Fermentation Quality Of Alfalfa Silage Research Articles

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.

The Effect of Lactobacillus planturum YQM48 Inoculation on the Quality and Microbial Community Structure of Alfalfa Silage Cultured in Saline-Alkali Soil

Alfalfa cultivated in salt–alkali soil was used for fermentation, to which an inoculum of Lactobacillus plantarum YQM48 was added, to assess its effect on the feed quality and the microbial community structure of the fermented silage. A control was included without inoculum. The nutritional components, fermentation quality, pH, and microbial community of the silage were measured after 30 and 60 days of anaerobic fermentation. The results showed that after 30 and 60 days of fermentation in the presence of the inoculum, the content of water-soluble carbohydrates, crude protein content, and dry matter were all significantly higher than those of the control silage, the pH and butyric acid content were lower, and the content of lactic acid and acetic acid were higher (p < 0.05 for all). There was no significant difference in pH and butyric acid content between 30 and 60 days of fermentation in the presence of the inoculum (p > 0.05), while the lactic acid and acetic acid contents were significantly lower in the 60-day silage compared to the 30-day silage (p < 0.05). Fermentation reduced the abundance of Cyanobacteria and Proteobacteria (the abundant phyla in the fresh alfalfa), while the abundance of Firmicutes increased, reaching 92.3% after 60 days of fermentation with inoculum. The dominant genus in that sample was Lactobacillus (70.0%), followed by Enterococcus (12.9%), while fermentation of 60 days without inoculum resulted in only 29.2% Lactobacillus, together with 27.8% Cyanobacteria, and 12.2% Enterococcus. In summary, the addition of L. plantarum YQM48 can improve the nutritional components and fermentation quality of alfalfa silage cultivated on a salt-alkali soil.

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 Bacillus coagulans and Lactobacillus plantarum on the Fermentation Characteristics, Microbial Community, and Functional Shifts during Alfalfa Silage Fermentation.

This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 × 106 CFU/g FW), Lactobacillus plantarum (LP, 1 × 106 CFU/g FW), and their combinations (LP+BC, 1 × 106 CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH3-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman's rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality.

Effect of lactobacilli inoculation on protein and carbohydrate fractions, ensiling characteristics and bacterial community of alfalfa silage.

Alfalfa (Medicago sativa L.) silage is one of the major forages with high protein for ruminants. The objective of this study was to investigate the effects of lactobacilli inoculants on protein and carbohydrate fractions, ensiling characteristics and bacterial community of alfalfa silage. Wilted alfalfa (35% dry matter) was inoculated without (control) or with Lactobacillus coryniformis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus pentosus and ensiled for 7, 15, and 60 days. Silage inoculated with L. pentosus was superior to L. coryniformis, L. casei, L. plantarum in improving the fermentation quality of alfalfa silage, as indicated by the lowest ammonia nitrogen content and silage pH during ensiling. There was minor difference in water soluble carbohydrates content among all silages, but L. pentosus inoculants was more efficient at using xylose to produce lactic acid, with lower xylose content and higher lactic acid content than the other inoculants. Compared with the control, L. pentosus inoculants did not affect true protein content of silage, but increased the proportions of buffer soluble protein and acid detergent soluble protein. The L. pentosus inoculants reduced the bacterial diversity In alfalfa silage with lower Shannon, Chao1, and Ace indices, and promoted relative abundance of lactobacillus and decreased the relative abundance of Pediococcus compared with the control. As well as L. pentosus inoculants up-regulated amino acid, carbohydrate, energy, terpenoids, and polypeptides metabolism, and promoted lactic acid fermentation process. In summary, the fermentation quality and nutrient preservation of alfalfa silage were efficiently improved by inoculated with L. pentosus.

Effect of Storage Period on the Fermentation Profile and Bacterial Community of Silage Prepared with Alfalfa, Whole-Plant Corn and Their Mixture

This study aimed to investigate the impact of storage time on the bacterial community and fermentation profile of silage prepared with alfalfa, whole-plant corn, and their mixture. Fresh alfalfa and whole-plant corn were chopped and combined in fresh weight ratios of 1:0 (alfalfa, control), 0.8:0.2 (M1), 0.6:0.4 (M2), and 0:1 (corn). Three silos of each treatment were analyzed after 30, 60, and 90 d of storage. With storage time, pH, acetic acid, propionic acid, butyric acid, and ammonia nitrogen levels increased in alfalfa silage (p &lt; 0.01), whereas lactic acid level decreased (p &lt; 0.01). Compared to alfalfa silage, M1, M2, and corn silages were better fermented and more stable during storage. The dominant bacteria in M1, M2, and corn silages shifted significantly from L. plantarum, L. buchneri, and L. brevis to L. acetotolerans and L. buchneri during 30 to 60–90 d of storage, and storage time decreased the bacterial diversity of these silages. In conclusion, storage time significantly decreased the fermentation quality of alfalfa silage and remarkably optimized the bacterial community structure of well-fermented M1, M2, and corn silages. Alfalfa should be ensiled with at least 20% whole-plant corn to improve silage fermentation quality and storage stability.

Bacterial Community and Fermentation Quality of Ensiling Alfalfa With Commercial Lactic Acid Bacterial Additives.

The aim of this study was to determine the effects of six common commercial lactic acid bacteria (LAB) additives [A1, Lactobacillus plantarum, L. buchneri, and Enterococcus faecalis; A2, L. plantarum and L. casei; A3, L. plantarum and L. buchneri; A4, L. plantarum, L. buchneri, L. casei, and Pediococcus acidilactici; A5, L. plantarum (producing feruloyl esterase); and A6, L. buchneri, P. acidilactici, β-glucanase, and xylanase] on the bacterial community and fermentation quality of alfalfa silage. Alfalfa was harvested at the squaring stage, wilted in the field for 24 h, and ensiled without any additives (Control) or with A1, A2, A3, A4, A5, or A6. Microbial counts, bacterial community, fermentation parameters, and nutritional composition were determined after ensiling for 90 days. The total abundance of LAB genera on alfalfa pre-ensiling was 0.38% in bacterial community. The abundances of Lactobacillus, Enterococcus, and Pediococcus in the Control silage were 42.18, 40.18, and 8.09% of abundance, respectively. The abundances of Lactobacillus in A1-, A2-, A3-, A4-, and A5-treatments were 89.32, 92.93, 92.87, 81.12, and 80.44%, respectively. The abundances of Pediococcus and Lactobacillus in A6-treatment were 70.14 and 24.86%, respectively. Compared with Control silage, LAB-treated silage had lower pH and less ammonia nitrogen and water-soluble carbohydrates concentrations (p < 0.05). Further, the A5- and A6-treatments contained lower neutral detergent fiber, acid detergent fiber, and hemicellulose than other treatments (p < 0.05). Overall, LAB genera were presented as minor taxa in alfalfa pre-ensiling and as dominant taxa in alfalfa silage. Adding LAB additives improved the fermentation quality and altered the bacterial community of alfalfa silage. The main bacterial genera in Control silage were Lactobacillus, Enterococcus, and Pediococcus. Lactobacillus dominated the bacterial communities of A1-, A2-, A3-, A4-, and A5-treatments, while Pediococcus and Lactobacillus were dominant bacterial genera in A6-treatment. Inoculating A5 and A6 degraded the fiber in alfalfa silage. It is necessary to ensile alfalfa with LAB inoculants.

Influence of Condensed and Hydrolysable Tannins on the Bacterial Community, Protein Degradation, and Fermentation Quality of Alfalfa Silage.

Simple SummaryProteolysis widely exists in alfalfa ensiling, and up to 88% of total nitrogen could be converted to non-protein nitrogen, leading to low protein utilization efficiency. In order to limit proteolysis, tannins have been used to protect protein from microbial degradation. Although there have been studies that evaluated the efficiency of hydrolysable tannin and condensed tannin to prevent proteolysis, the two were mostly considered individually. In addition, the effects of different types of tannins on plant proteases are unknown. We turned our attention to evaluating the effects of hydrolysable tannin and condensed tannin on the bacterial community and proteolysis of alfalfa silage. In this study, both hydrolysable tannin and condensed tannin were able to reduce proteolysis in alfalfa silages—especially for condensed tannin. The application of hydrolysable tannin decreased the abundance of Lactobacillus and increased the abundances of Enterococcus, while the opposite results were observed when condensed tannin was applied. In addition, condensed tannin reduced the activity of carboxypeptidases and aminopeptidases in treated silage, whereas silage treated with hydrolysable tannin showed lower acid protease and carboxypeptidases activities. Thus, both hydrolysable tannin and condensed tannin could limit proteolysis in alfalfa silage, but the mechanism and their effects on the bacterial communities were different.This study evaluated the effects of hydrolysable tannin (HT) and condensed tannin (CT) on the bacterial community, fermentation quality, and proteolysis of alfalfa silage. Alfalfa was wilted to a dry matter (DM) of 35% fresh weight and ensiled with or without 4% HT or 4% CT. The application rates of tannins were based on fresh weight, and each treatment was ensiled in triplicate. After 60 d of fermentation, the CT-treated group had lower concentrations of ammonia nitrogen (NH3-N) and free amino acid nitrogen (AA-N), but greater lactic acid concentration, than those in the control and HT-treated silage (p < 0.05). Compared to the control group, the application of tannins increased the abundance of Pseudomonas (negatively correlated with aminopeptidases activity), and decreased the abundance of Pediococcus—which was positively correlated with aminopeptidases activity—and the concentrations of non-protein nitrogen (NPN), NH3-N, and AA-N. The application of HT decreased the abundance of Lactobacillus and increased the abundances of Enterococcus, while the opposite results were observed in the CT-treated group. The application of HT and CT reduced the proteolysis in treated silages, but the two were different in terms of their mechanism and their effects on the bacterial communities of the alfalfa silage.

A Preliminary Investigation on Bacterial Diversity and Fermentation Quality of High-Moisture Alfalfa Silage Prepared with Biochar

The fermentation quality of alfalfa silage is poor but can be improved with additives. This study investigates the effects of biochar on the fermentation quality and bacterial diversity of high-moisture alfalfa silage. Alfalfa was treated with: (i) control without additive (CK); (ii) 1% biochar (1% carbon C); (iii) 2% biochar (2% C), and fermented for 15 or 30 d. Mixing alfalfa with biochar significantly decreased (p &lt; 0.05) pH, the number of coliform bacteria, nonprotein nitrogen content, and ammonia–nitrogen content, and significantly increased the contents of dry matter, lactic acid, and true protein. The addition of biochar also influenced bacterial community distribution. The relative abundance of Lactobacillus and Enterococcus increased while the abundance of Pantoea decreased with biochar treatment. In conclusion, alfalfa silage’s fermentation quality and microbial community structure are improved by adding biochar from the pyrolysis of waste furniture.

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.

Isolation, identification and utilization of lactic acid bacteria from silage in a warm and humid climate area

The study aimed to isolate and identify lactic acid bacteria (LAB) from silages and their application to improve the fermentation quality of alfalfa. Forty-nine LAB strains were isolated from silages, and two strains were screened for growth and acid production rates. Then two strains were selected for Physiological and morphological tests and 16S rRNA sequencing. They were Gram-positive and Catalase-negative and were able to grow at pH 3.5 and at 45 °C, were unable to grow different NaCl concentrations as 3.0% and 6.5%. Strain BDy3-10 was identified as Lactobacillus rhamnosus, while TSy1-3 was identified as L. buchneri. The selected strains were evaluated on fermentation of alfalfa silage. The highest crude protein content occurred in the BDy3-10 treatment group. The contents of neutral detergent fiber and acid detergent fiber in the TSy1-3 treatment were significantly lower than other treatment (P < 0.05). Compared to the control treatment, inoculation treatments deceased pH during ensiling (P < 0.001) and provided the most increased lactic acid content after ensiling for 10 days (P < 0.001). The acetic acid contents of all the inoculation groups were significantly increased (P < 0.001) during ensiling, and were lower than that of control group (P < 0.001). So, the TSy1-3 treatment most effectively improved the fermentation quality of alfalfa silage in warm and humid climate area.

Treatment of alfalfa silage with tannin acid at different levels modulates ensiling characteristics, methane mitigation, ruminal fermentation patterns and microbiota

The objective of this study was to evaluate the potential of tannin acid (TA) derived from Chinese gallnut, with different doses for modulating ensiling characteristics, methane (CH4) emission, ruminal fermentation profile and microbiota of alfalfa silage. Second-cut alfalfa was wilted for 6 h to dry matter (DM) 439 g/kg fresh weight and chopped to 2 cm length. The chopped forage was ensiled with 5 TA levels (0, 10, 20, 40, and 60 g/kg DM), and each treatment was ensiled in triplicate in vacuum-sealed polyethylene plastic bags for 60 d. All TA treatments improved the fermentation quality of alfalfa silage, indicated by lower (P < 0.05) DM losses, pH values, and non-protein nitrogen concentrations. The acetic acid concentration markedly decreased (P < 0.05) with the greater levels of TA, leading to a reduction in the aerobic stability of silage. Ruminal CH4 production decreased (P < 0.05) with increasing TA levels from 20 to 60 g/kg DM, by which the CH4 concentration decreased by 10.3 to 12.4% compared with control. The DM, crude protein and neutral detergent fiber degradation linearly decreased (P < 0.05) with increasing TA levels, while propionic acid molar proportion and microbial protein production linearly increased (P < 0.05) with increasing TA levels, but the greatest level of 60 g/kg DM did not further increase these variables. The abundance of methanogens, anaerobic fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Prevotella ruminicola as well as the activity of cellulase and protease linearly decreased (P < 0.05) with TA inclusion, whereas Fibrobacter succinogenes was enhanced. Spearman correlation analysis showed high correlations between ruminal microbial abundance and CH4 production/fermentation parameters. The results indicated that TA, used as an environmentally friendly modifier prior to ensiling alfalfa can improve silage quality and modulate rumen fermentation pattern. However, TA from Chinese gallnut with levels greater than 40 g/kg DM is not recommended for addition to alfalfa silage.

Silage Additives Usage in Improving Fermentation Quality of Alfalfa Silage: A Review

Alfalfa is difficult to ensile due to its high protein content, low water-soluble carbohydrate level, low dry matter content, and high buffering capacity. Therefore, alfalfa has recently increased its efforts to make silage with additives. Silage additives have been used to enhancing the silage quality of alfalfa in recent years. This review covers research studies that have investigated the efficacy of silage additives on fermentation quality, the bacterial diversity of alfalfa silage using the Next-Generation Sequencing (NGS) technique, the effects of microbial inoculant on these bacterial communities, silage's antioxidant enzymes, and unsaturated fatty acid levels. In recent studies, more clear information about bacterial communities in silage fermentation has begun to be obtained with the use of NGS technique. In studies using these techniques, it was reported that lactic acid bacteria (LAB) organic acid additions to alfalfa silage caused Lactobacillus, Pediococcus and Sporolactobacillus species to increase relatively in silage environment, the ratio of unwanted species decreased and silage quality increased. Although the amount of alpha-tocopherol, and beta carotene decreased by half in pure silage compared to fresh alfalfa, it was observed that LAB inoculation increased the ratio of alpha tocopherol, beta carotene and also polyunsaturated fatty acids. Future silage additives are expected to determine effects fermentation quality, the bacterial community, the antioxidant enzymes, and unsaturated fatty acid levels.

Lactobacillus plantarum Inoculants Delay Spoilage of High Moisture Alfalfa Silages by Regulating Bacterial Community Composition.

The objective of this study was to investigate the mechanism of Lactobacillus plantarum (L. plantarum) involved in improving fermentation quality of naturally ensiled alfalfa under poor conditions. High-moisture wilted alfalfa was ensiled without inoculants (CK) or with inoculation of two L. plantarum additives (LPI and LPII). The pH and fermentation products of silage were determined after 30 and 90 days of ensiling. Additionally, the bacterial community compositions were analyzed. The L. plantarum inoculants significantly promoted lactic acid accumulation, and Lactobacillus abundance for both periods. At 90 days, silage in CK exhibited a high pH, a loss in dry matter, and a high concentration of ammoniacal nitrogen. The inoculations of L. plantarum significantly inhibited the growth of Clostridia, and reduced ammoniacal nitrogen concentration in silage (P < 0.05). Thus, inoculation with L. plantarum improved the fermentation quality of alfalfa silage and inhibited the growth of spoilage microorganisms, and further delayed spoilage of alfalfa silage under adverse ensiling conditions.

Pectin Degradation is an Important Determinant for Alfalfa Silage Fermentation through the Rescheduling of the Bacterial Community.

This study aimed to evaluate the effects of the four kinds of additives on the silage quality and the relevant bacterial community diversity by Illumina HiSeq 16S rRNA sequencing. The four kinds of additives were Lactobacillus plantarum (LP), organic acids including gallic acid (GA) and phenyllactic acid (PA), pectin (PEC), and enzymes including pectinase (PEE) and cellulase (CE). After 30 d of fermentation, the pH value was shown to have the lowest value in the PEE and PEC groups, followed by the PA group, and then in CE and GA groups; the highest value of pH was found in both LP and control groups. The ammonia nitrogen concentration was lower in the PEE group compared to the other groups except for the PA group. In the comparisons among the seven groups, Lactobacillus was higher in the LP group, Paracoccus was higher in the GA group, Weissella was higher in the PA group, Leuconostoc was higher in the PEC group, Bacillus, Aeromonas, and Curvibacter were higher in the PEE group, and Coriobacteriaceae_UCG_002 was higher in the CE group compared to the other groups. This study proposed that the addition of PEC and PEE improved the fermentation quality of alfalfa silage compared to other additives by improving the bacterial community of Leuconostoc, and Bacillus and Aeromonas, respectively. Moreover, the enhanced fermentation quality of alfalfa silage by the supplementation of PEC and PEE might be attributed to other unclassified genera. This study provides an implication that pectin degradation is an important determinant for alfalfa silage fermentation through the rescheduling of bacterial community diversity.

Effect of Lactobacillus plantarum expressing multifunctional glycoside hydrolases on the characteristics of alfalfa silage.

For the first time, Lactobacillus plantarum strains carrying heterologous genes encoding multifunctional glycoside hydrolases were constructed and used as additives for alfalfa silage. The chemical characteristics, nonstructural carbohydrate composition, and fermentation quality of alfalfa silage were examined. The supernatant of L. plantarum expressing CbXyn10C and Bgxg1 (LP11AG) showed activities on xylan, Avicel, and carboxymethylcellulose (CMC), while the supernatant of the wild-type L. plantarum showed no activity. When LP11AG was used as silage additive, the water-soluble carbohydrate content of alfalfa silage increased by 72%, 55%, and 155% compared with control when the silage was stored at 20 °C, 30 °C, and 40 °C, respectively. With LP11AG being used as an additive for the alfalfa silage stored at 20 °C, the hemicellulose, cellulose, and acid detergent ligninin (ADL) contents decreased by 17%, 6%, and 14% compared with the control (p < 0.05), respectively. Compared with the corresponding original contents, the contents of glucose, arabinose, galactose, and fructose detected in silage treated with LP11AG after 45 days of ensiling increased by 55%, 1494%, 68%, and 5% , respectively, when stored at 40 °C. Raffinose and stachyose, originally present in alfalfa, disappeared after ensiling. In conclusion, our results suggest that LP11AG provides a substantial benefit as a silage additive.

Effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage.

To investigate the effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage, first-cut alfalfa was ensiled with or without two screened lactic acid bacteria (LAB) strains, Lactobacillus plantarum, LP, and Lactobacillus casei, LC. Each treatment was divided into three parts and stored at 20°C, 30°C, 40°C, respectively. After 60days ensiling, fermentation characteristics were measured and bacterial diversity was investigated by 16S ribosomal RNA gene sequencing using Illumina MiSeq platform. LP and LC decreased pH, coliform bacteria counts and increased lactic acid content at 20°C, and the two strains decreased pH, ammonia-N concentration, coliform bacteria counts at 30°C. When the environmental temperature was 40°C, silage treated with LC showed lower LAB and coliform bacteria counts and higher lactic acid content than the untreated and LP treated silages. Butyric acid mainly appeared in silages stored at 40°C. The relative abundance of Lactobacillus in alfalfa silages stored at 20°C and 30°C was highest and increased after LP and LC were added. Garciella was another dominant genus in silages stored at 40°C. In conclusion, LP and LC improved fermentation quality of alfalfa silage by increasing Lactobacillus proportions at 20°C and 30°C; ensiling alfalfa at 40°C was difficult because of Garciella.

Isolating and evaluating lactic acid bacteria strains with or without sucrose for effectiveness of silage fermentation

AbstractThe aim of this study was to screen and evaluate lactic acid bacteria strains for their ability to ferment alfalfa, Leymus chinensis and native grass into silage. We isolated 186 lactic acid bacteria strains from alfalfa, native grass and selected eight strains by acid production test and growth rate test. The eight stains were identified using morphological observations, Gram staining, physiological and biochemical tests, an acid tolerance test and 16S rDNA sequencing. All eight strains (LP1, LP2, LC1, LC2, PP1, PP2, EF1 and EF2) grew at pH 4.0 and 6.5%NaCl. Strains LP1 and LP2 were identified as Lactobacillus plantarum, LC1 and LC2 as Lactobacillus casei, PP1 and PP2 as Pediococcus pentosaceus, EF1 as Enterococcus faecalis, and EF2 as Enterococcus faecium based on their 16S rDNA sequences. The eight strains and two commercial inoculants (PS and CL) were added with or without sucrose to native grass, L. chinensis, and alfalfa to produce silage. All eight screened strains decreased the pH and ammonia nitrogen (N) content and increased the lactic acid content of alfalfa silages. No butyric acid was detected in the L. chinensis and alfalfa silages. Addition of sucrose with the screened strains resulted in lower pH and ammonia N content, and higher lactic acid content and total fermentation acids content in all three silages. The eight strains improved the fermentation quality of alfalfa silage. All strains were more effective when supplemented with sucrose. No efficient lactic acid bacteria strain was found for ensiling the native grass, but the high fermentation quality native grass silage could be obtained using the combination of lactic acid bacteria strains and 2% sucrose.