Haiping Li

Freeze–thaw cycles on the Qinghai–Tibetan Plateau inhibit microbial activity and challenge silage preservation. This paper aimed to elucidate how an indigenous, freeze–thaw-resistant Lactobacillus plantarum strain (LP160) improves oat silage quality under such stress. Oats were ensiled for 60 days under constant 20 °C (t) or freeze–thaw cycles (12 h at 20 °C/−5 °C; s) with or without LP160 inoculation. Samples after ensiling and 5-day aerobic exposure were analyzed for fermentation parameters, nutrients, microbiome, and non-targeted metabolomics using liquid chromatography–tandem mass spectrometry (LC-MS/MS). LP160 inoculation improved silage quality, as shown by the lower pH, ammoniacal nitrogen, neutral detergent fiber, acid detergent fiber contents as well as the greater amount of lactic acid. Key findings demonstrated that LP160 inoculation significantly enhanced Lactobacillus dominance, effectively curbed the growth of detrimental bacteria like Mucor, and regulated the microbial structure. During the aerobic exposure phase, the microbial community structures and successions varied under different temperature treatments. When inoculated under freeze–thaw conditions, the genus Bacillus increased, while Paenibacillus was not impeded. A total of 943 metabolites were identified, predominantly comprising amino acids, fatty acids, and the like. The expressions of metabolites with antioxidant and antibacterial properties were upregulated with LP160 inoculation. This led to the inhibition of protein hydrolysis and a reduction in ammonia–nitrogen production. The results of correlation analysis indicated that inoculating LP160 suppressed the proliferation of Mucor and enhanced the abundance of Torulaspora; meanwhile, the expression of L-palmitoylcarnitine involved in the fatty acid degradation pathway and fatty acid metabolism pathway was inhibited along with the generation of ammonia–nitrogen. Consequently, the degradation of fatty acids and proteins was restrained. The results of this paper provided new insights into the silage under freeze–thaw conditions.

This study explored the patterns and mechanisms influencing changes in silage quality, mycotoxin accumulation, and microbial community structure in oat silage after lodging. Upright oat forage (control, CK), lodging oat forage (upper layer (UL), lower layer (LL), and mixed layers (MLs) were harvested at 0, 7, 25, and 45 days after lodging and ensiled for 60 days. The results showed that the dry matter (DM) and water-soluble carbohydrate (WSC) content decreased significantly (p < 0.05), whereas crude protein (CP) and fiber content increased significantly compared to upright oats (p < 0.05). The WSC and CP content in silage decreased with increasing lodging duration. The fiber content increased in late harvest after lodging. The risk of mycotoxin infection increased after lodging, with aflatoxin levels exceeding EU limits. The mycotoxins in UL silage were the lowest when lodging lasted for seven days. Lodging oat silage was dominated by Lactobacillus, and the Pseudomonas in the lodging group was less than 4%. The fungi in lodging oat silage was lower, and the UL (upper layer) treatment was the lowest when lodging for 7 days. Overall, the transfer of microorganisms, especially Plectosphaerella, Fusarium, Alternaria, Cladosporium, and Botryotrichum, from soil to silage following oat collapse is of interest. The results suggest the soil–plant microbial interactions and their effects on silage fermentation and mycotoxins in lodging oats.

Planting oat forage in fallow fields during winter and producing total mixed ration (TMR) silage can effectively address issues of land wastage and forage shortages while maintaining forage quality. This study used oats and common vetch grown in winter fields in southern China as base materials, with additives including corn flour, soybean meal, corn lees, cottonseed meal, and premixes to formulate mixed feeds with roughage-to-concentrate ratios of 75:25, 70:30, and 65:35 on a dry matter basis. TMR silage was inoculated with a customized mixed lactic acid bacteria (LAB) additive composed of Lactobacillus plantarum 160 (patent number ZL202210218695.5), Lactobacillus pentosus 260 (patent number ZL202210204293), and Lactobacillus buchneri 225 (patent number ZL202210204293), at a ratio of 2:1:1, with addition rates of 4 × 10⁶, 2 × 10⁶, and 2 × 10⁶ cfu/g, respectively (IN), while sterile distilled water served as the control (CK). After a 60-day fermentation, the cornell net carbohydrate protein system (CNCPS) and in vitro digestion analysis were used to assess the effects of different roughage-to-concentrate ratios on the carbohydrate and protein components and ruminal degradation rate of fermented TMR (FTMR) silage, as well as to evaluate the impact of mixed LAB inoculation on FTMR nutritional quality, fermentation quality, and aerobic stability. The results indicated the following: (1) Regardless of the LAB addition, dry matter (DM), ether extract (EE), crude protein (CP), and Ash contents significantly decreased (p < 0.05) as the concentrate level decreased. In the IN group, as the concentrate level decreased, the water-soluble carbohydrate (WSC) content significantly increased (p < 0.05), the pH significantly decreased (p < 0.05), and the NH₃-N/TN significantly decreased (p < 0.05), with LAB counts significantly higher at a 65:35 roughage-to-concentrate ratio than in the other two groups. In the CK group, no significant changes (p > 0.05) were observed in the WSC content, pH, or LAB counts. (2) CNCPS analysis showed that in the IN group, the carbohydrate (CHO) content at a 75:25 roughage-to-concentrate ratio was significantly higher than in the other two groups (p < 0.05), while the non-utilizable carbohydrate (CC) content was significantly lower (p < 0.05). As the concentrate levels decreased, the non-protein nitrogen (PA) and moderately degradable true protein (PB2) content significantly increased (p < 0.05), whereas the rapidly degradable true protein (PB1) and slowly degradable true protein (PB3) content significantly decreased (p < 0.05). In the CK group, the CHO, PA, PB2, and PC content significantly increased (p < 0.05) as concentrate levels decreased, while the PB1 and PB3 content significantly decreased (p < 0.05). (3) In vitro digestibility characteristics indicated that gas production (GP) in the IN group was significantly lower than in the CK group (p < 0.05), with crude protein degradability increasing as concentrate levels decreased, regardless of the LAB addition. (4) At a 65:35 roughage-to-concentrate ratio, aerobic stability in the IN group was significantly higher than in the CK group (p < 0.05). In conclusion, higher concentrate ratios in total mixed rations (TMRs) with varying roughage-to-concentrate proportions improve the nutritional quality and promote the ruminal degradation of the FTMR. LAB inoculant addition could be an effective approach for addressing FTMR feed challenges.

To further explore the effects of heterofermentative lactic acid bacteria (LAB) on silage fermentation and aerobic stability, whole-plant corn at around the 1/2 milk-line stage was freshly chopped and ensiled in laboratory silos with deionized water (control), Lactobacillus buchneri (LB), or L. rhamnosus (LR). Each treatment was prepared in triplicate for 3, 14, and 60 d of fermentation, followed by 3 and 7 days of aerobic exposure. The dynamic changes in microbial community were studied by single molecule real-time (SMRT) sequencing. The results showed that the two LAB inoculants altered the microbial communities in different ways. Succession from L. plantarum to L. buchneri and L. rhamnosus was observed in LB- and LR-treated silage, respectively. Both silages improved aerobic stability (82 and 78 h vs. 44 h) by occupying the microbial niche to produce higher levels of acetic acid at terminal fermentation. Because Acetobacter fabarum dominated in the silages after aerobic exposure, beta diversity dramatically decreased. In this study, a. fabarum was reported for the first time in silage and was related to aerobic spoilage. The two heterofermentative LAB produced acetic acid and improved the aerobic stability of the corn silage by occupying the microbial niche at terminal fermentation. Inoculated L. rhamnosus had a greater pH for a longer period of time after opening and less DM loss at day 7.