Objective: This study aimed to assess the impact of a hydroethanolic extract of walnut green husks (WGH) on rumen fermentation and the diversity of bacteria, methanogenic archaea, and fungi in sheep fed a high-concentrate diet.Methods: Five healthy small-tailed Han ewes with permanent rumen fistula were selected and housed in individual pens. This study adopted a self-controlled and crossover design with a control period and an experimental period. During the control period, the animals were fed a basal diet (with a ratio of concentrate to roughage of 65:35), while during the treatment period, the animals were fed the basal diet supplemented with 0.5% hydroethanolic extract of WGH. Fermentation parameters, digestive enzyme activities, and microbial diversity in rumen fluid were analyzed.Results: Supplementation of hydroethanolic extract of WGH had no significant effect on feed intake, concentrations of total volatile fatty acids, isovalerate, ammonia nitrogen, and microbial protein (p>0.05). However, the ruminal pH, concentrations of acetate, butyrate and isobutyrate, the ratio of acetate to propionate, protozoa count, and the activities of filter paper cellulase and cellobiase were significantly increased (p<0.05), while concentrations of propionate and valerate were significantly decreased (p<0.05). Moreover, 16S rRNA gene sequencing revealed that the relative abundance of rumen bacteria Christensenellaceae R7 group, Saccharofermentans, and Ruminococcaceae NK4A214 group were significantly increased, while Ruminococcus gauvreauii group, Prevotella 7 were significantly decreased (p<0.05). The relative abundance of the fungus Pseudomonas significantly increased, while Basidiomycota, Fusarium, and Alternaria significantly decreased (p<0.05). However, there was no significant change in the community structure of methanogenic archaea.Conclusion: Supplementation of hydroethanolic extract of WGH to a high-concentrate diet improved the ruminal fermentation, altered the structure of ruminal bacterial and fungal communities, and exhibited beneficial effects in alleviating subacute rumen acidosis of sheep.

The development of probiotics capable of quickly colonizing the intestines of animals is important in promoting the healthy growth of livestock. The aim of this study was to screen lactic acid bacteria (LAB) from the intestinal microbiota of chickens with potential applications, and to evaluate their probiotic properties and antagonistic abilities against Salmonella pullorum, Staphylococcus aureus, and Escherichia coli. The results showed that a total of 79 strains with the characteristics of LAB were isolated from the chicken cecum microbiota, of which 7 strains exhibited strong inhibitory activity against S. pullorum, S. aureus, and E. coli. Performing 16s rDNA sequencing revealed that these seven strains were Lactiplantibacillus pentosus (n = 1), Lactiplantibacillus plantarum (n = 3), Lactiplantibacillus paraplantarum (n = 1), Lactiplantibacillus argentoratensis (n = 1), and Lactiplantibacillus fabifermentans (n = 1). Among them, L. pentosus R26 and L. plantarum R32 exhibited superior antibacterial activity. These two strains demonstrated high lactic acid production ability, with survival rates of 86.29% and 87.99% after 3 h of treatment at pH 1.5, 86.66% and 85.52% after 3 h of treatment with 0.5% bile salts, 90.03% and 88.16% after 2 h of treatment with simulated gastric fluid, and 98.92% and 98.22% after 2 h of treatment with simulated intestinal fluid, respectively. Co-cultivation with L. pentosus R26 for 24 h resulted in 50% of the pathogens being antagonized, while almost complete inhibition was observed following 72 h of co-cultivation. In conclusion, L. pentosus R26 and L. plantarum R32 exhibited high antibacterial activity and acid production capability, while also demonstrating satisfactory tolerance to low pH values and high concentrations of bile salts and digestive fluid. The probiotic characteristics and stress resistance of L. pentosus R26 were slightly superior to those of L. plantarum R32, indicating its potential for development as a probiotic.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography–tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

In this study, Candida utilis, Lactobacillus plantarum, and non-starch polysaccharide enzymes (cellulase, laccase, β-glucanase, xylanase, and mannanase) were employed to examine the effects of various microorganism–enzyme combinations on the nutritional composition, fiber structure, and fermentation quality of corn stover and wheat straw. Furthermore, the synergistic effects of these treatments were assessed through the use of in vitro rumen fermentation. The results showed that the microorganism–enzyme combinations significantly increased the crude protein content (p < 0.05), while reducing the acid detergent fiber and neutral detergent fiber levels (p < 0.05) in both substrates. The fermentation broth pH decreased (p = 0.06 for corn stover; p < 0.05 for wheat straw) as a result of the treatments, with a significant increase in the lactate concentration (p < 0.05). The reducing sugar levels varied across the treatments (p < 0.05). Mycotoxin analysis revealed trace amounts of zearalenone, well below the Chinese feed hygiene standard. Scanning electron microscopy showed structural modifications, including fiber breakage and surface wrinkling, in the treated substrates. In vitro rumen fermentation demonstrated significant changes in the NH₃-N production and volatile fatty acid profiles (p < 0.05). In conclusion, the addition of different microorganism–enzyme combinations can effectively improve the nutritional composition, fiber structure, and fermentation quality of corn stover and wheat straw. Among the treatments, the T3 group (25% each of C. utilis, L. plantarum, cellulase, and laccase, with a total addition ratio of 0.3% w/w) exhibited the most pronounced improvement in nutritional value for both corn stover and wheat straw. These findings suggest that microorganism–enzyme combinations effectively enhance the nutritional and fermentative quality of agricultural residues.

Ferulic acid esterase (FAE) plays an important role in plant fiber degradation by catalyzing the hydrolysis of lignocellulosic structures. FAE-producing lactic acid bacteria (LAB), as potential probiotics, can improve ruminant digestion and gut health. In this study, two LAB strains (Q2 and Q6) with FAE activity were isolated from sheep rumen. Based on 16S rDNA sequencing, they were identified as Lactobacillus mucosae and Streptococcus equinus, respectively. Compared to Q2, Q6 demonstrated higher enzyme production, lactic acid yield, broader carbohydrate utilization, and stronger antimicrobial activity. The whole genome sequencing revealed Q2 and Q6 possess genomes of 2.14 Mbp and 1.95 Mbp, with GC contents of 46.81% and 37.30%, respectively. Q2 and Q6 exhibited the highest average nucleotide identity (ANI) with L. mucosae DSM 13345 (97.30%) and S. equinus ATCC 33317 (97.92%), respectively. The strains harbored 2101 and 1928 predicted genes, including 1984 and 1837 coding sequences (CDSs), respectively. GO enrichment analysis showed the CDSs predominantly associated with membranes (or cells), catalytic activity, and metabolic processes. KEGG analysis revealed both strains enriched in metabolic pathways, with Q6 showing a notably higher number of proteins in the ABC transporters and quorum sensing than Q2. Carbohydrate-active enzymes database (CAZy) profiling identified 75 CAZymes in Q2 and 93 CAZymes in Q6, with each strain containing one novel fae gene. Safety assessment identified 1 and 33 pathogenic genes, along with 2 and 4 putative antimicrobial peptide genes, in Q2 and Q6, respectively. Notably, Q6 carried 12 virulence factor genes. These findings suggest Q2 exhibits a superior safety profile compared to Q6, indicating a higher probability of Q2 being an effective probiotic strain. In conclusion, both LAB strains produce FAE. L. mucosae Q2 demonstrates suitability as a direct-fed probiotic for livestock, while Q6 exhibits potential as a silage inoculant, though comprehensive safety evaluations are required prior to its application.