Hazardous risk of antibiotic resistance genes: Host occurrence, distribution, mobility and vertical transmission from different environments to corn silage.

Antibiotic resistance genes (ARGs) are one of the emerging contaminants posing a great deal of hazardous risk to public health. This study employed metagenomics and deciphered the potential risk of the antibiotic resistome and their vertical transfer to ensiled whole-crop corn silage harvested from six climate zones: 1. Warm temperate-fully humid-hot summer (Cfa), 2. Arid-desert-cold arid (BWk), 3. Snow-desert-cold summer (Dwc), 4. Snow-desert-hot summer (Dwa), 5. Arid-steppe-cold arid (BSk), and 6. Equatorial-desert (Aw) based on the Köppen-Geiger climate classification in China. The findings demonstrate a high diversity of ARGs, which is related to the drug classes of tetracycline, ciprofloxacin, lincosamide, fosfomycin, and beta lactam. Resistome variations are mostly related to variations in microbial composition and fermentation characteristics of the silages from different climate zones, which are indirectly influenced by environmental conditions. The most dominating ARGs in corn silage were tetM, acrA, H-NS, lnuA, emrR, and KpnG, which is primarily hosted by Klebsiella and Lactobacilli. There were 5 high-risk ARGs (tetM, bacA, SHV-1, dfrA17, and QnrS1) in silage from different climate zones, and the tetM was the most prevalent high-risk ARG. However, throughout the ensiling process, the abundance of ARGs, and mobile ARGs were reduced. The resistome contamination in silage from Tibet (Dwc) with high altitude and harsh environment was relatively low due to the low variety and abundance of ARGs, the low abundance of mobile ARGs and high-risk ARGs. In addition, most of the bacteria responsible for the silage fermentation were also found to be the hosts to the ARGs, although their abundance decreased after 90 d of silage fermentation. Hence, we alert the existence of ARGs-related biosafety risk in silages and call for more attention to the silage ARGs, their hosts, and mobile genetic elements in order to curtail their possible risk to public health.

Detoxifying mycotoxins and antifungal properties of two rumen-derived Enterococcus species in artificially contaminated corn silages.

BACKGROUND: Mycotoxins contamination in food and feed has emerged as an issue of serious concern because they pose serious health risks to both humans and livestock. The study aimed to evaluate the effects of two rumen-derived Enterococcus spp. on fermentation and hygienic quality of artificially contaminated corn silages. The toxigenic fungal-infested (FI) and non-fungal infested (NFI) corn was harvested at 1/2 milk line stage and ensiled without additives (CON) or with Enterococcus faecalis (E) or Enterococcus faecium (M). RESULTS: The pH of FI silages was higher than that of NFI silages, the pH in NFI-M was lower than in NFI-CON. Inoculating E. faecium markedly increased lactic acid concentration compared to CON and E silages. Both E. faecium and E. faecalis decreased the deoxynivalenol (DON) and zearalenone (ZEN) concentrations compared with the CON for FI silages, while E. faecium was more effective in eliminating aflatoxin B1 (AFB1 ). The FI silage had higher bacterial and fungal Shannon indexes than NFI silages. The relative abundance (RA) of Aspergillus and Fusarium marked a decline from day 5 to day 90. Inoculating E. faecium and E. faecalis reduced the RA of Penicillium compared to CON. In vitro mycotoxins removal assay indicated that E. faecium was more effective in AFB1 detoxification while having lower detoxifying ZEN capacity than E. faecalis. CONCLUSION: Inoculating rumen-derived Enterococcus spp. isolates alleviated the negative effects of fungal infestation on the fermentation and hygienic quality of corn silages by changing the microbial communities and detoxifying mycotoxins. © 2023 Society of Chemical Industry.

Altitudinal Distribution Patterns of Phyllosphere Microbial Communities and Their Contribution to Silage Fermentation of Kobresia pygmaea Along the Elevation Gradient on the Tibetan Plateau.

The study aimed to reveal altitudinal distribution patterns of phyllosphere microbial communities and silage fermentation of Kobresia pygmaea along the elevation gradient on the Tibetan Plateau. The K. pygmaea was individually collected from 2,500, 3,000, 4,000, 4,500, and 5,000 m above sea level (a.s.l.) on the Tibetan Plateau and ensiled for 60 days, respectively. The phyllosphere bacterial diversity increased while fungal diversity decreased along the elevation gradient, and bacterial and fungal richness showed a unimodal distribution with peak abundance at 4,000 and 3,000 m a.s.l., respectively. After 60 days of ensiling, the bacterial and fungal community composition changed but did not exhibit clear altitudinal distribution patterns. All K. pygmaea underwent a weak fermentation indicated by pH above 5.0 and low ratio of lactic/acetic acid (LA/AA). The S5000 and S3000 showed the highest and lowest pH, respectively. Although Lactobacillus dominated S4000 after 60 days of ensiling, S4000 still exhibited poor fermentation quality as well as silages from the other four regions. The higher ammonia N concentrations in S3000 and S4000 than the other silages were consistent with the detectable butyric acid in S3000 and S4000. The silage fermentation of K. pygmaea collected from five regions exhibited poor fermentation quality, thereby inoculating lactic acid bacteria to K. pygmaea before ensiling is highly recommended to improve fermentation quality on the Tibetan Plateau.

The Effect of Early and Delayed Harvest on Dynamics of Fermentation Profile, Chemical Composition, and Bacterial Community of King Grass Silage.

The objective of this study was to assess the effect of harvesting time on the fermentation characteristics, chemical composition, and microbial community of king grass silage. King grass was harvested at three growth periods of 90 days (KN90S), 110 days (KN110S), and 130 days (KN130S); chopped into 2-3-cm particle size; and ensiled in polyethylene bags (20 × 30 cm). The fermentation quality and chemical composition of silages were analyzed after 1, 3, 7, 14, 30, and 60 days of ensiling. Bacterial community of silage ensiled for 60 days was profiled using next generation sequencing (NGS) technology. The KN110S showed the most extensive lactic acid (LA) fermentation during 7 days of fermentation compared to KN90S and KN130S. After 60 days of fermentation, the KN110S showed the lowest pH and the highest lactic acid content among the three treatments. The butyric acid and ammonia nitrogen contents of KN90S and KN130S were significantly greater than those of KN110S (p < 0.05). After a timespan of 60 days of ensiling, the bacterial community of king grass silage was predominantly populated by Proteobacteria in phylum level, whereas unclassified Enterobacteriaceae genus remained dominant in all silages. A higher relative abundance of Clostridium was observed in KN90S and KN130S, but not in KN110S, and greater abundance of Lactococcus appeared in KN110S and KN130S silages than KN90S. It is concluded that harvesting time had an important effect on the fermentation quality and microbial community of king grass silage.

Mycotoxins in Maize Silage from China in 2019.

Animal feed (including forage and silage) can be contaminated with mycotoxins. Here, 200 maize silage samples from around China were collected in 2019 and analyzed for regulated mycotoxins, masked mycotoxins (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside), and emerging mycotoxins (beauvericin, enniatins, moniliformin, and alternariol). Deoxynivalenol and zearalenone were detected in 99.5% and 79.5% of the samples, respectively. Other regulated mycotoxins were detected in fewer samples. The highest deoxynivalenol and zearalenone concentrations were 3600 and 830 µg/kg, respectively. The most commonly detected masked mycotoxin was 15-acetyldeoxynivalenol, which was detected in 68.5% of the samples and had median and maximum concentrations of 61.3 and 410 µg/kg, respectively. The emerging mycotoxins beauvericin, alternariol, enniatin A, enniatin B1, and moniliformin were detected in 99.5%, 85%, 80.5%, 72.5%, and 44.5%, respectively, of the samples but at low concentrations (medians <25 µg/kg). The samples tended to contain multiple mycotoxins, e.g., the correlation coefficients for the relationships between the concentrations of beauvericin and deoxynivalenol, deoxynivalenol and zearalenone, and zearalenone and beauvericin were 1.0, 0.995, and 0.995, respectively. The results indicated that there needs to be more awareness of the presence of one or more masked and emerging mycotoxins in maize silage in China.

Effects of hexanoic acid on microbial communities, fermentation, and hygienic quality of corn silages infested with toxigenic fungi.

BACKGROUND: This study aims to reveal the effects of hexanoic acid on the microbial communities, fermentation, and the hygienic quality of corn silages with or without fungal infection. Fungal-infested (FI) and non-infested (NFI) whole-crop corn samples were separately ensiled without (control, CON) or with hexanoic acid (Hex, 90% purity) at 0.2 g·kg-1 fresh weight (FW). RESULT: The addition of Hex accelerated the pH decline during the first 5 days of ensiling regardless of fungal infestation. The lactic acid (LA) concentration in Hex silages was lower than that in CON during 45 days of ensiling; however the FI-Hex silage had the highest LA concentration among treatments on day 90. The Hex silage showed lower aflatoxin B1 (AFB1 ), zearalenone (ZEA), and deoxynivalenol (DON) concentrations than CON for FI silages. On day 5, the addition of Hex decreased the relative abundance of Klebsiella, Pantoea, and Enterobacter compared with CON, regardless of fungal infestation. This inhibitory effect lasted until day 90 for NFI silages but disappeared for FI silages on day 90. The fungal infestation resulted in the accumulation of Candida (34.05%) and Wickerhamomyces (19.46%). Hex decreased the relative abundance of Asperigillus, Issatchenkia, and Penicillium for NFI silages on day 5; however, its inhibitory effects were not observed in FI silages on day 5. CONCLUSION: Fungal infestation was associated with poor fermentation and hygienic quality of corn silage. Adding Hex accelerated the pH decline and maintained the antifungal activity until 90 days of ensiling, attenuating adverse effects of fungal infestation on the fermentation and preventing the accumulation of mycotoxins in corn silages. © 2021 Society of Chemical Industry.

Effect of Lactic Acid Bacteria on the Fermentation Quality and Mycotoxins Concentrations of Corn Silage Infested with Mycotoxigenic Fungi.

This study was conducted to evaluate the effect of lactic acid bacteria (LAB) on fermentation quality, mycotoxin concentrations, and microbial communities of whole-crop corn silages infested with mycotoxigenic fungi. Cultured spores (106 cfu/mL) of mycotoxigenic Aspergillus flavus and Fusarium graminearum were sprayed (5 mL) on corn forage on 27 July and 10 August 2018. On 21 August 2018, sprayed (FI; 3 plots) and unsprayed (NFI; 3 plots) corn forage were harvested at the 1/2 kernel milk line stage, followed by chopping and ensiling without inoculants (CON), or with Lactobacillus buchneri (LB, 1 × 106 cfu/g FW), Lactobacillus plantarum (LT, 1 × 106 cfu/g FW), or L. buchneri + L. plantarum (BT: both L. buchneri and L. plantarum applied at 0.5 × 106 cfu/g FW). After 90 d of ensiling, FI silages had a higher (p < 0.05) pH value and higher acetic acid (ACA), ethanol, and ammonia nitrogen (ammonia N) concentrations, but lower (p < 0.05) lactic acid (LA) concentrations than NFI silage. The inoculants decreased pH and increased LA concentration and LA/ACA compared with CON. The aflatoxin B1 (AFB1) was only detected in FI fresh corn and silages; ensiling decreased (p < 0.05) AFB1 concentration compared with fresh corn, and LB and BT decreased AFB1 concentration compared with CON. The zearalenone (ZEN), deoxynivalenol (DON), and fumonisin B1 (FB1) concentrations were similar (p < 0.05) for NFI silages, while ZEN concentration in BT was the lowest (p < 0.05) among all FI silages; DON and FB1 concentrations in LB, LT, and BT silages were significantly lower (p < 0.05) than those of CON in FI silages. The fungal infestation increased the bacterial and fungal diversity of silages compared with NFI silages. The FI silages had a higher relative abundance (RA) of Lactobacillus, Weissella, Wickerhamomyces, Pichia, and Epicoccum than the corresponding NFI silages. The RA of Aspergillus and Fusarium markedly decreased after 90 d of ensiling, and the inoculation expanded this trend irrespective of fungal infestation. The Penicillium in FI silages survived after 90 d of ensiling, while the inoculants decreased the RA of Penicillium. Inoculants mitigate the adverse effects of fungal infestation on corn silage quality by changing the bacterial and fungal communities.

Effect of hexanoic acid, Lactobacillus plantarum and their combination on the aerobic stability of napier grass silage.

AIMS: The objective of this study was to evaluate the potential of hexanoic acid (Hex) as a silage additive. METHODS AND RESULTS: The effect of Hex, Lactobacillus plantarum (Lp) and their combination (Hex + Lp) on the aerobic stability of napier grass silage was investigated. Napier grass was ensiled without additives (C) or with Lp, Hex or Hex + Lp for 60 days followed by 7 days of aerobic exposure. After 60 days of ensiling, the Lp silage had the lowest pH and the highest lactic acid (LA) concentration among all silage, whereas the highest water-soluble carbohydrates (WSC) content was observed in Hex + Lp silage, followed by Hex silage. After 60 days of ensiling, the population of yeasts in C and Lp silages was > 3·0 log10 CFU per g FW, while that of Hex and Hex + Lp was <2·0 log10 CFU per g FW. During aerobic exposure, the pH in the Lp and C silages increased (P < 0·05) above 7·0 on day 5. The pH of Hex silage was the lowest among all silages on day 3 and 5, followed by a significant (P < 0·05) increase until 7 days of aerobic exposure. There were no significant changes in pH and AA of Hex + Lp silage over the duration of aerobic exposure. The concentrations of LA in C, Lp and Hex silages decreased while that of Hex + Lp silage remained stable after 3 days of aerobic exposure. The Hex delayed the decline of WSC contents and the increase in yeasts over the aerobic exposure period. CONCLUSION: Addition of Hex (97 h) and Hex + Lp (>168 h) improved aerobic stability (P < 0·05) as compared to the control (83 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Hex or in combination with L. plantarum inhibited the proliferation of yeasts during aerobic exposure of napier grass. Thus, Hex is an alternative antifungal additive to improve aerobic stability.

Effect of alfalfa microbiota on fermentation quality and bacterial community succession in fresh or sterile Napier grass silages.

The aim of this study was to reveal the reconstitution dynamics of alfalfa microbiota and their contribution to the fermentation quality of Napier grass silages. Napier grass was harvested at approximately 20% dry matter content, chopped to a theoretical length of cut of 2 to 3 cm, and ensiled in laboratory polyethylene plastic bags (400 × 250 mm) for 30 d. The Napier grass was treated with the following: natural fermentation and distilled water (NG), inoculum of alfalfa microbiota (AM), gamma-ray irradiation and distilled water (IR), and gamma-ray radiation and alfalfa microbiota (IR+AM). Three milliliters of inoculum (containing 8.93 log cfu/mL lactic acid bacteria, 9.76 log cfu/mL Enterobacteriaceae, 5.94 log cfu/mL yeast, and 6.53 log cfu/mL mold) eluted from equivalent fresh alfalfa (450 g) was added to each silo of AM and IR+AM treatments, and 3 mL of distilled water was added to the silo of the NG and IR treatments. Three triplicate silos per treatment were opened on d 1, 3, 5, 7, 14, and 30 for sampling and analysis of fermentation quality and bacterial community. Relative to the NG silages, IR+AM silages exhibited a higher lactic acid concentration. The higher acetic acid concentration in NG than in IR+AM silages after 7 d of ensiling was attributed to the dominant genus of Leuconostoc (64.29-49.04%). Adding alfalfa microbiota to sterile Napier grass could increase ammonia-N concentration compared with NG and IR silages after 3 d of ensiling. Leuconostoc was the most predominant genus in NG silages, followed by Lactobacillus. Pediococcus had a greater relative abundance than the indigenous microorganisms and was exclusively found in AM and IR+AM silages, whereas Lactobacillus exhibited a slight increase after 30 d of ensiling (relative abundance in each silage: 6.29 vs. 3.82%, respectively). Lactobacillus was the predominant genus in IR silages since the onset of the ensiling. These results suggest that alfalfa microbiota affected bacterial community succession in Napier grass silages, which in turn affected the fermentation products. Adding alfalfa microbiota to sterile Napier grass could decrease acetic acid concentration compared with NG silages; however, it increased ammonia-N concentration compared with IR silages after 3 d of ensiling.

The reconstitution mechanism of napier grass microiota during the ensiling of alfalfa and their contributions to fermentation quality of silage.

To reveal the reconstitution mechanism of exogenous microbiota and their contributions to fermentation quality during the early stage of alfalfa ensiling. The chopped alfalfa was treated with the following: distilled water (A1); napier grass microbiota (A1N); γ-ray radiation + distilled water (A0); γ-ray radiation + napier grass microbiota (A0N). Inoculating napier grass microbiota to non-irradiated alfalfa decreased the LA concentration, while enhanced the LA production of irradiated alfalfa during the 7 d of ensiling. Inoculating napier grass microbiota increased AA and ammonia-N contents and enhanced the decline of WSC for both non-irradiated and irradiated alfalfa silages. Enterococcus and Pediococcus dominated A1 silage. Leuconostocs and Lactobacillus constituted the majority of bacterial community in A0N, Lactobacillus rapidly became the predominated genera, while Lactobacillus, Leuconostocs, Enterococcus, and Pediococcus constituted the majority of bacterial community in A1N. Thus forage microbiota transplantation may be a potential practice to improve fermentation quality of less readily fermentable forages.

Comparison of nitrogen transformation dynamics in non-irradiated and irradiated alfalfa and red clover during ensiling.

Objective: To study the contribution of plant enzyme and microbial activities on protein degradation in silage, this study evaluated the nitrogen transformation dynamics during ensiling of non- and irradiated alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.). Methods: Alfalfa and red clover silages were prepared and equally divided into two groups. One group was exposed to γ-irradiation at a recommended dosage (25Gky). Therefore, four types of silages were produced: (1) non-irradiated alfalfa silage; (2) irradiated alfalfa silage; (3) non-irradiated red clover silage; and (4) irradiated red clover silage. These silages were opened for fermentation quality and nitrogen components analyses after 1, 4, 8 and 30 days, respectively. Results: The γ-irradiation successfully suppressed microbial activity, indicated by high pH and no apparent increases in fermentation end products in irradiated silages. All nitrogen components, except for peptide-N, increased throughout the ensiling process. Proteolysis less occurred in red clover silages compared with alfalfa silages, indicated by smaller (P<0.05) increment in peptide-N and free amino acid N (FAA-N) during early stage of ensiling. The γ-irradiation treatment increased (P<0.05) peptide-N and FAA-N in alfalfa silage at day 1, whereas not in red clover silage; these two nitrogen components were higher (P<0.05) between day 4 and day 30 in non-irradiated silages than the irradiated silages. The ammonia nitrogen (NH3-N) and non-protein nitrogen (NPN) were highest in non-irradiated alfalfa silage and lowest in irradiated red clover silage after ensiling. Conclusion: The result of this study indicate that red clover and alfalfa are two forages varying in their nitrogen transformation patterns, especially during early stages of ensiling. Microbial activity plays a certain role in the proteolysis and seems little affected by the presence of PPO in red clover compared with alfalfa.

Dynamics of microbial community and fermentation quality during ensiling of sterile and nonsterile alfalfa with or without Lactobacillus plantarum inoculant.

To reveal the mechanism of the survival and adaption of inoculated Lactobacillus plantarum during ensiling. Alfalfa was ensiled directly (A1), after γ-ray irradiation (A0), and after inoculation of the sterile (A0L) or fresh alfalfa (A1L) with Lactobacillus plantarum. The A0L had the higher lactic acid content and lower pH than that in A1L from 3 days of ensiling. Pediococcus was the dominant microbes in A1 silage, followed by Enterococcus and Lactobacillus, while Lactobacillus in A1L outnumbered all other genera at 3 d. In A0L silage, the relative abundance of Lactobacillus increased to 99.13% at day 3. It indicated that Lactobacillus could dominated the fermentation of inoculated silages regardless of the γ-ray irradiation, although there was a short lag period for irradiated alfalfa.

The effects of fibrolytic enzymes, cellulolytic fungi and bacteria on the fermentation characteristics, structural carbohydrates degradation, and enzymatic conversion yields of Pennisetum sinese silage.

Biological inoculants were tested on Pennisetum sinese for their effects on fermentation characteristics, structural carbohydrates degradation, and enzymatic conversion yields. Pennisetum sinese was ensiled without additive, Lactobacillus plantarum (Lp), Trichoderma reesei (Tr), fibrolytic enzymes (E), and Enterococcus faecium (Y83) for 90 days. Y83 silages had higher LA and lower AA, ammonia-N and DM loss as compared to E and Tr silages. Tr and E had superior effects for degrading lignocellulose while Y83 had intermediate effects. The first-order exponential decay models (R2 = 0.928-0.998) predicted nonstructural carbohydrates kinetics and demonstrated high water soluble carbohydrate (g/kg DM) preservation potential in Y83 (21.40), followed by Tr (18.94) and E (16.74). Addition of Y83 improved the conversion efficiency of P. sinese silage than Tr and E, indicated by higher glucose and total reducing sugars yield (22.49 and 36.89 w/w % DM, respectively). In conclusion, Y83 can be exploited for the ensiling lignocellulosic biomass before grass processing.

Characterization of Enterococcus faecalis JF85 and Enterococcus faecium Y83 isolated from Tibetan yak (Bos grunniens) for ensiling Pennisetum sinese.

Two bacteria strains with cellulolytic potential isolated from Tibetan yak (Bos grunniens) rumen were identified as Enterococcus faecalis (JF85) and Enterococcus faecium (Y83). Isolates grow well within a range of temperature 15 to 55 °C and pH 3.0-7.0, respectively. Two strains were inoculated with or without Lactobacillus plantarum (Lp) to Pennisetum sinese silage for 90 days. All inoculants increased lactic acid content, decreased pH and lignocellulose contents compared with silage without additives (control). The lowest pH, highest lactic acid and largest reduction in lignocellulose contents were observed in JF85+Lp and Y83+Lp silages. Isolates alone or in combination with Lp significantly increased WSC, mono- and disaccharides contents as compared to the control. Combined addition efficiently improved enzymatic hydrolysis of Pennisetum sinese silage, indicated by higher glucose yield and cellulose convertibility. Pennisetum sinese ensiled with combined additives is a suitable storage and pretreatment method prior to sugars production from energy crop.

Effects of applying oil-extracted microalgae on the fermentation quality, feed-nutritive value and aerobic stability of ensiled sweet sorghum.

BACKGROUND: A laboratory-silo study was conducted to evaluate the fermentation quality, feed-nutritive value and aerobic stability of sweet sorghum silage with or without oil-extracted microalgae supplementation. Sweet sorghum was mixed with four microalgae levels (0%, 1%, 2% and 3% on a dry matter basis; control, M1, M2 and M3, respectively) and ensiled for 45 d. Further, the four experimental silages were subjected to an aerobic stability test lasting 7 d. RESULTS: All the silages except M3 silage had good fermentative characteristics with low pH and ammonia nitrogen concentrations, and high lactic acid concentrations and favorable microbial parameters. Meanwhile, oil-extracted microalgae supplementation improved the feed-nutritional value of sweet sorghum silage. Fibre (neutral detergent fibre, acid detergent fibre, acid detergent lignin and cellulose) concentrations decreased, while dry matter and crude protein levels markedly increased (P < 0.05). Compared with the control (69.7 h), treatments M2 and M3 improved the aerobic stability of sweet sorghum silage by 43.8% and more than 143% respectively, and decreased the clostridia spore counts during the stage of air exposure. CONCLUSION: Sweet sorghum silage produced with 2% oil-extracted microalgae addition was the most suitable for animal use due to the optimal balance of fermentation quality, feed-nutritional value and aerobic stability, which merits further in vivo studies using grazing ruminants. © 2018 Society of Chemical Industry.

Isolating and evaluating lactic acid bacteria strains for effectiveness on silage quality at low temperatures on the Tibetan Plateau.

Four lactic acid bacteria (LAB) strains isolated from straw silages on the Tibetan Plateau were characterized, and their effects on the fermentation quality of Italian ryegrass (Lolium multiflorum Lam.) at different temperatures (10°C, 15°C and 25°C) were studied. These LAB isolates were evaluated using the acids production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All the isolates (M1, LM8, LO7 and LOG9) could grow at 5-20°C, pH 3.5-7.0 and NaCl (3.0%, 6.5%). Strains M1, LM8, LO7 and LOG9 were identified as Lactobacillus plantarum, L. coryniformis, Pediococcus pentosaceus and P. acidilactici, respectively, by sequencing 16S ribosomal DNA. The four isolates were added to Italian ryegrass for ensiling for 30 days at various temperatures. Compared with the corresponding control, inoculating with isolates M1, LM8 and LO7 could improve the silage quality of Italian ryegrass at low temperatures, indicated by significantly (P < 0.05) higher lactic acid (LA) contents and ratios of lactic acid/acetic acid (LA/AA), and significantly (P < 0.05) lower pH and ammonia nitrogen/total nitrogen (AN/TN). Compared with other isolates, LM8 performed better at 10°C and 15°C, indicated by the higher (P < 0.05) LA content and ratio of LA/AA, and the lower (P < 0.05) pH and AN/TN.

Effects of calcium propionate on the fermentation quality and aerobic stability of alfalfa silage.

OBJECTIVE: To assess the potency of calcium propionate (CAP) used as silage additive, an experiment was carried out to evaluate the effect of CAP on the nitrogen transformation, fermentation quality and aerobic stability of alfalfa silages. METHODS: Alfalfa was ensiled with four levels of CAP (5, 10, 15, and 20 g/kg of fresh weight [FW]) in laboratory silos for 30 days. After opening, the silages were analyzed for the chemical and microbiological characteristics, and subjected to an aerobic stability test. RESULTS: The increasing proportion of CAP did not affect pH, lactic acid (LA) concentrations and yeast counts, while linearly decreased counts of enterobacteria (p = 0.029), molds (p<0.001) and clostridia (p<0.001), and concentrations of acetic acid (p<0.001), propionic acid (p<0.001), butyric acid (p<0.001), and ethanol (p = 0.007), and quadratically (p = 0.001) increased lactic acid bacteria counts. With increasing the proportion of CAP, the dry matter (DM) loss (p<0.001), free amino acid N (p<0.001), ammonia N (p = 0.004), and non-protein N (p<0.001) contents were linearly reduced, whereas DM (p = 0.048), water soluble carbohydrate (p<0.001) and peptide N (p<0.001) contents were linearly increased. The highest Flieg's point was found in CAP10 (75.9), represented the best fermentation quality. All silages treated with CAP improved aerobic stability as indicated by increased stable hours compared with control. CONCLUSION: The addition of CAP can suppress the undesirable microorganisms during ensiling and exposure to air, thereby improving the fermentation quality and aerobic stability as well as retarding the proteolysis of alfalfa silage. It is suggested that CAP used as an additive is recommended at a level of 10 g/kg FW.

Effects of sodium diacetate on the fermentation profile, chemical composition and aerobic stability of alfalfa silage.

OBJECTIVE: The objective of this study was to evaluate the effect of sodium diacetate (SDA) on fermentation profile, chemical composition and aerobic stability of alfalfa (Medicago sativa L.) silage. METHODS: Fresh alfalfa was ensiled with various concentrations of SDA (0, 3, 5, 7, and 9 g/kg of fresh forage). After 60 days of the ensiling, the samples were collected to examine the fermentative quality, chemical composition and aerobic stability. RESULTS: The application of SDA significantly (p<0.05) decreased silage pH with the lowest value in silage with 7 g/kg of SDA. The proliferations of enterobacteria, yeasts, molds and clostridia were inhibited by SDA, resulted in lower ethanol, propionic and butyric acid concentrations and dry matter loss in SDA treated silages than control. The increasing SDA linearly decreased free amino acid N (p<0.001), ammonia N (p = 0.018) and non-protein N (p<0.001), while linearly increased water soluble carbohydrate (p<0.001) and peptide N (p<0.001). It is speculated that SDA accelerated the shift from homofermentative to heterofermentative lactic acid bacteria during the silage fermentation, indicated by lower lactic acid production in SDA-9 than SDA-7 silages after 60 days of ensiling. Alfalfa silages treated with SDA at 7 g/kg had highest Flieg's point and remained stable more than 9 d during aerobic exposure under humid and hot conditions in southern China. CONCLUSION: SDA may be used as an additive for alfalfa silages at a level of 7 g/kg.

Effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage.

BACKGROUND: Short-chain fatty salts have been widely used as food and forage preservatives because of their antimicrobial properties. This study evaluated the effects of four chemical compounds with antimicrobial properties on nitrogen transformations and intrinsic protease activity of alfalfa silage. RESULTS: Potassium diformate (PD) and formic acid (FA) rapidly reduced silage pH. Silages treated with sodium diacetate (SD) and calcium propionate (CAP) had higher final peptide N concentrations than other silage. The free amino acid N contents in PD and FA treated silages were lower than other silages at all intervals of ensilage. The ammonia N concentrations in FA and PD silages were the lowest, followed by SD and CAP silages. As ensiling progressed, the aminopeptidase activity was completely lost by day 5 for FA and PD silages and inactive by day 7 for SD silage, while it remained active after day 7 for control and CAP silage. The carboxypeptidase activities in FA and PD silages were already reduced below 50% by day 1 of ensiling. CONCLUSION: Potassium diformate was as effective as formic acid in depressing the proteolysis, while sodium diacetate and calcium propionate were inferior to formic acid in protecting alfalfa proteins from being hydrolysed. © 2016 Society of Chemical Industry.

Characteristics of lactic acid bacteria isolates and their effect on the fermentation quality of Napier grass silage at three high temperatures.

BACKGROUND: The poor fermentation quality of silage is an important issue for silage production during the high temperatures of summer. Pediococcus acidilactici GG13 (GG13) and Lactobacillus rhamnosus GG26 (GG26) isolated from Italian ryegrass (Lolium multiflorum Lam.) silage were characterised by morphological and physiological tests and 16S rRNA sequencing analysis, and their effects, along with those of a commercial lactic acid bacteria (LAB) inoculant (CB), on the fermentation quality of facultative halophyte Napier grass (Pennisetum purpureum Schumach) ensiled at 30 °C, 40 °C and 50 °C were studied, respectively. RESULT: The strains GG13 and GG26 grew well at 50 °C and pH 3.5, and were tolerant to 6.5% NaCl. After ensiling for 50 days, the strains GG13 and GG26 and the CB decreased (P < 0.001) the pH and acetic acid and ammonia-N contents and increased (P < 0.001) the lactic acid contents at 30 °C, and decreased (P < 0.001) the ammonia-N contents at 40 °C in Napier grass. CB did not affect the fermentation quality at 50 °C, whereas both isolated strains improved the fermentation quality of Napier grass silage as indicated by the lower (P < 0.001) pH, butyric acid and ammonia-N contents and higher (P < 0.001) lactic acid contents. The strain GG13 is better than GG26 with regard to improvement in fermentation quality of Napier grass silage. CONCLUSIONS: The results of this study suggested that strain GG13 is a good LAB inoculant for producing well-fermented silages during the high temperatures of summer times. © 2016 Society of Chemical Industry.