Ensiled Alfalfa Research Articles

Genotypic and Phenotypic Characteristics of Lactic Acid Bacteria Associated with Forage Plants in the Native Grassland of Western Inner Mongolia and Their Application for Alfalfa Silage Fermentation.

This study was conducted to investigate the genotypic and phenotypic characteristics of lactic acid bacteria (LAB) associated with forage plants in the native grassland of western Inner Mongolia and to evaluate their effects on alfalfa silage fermentation. Forage plants and their spontaneous fermentation silages were analysed using culture-based techniques for LAB isolation; the phenotypic properties and 16S rDNA and pheS or rpoA gene sequences of the isolates were evaluated; alfalfa was ensiled with four additive combinations: Lactiplantibacillus plantarum subsp. plantarum (GI19), Lact. plantarum subsp. plantarum and Pediococcus pentosaceus (GI19+GI51), GI19 and 20 g/kg fresh matter of sucrose (GI19+S), and GI19+GI51+S, for 60 d. A total of 73 strains belonging to 16 species were isolated. All isolates grew at 5-45 °C and in 3.0% NaCl, and most of them grew in 6.5% NaCl. Enterococcus faecalis and Lact. plantarum were 26.03% and 17.81% of the total isolates, respectively. All additives improved the silage quality, while GI19+S was more effective for alfalfa ensiling with a higher lactic acid content and lower pH, undesirable microorganism counts, and acetic acid and NH3-N contents than remnant additives. In conclusion, the LAB species were diverse, and most of them possessed good cryotolerance and osmotolerance; GI19+S was the optimal inoculant for alfalfa fermentation improvement.

Investigation of fermentation profiles, bacterial community structure and bacterial β-carotene synthesis of alfalfa silage treated with propionic acid or its combination with squalene

Abstract This study examined the fermentation dynamics, bacterial community composition and bacterial β-carotene synthesis in alfalfa that was ensiled for 3, 15, 45 and 90 days without additives (CON), or with a chemical agent (propionic acid, PA); or a combination of PA and squalene, SQPA). The results showed that silage treated with PA had a lower (P < 0.01) pH value than the CON silage in the early ensiling phase (3–15 days). Meanwhile, silage treated with PA had the highest contents of lactic acid, acetic acid and PA after 90 days of ensiling (P < 0.01). The β-carotene in alfalfa was lost seriously in the initial ensiling phase (3 days) and epiphytic Pantoea agglomerans with the ability to produce β-carotene became extinct. With the extension of ensiling time, the loss of β-carotene was alleviated in all silages. PA and SQPA not only lowered bacterial diversity and simplified bacterial networks but also facilitated the emergence of new β-carotene-producing bacteria. The metabolic function prediction indicated that β-carotene synthesis tended to decrease initially and subsequently increase during ensiling. Furthermore, the variance of enzymes involved in the bacterial synthesis of β-carotene in silages was influenced by PA, SQPA and ensiling time. In summary, the impact of solely adding PA demonstrated superior effects on the fermentation quality of alfalfa silage compared to the effects observed with SQPA. Throughout the ensiling of alfalfa, the succession of different β-carotene-producing bacteria resulted in fluctuations in the levels of β-carotene.

Influences of Growth Stage and Ensiling Time on Fermentation Characteristics, Nitrite, and Bacterial Communities during Ensiling of Alfalfa.

This study examined the impacts of growth stage and ensiling duration on the fermentation characteristics, nitrite content, and bacterial communities during the ensiling of alfalfa. Harvested alfalfa was divided into two groups: vegetative growth stage (VG) and late budding stage (LB). The fresh alfalfa underwent wilting until reaching approximately 65% moisture content, followed by natural fermentation. The experiment followed a completely randomized design, with samples collected after the wilting of alfalfa raw materials (MR) and on days 1, 3, 5, 7, 15, 30, and 60 of fermentation. The growth stage significantly influenced the chemical composition of alfalfa, with crude protein content being significantly higher in the vegetative growth stage alfalfa compared to that in the late budding stage (p < 0.05). Soluble carbohydrates, neutral detergent fiber, and acid detergent fiber content were significantly lower in the vegetative growth stage compared to the late budding stage (p < 0.05). Nitrite content, nitrate content, nitrite reductase activity, and nitrate reductase activity were all significantly higher in the vegetative growth stage compared to the late budding stage (p < 0.05). In terms of fermentation parameters, silage from the late budding stage exhibited superior characteristics compared to that from the vegetative growth stage. Compared to the alfalfa silage during the vegetative growth stage, the late budding stage group exhibited a higher lactate content and lower pH level. Notably, butyric acid was only detected in the silage from the vegetative growth stage group. Throughout the ensiling process, nitrite content, nitrate levels, nitrite reductase activity, and nitrate reductase activity decreased in both treatment groups. The dominant lactic acid bacteria differed between the two groups, with Enterococcus being predominant in vegetative growth stage alfalfa silage, and Weissella being predominant in late budding stage silage, transitioning to Lactiplantibacillus in the later stages of fermentation. On the 3rd day of silage fermentation, the vegetative growth stage group exhibited the highest abundance of Enterococcus, which subsequently decreased to its lowest level on the 15th day. Correlation analysis revealed that lactic acid bacteria, including Limosilactobacillus, Levilactobacillus, Loigolactobacillus, Pediococcus, Lactiplantibacillus, and Weissella, played a key role in nitrite and nitrate degradation in alfalfa silage. The presence of nitrite may be linked to Erwinia, unclassified_o__Enterobacterales, Pantoea, Exiguobacterium, Enterobacter, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium.

Occurrence and fate of antibiotic-resistance genes and their potential hosts in high-moisture alfalfa silage treated with or without formic acid bactericide

Silage as the main forage for ruminants could be a reservoir for antibiotic resistance genes (ARGs) through which these genes got access into the animals' system causing a latent health risk. This study employed metagenomics and investigated the ARGs’ fate and transmission mechanism in high-moisture alfalfa silage treated with formic acid bactericide. The results showed that there were 22 ARGs types, in which multidrug, macrolide-lincosamide-streptogramine, bacitracin, beta-lactam, fosmidomycin, kasugamycin, and polymycin resistance genes were the most prevalent ARGs types in the ensiled alfalfa. The natural ensiling process increased ARGs enrichment. Intriguingly, after 5 days of ensiling, formic acid-treated silage reduced ARGs abundances by inhibiting host bacterial and plasmids. Although formic acid bactericide enhanced the fermentation characteristics of the high-moisture alfalfa by lowering silage pH, butyric acid concentration, dry matter losses and proteolysis, it increased ARGs abundances in alfalfa silage owing to increases in abundances of ARGs carriers and transposase after 90 days of ensiling. Notably, several pathogens like Staphylococcus, Clostridium, and Pseudomonas were inferred as potential ARGs hosts in high-moisture alfalfa silage, and high-moisture alfalfa silage may harbor a portion of the clinical ARGs. Fundamentally, microbes were distinguished as the foremost driving factor of ARGs propagation in ensiling microecosystem. In conclusion, although formic acid bactericide improved the fermentation characteristics of high-moisture alfalfa during ensiling and reduced ARGs enrichment at the initial ensiling stage, it increased ARGs enrichment at the end of ensiling.

Comparative Na+ and K+ Profiling Reveals Microbial Community Assembly of Alfalfa Silage in Different Saline-Alkali Soils

Alfalfa cultivated in saline-alkaline soils exhibits a high buffering capacity but low carbohydrate content, posing challenges in the production of high-quality silage feed. This study investigated alfalfa plants grown under varying conditions: mild (QE, salt content 1–2%), moderate (ZE, salt content 2–3%), severe (HE, salt content 3–4%), and non-saline-alkaline (CON, salt content &lt; 1%). Employing a two-factor experimental design, we analyzed the fluctuations in nutritional quality, microbial abundance, and community composition of different salt–alkaline alfalfa materials and silage feeds. Furthermore, we elucidated the fermentation mechanism involved in salt–alkaline alfalfa ensiling. Following a 60-day ensiling period, the ZE and HE treatments led to substantial reductions in pH, acetic acids (AA), branched-chain butyric acids (BA) content, facultative anaerobic bacteria, and Escherichia coli populations (p &lt; 0.05). Conversely, the ZE and HE treatments increased lactic acid (LA) content and the population of lactic acid bacteria (LAB) (p &lt; 0.05). Additionally, these treatments significantly mitigated protein losses in both raw alfalfa and silage feeds (p &lt; 0.05), while remarkably augmenting the water-soluble carbohydrates (WSC), Na+, and K+ content of alfalfa materials. Sodium ions were found to exert a considerable influence on bacterial community composition during salt–alkaline alfalfa ensiling, with Enterococcus, Lactococcus, and Lactobacillus identified as the predominant fermentative microorganisms. Overall, moderately salt-alkaline alfalfa materials displayed optimal nutritional and fermentation quality, ensuring favorable nutritional attributes and fermentation outcomes under such conditions.

Effect of Adding Flaxseed Essential Oil in Alfalfa Ensiling Process on Ruminal Fermentation Kinetics

The experiment's purpose was to evaluate the effect of adding different levels of essential flaxseed oils (FEO) on the chemical composition and in vitro degradability of alfalfa silage. Treatments were alfalfa silage with no additive (control) or treated with 60 or 120 ml/kg DM of essential flaxseed oils (FEO60, FEO120, respectively). Whole plant alfalfa was ensiled for 60 d in triplicate laboratory-scale tubes. Dry matter (DM) content was more significant for FEO120 than control. Compared with the control, neutral detergent fiber (NDF) concentration was decreased in FEO120. Adding essential oils to the silage significantly decreased silage pH compared with untreated silage (P &amp;lt; .0001). Interestingly increased (DM and OM digestibility) for all the silages containing essential oil compared with untreated silage. The addition of flaxseed essential oil to alfalfa silage increased the rate of disappearance of organic matter (OM) and dry matter (DM) in all treatments compared to the control treatment. The degradability potential of alfalfa silage has increased in treatments containing FEO (120 mg/kg DM). In general, the obtained data show that the FEO had a positive effect on the quality of alfalfa silage and its nutritional characteristics.

Dry matter content and inoculant alter the metabolome and bacterial community of alfalfa ensiled at high temperature.

Alfalfa silage fermentation quality, metabolome, bacterial interactions, and successions as well as their predicted metabolic pathways were explored under different dry matter contents (DM) and lactic acid bacteria (LAB) inoculations. Silages were prepared from alfalfa with DM contents of 304 (LDM) and 433 (HDM) g/kg fresh weight and inoculated with Lactiplantibacillus plantarum (L. plantarum, LP), Pediococcus pentosaceus (P. pentosaceus, PP), or sterile water (control). The silages were stored at a simulated hot climate condition (35°C) and sampled at 0, 7, 14, 30, and 60 days of fermentation. The results revealed that HDM significantly improved the alfalfa silage quality and altered microbial community composition. The GC-TOF-MS analysis discovered 200 metabolites in both LDM and HDM alfalfa silage, mainly consisting of amino acids, carbohydrates, fatty acids, and alcohols. Compared with LP and control, PP-inoculated silages had increased concentrations of lactic acid (P < 0.05) and essential amino acids (threonine and tryptophan) as well as decreased pH, putrescine content, and amino acid metabolism. However, alfalfa silage inoculated with LP had higher proteolytic activities than control and PP-inoculated silage, as revealed by a higher concentration of ammonia nitrogen (NH3-N), and also upregulated amino acid and energy metabolism. HDM content and P. pentosaceus inoculation significantly altered the composition of alfalfa silage microbiota from 7 to 60 days of ensiling. Conclusively, these results indicated that inoculation with PP exhibited great potential in enhancing the fermentation of silage with LDM and HDM via altering the microbiome and metabolome of the ensiled alfalfa, which could help in understanding and improving the ensiling practices under hot climate conditions. KEY POINTS: • HDM improved fermentation quality and declined putrescine content of alfalfa silage • P. pentosaceus inoculation enhanced the fermentation quality of alfalfa silage • P. pentosaceus is an ideal inoculant for alfalfa silage under high temperature.

Effects of inoculating feruloyl esterase-producing Lactiplantibacillus plantarum A1 on ensiling characteristics, in vitro ruminal fermentation and microbiota of alfalfa silage

BackgroundFerulic acid esterase (FAE)-secreting Lactiplantibacillus plantarum A1 (Lp A1) is a promising silage inoculant due to the FAE’s ability to alter the plant cell wall structure during ensiling, an action that is expected to improve forage digestibility. However, little is known regarding the impacts of Lp A1 on rumen microbiota. Our research assessed the influences of Lp A1 in comparison to a widely adopted commercial inoculant Lp MTD/1 on alfalfa’s ensilage, in vitro rumen incubation and microbiota.ResultsSamples of fresh and ensiled alfalfa treated with (either Lp A1 or Lp MTD/1) or without additives (as control; CON) and ensiled for 30, 60 and 90 d were used for fermentation quality, in vitro digestibility and batch culture study. Inoculants treated silage had lower (P < 0.001) pH, acetic acid concentration and dry matter (DM) loss, but higher (P = 0.001) lactic acid concentration than the CON during ensiling. Compared to the CON and Lp MTD/1, silage treated with Lp A1 had lower (P < 0.001) aNDF, ADF, ADL, hemicellulose, and cellulose contents and higher (P < 0.001) free ferulic acid concentration. Compared silage treated with Lp MTD/1, silage treated with Lp A1 had significantly (P < 0.01) improved ruminal gas production and digestibility, which were equivalent to those of fresh alfalfa. Real-time PCR analysis indicated that Lp A1 inoculation improved the relative abundances of rumen’s total bacteria, fungi, Ruminococcus albus and Ruminococcus flavefaciens, while the relative abundance of methanogens was reduced by Lp MTD/1 compared with CON. Principal component analysis of rumen bacterial 16S rRNA gene amplicons showed a clear distinction between CON and inoculated treatments without noticeable distinction between Lp A1 and Lp MTD/1 treatments. Comparison analysis revealed differences in the relative abundance of some bacteria in different taxa between Lp A1 and Lp MTD/1 treatments. Silage treated with Lp A1 exhibited improved rumen fermentation characteristics due to the inoculant effects on the rumen microbial populations and bacterial community.ConclusionsOur findings suggest that silage inoculation of the FAE-producing Lp A1 could be effective in improving silage quality and digestibility, and modulating the rumen fermentation to improve feed utilization.

Natural fermentation quality, bacteria, and functional profiles of three cuttings of alfalfa silage in a year in Inner Mongolia, China.

Alfalfa is harvested two or three times a year in central and western Inner Mongolia, China. However, the variations in bacterial communities as affected by wilting and ensiling, and the ensiling characteristics of alfalfa among the different cuttings, are not fully understood. To enable a more complete evaluation, alfalfa was harvested three times a year. At each time of cutting, alfalfa was harvested at early bloom, wilted for 6 h, and then ensiled in polyethylene bags for 60 days. The bacterial communities and nutritional components of fresh alfalfa(F), wilted alfalfa(W) and ensiled alfalfa(S), and the fermentation quality and functional profile of bacterial communities of the three cuttings alfalfa silage, were then analyzed. Functional characteristics of silage bacterial communities were evaluated according to the Kyoto Encyclopedia of Genes and Genomes. The results showed that all nutritional components, fermentation quality, bacterial communities, carbohydrate, amino acid metabolism and key enzymes of bacterial communities were influenced by cutting time. The species richness of F increased from the first cutting to the third cutting; it was not changed by wilting, but was decreased by ensiling. At phylum level, Proteobacteria were more predominant than other bacteria, followed by Firmicutes (0.063-21.39%) in F and W in the first and second cuttings. Firmicutes (96.66-99.79%) were more predominant than other bacteria, followed by Proteobacteria (0.13-3.19%) in S in the first and second cuttings. Proteobacteria, however, predominated over all other bacteria in F, W, or S in the third cutting. The third-cutting silage showed the highest levels of dry matter, pH and butyric acid (p < 0.05). Higher levels of pH and butyric acid were positively correlated with the most predominant genus in silage, and with Rosenbergiella and Pantoea. The third-cutting silage had the lowest fermentation quality as Proteobacteria were more predominant. This suggested that, compared with the first and second cutting, the third cutting is more likely to result in poorly preserved silage in the region studied.

Quality of alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) mixture silages depending on the share in the mixture and additives

Alfalfa silage has a high potential for improving ruminant nutrition. A problem that frequently occurs during its preparation is the process of proteolysis, which could partially be avoided with the use of certain techniques and materials. Red clover, often used in form of silage, expresses weaker proteolysis due to the possession of certain chemical compounds. This research was conducted to study the effects of ensiled alfalfa and red clover mixtures, mixed at different ratios (100:0, 90:10, 70:30, 50:50, 30:70, and 0:100), as well as the influence of additives – two doses of oak tannin extract (6 g kg-1 DM and 12 g kg-1 DM) and bacterial inoculant (Enterococcus faecium, Bacillus plantarum, and Bacillus brevis) on fermentation parameters and protein fractions of the silages. The treatments which contained any of the used percentages of red clover, as well as the ones which received the higher dose of oak tannin (12 g kg-1 DM), reduced the ammonia nitrogen content, which is one of the main indicators of proteolysis. On the other hand, considering non-protein nitrogen, as another of the indicators of proteolysis, there was no positive sign of the contribution of red clover to the reduction of proteolysis in alfalfa, and neither there was a significant impact of additives that were applied in this experiment.

Metagenomics insights into the effects of lactic acid bacteria inoculation on the biological reduction of antibiotic resistance genes in alfalfa silage

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.

Effects of Different Additives on Fermentation Quality, Microbial Communities, and Rumen Degradation of Alfalfa Silage

This study examined the effects of different additives on the fermentation quality, nutrient composition, microbial communities, and rumen degradation of ensiled alfalfa. Six treatments were employed in which additives were applied to alfalfa on a fresh weight basis: CK (no additive), FA (0.6% formic acid), CaO (3% calcium oxide and 3% urea), LB (1 × 106 cfu/g Lentilactobacillus buchneri), GLB (2% glucose and 1 × 106 cfu/g L. buchneri), and FLB (2% fucoidan and 1 × 106 cfu/g L. buchneri). After 60 days of ensiling, all treatments altered the bacterial communities, improved the fermentation quality, reduced dry matter (DM) and crude protein (CP) losses, and enhanced the rumen degradation of nutrients. The addition of LB increased the relative abundance of Lactobacillus spp. (p &lt; 0.05), whereas GLB reduced (p &lt; 0.05) the NH3-N:TN ratio and elevated (p &lt; 0.05) the concentrations of Lactobacillus and lactic acid content. The FA treatment reduced (p &lt; 0.05) the pH, as well as the DM and CP degradations, while the CaO treatment increased the degradations of DM, acid detergent fiber, and neutral detergent fiber. We concluded that FA, LB, GLB, and FLB had beneficial effects on alfalfa fermentation, and that CaO increased alfalfa silage rumen degradation.

Photosensitisation and green egg yolks in laying hens caused by the feeding of ensiled alfalfa leaves

ABSTRACT 1. The present study was carried out to determine the effects of feeding ensiled alfalfa leaves (ALS) as an alternative protein source to laying hens under the terms of an organic diet. Due to the occurrence of unexpected negative health effects and undesirable egg yolk pigmentation in the test groups the trial was prematurely stopped and further analysis was conducted to evaluate the responsible substances. 2. Body weights of the test groups decreased significantly already in week 2 of the trial. Performance variables dropped. Olive green pigmented egg yolks were found in groups fed diets containing ALS. Severe comb necrosis occurred in the experimental group receiving the highest level of ALS (20%) combined with the option of free-range access and therefore natural light exposure. 3. The noxious agent found in ALS, blood serum and egg yolk was the photosensitising chlorophyll derivate pheophorbid a (PPBa), deriving from a strong depletion of chlorophyll contained in the alfalfa leaves. PPBa caused the olive-green pigmentation found in yolks and led to photosensitivity in groups with the highest level of ALS in the diet in combination with light exposure. 4. By aiming for high protein and amino acid levels, harvesting and processing have, unintentionally and initially unnoticed, led to a strong accumulation of phototoxic PPBa. From these results it is strongly advised not to include ensiled alfalfa leaves as a protein source in organic laying hen diets.

Exploiting the anaerobic fermentation of alfalfa as a renewable source of squalene.

The use of alfalfa is a promising response to the increasing demand for squalene. Ensiling could enhance the squalene content of fresh alfalfa and silage. To investigate and exploit the anaerobic fermentation of forage as a new squalene source, alfalfa was ensiled without (CON) or with molasses (ML) and sunflower seed oil (SSL) for 10, 40, and 70 days. Naturally ensiled alfalfa was of poor quality but had up to 1.93 times higher squalene content (P < 0.001) than fresh alfalfa. The squalene-producing bacteria were found to be cocci lactic acid bacteria (LAB). Adding ML and SSL decreased squalene content (P=0.002 and P < 0.001) by 6.89% and 11.6%, respectively. Multiple linear regression models and correlation analysis indicated that squalene synthase was the key enzyme for squalene synthesis. The addition of ML and SSL altered the structure of LAB communities, mainly decreasing the relative abundance of cocci LAB, which was responsible for squalene synthesis, and changing the fermentation products (lactic acid, propionic acid, and ammonia-N) influencing the squalene-related enzymes, thereby decreasing squalene production. Compared with squalene production from the reference bacteria (Pediococcus acidilactici Ch-2, Rhodopseudomonas palustris, Bacillus subtilis, engineered Escherichia coli), alfalfa silage had the potential to be a new squalene source. Natural ensiled alfalfa was a promising source for squalene, and ensiling was a potential pathway to obtain novel high-yield squalene bacteria. © 2022 Society of Chemical Industry.

Effects of urea supplementation on the nutritional quality and microbial community of alfalfa (Medicago sativa L.) silage.

The objectives of this study were to evaluate the contribution of urea on the nutritional quality and microbial community of ensiled alfalfa (Medicago sativa L.). Alfalfa silage was control group without urea (AL), supplementation with 0.5% urea (AU1), or supplementation with 1% urea (AU2). The silage tanks were opened and sampled after silage at 0, 15, 30, and 60 d. Results showed that AU2 had higher pH, ratio of (ammonia-N)/(total nitrogen) (NH3-N/TN) and crude protein (CP) content than those in AL and AU1, while AU1 had higher acid detergent fiber (ADF) than that in AL and AU2 after 15 d silage. Richness and diversity indices of microbial communities in silage were no significant differences among AL, AU1 and AU2 group. Proteobacteria (58.23%) and Firmicutes (40.95%) were the predominant phylum in three groups during the silage process. The percent of community abundances on genera level of Enterobacteriaceae (37.61%) and Klebsiella (41.78%) in AL were a little higher than those in AU1 (30.39%, 25.02%) and AU2 (33.48%, 26.92%). These results showed that silage with urea alone could not improve the quality of alfalfa.

Innovative utilization of herbal residues: Exploring the diversity of mechanisms beneficial to regulate anaerobic fermentation of alfalfa

In order to increase the utilization of herbal residues, realize efficient utilization of resources, the bacterial community and anaerobic fermentation characteristics of alfalfa ensiling treated with 36 kinds of herbal residues were studied. All the herbal residues improved the anaerobic fermentation quality in different degrees, indicated by lower pH, NH3-N and butyric acid concentrations. However, the contents of lactic and acetic acids varied widely in silage with different herbal residues. Pearson’s correlation analysis showed that the improved fermentation quality was closely associated with the variation of lactic acid bacteria community. Consequently, the herbal residues could improve anaerobic fermentation quality by stimulating desirable Lactobacillus species and inhibiting undesirable microbes. This study provides new insights for efficient utilization of herbal residues.

Antioxidant, flavonoid, α-tocopherol, β-carotene, fatty acids, and fermentation profiles of alfalfa silage inoculated with novel Lactiplantibacillus Plantarum and Pediococcus acidilactici strains with high-antioxidant activity

Six lactic acid bacteria strains with high-antioxidant activity were screened and applied to alfalfa silage to develop antioxidant probiotics for silage production. Alfalfa was ensiled without inoculants (control) or with a commercial strain (Lactiplantibacillus plantarum GFG) and antioxidant strains L. plantarum 24-7 (24-7), L. plantarum BX62 (BX62), L. plantarum AS21 (AS21), L. plantarum FM15 (FM15), Pediococcus acidilactici 13-7 (13-7), P. acidilactici J17 (J17) at dry matter concentration of 400 g/kg for 60 d, respectively. Alfalfa silage was investigated on silage fermentation, chemical and microbial compositions, antioxidant enzymes, total flavonoid, α-tocopherol, β-carotene and fatty acid. The results showed that AS21, FM15, 13-7, and J17 inoculations improved (P < 0.001) lactic acid concentration in alfalfa silage compared with control and GFG-treated silages. Compared with control and GFG-treated silages, AS21- (P < 0.001), FM15- (P = 0.007), 13-7- (P = 0.03), and J17-treated (P < 0.001) silages decreased ammonia nitrogen concentration. All inoculants with high-antioxidant activity reduced the losses of α-tocopherol (P < 0.001) and β-carotene (P < 0.001). Higher total flavonoid concentration was found in 24-7- (P = 0.004), BX62- (P < 0.001), FM15- (P = 0.03), J17-treated (P < 0.001) silages versus control. In comparison to control and GFG-treated silages, 24-7- (P < 0.001), BX62- (P < 0.001), AS21- (P = 0.004), FM15- (P = 0.002), J17-treated (P < 0.001) silages had higher proportion of polyunsaturated fatty acid. Except for strain J17, inoculations with the screened strains had higher (P < 0.001) total antioxidant capacity (T-AOC) activity in alfalfa silage compared with control and GFG-treated silages. The highest T-AOC (P < 0.001, 200 U/g FW) was observed in AS21-treated silage. Inoculated silages had higher (P < 0.001) glutathione peroxidase activity versus control. Conversely, compared with control and GFG-treated silages, lower superoxide dismutase activity was observed in FM15- (P < 0.001) and J17-treated (P < 0.001) silages. Lower lipoxygenase (LOX) activity (P < 0.001) was observed in screened strains treated-silages compared with control and GFG-treated silages. Pearson correlation analysis showed that T-AOC was positively correlated with α-tocopherol (r = 0.66, P = 0.05) and β-carotene (r = 0.76, P = 0.02), but negatively correlated with LOX (r = − 0.67, P = 0.05). Conclusively, all the tested strains with high-antioxidant activity can be used as candidate strains to improve the antioxidant status of ensiled alfalfa, and strain AS21 is the most preferred strain.

Dynamics of the bacterial communities and predicted functional profiles in wilted alfalfa silage.

To investigate the fermentation characteristics, bacterial community and predicted functional profiles during the ensiling of wilted alfalfa (Medicago sativa L.). First-cutting alfalfa was harvested at the early bloom stage, wilted for 6h, and ensiled in laboratory-scale silos (1L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60days of ensiling, respectively. The bacterial communities of wilted alfalfa and silages on day3 and 60 were assessed through high throughput sequencing technology, and their functional characteristics were evaluated according to the Kyoto Encyclopedia of Genes and Genomes using Tax4Fun. After 60days of ensiling, alfalfa silage showed a moderate fermentation quality, indicated by high lactic acid (56.7gkg-1 dry matter [DM]) and acetic acid (39.4gkg-1 DM) contents, and low concentrations of butyric acid (2.12gkg-1 DM) and ammonia nitrogen (128gkg-1 total nitrogen). Lactobacillus rapidly became predominant on day3 and increased to 60.4% on day60. Results of functional prediction analyses showed that the metabolism of amino acid, energy, cofactors and vitamins were reduced, while metabolism of nucleotide and carbohydrate were increased during ensiling. Fructokinase, 1-phosphofructokinase and pyruvate kinase played important roles in producing lactic acid. The production of acetic acid may be correlated with the enhancement of 6-phosphogluconate dehydrogenase and acetyl-CoA synthetase. Knowledge regarding bacterial dynamics and their metabolic pathways during alfalfa ensiling is important for understanding the fermentation process and may contribute to the production of nutritious and stable alfalfa silage. High throughput sequencing technology combined with 16S rRNA gene-predicted functional analyses could provide a new and comprehensive insight into bacterial community dynamics and functional profiles to further improve the silage quality.

Microbial mechanisms of using feruloyl esterase-producing Lactobacillus plantarum A1 and grape pomace to improve fermentation quality and mitigate ruminal methane emission of ensiled alfalfa for cleaner animal production

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 (CH4) emission, and the microbiota of ensiled alfalfa. Alfalfa at 42% dry matter (DM) was treated in a 2 × 2 factorial design: with the application of Lp A1 at 0 (control) or 1 × 106 cfu/g of fresh forage, and GP at 0 or 5% of fresh forage. After 60 d of ensiling, a decrease in nonprotein nitrogen (NPN) was observed in GP treated silage. Lp A1 inoculated silage had a lower fiber content than silages without Lp A1. The lowest NPN was found in silage treated with LG, and an obvious increase in the relative abundance of Lactobacillus paracasei was detected in silages treated with Lp A1 and LG, respectively. In vitro ruminal experiments indicated that, although the application of GP deceased ruminal total gas, CH4 production, nitrogen degradation and the number of methanogenic archaea in alfalfa silage, it also reduced silage DM digestibility. In contrast, inoculation with Lp A1 not only increased DM digestibility and populations of ruminal Ruminococcus flavefaciens and fungi, but also improved ruminal total gas and CH4 production. As expected, LG treatment decreased alfalfa silage ruminal total gas and CH4 production relative to Lp A1 treatment alone, and increased silage DM digestibility compared with GP treated silage. In conclusion, the application of LG before ensiling alfalfa, balanced silage proteolysis, feed digestibility, and CH4 emission, and could be a promising strategy for using food industry by-products to produce a nutritional and environmentally-friendly legume silage that will mitigate N and greenhouse gas emissions from ruminants.

Characterization of a novel beta-cypermethrin-degrading strain of Lactobacillus pentosus 3-27 and its effects on bioremediation and the bacterial community of contaminated alfalfa 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.