The Development and Characterization of Novel Ionic Liquids Based on Mono- and Dicarboxylates with Meglumine for Drug Solubilizers and Skin Permeation Enhancers.

In this study, we synthesized a family of novel ionic liquids (ILs) with meglumine (MGM) as cations and tartaric acid (TA), azelaic acid (AA), geranic acid (GA), and capric acid (CPA) as anions, using pharmaceutical additives via simple acid-base neutralization reactions. The successful synthesis was validated by attenuated total reflection-Fourier transform infrared (ATR-FTIR) and powder X-ray diffraction (PXRD). Thermal analysis using differential scanning calorimetry confirmed the glass transition temperature of MGM-ILs to be within the range of -43.4 °C--13.8 °C. We investigated the solubilization of 15 drugs with varying pKa and partition coefficient (log P) values using these ILs and performed a comparative analysis. Furthermore, we present MGM-IL as a new skin permeation enhancer for the drug model flurbiprofen (FRP). We confirmed that AA/MGM-IL improves the skin permeation of FRP through hairless mouse skin. Moreover, AA/MGM-IL enhanced drug skin permeability by affecting keratin rather than stratum corneum lipids, as confirmed by ATR-FTIR. To conclude, MGM-ILs exhibited potential as drug solubilizer and skin permeation enhancers of drugs.

High-Performance Photodynamic Therapy of Tongue Squamous Cell Carcinoma with Multifunctional Nano-Verteporfin.

Background: The photodynamic therapy (PDT) showed promising potential in treating tongue squamous cell carcinoma (TSCC). The Food and Drug Administration approved Verteporfin (Ver) is a powerful alternative in this field for its penetrating power and high production of reactive oxygen species (ROS). However, its applications in the treatment of TSCC are still rare. Methods: Ver was loaded onto Poly (lactic-co-glycolic acid) (PLGA) nanoparticles, followed by the modification with RGD peptide as the ligand. The nanostructured was named as RPV. In vitro assessments were conducted to evaluate the cytotoxicity of RPV through the Live/Dead assay analysis and Cell Counting Kit-8 (CCK-8) assay. Using the reactive oxygen species assay kit, the potential for inducing targeted tumor cell death upon laser irradiation by promoting ROS production was investigated. In vivo experiments involved with the biological distribution of RPV, the administration with RPV followed by laser irradiation, and the measurement of the tumor volumes. Immunohistochemical analysis was used to detect the Ki-67 expression, and apoptosis induced by RPV-treated group. Systemic toxicity was evaluated through hematoxylin-eosin staining and blood routine analysis. Real-time monitoring was employed to track RPV accumulation at tumor sites. Results: The in vitro assessments demonstrated the low cytotoxicity of RPV and indicated its potential for targeted killing TSCC cells under laser irradiation. In vivo experiments revealed significant tumor growth inhibition with RPV treatment and laser irradiation. Immunohistochemical analysis showed a notable decrease in Ki-67 expression, suggesting the effective suppression of cell proliferation, and TUNEL assay indicated the increased apoptosis in the RPV-treated group. Pathological examination and blood routine analysis revealed no significant systemic toxicity. Real-time monitoring exhibited selective accumulation of RPV at tumor sites. Conclusion: The findings collectively suggest that RPV holds promise as a safe and effective therapeutic strategy for TSCC, offering a combination of targeted drug delivery with photodynamic therapy.

Nano-Photosensitizer Directed Targeted Phototherapy Effective Against Oral Cancer in Animal Model.

Background: Photodynamic therapy (PDT) has emerged as a promising strategy for oral cancer treatment. Verteporfin is a powerful photosensitizer and widely used in the treatment of macular degeneration. However, rare work has reported its potential in the treatment of oral cancer. Methods: In this study, we introduce an innovative approach of nano-photosensitizer based on Verteporfin, which was prepared by utilizing macrophage membrane to coat Verteporfin-loaded zeolitic imidazolate framework 8 (ZIF-8) for effective photodynamic therapy against oral cancer. Nanoparticle characteristics were assessed including size, zeta potential, and PDI. Cellular uptake studies were conducted using CAL-27 cells. Furthermore, inhibitory effects in both in vitro and in vivo settings were observed, ensuring biosafety. Assessment of anticancer efficacy involved tumor volume measurement, histological analyses, and immunohistochemical staining. Results: In vitro experiments indicated that the nano-photosensitizer showed efficient cellular uptake in the oral cancer cells. Upon the laser irradiation, the nano-photosensitizer induced the generation of reactive oxygen species (ROS), leading to cancer cell apoptosis. The in vivo experiments indicated that the coating with cell membranes enhanced the circulation time of nano-photosensitizer. Moreover, the specificity of the nano-photosensitizer to the cancer cells was also improved by the cell membrane-camouflaged structure in the tumor-bearing mouse model, which inhibited the tumor growth significantly by the photodynamic effect in the presence of laser irradiation. Conclusion: Overall, our findings demonstrate the potential of macrophage membrane-coated ZIF-8-based nanoparticles loaded with Verteporfin for effective photodynamic therapy in oral cancer treatment. This nano-system holds promise for synergistic cancer therapy by combining the cytotoxic effects of PDT with the activation of the immune system, providing a novel therapeutic strategy for combating cancer.

The mirrored cationic peptide as miRNA vehicle for efficient lung cancer therapy.

Gene therapy has emerged as a potential approach for lung cancer therapy. However, the application of gene therapy is still limited by their properties, such as low specificity to the cancer cells, negatively charged groups, short systemic circulation time, and rapid degradation by nucleases. The progression of lung adenocarcinoma (LUAD) can be promoted through the methylation process of miR-148a-3p promoter, as confirmed by our previous research. In the current study, we are the first to design a mirrored Arg-Gly-Asp (RGD)-modified cationic peptide (RD24) as a microRNA (miRNA) vehicle, which enabled to pack the miRNA (miR-148a-3p) efficiently and generate RD24/miR-148a-3p nanoparticles (RPRIN) by self-assembling. RPRIN exhibited a high transfection efficiency in lung cancer cells via the conjugation between RGD and integrins on the surface of lung cancer cells. Furthermore, RD24 showed matrix metallopeptidase 2 (MMP2) responsiveness, which improved lung cancer cell inhibition induced by the miRNA intracellularly. In addition, RPRIN exhibits several advantages, such as prolonged circulation duration, reduced toxicity, and immune escape. Experiments conducted both in vitro and in vivo revealed that RPRIN effectively suppressed the growth and progression of lung cancer. Thus, the mirrored RGD-modified cationic peptide showed great potential in transducing miRNA for lung cancer therapy.

Inhibition of YAP1 activity ameliorates acute lung injury through promotion of M2 macrophage polarization.

The balance of M1/M2 macrophage polarization plays an important role in regulating inflammation during acute lung injury (ALI). Yes-associated protein (YAP1) is a key protein in the Hippo-YAP1 signaling pathway and is involved in macrophage polarization. We aimed to determine the role of YAP1 in pulmonary inflammation following ALI and regulation of M1/M2 polarization. Pulmonary inflammation and injury with upregulation of YAP1 were observed in lipopolysaccharide (LPS)-induced ALI. The YAP1 inhibitor, verteporfin, attenuated pulmonary inflammation and improved lung function in ALI mice. Moreover, verteporfin promoted M2 polarization and inhibited M1 polarization in the lung tissues of ALI mice and LPS-treated bone marrow-derived macrophages (BMMs). Additionally, siRNA knockdown confirmed that silencing Yap1 decreased chemokine ligand 2 (CCL2) expression and promoted M2 polarization, whereas silencing large tumor suppressor 1 (Lats1) increased CCL2 expression and induced M1 polarization in LPS-treated BMMs. To investigate the role of inflammatory macrophages in ALI mice, we performed single-cell RNA sequencing of macrophages isolated from the lungs. Thus, verteporfin could activate the immune-inflammatory response, promote the potential of M2 macrophages, and alleviate LPS-induced ALI. Our results reveal a novel mechanism where YAP1-mediated M2 polarization alleviates ALI. Therefore, inhibition of YAP1 may be a target for the treatment of ALI.

Dynamic covalent nano-networks comprising antibiotics and polyphenols orchestrate bacterial drug resistance reversal and inflammation alleviation.

New antimicrobial strategies are urgently needed to meet the challenges posed by the emergence of drug-resistant bacteria and bacterial biofilms. This work reports the facile synthesis of antimicrobial dynamic covalent nano-networks (aDCNs) composing antibiotics bearing multiple primary amines, polyphenols, and a cross-linker acylphenylboronic acid. Mechanistically, the iminoboronate bond drives the formation of aDCNs, facilitates their stability, and renders them highly responsive to stimuli, such as low pH and high H2O2 levels. Besides, the representative A1B1C1 networks, composed of polymyxin B1(A1), 2-formylphenylboronic acid (B1), and quercetin (C1), inhibit biofilm formation of drug-resistant Escherichia coli, eliminate the mature biofilms, alleviate macrophage inflammation, and minimize the side effects of free polymyxins. Excellent bacterial eradication and inflammation amelioration efficiency of A1B1C1 networks are also observed in a peritoneal infection model. The facile synthesis, excellent antimicrobial performance, and biocompatibility of these aDCNs potentiate them as a much-needed alternative in current antimicrobial pipelines.

Dynamics of Phyllosphere Microbiota and Chemical Parameters at Various Growth Stages and Their Contribution to Anaerobic Fermentation of Pennisetum giganteum.

This work evaluated the dynamic changes of phyllosphere microbiota and chemical parameters at various growth stages of Pennisetum giganteum and their effects on the bacterial community, cooccurrence networks, and functional properties during anaerobic fermentation. P. giganteum was collected at two growth stages (early vegetative stage [PA] and late vegetative stage [PB]) and was naturally fermented (NPA and NPB) for 1, 3, 7, 15, 30, and 60 days, respectively. At each time point, NPA or NPB was randomly sampled for the analysis of chemical composition, fermentation parameter, and microbial number. In addition, the fresh, 3-day, and 60-day NPA and NPB were subjected to high-throughput sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional prediction analyses. Growth stage obviously affected the phyllosphere microbiota and chemical parameters of P. giganteum. After 60 days of fermentation, NPB had a higher lactic acid concentration and ratio of lactic acid to acetic acid but a lower pH value and ammonia nitrogen concentration than NPA. Weissella and Enterobacter were dominant in 3-day NPA and Weissella was dominant in 3-day NPB, while Lactobacillus was the most abundant genus in both 60-day NPA and NPB. The complexity of bacterial cooccurrence networks in the phyllosphere decreased with P. giganteum growth. The ensiling process further decreased the complexity of bacterial networks, with the simplest bacterial correlation structures in NPB. There were great differences in the KEGG functional profiles of PA and PB. Ensiling promoted the metabolism of lipid, cofactors, vitamins, energy, and amino acids but suppressed the metabolism of carbohydrates and nucleotides. Storage time had a greater influence than growth stage on bacterial community diversity, cooccurrence networks, and functional profiles of P. giganteum silage. Differences in bacterial diversity and functionality of P. giganteum silage caused by growth stage appear to be offset by long-term storage. IMPORTANCE The phyllosphere microbiota consists of various and complex microbes, including bacteria with crucial relevance to the quality and safety of fermented food and feed. It initially derives from soil and becomes specific to its host after interaction with plants and climate. Bacteria associated with the phyllosphere are highly abundant and diverse, but we know little about their succession. Here, the phyllospheric microbiota structure was analyzed within the growth of P. giganteum. We also evaluated the effects of phyllosphere microbiota and chemical parameter changes on the anaerobic fermentation of P. giganteum. We observed remarkable differences in bacterial diversity, cooccurrence, and functionality of P. giganteum at various growth stages and storage times. The obtained results are important for understanding the fermentation mechanism and may contribute to high-efficient production without additional cost.

Sustainable utilization of residual grass: effect of anaerobic storage days on chemical composition, fermentation performance, microbial community, and functional profiles of Pennisetum giganteum.

The seasonal surplus and putrefactive property of moist forages inevitably increase the pressure on environmental protection and residual grass disposal. In the current work, the anaerobic fermentation approach was adopted to assist the sustainable recycling of leftover Pennisetum giganteum (LP), and its chemical composition, fermentation performance, bacterial community and functional profiles during anaerobic fermentation were studied. Fresh LP was spontaneously fermented for up to 60 d. At the end of anaerobic fermentation, fermented LP (FLP) displayed homolactic fermentation with low pH value, ethanol, and ammonia nitrogen concentrations but high lactic acid concentration. Weissella was dominant in 3-day FLP, yet Lactobacillus was the overwhelming genus (92.6%) in 60-day FLP. The anaerobic fermentation process promoted (P < 0.05) the metabolism of carbohydrate and nucleotide while suppressing (P < 0.05) that of lipid, cofactors, vitamins, energy, and amino acid. The results showed that the residual grass with LP as an example could be successfully fermented even if no additives were added, without signs of clostridial and fungal contamination.

Assessing the impact of phyllosphere microbiota on dynamics of in-silo fermentation of Italian ryegrass harvested at heading and blooming stages.

BACKGROUND: The present study aimed to investigate the relationship between the phyllosphere microbiota of Italian ryegrass (Lolium multiflorum Lam.) harvested at heading (H) [> 50% earing rate or 216 g kg-1 fresh weight (FW)] and blooming (B) (> 50% bloom or 254 g kg-1 FW) stages and in-silo fermentation products, and the composition, abundance, diversity and activity of bacterial community. In total, 72 (4 treatments × 6 ensiling durations × 3 replicates) laboratory scale (400 g) silages of Italian ryegrass were prepared: (i) irradiated heading stage silages (IRH) (n = 36) were inoculated with phyllosphere microbiota inoculum (2 mL) eluted from fresh Italian ryegrass at either heading (IH) (n = 18) or blooming (IB) (n = 18) stages; (ii) irradiated blooming stage silages (IRB) (n = 36) were inoculated with either IH (n = 18) or IB (n = 18). Triplicate silos of each treatment were analyzed after 1, 3, 7, 15, 30 and 60 days of ensiling. RESULTS: In fresh forage, Enterobacter, Exiguobacterium and Pantoea were the three major genera at heading stage, and Rhizobium, Weissella and Lactococcus were the most abundant genera at blooming stage. Higher metabolic activity was found in IB. After 3 days of ensiling, the large amounts of lactic acid in IRH-IB and IRB-IB can be attributed to the higher abundances of Pediococcus and Lactobacillus, 1-phosphofructokinase, fructokinase, l-lactate dehydrogenase and glycolysis I, II and III. CONCLUSION: The composition, abundance, diversity and functionality of the phyllosphere microbiota of Italian ryegrass at different growth stages could remarkably affect silage fermentation characteristics. © 2023 Society of Chemical Industry.

Dynamics in fermentation quality, bacterial community, and metabolic profile during silage fermentation of late-harvested elephant grass.

The present study aimed to evaluate the effects of delayed harvest and storage length on fermentation products, bacterial community, and metabolic shifts of elephant grass silage. The late-harvested elephant grass (LG) was naturally fermented (NLG) for 1, 3, 7, 15, 30, and 60 days, respectively. After 60-day ensiling, NLG displayed homolactic fermentation with low pH value, butyric acid, and ammonia nitrogen concentrations, and high lactic acid concentration, and ratio of lactic acid to acetic acid. Pseudomonas, Sphingomonas, and Pantoea dominated the bacterial community in LG, but Lactobacillus, Lactococcus, and Pediococcus were the advantageous genera in a 3-day and 60-day NLG. The correlation heatmap revealed that Acetobacter was positively related to acetic acid, ethanol, ammonia nitrogen, and butyric acid concentrations. There were distinct differences in the KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic profiles of fresh and ensiled LG. Ensiling suppressed the metabolism of amino acid, vitamins, and energy, while promoted the metabolism of carbohydrate. The LG can be well-fermented without additives, but its low crude protein content should not be ignored when applied in agricultural practice. The ensiling process remarkably affected the fermentation quality, bacterial community, and metabolic profiles of NLG.

The effects of epiphytic microbiota and chemical composition of Italian ryegrass harvested at different growth stages on silage fermentation.

BACKGROUND: The influence of epiphytic microbiota and chemical composition on fermentation quality and microbial community of Italian ryegrass silage was evaluated. Italian ryegrass harvested at the filling stage (FS) and the dough stage (DS) was sterilized by gamma-ray irradiation and inoculated as follows: (I) FS epiphytic microbiota + irradiated FS (FF); (II) FS epiphytic microbiota + irradiated DS (FD); (III) DS epiphytic microbiota + irradiated DS (DD); (IV) DS epiphytic microbiota + irradiated FS (DF). RESULTS: After 60 days of ensiling, silage made from irradiated FS had a lower pH and ammonia nitrogen (NH3 -N) content and a higher lactic acid (LA) content than that made from irradiated DS. Similarly, silage inoculated with the epiphytic microbiota of DS had a lower pH and NH3 -N content and a higher LA content than that inoculated with the epiphytic microbiota of FS. However, LA-type fermentation (lactic acid:acetic acid > 2:1) was presented at DF and DD. The principal coordinates analysis showed that the distance between FF and DF and FD and DD was closer than other treatments, suggesting that the microbial community of silages made from irradiated FS (or DS) was more similar. CONCLUSION: The epiphytic microbiota played a more important role in the fermentation type, whereas the chemical composition had a great influence on the contents of fermentation end-products. However, chemical composition had a stronger effect on the microbial community of silage than the epiphytic microbiota. © 2022 Society of Chemical Industry.

Diurnal Variation of Epiphytic Microbiota: an Unignorable Factor Affecting the Anaerobic Fermentation Characteristics of Sorghum-Sudangrass Hybrid Silage.

Forage epiphytic microbiota exhibits pronounced changes in composition and function throughout the day. However, the effects of these changes on silage fermentation are rarely explored. Here, we transplanted the epiphytic microbiota of sorghum-sudangrass hybrid (SSG) harvested at 7:00 h (AM), 12:00 h (M), and 17:00 h (PM) to sterilized SSG to evaluate the effects of diurnal variation of epiphytic microbiota on fermentation characteristics. During fermentation, remarkable differences in bacterial community successions were observed between silages inoculated with AM and M microbiota. Compared to AM microbiota, M microbiota inoculation increased the proportions of Pantoea dispersa, Leuconostoc lactis, Enterobacter, and Klebsiella variicola, whereas it decreased the proportions of Weissella cibaria and Lactobacillus plantarum during fermentation. This led to the most rapid pH declines and organic acid production in AM silage and the slowest in M silage. Both M and PM microbiota affected the bacterial cooccurrence patterns, indicated by decreased complexity and stability in the community structures of M and PM silages compared to that of AM silage. The predicted functions indicated that some key carbohydrate metabolism pathways related to lactic acid synthesis were downregulated, while some competing pathways (ascorbate and aldarate metabolism and C5-branched dibasic acid metabolism) were upregulated in M silage compared to AM silage after 3 days of fermentation. Correlation analysis revealed positive correlations between competing pathways and enterobacterial species. The current study highlights the importance of diurnal variation of epiphytic microbiota in affecting the silage bacterial community, potentially providing an effective strategy to improve silage quality by optimizing harvest time. IMPORTANCE Ensiling is a way to preserve wet biomass for animal and bioenergy production worldwide. The fermentation quality of silage is largely dependent on the epiphytic microbiota of the material. Plant epiphytic microbiota exhibit diurnal changes in composition and function. However, the effects of these changes on silage fermentation are rarely explored. The results presented here demonstrated that diurnal variation of epiphytic microbiota could affect the fermentation characteristics and bacterial community during SSG fermentation. Marked bacterial community differences were observed between AM and M silages during the initial 3 days of fermentation. The dominance rate of Lactobacillus plantarum was highest in AM silage, whereas enterobacterial species were more abundant in M silage. The predicted function revealed downregulated lactic acid synthesis pathways and upregulated competing pathways in M silage compared to those in AM silage. This study provides clues for technological-parameter optimization of the fermentation process by the selection of harvest time.

Exploring the Epiphytic Microbial Community Structure of Forage Crops: Their Adaptation and Contribution to the Fermentation Quality of Forage Sorghum during Ensiling.

In this study, the effects of epiphytic microbiota from different forages on the fermentation characteristics and microbial community structure of forage sorghum silage were investigated. The gamma irradiated sterilized forage sorghum was treated through sterile water, epiphytic microbiota of forage sorghum (FSm), Sudan grass (SDm), Napier grass (NPm), and maize (MZm). NPm and SDm inoculated silages showed similar pH value and lactic acid (LA) and acetic acid (AA) contents at day 3 and 60 of ensiling. The final silage of FSm and MZm showed lower (p < 0.05) pH and AA content and a higher LA content compared to the NPm and SDm silages. Bacterial species from the Weisella genus were predominantly present in FSm, NPm, and SDm, while Lactococcus dominated the MZm silage during early ensiling. Lactobacillus was predominant in all inoculated terminal silages. Overall, the four inoculated microbiota decreased the pH value of silage and were dominated by lactic acid bacteria (LAB); however, the NPm and SDm treatments resulted in comparatively higher AA contents which could have an inhibitory effect on the secondary fermentation developed by the yeast and enhanced the aerobic stability of forage sorghum silage.

The reversion of DNA methylation-induced miRNA silence via biomimetic nanoparticles-mediated gene delivery for efficient lung adenocarcinoma therapy.

BACKGROUND: Lung cancer is one of the fatal cancers worldwide, and over 60% of patients are lung adenocarcinoma (LUAD). Our clinical data demonstrated that DNA methylation of the promoter region of miR-126-3p was upregulated, which led to the decreased expression of miR-126-3p in 67 cases of lung cancer tissues, implying that miR-126-3p acted as a tumor suppressor. Transduction of miR-126-3p is a potential therapeutic strategy for treating LUAD, yet the physiological environment and properties of miRNA challenge current transduction approaches. METHODS: We evaluated the expression of miR-126-3p in 67 pairs of lung cancer tissues and the corresponding adjacent non-tumorous tissues by Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The relationship between the overall survival of lung cancer patients and miR-126-3p was analyzed by the Cancer Genome Atlas cohort database (Oncolnc, http://www.oncolnc.org ). We analyzed DNA methylation Methylation-specific PCR (MSP) analysis. To determine whether ADAM9 is the direct target of miR-126-3p, we performed the 3'-UTR luciferase reporter assay. The protein levels in the cells or tissues were evaluated with western blotting (WB) analysis. The biodistribution of nanoparticles were monitored by in vivo tracking system. RESULTS: We describe the development of novel stealth and matrix metalloproteinase 2 (MMP2)-activated biomimetic nanoparticles, which are constructed using MMP2-responsive peptides to bind the miR-126-3p (known as MAIN), and further camouflaged with red blood cell (RBC) membranes (hence named REMAIN). REMAIN was able to effectively transduce miRNA into lung cancer cells and release them via MMP2 responsiveness. Additionally, REMAIN possessed the advantages of the natural RBC membrane, including extended circulation time, lower toxicity, better biocompatibility, and immune escape. Moreover, in vitro and in vivo results demonstrated that REMAIN effectively induced apoptosis of lung cancer cells and inhibited LUAD development and progression by targeting ADAM9. CONCLUSION: The novel style of stealth and MMP2-activated biomimetic nanoparticles show great potential in miRNA delivery.

Changes in the fermentation products, taxonomic and functional profiles of microbiota during high-moisture sweet sorghum silage fermentation.

The purpose of this study was to evaluate the fermentation quality, microbial community, and functional shifts of sweet sorghum during ensiling. The high-moisture sweet sorghum (SS) was naturally ensiled for 1, 3, 7, 15, 30, and 60 days. After 60 days of ensiling, sweet sorghum silage (SSS) showed homolactic fermentation with absent butyric acid, low pH value, acceptable concentrations of propionic acid, ethanol, and ammonia nitrogen and high lactic acid concentration. Acinetobacter, Sphingomonas, and Pseudomonas were the advantage genera in SS. While, Lactococcus, Weissella, and Pediococcus were dominant in 3-day SSS and subsequently replaced by Lactobacillus in 60-day SSS. Spearman's correlation heatmap showed that Pediococcus and Leuconostoc were negatively related to the pH value of SSS. There were great differences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional profiles of SS and SSS. Ensiling process downregulated the metabolism of amino acid, energy, cofactors, and vitamins, but upregulated the metabolism of nucleotides and carbohydrates. Overall, next-generation sequencing in conjunction with KEGG functional prediction revealed the distinct differences in the initial and late phases of ensiling in terms of both community succession and functional shifts. The knowledge regarding bacterial community dynamics and functional shifts of SS during ensiling is important for understanding the fermentation mechanism and may contribute to the production of high-quality sweet sorghum silage.

Assessment of organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration silage.

The work aimed to investigate the effects of four organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration (TMR) silage prepared with citric acid residue, wet brewers' grains, and Napier grass. The TMR was ensiled with the following: (1) no additives (control), (2) 0.1% sodium benzoate (SB), (3) 0.1% potassium sorbate (PS), (4) 0.5% sodium diacetate (SDA), (5) 0.5% calcium propionate (CAP) on a fresh weight basis. All silos (10 L) were opened after 60 days of ensiling to determine fermentation profiles and in vitro rumen digestibility, and then were subjected to a 9-day aerobic stability test. Four organic acid salts significantly (p < 0.05) increased dry matter contents, lactic acid bacteria count, and decreased ethanol content and yeast count compared with the control. The SDA and CAP significantly (p < 0.05) increased water-soluble carbohydrates, lactic acid, and crude protein contents, and decreased pH, ammonia nitrogen, neutral detergent fiber, and hemicellulose contents compared with other TMR silages after 60 days of ensiling. Organic acid salts significantly (p < 0.05) prolonged the hours of aerobic stability and significantly (p < 0.05) increased cumulative gas production and potential gas production compared with the control. The treatments of SDA and CAP significantly (p < 0.05) improved aerobic stability as indicated by higher (p < 0.05) lactic acid and water-soluble carbohydrates contents, and lower (p < 0.05) pH, ammonia nitrogen, ethanol contents, and yeast count compared with the control. The treatments of SDA and CAP significantly (p < 0.05) increased in vitro rumen parameters, as indicated by higher (p < 0.05) in vitro digestibility of dry matter, crude protein, and neutral detergent fiber after 60 days of ensiling. Overall, these results indicated that the addition of SDA and CAP could ensure the good fermentation quality and improve aerobic stability of TMR silages. By comprehensive consideration, CAP was recommended for improving fermentation quality, aerobic stability, and in vitro rumen digestibility of TMR silages prepared with wet brewers' grains, citric acid residue, and Napier grass.

Time of Day for Harvest Affects the Fermentation Parameters, Bacterial Community, and Metabolic Characteristics of Sorghum-Sudangrass Hybrid Silage.

To characterize the effects of time of day for harvest on the fermentation parameters, bacterial community, and metabolic characteristics of sorghum-sudangrass hybrid (SSG) silage, SSG (vegetative stage) harvested at 7:00 (AM), 12:00 (M), and 17:00 (PM) on three sunny days were ensiled for 1, 3, 7, 14, 30, and 60 days. Compared to AM silage, M and PM silages were characterized by delayed fermentation, unnormal lower final pH, and lower acetic acid production. In addition, PM silage contained higher residual water-soluble carbohydrates than other silages. After 60 days of ensiling, AM silage was dominated by Lactobacillus, whereas the bacterial communities of M and PM silages were complex and mainly composed of bacteria such as Delftia, Methylobacterium-Methylorubrum, Enhydrobacter, Acinetobacter, and Bacillus. The harvest time affected a wide range of metabolic pathways including "Metabolism" and "Cellular Processes" and "Organismal Systems" in SSG silage. Particularly, at the late stage of ensiling M silage exhibited highest relative abundances of amino acid metabolisms including "glycine, serine, and threonine metabolism," "phenylalanine metabolism," and lowest relative abundances of "lysine biosynthesis." These results suggest that the time of day for harvest could affect the fermentation parameters, bacterial community, and metabolic characteristics of SSG silage. Better SSG silage characteristics could be achieved through morning harvest. IMPORTANCE Ensiling is a common way for preserving green forages worldwide. Silage fermentation quality can vary greatly depending on the chemical and microbial characteristics of forage crop being ensiled. It is well documented that forages exhibit considerable variations in chemical composition and epiphytic microbiota during daylight. However, the effects of the time of day for harvest on silage fermentation is less investigated. Our results demonstrate that the time of day for harvest could affect the fermentation parameters, bacterial community, and metabolic characteristics of SSG hybrid silage. Harvesting SSG late in the day delayed fermentation process, lowered acetic acid production and final pH, and increased the residual water-soluble carbohydrates content in silage. Moreover, the delayed harvest time increased the relative abundances of bacteria such as Delftia, Methylobacterium-Methylorubrum, Acinetobacter, Enhydrobacter, and Bacillus, and amino acid metabolisms at the late stage of SSG ensiling. This study highlights the importance of diurnal changes in forage to fermentation characteristics, providing a strategy to improve silage quality through optimizing the harvest time.

Antimicrobial effects of four chemical additives on fermentation quality, aerobic stability, and in vitro ruminal digestibility of total mixed ration silage prepared with local food by-products.

The objective of the work is to evaluate the effects of four chemical additives on fermentation quality, aerobic stability, and in vitro ruminal digestibility of total mixed ration (TMR) silage. TMR containing 15% spent mushroom substrate, 25% soybean sauce residue, 45% napiergrass (Pennisetum purpureum (L.) Schum.), and 15% concentrate was ensiled with the following: (1) no additives (control), (2) potassium sorbate (PS, 0.1%), (3) sodium benzoate (SB, 0.1%), (4) sodium diacetate (SDA, 0.5%), and (5) calcium propionate (CAP, 0.5%) based on fresh weight. All silos (10 L) were opened for fermentation quality, in vitro ruminal digestibility analysis after 60 days of ensiling, and then subjected to aerobic stability test for 9 days. All TMR silages were well-conserved, as indicated by low pH, butyric acid, and ammonia nitrogen contents. During aerobic exposure, SDA was more stable with higher (p < 0.05) lactic acid and acetic acid contents and lower (p < 0.05) yeast counts than other TMR silages. In addition, SDA significantly (p < 0.05) increased cumulate gas production and in vitro dry matter digestibility compared with the control. Overall, SDA is recommended as additives to improve fermentation quality, in vitro ruminal digestibility, and aerobic stability of TMR silage prepared with local food by-products.

Fermentative products and bacterial community structure of C4 forage silage in response to epiphytic microbiota from C3 forages.

OBJECTIVE: The observation that temperate C3 and tropical C4 forage silages easily produce large amounts of ethanol or acetic acid has puzzled researchers for many years. Hence, this study aimed to assess the effects of epiphytic microbiota from C3 forages (Italian ryegrass and oat) on fermentative products and bacterial community structure in C4 forage (sorghum) silage. METHODS: Through microbiota transplantation and γ-ray irradiation sterilization, the irradiated sorghum was treated: i) sterile distilled water (STSG); ii) epiphytic microbiota from sorghum (SGSG); iii) epiphytic microbiota from Italian ryegrass (SGIR); iv) epiphytic microbiota from oat (SGOT). RESULTS: After 60 days, all the treated groups had high lactic acid (>63.0 g/kg dry matter [DM]) contents and low pH values (<3.70), acetic acid (<14.0 g/kg DM) and ammonia nitrogen (<80.0 g/kg total nitrogen) contents. Notably, SGIR (59.8 g/kg DM) and SGOT (77.6 g/kg DM) had significantly (p<0.05) higher ethanol concentrations than SGSG (14.2 g/kg DM) on day 60. After 60 days, Lactobacillus were predominant genus in three treated groups. Higher proportions of Chishuiella (12.9%) and Chryseobacterium (7.33%) were first found in silages. The ethanol contents had a positive correlation (p<0.05) with the abundances of Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium. CONCLUSION: The epiphytic bacteria on raw materials played important roles in influencing the silage fermentation products between temperate C3 and tropical C4 forages. The quantity and activity of hetero-fermentative Lactobacillus, Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium may be the key factors for the higher ethanol contents and DM loss in silages.