This study was conducted to study the effects of ensiled alfalfa (Medicago sativa) and red clover (Trifolium pratense) at different ratios on dynamics of fermentation parameters, N distribution, protein fractions, and protease activities during ensiling. Alfalfa and red clover were harvested and wilted to 35 and 25% dry matter, respectively, chopped to 1 cm, mixed, weighed into 1.0-L buckets at a density of 700 g/L, and ensiled for 1, 3, 7, 15, and 30 d at 30°C. The treatments were mixing ratio of alfalfa to red clover at 100:0, 70:30, 50:50, 30:70, and 0:100 (R0, R30, R50, R70, and R100, respectively; fresh weight). For each ensiling duration, 3 replicates of each treatment were prepared. With increasing proportion of red clover in silage, total N content and proportions of nonprotein N, peptide N, free amino acid N, and NH₃-N decreased linearly, and PC (indigestible true protein, acid detergent insoluble N) proportion increased linearly after ensiling. Moreover, the final pH was lower in R50 and R100 than R0 (4.29, 4.20 vs. 4.48, respectively) on d 30. Also, lactic acid concentration on d 30 was higher in R50, R70, and R100 silage compared with R0 (7.77, 7.66, and 8.76 vs. 6.34, % of dry matter, respectively). The proportion of NH₃-N in R50 was lower than in R0 but closer to R100 after ensiling. During ensiling, proteases including carboxypeptidase, aminopeptidase, and acid proteinase activities decreased as red clover proportion increased. However, no differences were detected in aminopeptidase and acid proteinase activities among R50, R70, and R100 during ensiling. Overall, 50:50 was the optimal mixing ratio of alfalfa with red clover, showing good fermentation quality with lower pH and higher lactic acid concentration, reduced protease activities and proteolysis compared with pure alfalfa silage, and also more total N content than pure red clover silage.

Perilla frutescens (L.) Britt., an annual herbaceous plant, has antibacterial, anti-inflammation, and antioxidant properties. To understand the effects of P. frutescens leaf on the ruminal microbial ecology of cattle, Illumina MiSeq 16S rRNA sequencing technology was used. Fourteen cows were used in a randomized complete block design trial. Two diets were fed to these cattle: a control diet (CON); and CON supplemented with 300 g/d P. frutescens leaf (PFL) per cow. Ruminal fluid was sampled at the end of the experiment for microbial DNA extraction. Overall, our findings revealed that supplementation with PFL could increase ruminal fluid pH value. The ruminal bacterial community of cattle was dominated by Bacteroidetes, Firmicutes, and Proteobacteria. The addition of PFL had a positive effect on Firmicutes, Actinobacteria, and Spirochaetes, but had no effect on Bacteroidetes and Proteobacteria compared with the CON. The supplementation with PFL significantly increased the abundance of Marvinbryantia, Acetitomaculum, Ruminococcus gauvreauii, Eubacterium coprostanoligenes, Selenomonas_1, Pseudoscardovia, norank_f__Muribaculaceae, and Sharpea, and decreased the abundance of Treponema_2 compared to CON. Eubacterium coprostanoligenes, and norank_f__Muribaculaceae were positively correlated with ruminal pH value. It was found that norank_f__Muribaculaceae and Acetitomaculum were positively correlated with milk yield, indicating that these different genera are PFL associated bacteria. This study suggests that PFL supplementation could increase the ruminal pH value and induce shifts in the ruminal bacterial composition.

Investigating the microbial communities and biogenic amine (BA) formation in silage is of vital for improving the quality and safety of oat silage. The present study evaluated the effects of propionic acid (P) and sodium benzoate (SB) on the quality properties, microbial communities, and BA formation in oat silage. Oat was harvested at boot stage and ensiled using P and SB as additives in mini silos, followed by 14 days of aerobic exposure. The results showed that P and SB improved fermentation quality of oat silage, increased the lactic acid content, and decreased pH value and ammonia nitrogen content. Putrescine, cadaverine, and tyramine were the dominant BAs in oat silage; spermidine and spermine were not detected. The control silage had the highest content of total biogenic amine (TBA, 2506.7 mg kg^–1 DM), and decreased by 51.1 and 57.7% after adding P and SB, respectively. Moreover, a lower putrescine, cadaverine, and tyramine content and undesirable microbes, such as Caproiciproducens, Stenotrophomonas, Herbinix, and Enterobacter genera, were observed in P and SB silages, which was beneficial for oat silage quality. The fungal community of P silage was dominated by Monascus fuliginosus, and the temperature, pH and ammonia nitrogen content increased after exposure to air. Sedimentibacter, Herbinix, Caproiciproducens, Enterobacter, and Escherichia-Shigella were found to be positively correlated with BA formation in oat silage. Overall, P and SB effectively inhibit the undesirable microbes and BA formation in oat silage, the P silage exhibited lower aerobic stability than the SB silage.

Two lactic acid bacteria (LAB) strains with different ferulic acid esterase (FAE) activities were isolated: Lactobacillus farciminis (LF18) and Lactobacillus plantarum (LP23). The effects of these strains on the fermentation quality, in vitro digestibility and phenolic acid extraction yields of sorghum (Sorghum bicolor L.) silage were studied at 20, 30 and 40 °C. Sorghum was ensiled with no additive (control), LF18 or LP23 for 45 days. At 40 °C, the lactic acid content decreased, whereas the ammonia nitrogen (NH₃-N) content significantly increased (p < 0.05). At all three temperatures, the inoculants significantly improved the lactic acid contents and reduced the NH₃-N contents (p < 0.05). Neither LP23 nor LF18 significantly improved the digestibility of sorghum silages (p > 0.05). The LP23 group exhibited higher phenolic acid extraction yields at 30 °C (p < 0.05), and the corresponding yields of the LF18 and control groups were improved at 40 °C (p < 0.05). FAE-producing LABs might partially ameliorate the negative effects of high temperature and improve the fermentation quality of sorghum silage. The screened FAE-producing LABs could be candidate strains for preserving sorghum silage at high temperature, and some further insights into the relationship between FAE-producing LABs and ensiling temperatures were obtained.