel pretreatment of CGM provided a proof of concept for using B. velezensis CL-4 to degrade lignocellulose components, improve nutritional characteristics of CGM, and expand CGM lignocellulosic biological feed production. KEY POINTS: • Sodium bicarbonate (baking soda) can be used as an economical and green additive to pretreat corn germ meal; • Fermentation with B. velezensis degrades the cellulose and hemicellulose component of corn germ meal and improves its feed quality; • As a novel qualified presumption of safety (QPS) strain, B. velezensis should have broad potential applications in food and feed industries.

The purpose of this experiment was to investigate the changes of carbohydrate composition in fermented total mixed diet and its effects on rumen fermentation, methane production, and rumen microbiome in vitro. The concentrate-to-forage ratio of the total mixed ration (TMR) was 4:6, and TMR was ensiled with lactic acid bacteria and fibrolytic enzymes. The results showed that different TMRs had different carbohydrate compositions and subfractions, fermentation characteristics, and bacterial community diversity. After fermentation, the fermented total mixed ration (FTMR) group had lower contents of neutral detergent fiber, acid detergent fiber, starch, non-fibrous carbohydrates, and carbohydrates. In addition, lactic acid content and relative abundance of Lactobacillus in the FTMR group were higher. Compared with the TMR group, the in vitro ammonia nitrogen and total volatile fatty acid concentrations and the molar proportion of propionate and butyrate were increased in the FTMR group. However, the ruminal pH, molar proportion of acetate, and methane production were significantly decreased in the FTMR group. Notably, we found that the relative abundance of ruminal bacteria was higher in FTMR than in TMR samples, including Prevotella, Coprococcus, and Oscillospira. At the same time, we found that the diversity of methanogens in the FTMR group was lower than that in the TMR group. The relative abundance of Methanobrevibacter significantly decreased, while the relative abundances of Methanoplanus and vadinCA11 increased. The relative abundances of Entodinium and Pichia significantly decreased in the FTMR group compared with the TMR group. These results suggest that FTMR can be used as an environmentally cleaner technology in animal farming due to its ability to improve ruminal fermentation, modulate the rumen microbiome, and reduce methane emissions.

The features and genome annotation of a newly bacteriophage v B_EcoP_E212 (referred to as E212) which isolated from farm sewage collected in Jilin, China was describes in this study. Bacteriophage E212 belongs to the family Podoviridae, order Caudovirales through transmission electron microscopy. This phage specifically infects enterotoxigenic Escherichia coli K88. The dsDNA molecule of phage E212 was 38252 bp in length and contained 46.98% G + C content. It has been predicted to contain 53 ORFs, and no tRNAs. Phage E212 carried the integrase gene, and no homologues of virulence factors or antimicrobial resistance genes were found in this phage. Phage E212 was assigned to the genus Lederbergvirus in accordance with nucleotide sequence alignment and phylogenetic analysis.

The purpose of this experiment was to investigate the changes of carbohydrate composition in fermented total mixed diet and its effects on rumen fermentation, methane production, and rumen microbiome in vitro. The concentrate-to-forage ratio of the total mixed ration (TMR) was 4:6, and TMR was ensiled with lactic acid bacteria and fibrolytic enzymes. The results showed that different TMRs had different carbohydrate compositions and subfractions, fermentation characteristics, and bacterial community diversity. After fermentation, the fermented total mixed ration (FTMR) group had lower contents of neutral detergent fiber, acid detergent fiber, starch, non-fibrous carbohydrates, and carbohydrates. In addition, lactic acid content and relative abundance of Lactobacillus in the FTMR group were higher. Compared with the TMR group, the in vitro ammonia nitrogen and total volatile fatty acid concentrations and the molar proportion of propionate and butyrate were increased in the FTMR group. However, the ruminal pH, molar proportion of acetate, and methane production were significantly decreased in the FTMR group. Notably, we found that the relative abundance of ruminal bacteria was higher in FTMR than in TMR samples, including Prevotella, Coprococcus, and Oscillospira. At the same time, we found that the diversity of methanogens in the FTMR group was lower than that in the TMR group. The relative abundance of Methanobrevibacter significantly decreased, while the relative abundances of Methanoplanus and vadinCA11 increased. The relative abundances of Entodinium and Pichia significantly decreased in the FTMR group compared with the TMR group. These results suggest that FTMR can be used as an environmentally cleaner technology in animal farming due to its ability to improve ruminal fermentation, modulate the rumen microbiome, and reduce methane emissions.

This work describes the characterization and genome annotation of a newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscope revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects the Shigella sonnei, Shigella dysenteriae and Escherichia coli. The genome of the phage Z31 is an 89,355 bp length dsDNA molecule with a G + C content of 38.87%. It has been predicted to contain 133 ORFs, and 24 tRNAs. No homologs of virulence factors or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirnae.

Bacillus velezensis is widely used for agricultural biocontrol, due to its ability to enhance plant growth while suppressing the growth of microbial pathogens. However, there are few reports on its application in fermented feed. Here, a two-stage solid-state fermentation process using Bacillus velezensis followed by Lactobacillus plantarum was developed to degrade antinutritional factors (ANFs) and improve soybean meal (SBM) nutrition for animal feed. The process was evaluated for performance in degrading SBM antinutritional factors, dynamic changes in physicochemical characteristics, microorganisms and metabolites. After two-stage fermentation, degradation rates of glycinin and β-conglycinin contents reached 78.60% and 72.89%, respectively. The pH of fermented SBM (FSBM) decreased to 4.78 ± 0.04 and lactic acid content reached 183.38 ± 4.86 mmol/kg. NSP-degrading enzymes (Non-starch polysaccharide, NSPases) and protease were detected from the fermented product, which caused the changed microstructure of SBM. Compared to uninoculated SBM, FSBM exhibited increased proportions of crude protein (51.97 ± 0.44% vs. 47.28 ± 0.34%), Ca, total phosphorus (P), and trichloroacetic acid-soluble protein (11.79 ± 0.13% vs. 5.07 ± 0.06%). Additionally, cellulose and hemicellulose proportions declined by 22.10% and 39.15%, respectively. Total amino acid content increased by 5.05%, while the difference of AA content between the 24 h, 48 h and 72 h of fermentation was not significant (P > 0.05). Furthermore, FSBM also showed antibacterial activity against Staphylococcus aureus and Escherichia coli. These results demonstrated that two-stage SBM fermentation process based on Bacillus velezensis 157 and Lactobacillus plantarum BLCC2-0015 is an effective approach to reduce ANFs content and improve the quality of SBM feed.

Additional file 1: Figure S1. Protein degradation capacity of B. velezensis 157 and L. plantarum BLCC2-0015. Figure S2. Antimicrobial activity of the fermented product

This work describes the characterization and genome annotation of the newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscopy revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects Shigella sonnei, Shigella dysenteriae, and Escherichia coli. The genome of the phage Z31 is an 89,355-bp-long dsDNA molecule with a G+C content of 38.87%. It was predicted to contain 133 ORFs and encode 24 tRNAs. No homologs of virulence factor genes or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirinae.

This study describes the characterization and genomic analysis of six lytic Salmonella phages. To examine the feasibility of using these phages as biocontrol agents, we analyzed their genomes and compared them to those of similar phages. These six phages belong to genus Epseptimavirus, family Demerecviridae. We identified the genes of these six phages by comparing their genomes with those of three type phages in subfamily Markadamsvirinae. All six phages examined in this study were obligately lytic and did not carry undesirable genes. Two phages (vB_SalS_1-23 and vB_SalS_3-29) were selected as the representative phages for general characterization and physiological tests. The biocontrol efficacy of the representative phages was determined by comparing the viable counts of recovered host Salmonella ser. Newlands ZC-S1 from treatment and phage-free control samples. The biocontrol experiment showed that the representative phages were able to reduce the counts of ZC-S1 to below 2 log10 CFU/mL (~4.3 log10 CFU/mL reduction) at 3 h post-infection at 37 °C. Furthermore, we investigated the application of these two phages in the control of ZC-S1 contamination in chicken products and on eggshells. When applied to the surfaces of the samples, the phage cocktail (MOI = 100) reduced the ZC-S1 count to below 2 log10 CFU/mL on chicken skin and to undetectable levels (1 log10 CFU/mL) in chicken breast meat, ground chicken meat and eggshell samples (p

This study investigated the effects of mannan oligosaccharides (MOS) on in vitro and in vivo gas emission, utilization of crude protein (CP) and energy, and relative parameters of sheep under fasting metabolism conditions. In vitro gas productions were evaluated over 12 h in sheep diets containing different amounts of MOS (from 0% to 6.0%/kg, the increment was 0.5%). A control experiment was used to assess the gas emission, utilization of CP and energy, and fasting metabolism in control sheep and sheep treated with 2.0% MOS over 24 days (d). The results showed that 2.0% MOS supplementation led to the lowest in vitro CO2 production and less CH4 production, while also leading to decrease in vivo nutrients intake, CP and energy excretion, digested and retained CP, and energy released as CH4 (p &lt, 0.05). Furthermore, 2.0% MOS supplementation appeared to decrease in vivo O2 consumption and CH4 production per metabolic body weight (BW0.75), and increase the CP retention rate of sheep (p &lt, 0.074). MOS did not affect other parameters, along with the same parameters of sheep under fasting metabolism conditions (p &gt, 0.05). The findings indicate MOS has only slight effects on the gas emission and nutrients and energy metabolism of sheep.

The objective of this study was to investigate the effects ofl-carnitine and/or maize distillers dried grains with solubles (DDGS) in diets of gestating and lactating sows on the intestinal barrier functions of their offspring. The experiment was designed as a 2×2 factorial with two dietary treatments (soyabean mealv.DDGS) and twol-carnitine levels (0v.100 mg/kg in gestating diets and 0v.200 mg/kg in lactating diets). Sows (Landrace×Large White) with an average parity of 4·2 with similar body weight were randomly assigned to four groups of thirty each. Dietary supplementation withl-carnitine increased the total superoxide dismutase activity but decreased the concentration of malondialdehyde of the jejunal mucosa in newborn piglets and weaning piglets on day 21. Dietary supplementation withl-carnitine decreased the concentrations of IL-1β, IL-12 and TNF-αin the jejunal mucosa of newborn piglets and decreased the concentrations of IL-6 and TNF-αin the jejunal mucosa of weaning piglets on day 21. There was an interaction between dietary treatment andl-carnitine on the bacterial numbers of total eubacteria in the digesta of caecum in weaning piglets on day 21. Bacterial numbers of total eubacteria in weaning piglets on day 21 were significantly increased byl-carnitine only in soyabean meal diet, but there was no significant effect ofl-carnitine in DDGS-based diet. Dietary supplementation withl-carnitine increased the bacterial numbers ofLactobacillusspp. and bifidobacteria spp. in the digesta of caecum in weaning piglets on day 21. Dietary supplementation withl-carnitine in sows affected the expression of tight junction proteins (claudin 1, zonula occludens-1 (ZO-1) and occludin) in the jejunal mucosa of their offspring by increasing the expression of ZO-1 mRNA in the jejunal mucosa of newborn piglets, and by increasing the expression of ZO-1 and occludin mRNA in the jejunal mucosa of weaning piglets on day 21. In conclusion, dietary supplementation withl-carnitine in gestating and lactating sows had positive effects on intestinal barrier functions of newborn piglets and weaning piglets on day 21, but it did not have effects on intestinal barrier functions of growing–finishing pigs in the filial generation. There were no effects of dietary treatment of sows on intestinal barrier functions in their offspring.