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This study evaluated the effects of two organic acids on the fermentation of alfalfa silages at different dry matter (DM) contents. Alfalfa was wilted to DM contents of 30% (moderately low) and 38% (normal) and ensiled without additives (control) or treated with 0.6% fresh matter DL-malic acid (MA) or 0.6% fresh matter citric acid (CA) for 60 days. After ensiling, silages with a normal DM were higher in pH, water-soluble carbohydrates (WSC) and DM loss (p

The possibilities of using high‐quality forages in incorporation with total mixed ration (TMR) during the fattening period of beef cattle have been investigated. A total of 30 Simmental bulls (438.94 ± 11.45 kg) were selected and randomly divided into two groups, TMR with single corn silage (SS) and TMR with various silage (MS). The whole experiment consisted of 15 days preparation period and 69 days experimental period. Rumen fluid and blood samples were taken from six beef cattle per treatment at the end of the experiment. The results showed that the average daily gain of the MS group (1.56 kg/day) was higher than (p, We investigated the effects of total mixed ration (TMR) with various silage on growth performance, serum parameters, ruminal fermentation, and microbial profiles of beef cattle. The results showed that the beef cattle fed TMR with various silage was a feasible strategy in the intensive feeding system. High‐quality forage can be used in beef cattle diets instead of concentrates, and no negative effect on growth performance and rumen fermentation of beef cattle.

Summary The objectives of this study were to investigate the adaptation and competition of Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts using single‐molecule real‐time (SMRT) sequencing technology. Before ensiling, alfalfa was inoculated with L. plantarum (Lp), P. pentosaceus (Pp), E. faecalis (Ef) or their combinations (LpPp, LpEf, LpPpEf) and sampled at 1, 3, 7, 14 and 60 days. After 60‐days fermentation, the Lp‐, Pp‐ and LpPp‐inoculated silages had lower pH but greater concentrations of lactic acid were observed in Pp, LpEf and LpPpEf‐inoculated silages. The inoculants altered the keystone taxa and the bacterial community dynamics in different manners, where L. plantarum, Weissella cibaria and L. pentosaceus dominated the bacterial communities after 14 days‐fermentation in all treatments. The silages with better fermentation quality had simplified bacterial correlation structures. Moreover, different inoculants dramatically changed the carbohydrate, amino acid, energy, nucleotide and vitamin metabolism of bacterial communities during ensiling. Results of the current study indicate that effect of different inoculants on alfalfa silage fermentation was implemented by modulating the succession of bacterial community, their interactions and metabolic pathways as well during ensiling., This study were investigated the adaptation and competition of Lactobacillus plantarum, Pediococcus pentosaceus, and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts of alfalfa silage. The inoculants altered the keystone taxa, dynamics of bacterial community and their functions in different manners, and the silages with better fermentation quality had simplified bacterial correlation structures.

Summary Multi‐omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Inoculations of the two different inoculants resulted in substantial differences in microbial community and metabolic composition as well as their dynamics in ensiled corn. Inoculants also altered the correlations of microbiota in different manners, and various keystone species were identified in corn silages with different treatments. Many metabolites with biofunctional activities like bacteriostatic, antioxidant, central nervous system inhibitory and anti‐inflammatory were found in the present silage. A constitutive difference in microbiota dynamics was found for several pathways, which were upregulated by specific taxa in middle stage of fermentation, and widespread associations between metabolites with biofunctions and the species of lactic acid bacteria dominated in silage were observed. Multiple microbial and metabolic structures and dynamics were correlated and affected the fermentation process of the corn ensiling systems. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation and provide a framework to re‐evaluate silages with biofunctions, which may contribute to target‐based regulation methods to produce functional silage for animal production., Multi‐omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation, and provides a framework to re‐evaluate silages with biofunctions, which may contribute to target‐based reglation methods to produce functional silage for animal production.

Increased understanding of effects of regulation measures on whole-plant corn silage is important from bacterial community succession, interaction network and predicted functions. According to alpha diversity and meta co-occurrence network, the bacterial communities were more sensitive to storage temperature than LAB inoculants during whole-plant corn ensiling.

Antibiotic resistance genes (ARGs) are a new type of pollutant and pose major threats to public health. However, the distribution and transmission risk of ARGs in alfalfa silage as the main forage for ruminants have not been studied. This study first deciphered the effects of Lactobacillus plantarum MTD/1 or Lactobacillus buchneri 40788 inoculations on distribution and transmission mechanism of ARGs in alfalfa silage by metagenomics. Results showed that multidrug and bacitracin resistance genes were the dominant ARGs in ensiled alfalfa. The natural ensiling process increased the abundances of bacitracin, beta_lactam, and aminoglycoside in alfalfa silage with 30% DM, and vancomycin in alfalfa silage with 40% DM. Meanwhile, prolonged wilting increased ARG enrichment in fresh alfalfa. Interestingly, alfalfa silage inoculated with L. plantarum MTD/1 or L. buchneri 40788 reduced the abundances of total ARG, and multidrug, MLS, vancomycin, aminoglycoside, tetracycline, and fosmidomycin resistance genes by reductions of the host bacteria and the enrichment of ARGs located in the plasmid. The hosts of ARG in alfalfa silage were mainly derived from harmful bacteria or pathogens, and some of the clinical ARGs were observed in alfalfa silage. Basically, the combined effect of microbes, MGEs, and fermentation quality was the major driver of ARG transfer and dissemination in microecosystem of ensiling, where the microbes appeared to be the crucial factor. In summary, inoculation with the present lactic acid bacteria could reduce ARG abundance in ensiled alfalfa, and a better effect was observed in L. plantarum-treated silage than in L. buchneri treated silage.

The study was conducted to screen high 1,2-propanediol produced by Lactobacillus buchneri strains, isolated from baled silages stored for 1 or 2 years, and to evaluate their effects on fermentation quality and aerobic stability of whole-plant corn silage. In total, 31 L. buchneri strains were isolated from alfalfa, whole-plant corn and oat silages. Based on growth performance and 1,2-propanediol and acetic acid production, two strains, L. buchneri 9-2 and L. buchneri 10-1, from alfalfa silage, were further assessed in an ensiling trial on whole-plant corn. The corn silage inoculated with L. buchneri 9-2 or L. buchneri 10-1 had a higher concentration of 1,2-propanediol (34.7 or 34.6 g/kg dry matter (DM)) and acetic acid (47.2 or 45.9 g/kg DM) in comparison with L. buchneri 40788 (reference strain) treated silage (19.5 and 35.9 g/kg DM) after 90 d of fermentation. In addition, these two strains performed better in improving silage aerobic stability relative to control and L. buchneri 40788. The results above indicated that L. buchneri 9-2 and L. buchneri 10-1 could be candidate strains to increase 1,2-propanediol and acetic acid concentrations and improve the aerobic stability of whole-plant corn silage.

In this study, a novel beta-cypermethrin (beta-cyp)-degrading strain Lactobacillus pentosus 3–27 (LP3–27) was screened from beta-cyp-contaminated silage. The strain could degrade 96% of beta-cyp (50 mg/L) in MSM medium after 4 d of culture, while the strain lost its degradation ability when the beta-cyp concentration reached 250 mg/L. The effects of LP 3–27 on fermentation, bacterial community, and bioremediation of contaminated alfalfa silage at two dry matter (DM) contents were studied. The results showed that inoculation with LP3–27 not only degraded beta-cyp, but also improved the fermentation quality of alfalfa silage after 60 d of ensiling. Meanwhile, L. pentosus dominated the bacterial community during ensiling in LP3–27 inoculated silages, whereas Pediococcus acidilactici was the dominant species in the control silage. LP3–27 inoculation also simplified the bacterial interaction networks of ensiled alfalfa. Beta-cyp degradation was positively correlated with L. pentosus in LP- inoculated silages, which confirmed the function of beta-cyp degradation by L. pentosus. In addition, higher beta-cyp degradation was observed in silage with 35% versus 43% DM. In summary, strain LP3–27 could be used as a candidate inoculum for bioremediation of beta-cyp-contaminated silage and to produce safe silage for animal production.

Aims Ferulic acid esterase (FAE)-producing Lactobacillus species isolated from ensiled Elymus nutans growing on the Qinghai-Tibetan plateau were characterized, and effects of their application to the alfalfa ensiling process and the evidence to synergic effect between cellulase and FAE were investigated. Methods and results The results of 16S rRNA gene sequence and species-specific polymerase chain reaction amplification showed that two screened strains with high FAE activity were Lactobacillus plantarum A1 (LP) and L. brevis A3 (LBr). The optimum temperature and pH for the LP and LBr was 37°C and 6·4 respectively. The FAE exhibited a good stability at temperatures between 25 and 50°C and at pH values of 5·0-7·0. The two strains and a commercial cellulase (CE) were applied as additives to alfalfa silage. After 60 days of ensiling, the lactic acid in the control and CE groups were significantly lower than those of the other treatment groups. The neutral detergent fibre and acid detergent fibre contents in the LP group were significantly lower than those observed in the other groups. At the same time, the combination of CE and FAE-producing lactic acid bacteria synergistically improved the fermentation quality of the silage. Conclusions The addition of the FAE-producing strain of L. plantarum A1 to alfalfa silage improved its fermentation quality, and reduced the fibre content of the silage. Significance and impact of the study The screened homo-fermentative and FAE-producing strain of L. plantarum A1 could be a candidate strain in improving fermentation quality and fibre digestibility of ensiled forages.

The effects of Acremonium cellulase and L. plantarum A1 with ferulic acid esterase activity on corn stalk silage fermentation characteristics, carbohydrate composition and enzymatic saccharification were studied at 25 and 40 °C, respectively. Corn stalk was ensiled without additive (C), Acremonium cellulase (AC), L. plantarum A1 (Lp) and AC + Lp for 60 days. Pretreatment with Lp or AC + Lp promoted the better silage fermentation and the degradation of lignocellulose as indicated by high lactic acid and low pH and lignocellulose content compared to control silages at 25 °C. AC + Lp performed better in reducing lignocellulose and DM loss. In addition, Lp alone enhanced enzymatic saccharification of corn stalk silage. However, the influence of L. plantarum A1 on corn stalk silage was not obvious at 40 °C. Corn stalk ensiled with combined additive is a suitable pretreatment method for subsequent biofuel production at 25 °C, but addition of Acremonium cellulase alone at 40 °C may be a promising method.

This study was conducted to investigate the influence of feruloyl esterase-producing Lactobacillus plantarum A1 (Lp A1) and grape pomace (GP) alone, or in combination (LG) on ensiling characteristics and bacterial community, in vitro ruminal fermentation, methane (CH

Feed deficiency during the long cold period of a year is one of the major problems that the traditional year-round animal grazing system has faced on the Qinghai-Tibetan plateau (QTP) since ancient time. Therefore, ensiling the grasses from grassland could be a desirable feeding regime to preserve high quality forage and to alleviate the seasonal unbalanced feed supply problem on this plateau. The present study was designed to investigate dynamics of bacterial community and natural fermentation quality of ensiled Elymus nutans collected from grasslands in four different areas with different elevations [Tianzhu County (TZ), 2965 m; Golog Prefecture (GL), 3763 m; Damxung County (DX), 4228 m, and Nagqu Prefecture (NQ), 4752 m] on the QTP. The bacterial community was characterized by using the PacBio single molecule with real-time sequencing technology (SMRT). The harvested fresh E. nutans grasses were ensiled in vacuum-sealed polyethylene bags for 14, 30, 60, and 90 days. Obvious differences in the epiphytic bacterial community of the fresh E. nutans samples from the four areas were observed, which resulted in various bacterial community dynamics and fermentation qualities of ensiled E. nutans. Higher fermentation quality was observed in silage samples from Nagqu than in those from the other areas (P < 0.05). Lactic acid bacteria (LAB) involved in fermentation of E. nutans from low altitude areas consisted of Pediococcus pentosaceus, Lactobacillus sp., Leuconostoc mesenteroides, and Lactobacillus coryniformis, whereas major LAB species involved in the fermentation of E. nutans silage from high altitudes included L. mesenteroides, Lactobacillus brevis, and Lactobacillus sp. Correlation analysis between bacterial composition and fermentation quality of E. nutans silages made from the four different areas in the QTP indicated that the LAB species responsible for silage fermentation in different areas were totally different, which was mainly due to the different epiphytic bacterial compositions in fresh E. nutans before ensiling. The present results provide important information on revealing the bacterial communities and fermentation quality of ensiled E. nutans, and on future screening of LAB isolates for making high quality silage in order to alleviate feed shortage of the traditional year-round grazing system on the QTP.

The effects of feruloyl esterase-producing Lactobacillus plantarum A1, cellulase, or their combination on the fermentation characteristics, carbohydrate composition, and enzymatic hydrolysis of mixed corn stalk and potato pulp silage were investigated. Two mixture ratios were used: a weight ratio of rehydrated corn stalk to potato pulp of 35:1 (HD) and a weight ratio of dry corn stalk to potato pulp of 5:11 (LD). No advantage was observed with the addition of strain A1 alone for lignocellulosic degradation and cellulose conversion, while its combination with cellulase enhanced the lignocellulosic degradation and preserved more fermentable carbohydrates in co-ensiled corn stalk and potato pulp. The enzymatic hydrolysis results indicated a potential benefit of pretreatment for biogas production, as the co-ensiled HD ratio mixture without additive treatment showed high glucose yield after enzymatic hydrolysis following 60 d of fermentation.

Feeding ruminant animals with legume silage, rich in non-protein nitrogen, not only results in serious groundwater pollution but also induces a large amount of methane (CH4) releasing into atmospheric environment. Co-ensiling alfalfa with sainfoin containing condensed tannins in five ratios was investigated on silage fermentation, chemical and bacterial community composition, in vitro ruminal fermentation characteristics, CH4 emission and archaeal and protozoal community. Fresh alfalfa and sainfoin were wilted to dry matter concentrations of 381 and 390 g/kg wet weight, respectively, and chopped to 2–3 cm. Chopped alfalfa and sainfoin were combined in proportions of 1:0 (S0, Control), 0.75:0.25 (S25), 0.5:0.5 (S50), 0.25:0.75 (S75) and 0:1 (S100) on a wet weight basis, respectively. Each treatment was prepared in quadruplicate and fermented for 60 days. After 60 days of fermentation, incorporation of sainfoin into alfalfa before ensiling led to an improvement in silage fermentation and inhibited silage proteolysis in a proportion-dependent manner. Excellent fermentation profiles were observed in S50 and S75 silages, in which the non-protein nitrogen decreased by 11.33% and 13.01%, respectively, compared with S0 silage. As sainfoin proportion increased, the relative abundance of Pediococcus acidilactici linearly decreased (P

The study was conducted to investigate the effects of different malic acid (MA) isomers (D-MA, L-MA and DL-MA) and their application levels (0, 0.2, 0.6 and 1% of fresh weight, FW) on the fermentation and biochemical characteristics of alfalfa silage, as well as on the degradation of these MA isomers in silage. Alfalfa was harvested at early bloom stage, and after wilting to approximately 35% dry matter (DM), the chopped forages were ensiled in vacuum-sealed polyethylene bags containing 200–230 g wilted forage for 60 d, with 3 replications for each treatment. Silage pH decreased with the increase of the application level of MA. Lactic acid in silage also increased when the application level of MA ranged from 0.2% to 0.6%. However, a decline in lactic acid was observed when the application level of MA reached 1% (FW). On average silage treated with DL-MA had greater lactic acid concentration compared to D-MA or L-MA treated silages. Treatment with MA lowered non-protein nitrogen (NPN) concentration in ensiled forage as application level of the MA increased. In comparison with the control silage, inclusion of MA at ensiling effectively decreased the proportion of saturated fatty acids (SFA) in total fatty acid, whereas it markedly increased the proportion of α-linolenic acid and polyunsaturated fatty acids (PUFA), and silages treated with DL-MA had greater PUFA and lower SFA. After 60 d of ensiling, the concentration of MA decreased from 22.4 g/kg DM in fresh alfalfa to 6 g/kg DM in silage. On average the greatest percent of MA degraded was observed in L-MA followed by DL-MA, and the averaged residues of MA in D-MA, L-MA and DL-MA-treated silages were 19.8, 12.6, and 16.1 g/kg DM, respectively. Although the amount of degraded MA in ensiled alfalfa increased with the elevated application levels, the percent of MA degraded decreased constantly. Overall, silage microbes used more of L-MA or DL- MA than D-MA, and application of 0.6% DL-MA at the ensiling of alfalfa effectively improved silage fermentation, limited proteolysis, lipolysis and biohydrogenation in ensiled alfalfa.

Aims Characteristics of a strain Pediococcus pentosaceus Q6 isolated from Elymus nutans growing on the Tibetan plateau and its effects on E. nutans silage fermentation stored at low temperature were investigated. Methods and results Sugar fermentation pattern and growth profiles of the strain Q6 and its reference strain APP were characterized. The strain Q6 and APP were inoculated to E. nutans at ensiling respectively; and ensiled at different temperatures (10, 15 and 25°C) for 30, 60 and 90 days. The results indicated that Q6 could grow at pH 3·0 and at 4°C. In contrast to APP, Q6 could ferment mannitol, saccharose, sorbitol and rhamnose. Lower pH in Q6-treated silages fermented for 60 days at 10 and 15°C was found compared with the control and APP-treated groups. For the silages that were stored at 10 or 15°C, the greatest lactic acid content were detected in Q6-inoculated silages ensiled for 30 and 60 days respectively. There were no differences in pH and lactic acid content between Q6- and APP-treated silages ensiled at 10 and 15°C for 90 days respectively. Conclusions Inoculation of the strain P. pentosaceus Q6 could improve fermentation quality of ensiled E. nutans at the early stage of ensiling stored at low temperature (10 or 15°C). Significance and impact of the study The selection of P. pentosaceus inoculants for improving silage quality at low temperature, which provides a candidate strain to make high-quality silage in regions with frigid climate.

We investigated the long-term effects of total mixed rations (TMR) containing various forage silages on the rumen community structure in beef cattle. Fifteen Simmental bulls (437.73 ± 4.01 kg) were fattened for 180 d, split into three trial stages (P1: 1–60 d, P2: 61–120 d, P3: 121–180 d). The diets were composed of corn silage, oat silage, alfalfa silage, wheat straw, and concentrates, formulated into a TMR. Rumen fluid was collected separately on the 60th, 120th, and 180th days among the three fattening stages. High-throughput 16S rRNA gene sequencing was used to analyse rumen bacterial composition and function, and the correlation between rumen bacteria and fermentation parameters was determined using redundant analysis. The results showed that rumen fermentation parameters were stable during long-term fattening. The alpha diversity index indicated that bacterial diversity was higher during the late fattening phase than in the early phase. The most abundant bacteria were Bacteroidetes, Firmicutes, and Prevotella_1 at the three fattening stages. The bacterial communities were clearly separated in the different stages, as indicated by the Box diagram. The thirteen bacterial taxa identified by LEfSe were significantly enriched in the rumen during the three stages. KEGG pathway fractions indicated that carbohydrate metabolism, amino acid metabolism, and energy metabolism pathways were enriched in the rumen during the three stages. In conclusion, we found that long-term feeding of beef cattle with high-quality forage + low levels of concentrate was a healthy and efficient feeding method, promoting a healthy and suitable long-term change in the rumen microbial environment.

The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation.

The objective of this study was to evaluate the effects of a novel Lactobacillus plantarum strain with high-antioxidant activity on antioxidant status, chemical composition and fermentation profiles of alfalfa silage ensiled at two dry matter (DM) contents. Fresh chopped alfalfa was wilted to two targeted dry matter (DM) contents of approximately 300 (low DM) and 400 (high DM) g/kg fresh weight and treated without or with a strain of L. plantarum 24−7 with high antioxidant activity. Forages were ensiled in vacuum-sealed polyethylene bags containing ∼600 g wilted forage for 60 d, with 5 replicates for each treatment. Alfalfa silage ensiled at low DM had higher concentrations of acetic acid and non-protein nitrogen (NPN) and lower concentrations of lactic acid, propionic acid and water soluble carbohydrates (WSC) than silages with high DM. Inoculation of L. plantarum 24−7 led to lower silage pH and lower concentrations of NPN, neutral detergent fiber (aNDF), acid detergent fiber (ADF), and higher WSC compared to the control silage at two DM contents. Inoculation increased aerobic stability of the forage ensiled at low DM but decreased that ensiled at high DM. In control silages, almost half of the α-tocopherol and β-carotene was degraded during ensiling, whereas inoculation with L. plantarum 24−7 increased α-tocopherol and β-carotene concentrations as well as the proportion of polyunsaturated fatty acids (PUFA) in ensiled alfalfa at both DM contents. Greater concentrations of β-carotene were observed in L. plantarum 24−7 treated-silage at low DM compared to silage at high DM. The application of L. plantarum 24−7 strain also increased total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) and catalase (CAT) activities of the alfalfa silage but decreased superoxide dismutase (SOD) and lipoxygenase (LOX) activities. These results indicate that L. plantarum 24−7 improved the fermentation quality and antioxidant status of ensiled alfalfa forage relative to the control silage.

The present study investigated the bacterial community, metabolome and biotransformation of phenolic compounds in sainfoin silage inoculated either with or without Lactobacillus plantarum. Our results revealed that the epiphytic microbiota of wilted sainfoin mainly comprised Acinetobacter, Spingomonas, Pseudomonas, Lactobacillus, Delftia and Aquabacterium. After ensiling, L. acetotolerans, L. buchneri, L. plantarum, L. pentosus and Clostridium tyrobutricum were the dominant species. Compared to the control, the inoculant L. plantarum increased the relative abundance of L. buchneri while decreased that of the other dominant species. The ensiling process increased the concentrations of 21 flavones, 16 flavonols, 16 flavonoids, 14 flavanones and 9 isoflavones while decreased other 32 flavones and 11 anthocyanins, which mainly modulated by the "isoflavonoid biosynthesis" and "flavonoid biosynthesis" pathways. The inoculant L. plantarum mainly reduced the concentrations of 10 flavones. Compared with the control, the inoculant L. plantarum mainly affected the metabolism pathways related to carbohydrates and nitrogen.

This study assessed the effects of antibacterial peptide-producing Bacillus subtilis (BS), Lactobacillus buchneri (LB), or their combination on fermentation, proteolysis, aerobic stability, and microbial communities during ensiling and aerobic exposure phases of alfalfa silage. The results showed that the BS-treated silage displayed a higher lactic acid concentration, less proteolysis, and higher aerobic stability than those in the control silage. Both LB and BS treatments increased Lactobacillus and Ascochyta abundance, and decreased Enterococcus and Sporormiacea abundance after 60 d of fermentation. LB and BS also inhibited the growth of Enterococcus after 3 d of aerobic exposure but similar to the control silage, the fungal community of BS silage was dominated by Candida and Pichia after 9 d of aerobic exposure. Therefore, inoculation of BS improved silage fermentation quality, aerobic stability and bacterial community during ensiling and after 3 d of aerobic exposure.

The effects of inoculation of strain Pediococcus acidilactici J17 with high antioxidant activity and a widely used commercial inoculum of Lactobacillus plantarum MTD-1, on antioxidant status, concentrations of α‑tocopherol and β‑carotene, fatty acid composition and fermentation profiles of ensiled alfalfa prepared at two dry matter (DM) contents were investigated. Chopped fresh alfalfa was ensiled at two DM contents of approximately 300 (low DM) and 400 (high DM) g/kg fresh weight, and treated with distilled water (control), L. plantarumMTD-1 and P. acidilactici J17, respectively. Forages were ensiled in vacuum-sealed polyethylene bags containing 500 g wilted forage for 60 d, with 4 replicates for each treatment. All inoculants effectively improved fermentation profiles of silages and the silage with a high DM exhibited better effects on inhibiting proteolysis and lipid oxidation; however, the antioxidant strain P. acidilactici J17 had little influence on the fatty acid composition of alfalfa silage. Silages with a low DM had higher proportions of saturated fatty acid and monounsaturated fatty acid, and lower proportions of polyunsaturated fatty acid compared with the silages with a high DM. Extensive degradation of α-tocopherol and β-carotene in alfalfa occurred after ensiling. The concentration of α-tocopherol in control silage declined to almost half of its original concentration in fresh alfalfa. Concentrations of β-carotene in the control silages with the low and high DM were accounted for 1/3 and 1/2 of their original concentrations in fresh alfalfa, respectively. In comparison with control silage and silage treated with L. plantarum MTD-1, inoculation of P. acidilactici J17 with high-antioxidant activity markedly increased the total antioxidant capacity (T-AOC), catalase (CAT) activity and concentrations of α-tocopherol and β-carotene of alfalfa silage ensiled at both DM contents. On average, higher T-AOC was observed in silage with a high DM than silage with a low DM. However, both inoculants decreased superoxide dismutase (SOD) activity of ensiled alfalfa. These results indicated that the P. acidilactici J17 strain could be used as a candidate antioxidant inoculum to improve the antioxidant status of alfalfa silage.

The effects of two strains of class IIa bacteriocin-producing lactic acid bacteria, Lactobacillus delbrueckii F17 and Lactobacillus plantarum (BNCC 336943), or a non-bacteriocin Lactobacillus plantarum MTD/1 (NCIMB 40027), on fermentation quality, microbial counts, and aerobic stability of alfalfa silage were investigated. Alfalfa was harvested at the initial flowering stage, wilted to a dry matter concentration of approximately 32%, and chopped to 1 to 2 cm length. Chopped samples were treated with nothing (control, CON), Lactobacillus delbrueckii F17 (F17), Lactobacillus plantarum (BNCC 336943) (LPB), or Lactobacillus plantarum MTD/1 (NCIMB 40027) (LPN), each at an application rate of 1 &times, 106 colony-forming units/g of fresh weight. Each treatment was ensiled in quadruplicate in vacuum-sealed polyethylene bags packed with 500 g of fresh alfalfa per bag and ensiled at ambient temperature (25 &plusmn, 2 &deg, C) for 3, 7, 14, 30, and 60 days. The samples were then subjected to an aerobic stability test after 60 days of ensiling. Compared with the CON silage, the inoculants reduced the pH after 14 days of ensiling. After 60 days, pH was lowest in the LPB-treated silage, followed by the F17 and LPN-treated silages. Inoculation of F17 increased concentrations of lactic acid in silages fermented for 7, 14, 30, and 60 days relative to other treatments, except for the LPN-treated silages ensiled for 30 and 60 days, in which the lactic acid concentrations were similar to that of F17 silage. Application of F17 and LPB decreased the number of yeast and mold relative to CON and LPN-treated silages. Compared with the CON silage, inoculant-treated silages had greater aerobic stability, water-soluble carbohydrate, and crude protein concentrations, and lower neutral detergent fiber, amino acid nitrogen, and ammonia nitrogen concentrations. The LPB-treated silage had the greatest aerobic stability followed by the F17-treated silage. Both class IIa bacteriocin producing inoculants improved alfalfa silage fermentation quality, reduced the growth of yeasts and molds, and improved the aerobic stability of the ensiled forage to a greater extent than the proven LPN inoculant. However, higher crude protein concentration and lower ammonia nitrogen concentration were observed in LPN-treated silage relative to other treatments.

The impact of Lactobacillus plantarum A1 producing ferulic acid esterase and Acremonium cellulase on Pennisetum sinese silages was investigated at two dry matter contents by studying the ensiling characteristics, lignocellulosic degradation and enzymatic saccharification properties. The P. sinese was treated with nothing (control), Acremonium cellulase (AC), L. plantarum A1 (Lp) and AC + Lp and ensiled for 60 d. All additive treatments effectively preserved P. sinese and promoted the degradation of lignocellulose in comparison with control. Pretreatment with AC exhibited better effects in degradation of lignocellulose and enhancing enzymatic saccharification of P. sinese silage with low dry matter content (L-DM), while AC + Lp performed better in lignocellulose degradation in silages with high dry matter content (H-DM). Application of Lp exhibited a better performance in reducing the concentration of acid detergent lignin in treated silages. In addition, Lp enhanced enzymatic saccharification at 72 h in H-DM silage relative to other treatments.

The aim of this study was to evaluate the effect of two organic acids [malic acid (MA) and citric acid (CA)], Lactobacillus plantarum (LP) and their mixtures on the fermentation of alfalfa silage. The wilted and chopped alfalfa was ensiled in vacuum-sealed plastic bags (200-230 g per bag) with following additives: 0.5% distilled water (control), 0.5% DL-MA, 0.5% CA, LP (1 · 10

Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiled Medicago sativa (alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation of L. buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed in L. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.

The effects of adding 100 g of either dried apple pomace (AP), unground grape pomace (GP) or ground grape pomace (GGP) per kg wilted alfalfa (wet basis) on silage characteristics, aerobic stability, proteolysis and lipid composition were studied. The three combinations as well alfalfa without addition were ensiled in 1.8 L- jars for 60 d, with 4 jars per treatment. Addition of AP and GGP decreased pH (P

We studied the effects on alfalfa preservation and chemical composition of the addition of different levels of malic acid and citric acid at ensiling as well as the utilization efficiency of these 2 organic acids after fermentation. Alfalfa was harvested at early bloom stage. After wilting to a dry matter content of approximately 40%, the alfalfa was chopped into 1- to 2-cm pieces for ensiling. Four levels (0, 0.1, 0.5, and 1% of fresh weight) of malic acid or citric acid were applied to chopped alfalfa at ensiling with 4 replicates for each treatment, and the treated alfalfa forages were ensiled for 60 d in vacuum-sealed polyethylene bags (dimensions: 200 mm × 300 mm) packed with 200 to 230 g of fresh alfalfa per mini silo and an initial density of 0.534 g/cm3. The application of malic or citric acids at ensiling for 60 d led to lower silage pH than was observed in the control silage (0% of malic or citric acids). Application of the 2 organic acids led to higher lactic acid concentration in alfalfa silage than in the control silage except with the application rate of 1% of fresh weight. Silages treated with both organic acids had lower nonprotein nitrogen concentrations than the control silages, and the nonprotein nitrogen concentrations in ensiled forages decreased with the increase in malic or citric acid application rates. The application of the 2 organic acid additives led to lower saturated fatty acid proportions and higher polyunsaturated fatty acid proportions in ensiled alfalfa than in the control silage. The amount of malic and citric acids degraded during ensiling of alfalfa was 1.45 and 0.63 g, respectively. At the application rate of 0.5% of fresh weight, residues of malic acid and citric acid in alfalfa silage were 11.1 and 13.6 g/kg of dry matter. These results indicate that including malic or citric acids at the ensiling of alfalfa effectively improved silage fermentation quality, limited proteolysis, improved fatty acid composition of the ensiled forage, and could provide animals with additional feed additives proven to promote animal performance. However, when the application rate of both organic acids reached 1%, the concentration of lactic acid in silages decreased notably. Additionally, 0.5 and 1% application rates also increased the yeast count in ensiled alfalfa.

Effects of different chemical additives on peptide composition in ensiled alfalfa (Medicago sativa L.) were investigated by using gel filtration and determination of N characteristics. The alfalfa silages were prepared untreated (control) or with formic acid, formaldehyde or tannic acid as additives at ensiling. All additives reduced non-protein N (NPN), ammonia nitrogen (NH3 -N) and amino acid N (AA-N) in the ensiled forage, and the most effective reduction of NPN and AA-N was observed in the formaldehyde-treated silages. Peptides in the control silage were mainly dipeptides to peptides with five amino acid residues. Most peptides in the formic acid-treated silage contained 4-12 amino acid residues. Although most peptides in the formaldehyde-treated silages contained 4-6 amino acid residues, there was a considerable proportion of peptides with 7-11 amino acid residues. Tannic acid had little effect on peptide size of ensiled alfalfa extract in which most peptides contained 5-6 amino acid residues. Peptide size in formic acid-treated alfalfa silage was greater than that in the other treatments. Addition of formic acid and formaldehyde not only increased the peptide concentration in alfalfa silage, but enlarged the peptide size.

This experiment studied the contribution of plant enzymes and microbial activity on lipolysis and proteolysis in ensiled alfalfa. Before ensiling, the wilted alfalfa was treated with plant enzyme inactivation by autoclaving or with sterilization by γ-ray irradiation. The treated alfalfa was then inoculated with commercial lactic acid bacteria inoculants and ensiled for 40 d. Alfalfa without treatment was ensiled as the control. The content of total fatty acid (FA) after ensiling decreased 43% in the control silage and 28% in the γ-ray-treated silage, but did not change in the autoclave-treated silage. Among the major FA (C16:0, C18:2n-6, C18:3n-3), a considerable increase was observed in proportion of C16:0 in the control silage as compared with fresh alfalfa; conversely, decreases in proportions of C18:2n-6 and C18:3n-3 occurred during ensilage. Silage treated with γ-ray radiation at ensiling had a smaller proportion of C16:0 and greater proportions of C18:2n-6 and C18:3n-3 than control silage. Autoclave treatment further decreased proportions of C16:0 and most of the other FA, and increased C18:2n-6 and C18:3n-3 proportions in comparison with γ-ray treatment. Proportions of C16:0, C18:2n-6, C18:3n-3 and other detected FA (except for the proportion of C15:0) did not differ between fresh forage and autoclave-treated silage. Remarkably, smaller nonprotein nitrogen content was observed in the autoclave-treated silage compared with the γ ray-treated silage or the control silage. These results indicated that an extensive lipolysis occurred during ensiling of alfalfa, and plant enzymes played a major role in lipolysis and proteolysis.

In order to detect the effect of lactic acid bacteria isolated from Tibetan Plateau on silage fermentation quality of Elms nutans.We used 3 isolated lactic acid bacteria with better growth at low temperatures of 10 and 15 degrees C at ensiling of Elymus nutans. Subsequently, effects of the selected lactic acid bacteria on fermentation profiles of Elymus nutans silages stored at 15 and 25 degrees C were evaluated by using the same species of commercial inoculants as the control.PP-6 isolated from Tibetan Plateau could ferment raffinose, lactose, sorbitol, melibiose and sucrose, and LS-5 could ferment cottonseed sugar, laetrile, rhamnose, lactose, sorbitol, xylose, arabinose, melibiose and sucrose, but the same species of commercial strains could not use these sugars. Inoculation of these three strains into Elymus nutans at 15 and 25 degrees C ensiled for 50 d, we found that LS-5 significantly reduced silage pH, propionic acid concentration and ratio of ammonia nitrogen/total nitrogen at 15 degrees C (P0.05), salvaged more water-soluble carbohydrate and crude protein; Application of LP-2 and PP-6 as a combined inoculant to Elymus nutans significantly improved lactic acid concentration (P0.05), resulting in a lower ratio of ammonia nitrogen/total nitrogen, saved more crude protein and significantly reduced neutral detergent fiber content (P0.05) as compared with the commercial strains.The three isolated strains can improve silage quality of Elymus nutans growing on the Qinghai-Tibetan Plateau at low temperature, but these strains have no obvious advantages at 25 degrees C in comparison with the commercial inoculants.

The pH optimum and thermostability of both exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.

The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH(3)-N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH(3)-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.

Formic acid, formaldehyde, tannic acid or mixtures of two were studied on their effects on ensiled alfalfa ( Medicago sativa L.) amino acids and N fractions by the Cornell Net Carbohydrate and Protein System (CNCPS). The alfalfa forage was a second cut and was wilted to a mean over-dry dry matter (DM) content of 330 g/kg. All silages were prepared as mini-silos using 100 ml polypropylene centrifuge tubes (50 g) on a small laboratory-scale, with the additives added in 20 ml aliquots/kg herbage fresh weight (FW). After 35 d of ensiling, most of forage true protein was converted to fraction A and all of the added additives reduced fraction A content in the ensiled forages (P 1 in all of the additive-treated silages was higher (P 2 and B 3 , respectively. No difference was observed on fraction C content between the control silage and silages treated with additives except for the formaldehyde or tannic acid-treated silages. Amino acids were well preserved in additive-treated silages compared with the control silage. Concentration of total amino acid was higher in formic acid-treated silages than that in the control and the other additive-treated silages (P

Effects of formic acid, formaldehyde and two levels of tannic acid on changes in the distribution of nitrogen (N) and plant enzymatic activity during ensilage of lucerne (Medicago sativa) were studied. Lucerne [300 g dry matter (DM) kg−1 forage] silages were prepared untreated (control) and with formic acid (4 g kg−1 DM), formaldehyde (1 g kg−1 DM) and two levels of tannic acid (20 and 50 g kg−1 DM) as additives. Inhibition of proteolysis by formic acid was more effective than the other additives during the first 7 d of ensiling. Tannic acid was as effective at inhibiting production of non-protein-N, ammonia-N and free amino acid-N as formic acid and formaldehyde. However, increased concentrations of non-protein-N and free amino acid-N in silage from day 1 to 35 of ensiling were less with the higher level of tannic acid than that in the control and other additive-treated silages. Carboxypeptidase lost its activity slowly with increasing time of ensiling. At day 2, it still had 0·79 of the original activity in the control silage. After 21 d of ensiling, high levels of carboxypeptidase activity, proportionately 0·41, 0·49, 0·10, 0·35 and 0·30 of the original activity, remained in the control silage, and silages made with formic acid, formaldehyde, and low and high levels of tannic acid respectively. There were higher levels of activity of acid proteinase in formic acid-treated silage than in the control silage until day 2 of ensilage indicating that the reduction of proteolysis by formic acid was probably due to acidifying the forage below the pH optima of plant protease. Aminopeptidase activity in all silages declined rapidly after ensiling.

Effects of different chemical additives on peptide composition in ensiled alfalfa (Medicago sativa L.) were investigated by using gel filtration and determination of N characteristics. The alfalfa silages were prepared untreated (control) or with formic acid, formaldehyde or tannic acid as additives at ensiling. All additives reduced non-protein N (NPN), ammonia nitrogen (NH3 -N) and amino acid N (AA-N) in the ensiled forage, and the most effective reduction of NPN and AA-N was observed in the formaldehyde-treated silages. Peptides in the control silage were mainly dipeptides to peptides with five amino acid residues. Most peptides in the formic acid-treated silage contained 4-12 amino acid residues. Although most peptides in the formaldehyde-treated silages contained 4-6 amino acid residues, there was a considerable proportion of peptides with 7-11 amino acid residues. Tannic acid had little effect on peptide size of ensiled alfalfa extract in which most peptides contained 5-6 amino acid residues. Peptide size in formic acid-treated alfalfa silage was greater than that in the other treatments. Addition of formic acid and formaldehyde not only increased the peptide concentration in alfalfa silage, but enlarged the peptide size.

This study evaluated the effects of Lactobacillus plantarum with or without Lactobacillus buchneri on the fermentation and aerobic stability of mixed tall fescue (Festuca arundinacea Schreb) and meadow fescue (Festuca pratensis Huds.) silage ensiled at different dry matter (DM) contents. The first cut was harvested at boot stage and second-cut grasses were harvested when 30- to 35-cm tall. Four DM content treatments of the first cut were 17.9, 24.9, 34.6, and 48.7%; and of the second cut were 29.1, 36.3, 44.1, and 49.2%. Chopped grasses at each DM content were treated with (1) deionized water (control), (2) Lb. plantarum MTD-1 (LP), or (3) a combination of Lb. plantarum MTD-1 and Lb. buchneri 40788 (LP+LB). The application amount of each inoculant to the fresh forage was 1 × 10(6) cfu/g. Grasses were ensiled in vacuum-sealed polyethylene bags containing 150 g of DM for 60 d, with 4 replicates for each treatment. Silages inoculated with LP+LB had greater pH compared with untreated or LP-treated silages. Lactate was greater in LP silage than control or LP+LB silages. As silage DM increased, lactate in untreated and LP-treated silages decreased, but increased in LP+LB-treated silage. Acetate concentration decreased with increased DM in all silages. The LP+LB-treated silage had the longest and control silage the shortest aerobic stability for both harvests. The greatest values in aerobic stability were observed in silages with highest DM content. In this study, aerobic stability of grass mixes ensiled between 18 and 44% DM content increased as the percentage of DM increased. The LP and LP+LB inoculants improved aerobic stability of silages harvested between 18 and 44% DM content.

The effects of different additives, including lactic acid bacteria inoculants, acremonium cellulase and mixtures of lactic acid bacteria inoculants and acremonium cellulase on changes of nutritive components and in vitro digestibility of Ceratoides arborescens during ensiling were investigated. The silages of each treatment were prepared in plastic film bags, kept in an incubator at 30°C and bags were opened after 1, 3, 5, 10, 30 and 45 days at room temperature. The results show that addition of additives and ensiling time did not affect acid detergent lignin content during ensiling (P>0.05). With ensiling time, pH value decreased (P