Alfalfa (Medicago sativa L.) is one of the most important forage resources in the world due to its high nutritive value. However, its nutritional quality decreases during the transition from budding to flowering. Previous research revealed a decreased crude protein content and increased fibre content in alfalfa forage harvested at later maturity stages, leading to a reduction in nutritional quality. However, the reasons for this phenomenon have not been explained at the molecular level. In this study, leaves from the WL319HQ alfalfa cultivar were harvested at two developmental stages (budding and mid-flowering). The leaves were used to test the variable expression of proteins and metabolites during these stages. TMT-based quantitative proteomics and LC-MS/MS-based untargeted metabolomics methods were employed in this study. A total of 415 proteins and 49 metabolites showed at least a 1.2-fold difference in abundance during these stages. Most of the differentially expressed proteins and metabolites were involved in metabolic processes, including carbohydrate metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, and biosynthesis of amino acids. Alfalfa leaves in mid-flowering contain less crude protein due to the decrease in L-glutamic acid content. Carbohydrate metabolism provides the raw material for the synthesis of hemicellulose, resulting in an increase in the hemicellulose content of the alfalfa leaves, leading to an increase in the NDF content. In addition, the increase in L-phenylalanine content could have provided the conditions necessary for lignin synthesis. These are the main factors leading to reductions in alfalfa relative feed value (RFV) and quality. This study used joint proteomic and metabolomic analyses to elucidate the relationship between the reduction in the nutritional value of alfalfa and complex biological processes. This provides a theoretical basis for producing high-quality alfalfa hay and sets the stage for further research.

In this study, the ultrasound-assisted extraction (UAE) conditions of flavonoids from Lactuca indica L.cv. Mengzao (LIM) leaves was optimized, and the flavonoids content and their antioxidant potential in different parts were analyzed. The optimal extraction parameters to obtain the highest total flavonoids content (TFC) were a a ratio of liquid to solid of 24.76 mL/g, ultrasonic power of 411.43 W, ethanol concentration of 58.86% and an extraction time of 30 min, the average TFC of LIM leaves could reach 48.01 mg/g. For the yield of flavonoids, the UAE method had the best extraction capacity compared with solvent extraction and microwave-assisted extraction (MAE). In general, the TFC in different parts of LIM followed the order flower > leaf > stem > root, the flowering period is the most suitable harvesting period. From ultra-high performance liquid chromatography-mass spectrometry (UPLC–MS) quantification, the flower samples showed significantly higher six flavonoids and had the highest radical scavenging capacities compared to other samples. A high positive correlation was observed between the antioxidant activity and TFC, luteolin-7-O-glucoside and rutin were significantly (p < 0.05) correlated with all antioxidant evaluations. This study provides valuable information for the development and utilization of flavonoids in Lactuca indica as ingredients in food, feed and nutritional health products.

In this study, we analyzed the fermentation quality, microbial community, and metabolome characteristics of ryegrass silage from different harvests (first harvest-AK, second harvest-BK, and third harvest-CK) and analyzed the correlation between fermentative bacteria and metabolites. The bacterial community and metabolomic characteristics were analyzed by single-molecule real-time (SMRT) sequencing and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), respectively. After 60 days of ensiling, the pH of BK was significantly lower than those of AK and CK, and its lactic acid content was significantly higher than those of AK and CK. Lactiplantibacillus and Enterococcus genera dominate the microbiota of silage obtained from ryegrass harvested at three different harvests. In addition, the BK group had the highest abundance of Lactiplantibacillus plantarum (58.66%), and the CK group had the highest abundance of Enterococcus faecalis (42.88%). The most annotated metabolites among the differential metabolites of different harvests were peptides, and eight amino acids were dominant in the composition of the identified peptides. In the ryegrass silage, arginine, alanine, aspartate, and glutamate biosynthesis had the highest enrichment ratio in the metabolic pathway of KEGG pathway enrichment analysis. Valyl-isoleucine and glutamylvaline were positively correlated with Lactiplantibacillus plantarum. D-Pipecolic acid and L-glutamic acid were positively correlated with Levilactobacillus brevis. L-phenylalanyl-L-proline, 3,4,5-trihydroxy-6-(2-methoxybenzoyloxy) oxane-2-carboxylic acid, and shikimic acid were negatively correlated with Levilactobacillus brevis. In conclusion, this study explains the effects of different harvest frequencies on the fermentation quality, microbial community, and metabolites of ryegrass, and improves our understanding of the ensiling mechanisms associated with different ryegrass harvesting frequencies.

The aim of this study was to evaluate the effects of cellulase (CE) and lactic acid bacteria (LAB) on Caragana korshinskii silage by analyzing the fermentation parameters, chemical composition, and bacterial community. The Caragana korshinskii was harvested at the fruiting period and treated with cellulase and LAB alone as a control treatment with no additive (CK). The ensiling performance and bacterial community were determined after 3, 7, 15, 30, and 60 days of fermentation process. Compared with the CK group, the pH, dry matter loss, and ammonia nitrogen content were significantly (p < 0.05) decreased in the LAB and CE treatments. Compared with the CK and LAB group, the contents of acid detergent fiber, neutral detergent fiber, and acid detergent lignin in the CE group decreased significantly (p < 0.05), and the water-soluble carbohydrates, acetic acid, and lactic acid concentrations increased significantly (p < 0.05). At the genus level of microorganisms, the addition of cellulase and LAB significantly reduced the microbial diversity. Compared with the CK group (78.05%), the relative abundance of Lactiplantibacillus in the CE group (90.19%) and LAB group (88.40%) significantly (p < 0.05) increased. The relative abundance of Pediococcus in the CE group (3.66%) and LAB group (2.14%) was significantly (p < 0.05) lower than that in the CK group (14.73%). Predicted functional profiling of 16S rRNA genes revealed that the addition of cellulase and LAB increased the pyruvate metabolic pathway during Caragana korshinskii silage, thereby increasing the accumulation of lactic acid concentration. The addition of cellulase expressed a better advantage in the biosynthetic capacity of lysine. In summary, the addition of cellulase and LAB could adjust the bacterial community to improve the silage quality of Caragana korshinskii, and the addition of cellulase exhibited better results than the LAB additives.

This study investigated the influence of different temperatures (35℃ High temperature and average indoor ambient temperature of 25℃) and lactic acid bacterial additives (Lactiplantibacillus plantarym, Lentilactobacillus buchneri, or a combination of Lactiplantibacillus plantarym and Lentilactobacillus buchneri) on the chemical composition, fermentation quality, and microbial community of alfalfa silage feed. After a 60-day ensiling period, a significant interaction between temperature and additives was observed, affecting the dry matter (DM), crude protein (CP), acid detergent fiber (ADF), and neutral detergent fiber (NDF) of the silage feed (p < 0.05). Temperature had a highly significant impact on the pH value of the silage feed (p < 0.0001). However, the effect of temperature on lactic acid, acetic acid, propionic acid, and butyric acid was not significant (p > 0.05), while the inoculation of additives had a significant effect on lactic acid, acetic acid, and butyric acid (p > 0.05). As for the dynamic changes of microbial community after silage, the addition of three kinds of bacteria increased the abundance of lactobacillus. Among all treatment groups, the treatment group using complex bacteria had the best fermentation effect, indicating that the effect of complex lactic acid bacteria was better than that of single bacteria in high temperature fermentation. In summary, this study explained the effects of different temperatures and lactic acid bacterial additives on alfalfa fermentation quality and microbial community, and improved our understanding of the mechanism of alfalfa related silage at high temperatures.

This study aimed to isolate and identify lactic acid bacteria (LAB) from the native grass and naturally fermented silage from the Mongolian Plateau. The effect of selected strains on bacterial community and quality of native grass silage was also studied. Strains XM2, 265, and 842 could grow normally at 15°C–30°C, pH 4.0–8.0, and NaCl 3 and 6.5%; they were identified as Lactiplantibacillus plantarum subsp. plantarum, Pediococcus acidilactici, and Latilactobacillus graminis, by sequencing 16S rRNA, respectively. The three strains (XM2, 265, and 842) and one commercial additive (L) were used as inoculants and singularly added to the native grass. Compared to the control, the dry matter content was significantly (p < 0.05) lower in L and XM2 groups. The water-soluble carbohydrate content was significantly (p < 0.05) higher in control than in other groups. Compared with the control, the crude protein and ammonia nitrogen contents were significantly (p < 0.05) higher and lower in the LAB-treated groups, and the acid and detergent fiber contents were significantly (p < 0.05) reduced in the L and XM2 groups than those in other groups. There was a significant (p < 0.05) difference in the pH value, lactic acid content, and lactic acid-to-acetic acid ratio in L and XM2 groups than in other groups. Compared with the control, the number of LAB was significantly (p < 0.05) higher in LAB-treated silages, whereas no significant (p > 0.05) differences were observed in yeast and aerobic bacteria in all groups. Compared to the control, the Shannon index was significantly (p < 0.05) reduced. Simpson and Chao1 were significantly (p < 0.05) increased. Principal coordinate analysis based on the unweighted UniFrac distance showed clear separation of the bacterial community in fresh materials and LAB-treated silages. Besides, compared to the control, the principal coordinate analysis of LAB-treated silages was also separate. After 30 days of fermentation, the relative abundance of Firmicutes increased and was the primary phylum in all silages. Compared with the control, the abundance of Firmicutes and Proteobacteriawas significantly (p < 0.05) higher and lower in L and XM2 groups. In contrast, no significant differences were observed among control, 265, and 842 groups. At the genus level, the relative abundance of Lactobacillus, Enterobacter, Pediococcus, and Weissella was increased and dominated the native grass fermentation. Compared with the control, the abundance of Lactobacillus was significantly (p < 0.05) higher in L, XM2, and 842 groups, while no significant (p > 0.05) differences were observed between the control and 265 groups. The abundance of Pediococcus was higher than that in other groups. Consequently, the results demonstrated that LAB significantly influenced silage fermentation by reconstructing microbiota, and Lactobacillus was the dominant genus in the native grass silages. Furthermore, the results showed that strain XM2 could effectively improve the silage quality, and it is considered a potential starter for the native grass silage.