Chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage ensiling with lactic acid bacteria.

Mulberry has high crude protein and biologically active compounds but is difficult to be ensiled due to the lack of adequate epiphytic LAB. This study aimed to investigate the effects of inoculation with Lactiplantibacillus plantarum and Pediococcus pentosaceus isolated from mulberry with higher antioxidant capacity alone or in combination with Streptococcus bovis on chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage. The results showed that all inoculation groups had higher dry matter and lower pH than the control group, particularly in LP (dry matter, DM, 32.03% and pH = 4.44) and LP_PP_SB (DM, 31.68% and pH = 4.26) after 60 days of ensiling. Ammonia nitrogen (AN) content was the lowest in both LP_SB and LP_PP_SB groups, which were 1.86 g/kg FM and 1.05 g/kg FM, respectively, (P < 0.05). Only the LP_PP_SB group showed increased polyunsaturated fatty acids (PUFA, 1.2851 g/kg DM, P < 0.05) than the control group. Ferric-reducing antioxidant power (FRAP) values were increased in all inoculation-treated groups compared with the control group (P < 0.05). 2,2-Diphenyl-1-picrylhydrazyl (DDPH), 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and FRAP exhibited the highest levels in the LP_PP- and LP_PP_SB-treated groups. Enterobacter was dominant in both the control and SB-treated groups, and the relative abundance was 41.18% and 32.35%, respectively (P < 0.05). The relative abundance of Lactiplantibacillus was higher in the LP-, LP_PP-, and LP_SB-treated groups (81.84%-82.69%). Relative abundance of Pediococcus was higher in the PP-, PP_SB-, and LP_PP_SB-treated groups (74.27%-85.27%). Untargeted metabolomics analysis results showed that five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside)were upregulated in all inoculation groups (except for the SB-treated groups). Among them, eriodictyol was both positively correlated with ABTS and FRAP and also showed the highest relative abundance in the LP_PP- and LP_PP_SB-treated groups. To the best of our knowledge, this study was the first to investigate the relationship between inoculants of epiphytic lactic acid bacteria and antioxidant capacity by 16s rRNA Illumina sequencing technology and untargeted metabolomics analysis, respectively. Consequently, inoculated L. plantarum, P. pentosaceus alone, respectively, or in combination with S. bovis increased the relative abundance of Lactiplantibacillus and Pediococcus and decreased the relative abundance of Enterobacter, particularly in the LP_PP_SB-treated group. In addition, inoculants could increase the relative abundance of five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside), especially eriodictyol to improve the antioxidant capacity of mulberry silage.

Effects of Condensed Tannins on Bacterial and Fungal Communities during Aerobic Exposure of Sainfoin Silage.

BACKGROUND: Sainfoin is a forage legume that is widely distributed around the world and is beneficial for animals owing to the characteristics of its condensed tannins (CTs), which, from certain plants, can prolong the aerobic stability of silage. METHODS: The present study investigated whether sainfoin CTs can prolong aerobic stability by adding polyethylene glycol (PEG) to inactivate CT activity in the silage system. RESULTS: The results showed that aerobic stability increased under the PEG treatment (p < 0.05). Ammonia nitrogen (0.71 g/kg DM vs. 0.94 g/kg DM; p < 0.05) was higher in the PEG-treated group compared with the control after 3 d of aerobic exposure. BA was detected only in the PEG-treated group upon aerobic exposure. Yeasts were more abundant in the control compared with the PEG-treated group after 7 d of aerobic exposure, after which the relative abundance of Lactobacillus was lower in the PEG-treated group (65.01% vs. 75.01% in the control; p < 0.05), while the relative abundance of Pediococcus was higher in the PEG-treated group compared with the control (10.9% vs. 4.49%, respectively; p < 0.05).The relative abundances of Apiotrichum and Aspergillus were lower in the control than in the PEG-treated group after 7 d of aerobic exposure. CONCLUSIONS: The results suggested that sainfoin CTs decreased aerobic stability, but could inhibit certain bacteria and fungi, such as Pediococcus and Apiotrichum, and preserve the protein content during the aerobic exposure of silage.

Effects of combined nitrogen and phosphorus application on protein fractions and nonstructural carbohydrate of alfalfa.

Nitrogen (N) and phosphorus (P) fertilization significantly affect alfalfa production and chemical composition; however, the effect of combined N and P application on protein fractions and the nonstructural carbohydrate content of alfalfa is not fully understood. This two-year study investigated the effects of N and P fertilization on the protein fractions, nonstructural carbohydrates (NSC), and alfalfa hay yield. Field experiments were carried out using two nitrogen application rates (N60, 60 and N120, 120 kg N ha - 1) and four phosphorus application rates (P0, 0; P50, 50; P100, 100; and P150, 150 kg P ha - 1), total 8 treatment (N60P0, N60P50, N60P100, N60P150, N120P0, N120P50, N120P100 and N120P150). Alfalfa seeds were sown in the spring of 2019, uniformly managed for alfalfa establishment, and tested in the spring of 2021-2022. Results indicated that P fertilization significantly increased the hay yield (3.07-13.43% ranges), crude protein (6.79-9.54%), non-protein nitrogen of crude protein (fraction A) (4.09-6.40%), and NSC content (11.00-19.40%) of alfalfa under the same treatment of N application (p < 0.05), whereas non-degradable protein (fraction C) decreased significantly (6.85-13.30%, p < 0.05). Moreover, increasing N application resulted in a linear increase the content of non-protein N (NPN) (4.56-14.09%), soluble protein (SOLP) (3.48-9.70%), and neutral detergent-insoluble protein (NDIP) (2.75-5.89%) (p < 0.05), whereas acid detergent-insoluble protein (ADIP) content was significantly decreased (0.56-5.06%, p < 0.05). The regression equations for nitrogen and phosphorus application indicated a quadratic relationship between yield and forage nutritive values. Meanwhile, the comprehensive evaluation scores of NSC, nitrogen distribution, protein fractions, and hay yield by principal component analysis (PCA) revealed that the N120P100 treatment had the highest score. Overall, 120 kg N ha - 1 coupled with 100 kg P ha - 1 (N120P100) promoted the growth and development of perennial alfalfa, increased soluble nitrogen compounds and total carbohydrate content, and reduced protein degradation, thus improving the alfalfa hay yield and nutritional quality.

Effects of Lactiplantibacillus plantarum and Lactiplantibacillus brevis on fermentation, aerobic stability, and the bacterial community of paper mulberry silage.

The present study investigated the dynamic profiles of fermentation quality, aerobic stability, and the bacterial community of paper mulberry silage inoculants without (Control) or with Lactiplantibacillus plantarum (LP), Lactiplantibacillus brevis (LB), or their combination (LPLB), which was screened from naturally fermented paper mulberry. The results showed that the inoculated treatments had significantly reduced neutral detergent fiber, water-soluble carbohydrates, and ammoniacal nitrogen contents compared with the control after 60 days of ensiling (the decreased proportion of LP, LB, and LPLB treatments ranged from 7.33%-11.23%, 9.60%-21.44%, and 21.53%-29.23%, respectively, p < 0.05). The pH value of the LP and LB treatments was significantly lower than that of the control after 60 days of ensiling (4.42 and 4.56 vs. 4.71, p < 0.05). The LP treatment promoted lactic acid accumulation and LAB number compared with the control (66.59% vs. 54.12% and 8.71 log10 CFU/g vs. 8.52 log10 CFU/g, respectively, p < 0.05), and the LB and LPLB treatments inhibited the growth of yeast and mold after 14 days of fermentation. After 5 days of aerobic exposure, both the LB and LPLB treatments increased the aerobic stability time and acetic acid content (from 29 to 75 h and 16.14%-48.72%, respectively, p < 0.05), inhibited the growth of yeast and mold, and did not detect butyric acid. Additionally, the bacteria community of each treatment was dominated by Aerococcus on day 3 of ensilage (accounting for 54.36%-69.31%), while the inoculated treatments reduced the abundance of Aerococcus on day 60 (from 59.73% to 85.16%, p < 0.05), and Lactobacillus became the dominant genus (accounting for 54.57%-70.89%). Inoculation of L. plantarum effectively maintained the acidic environment at the end of the fermentation system by maintaining the abundance of Lactobacillus, maximizing the preservation of dry matter and protein, and reducing protein corruption. Inoculation of L. brevis alone or in combination with L. plantarum significantly inhibited the growth of mold and improved the aerobic stability of paper mulberry silage.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

Effect of Intrinsic Tannins on the Fermentation Quality and Associated with the Bacterial and Fungal Community of Sainfoin Silage.

Sainfoin (Onobrychis viciifolia) is rich in condensed tannins (CT). CT function includes inhibiting bacterial and fungi activity during the ensiling process. We used polyethylene glycol (PEG) to deactivate tannin activity to find out the effects of CT. The results show that the addition of PEG increased dry-matter loss (8.32% vs. 14.15%, on a dry-matter basis) after 60 d of ensiling, and also increased lactic acid (10.90% vs. 15.90%, on a dry-matter basis) and acetic-acid content (7.32% vs. 13.85%, on a dry-matter basis) after 30 d of ensiling. The PEG-treated group increased its Pediococcus relative abundance (0.37−3.38% vs. 7.82−23.5%,) during the ensiling process, increased its Gibellulopsis relative abundance after 3 d of ensiling (5.96% vs. 19.52%), increased its Vishniacozyma relative abundance after 3 d and 7 d of ensiling (2.36% vs. 17.02%, 3.65% vs. 17.17%), and increased its Aspergillus relative abundance after 7 d, 14 d and 60 d of ensiling (0.28% vs. 1.32%, 0.49% vs. 2.84% and 1.74% vs. 7.56%). However, the PEG-treated group decreased its Alternaria relative abundance during entire ensiling process (14.00−25.21% vs. 3.33−7.49%). These results suggest that condensed tannins inhibit lactic-acid bacteria fermentation though reducing Pediococcus activity, and inhibiting fungi activity depending on different strains.

Potential factors causing failure of whole plant nettle (Urtica cannabina) silages.

Introduction: Nettle is kind of new feed resources and benefit for animal production. However, a few studies observed that quality of nettle silage was poor under naturally fermentation. Consider of microbial activity was the mainly factors for fermentation characteristics of silage. Methods: Thus, the present study investigated the potential factors causing nettle silage failure through metabolome and bacterial community composition analyses during ensiling. Results: During ensiling, the pH was >6.22, and water-soluble carbohydrate and organic acid contents stabilized after 7 d. At the genus level, Enterococcus, Weissella, and Pediococcus were the dominant bacteria (relative abundance were 30.06-39.39, 17.29-23.34, and 3.13-7.22%, respectively), with stable trends, whereas Lactococcus and Enterobacter relative abundance decreased significantly over time (relative abundance were 5.68-13.96 and 3.86-24.1%, respectively). Lactobacillus relative abundance was <1% during the entire ensiling period, and malic acid metabolic pathway was the most important pathway. Enterococcus, Pediococcus, and Weissella were negatively correlated with malic acid, with Lactobacillus displaying an opposite trend. Discussion: The results suggested that Lactobacillus activity was the lowest among lactic acid bacteria (LAB) during ensiling, which is the main reason for nettle ensiling failure, and attributable to a low capacity to compete for fermentation substrates such as malic acid against other LAB during ensiling. Additionally, anti-bacteria activity of nettle probably inhibited Enterobacter activity during ensiling. Present study probably given a solution for improve nettle silage quality through addition with malic acid.