A Multi-Sensor Mini-Bioreactor to Preselect Silage Inoculants by Tracking Metabolic Activity in situ During Fermentation

The microbiome in silage may vary substantially from the onset to the completion of fermentation. Improved additives and inoculants are being developed to accelerate the ensiling process, to enhance fermentation quality, and to delay spoilage during feed-out. However, current methods for preselecting and characterizing these amendments are time-consuming and costly. Here, we have developed a multi-sensor mini-bioreactor (MSMB) to track microbial fermentation in situ and additionally presented a mathematical model for the optimal assessment among candidate inoculants based on the Bolza equation, a fundamental formula in optimal control theory. Three sensors [pH, CO2, and ethanol (EtOH)] provided data for assessment, with four additional sensors (O2, gas pressure, temperature, and atmospheric pressure) to monitor/control the fermentation environment. This advanced MSMB is demonstrated with an experimental method for evaluating three typical species of lactic acid bacteria (LAB), Lentilactobacillus buchneri (LB) alone, and LB mixed with Lactiplantibacillus plantarum (LBLP) or with Enterococcus faecium (LBEF), all cultured in De Man, Rogosa, and Sharpe (MRS) broth. The fermentation process was monitored in situ over 48 h with these candidate microbial strains using the MSMB. The experimental results combine acidification characteristics with production of CO2 and EtOH, optimal assessment of the microbes, analysis of the metabolic sensitivity to pH, and partitioning of the contribution of each species to fermentation. These new data demonstrate that the MSMB associated with the novel rapid data-processing method may expedite development of microbial amendments for silage additives.

Influence of anaerobic rumen fungal inoculation on the fermentation and nutritive content of selected forage silages

This study investigated the ability of isolated anaerobic fungi to facilitate silage fermentation characteristic and nutritive content. To achieve this, two fungal isolates out of fourteen obtained in our laboratory from sheep rumen content were selected as silage inoculants. Selection of the isolates was based on their ability to present the highest growth characteristics and they belong to the genus Orpinomyces, and Neocallimastix (as identified with microscope). The two fungi alongside the control (i.e. no fungi) were used to inoculate different silages prepared from four selected forages i.e. two tropical grasses (i.e. Andropogon gayanus- AG, Brachiaria decumbens- BD) and two temperate forages (i.e. Triticum aestivum- TA straw and Lolium perenne- LP) in order to examine their effect on fermentation (i.e. pH) and nutritive content of these silages over 14 and 28 days of inoculation, respectively. The anaerobic fungal inoculated silages showed significant (P < 0.05) improvement in quality than un-inoculated silages which was reflected in terms of increase in soluble fraction i.e. CP content; reduction in fibre content; increase in metabolites, increase in total antioxidant content (TAC); and reduction in pH with minimal nutrient loss. However, the rate of the anaerobic fungal improvement was influenced by the forages used for the silage and the length of inoculation. The highest and lowest increase in quality was recorded in LP and TA silages, respectively, while more extended inoculation time supported more quality improvement in the silages. The use of anaerobic fungi as inoculants improved AG, BD, TA and LP silages quality through facilitation of the fermentation characteristics (i.e. pH and nutritive content).

Potential of Lactic Acid Bacteria Isolated From Different Forages as Silage Inoculants for Improving Fermentation Quality and Aerobic Stability

We aimed at isolating lactic acid bacteria (LAB) from different plant materials to study their crossed-fermentation capacity in silos and to find strains able to confer enhanced aerobic stability to silage. A total of 129 LAB isolates were obtained from lucerne (alfalfa), maize, sorghum, ryegrass, rice, barley, canola, Gatton panic, Melilotus albus, soy, white clover, wheat, sunflower, oat, and moha. Four Lactiplantibacillus plantarum subsp. plantarum strains (isolated from oat, lucerne, sorghum, or maize) were selected for their growth capacity. Identity (16S sequencing) and diversity (RAPD-PCR) were confirmed. Fermentative capacity (inoculated at 104, 105, 106, 107 CFU/g) was studied in maize silage and their cross-fermentation capacity was assessed in oat, lucerne, sorghum, and maize. Heterofermentative strains with the highest acetic acid production capacity conferred higher aerobic stability to maize silages. Regardless the source of isolation, L. plantarum strains, inoculated at a rate of 106 CFU/g, were effective to produce silage from different plant materials. From more than 100 isolates obtained, the application of a succession of experiments allowed us to narrow down the number of potential candidates of silage inoculants to two strains. Based on the studies made, L. plantarum LpM15 and Limosilactobacillus fermentum LfM1 showed potential to be used as inoculants, however further studies are needed to determine their performance when inoculated together. The former because it positively influenced different quality parameters in oat, lucerne, sorghum, and maize silage, and the latter because of its capacity to confer enhanced aerobic stability to maize silage. The rest of the strains constitute a valuable collection of autochthonous strains that will be further studied in the future for new applications in animal or human foods.

Using Lactic Acid Bacteria as Silage Inoculants or Direct-Fed Microbials to Improve In Vitro Degradability and Reduce Methane Emissions in Dairy Cows

The current study has two objectives: (1) To determine the effect of different lactic acid bacteria (LAB) strains’ inoculant on silage quality of fresh ryegrass (FR) and rain-treated ryegrass (RTR), and (2) to find the optimal way (silage inoculant vs. direct-fed microbial (DFM)) to use LAB strains in order to improve nutrient digestibility and reduce methane emission (CH4) in ruminant production. Five LAB strains were tested, Lactiplantibacillus plantarum AGR-1, L. plantarum AGR-2, Lactococcus lactis subsp. lactis biovar diacetylactis AGR-3, L. lactis subsp. lactis AGR-4 and L. lactis subsp. lactis AGR-5. Each LAB strain was inoculated at 106 cfu/g fresh weight into the FR and the RTR and ensiled for 60 days. After ensiling, the effect of LAB strains included as a DFM or silage inoculant on rumen digestibility and CH4 production were measured using an in vitro gas production system with three separate runs. The in vitro experiment consisted of 24 treatments (2 grasses (FR and RTR) × 2 ways (inoculant or DFM) × 6 strains (5 LAB strains + 1 Control)). The results indicated that the LAB strains’ inoculant treatments reduced (p < 0.0001) the dry matter (DM) losses, the NH3 concentration (p < 0.0001) and the pH (p = 0.0019) upon ensiling in both the FR and the RTR. The lowest values in dry matter (DM) loss and NH3 concentration were found in the L. plantarum (AGR-2) and L. lactis (AGR-5). The in vitro CH4 production was lower for silages inoculated with L. plantarum (AGR-1, p = 0.0054), L. lactis (AGR-4, p = 0.026), L. lactis (AGR-5, p = 0.029) and L. plantarum (AGR-2, p = 0.090), compared to the control. Methane production was lower (p = 0.0027) for LABs when used as silage inoculants, compared to being used as DFM. Lactic acid bacteria used as silage inoculants increased (p ≤ 0.0001) the in vitro DM and organic matter (OM) degradability both in the FR and the RTR, whereas LAB strains used as DFM showed no such effect. The DM and OM digestibility were highest in the L. plantarum (AGR-1, p = 0.0175). Among the five LAB strains used in the current study, L. plantarum (AGR-2) was the best candidate to improve silage quality. Our observations suggest that these LAB strains are most promising when used as silage inoculants and to be confirmed in vivo.

Dual-Purpose Inoculants and Their Effects on Corn Silage

This study was conducted to screen dual-purpose lactic acid bacteria (LAB) from uncontrolled farm-scale silage, and then we confirmed their effects on corn silage. The LAB were isolated from eight farm-scale corn silages, and then we screened the antifungal activity against Fusarium graminearum and the carboxylesterase activity using spectrophotometer with p-nitrophenyl octanoate as substrate and McIlvane solution as buffer. From a total of 25 isolates, 5M2 and 6M1 isolates were selected as silage inoculants because presented both activities of antifungal and carboxylesterase. According 16S rRNA gene sequencing method, 5M2 isolate had 100.0% similarity with Lactobacillus brevis, and 6M1 isolate had 99.7% similarity with L. buchneri. Corn forage was ensiled in bale silo (500 kg) for 72 d without inoculant (CON) or with mixture of selected isolates at 1:1 ratio (INO). The INO silage had higher nutrient digestibility in the rumen than CON silage. Acetate was higher and yeasts were lower in INO silage than in CON silage on the day of silo opening. In all days of aerobic exposure, yeasts were lower in INO silage than CON silage. The present study concluded that Lactobacillus brevis 5M2 and L. buchneri 6M1 confirmed antifungal and carboxylesterase activities on farm-scale corn silage.

The role of homofermentative and heterofermentative lactic acid bacteria for alfalfa silage: a meta-analysis

Lactic acid bacteria (LAB) are usually employed as alfalfa silage inoculants to obtain high-quality feed for animal husbandry. However, the effects of these inoculants are still unclear and need to be studied extensively. Therefore, the objective of this meta-analysis was to quantitatively summarize published research studies that assess the effects of homofermentative (HoLAB) and heterofermentative lactic acid bacteria (HeLAB) on fermentation parameters, nutritive value, microbiological composition and aerobic stability of alfalfa silage. PubMed, ScienceDirect and Scopus have been screened for articles published from 1980 to 2018. The criteria for inclusion were: randomized and controlled trials using alfalfa silage and published in peer-reviewed journals. It was found that inoculation with LAB decreased silage pH, neutral detergent fibre, acid detergent fibre and ammoniacal nitrogen, while it increased dry matter and crude protein compared to control in the pooled raw mean difference random-effect model. Additionally, LAB inoculation decreased acetate, propionate, ethanol and butyrate concentrations, whereas it increased lactate. In addition, inoculants reduced the counts of yeasts and moulds. Lastly, LAB inoculation improved aerobic stability. To the best of our knowledge, this is the first meta-analysis that aims at comparing the application of HoLAB and HeLAB for alfalfa silage. In the pool estimate, positive effects attributable to the application of microbial silage inoculants were found in most of the evaluated parameters; supporting the importance of applying both types of inoculants to improve forage preservation for the livestock industry.

97 Nutritive value and fermentation characteristics of cool-season annual baleage

Baleage production is gaining popularity in the Southeast due to the ability for more timely harvests and more consistent forage quality compared with hay. The objective of this study was to determine the feasibility of cool-season mixtures for baleage production. Wheat (Triticum aestivum) with T-Raptor (Brassica rapa × napus; WT) or crimson clover (Trifolium incarnatum; WC) were planted at the E.V. Smith Research Center in Shorter, AL as a 2 × 2 factorial design (n = 3). Forage was treated or un-treated with silage inoculant to determine its efficacy in promoting proper fermentation. Samples were taken at 7 different time intervals to determine fermentation characteristics and quality. There was no difference (P ≥ 0.15) in ADF concentration among treatments, but day 120 after ensiling was greater (P ≤ 0.002) than all other sampling dates. There was no difference (P ≥ 0.11) in NDF between forages, silage inoculants, or among days and averaged 50.75%. The CP of the WC treatment was greater (P ≤ 0.0001) than WT by 3.6 units. Inoculated forage was greater (P ≤ 0.0001) than non-inoculated by 1.06 units. Crude protein was greater (P = 0.005) on day 120 than day 0. Inoculated WT had greater (P < 0.0001) total acid content than non-inoculated (8.35 vs. 6.19%, respectively). Wheat+clover had 1.5 units more total acid than WT. Inoculated forage had greater (P ≤ 0.0006) total acid present than non-inoculated forage. Butyric acid concentration increased (P ≤ 0.0001) with increasing days after ensiling. Butyric acid tended to be greater (P = 0.05) in WC than WT, likely due to increased pH in clover treatments as a result of legume buffering capacity. The results indicate that silage inoculants offer minimal benefits to baleage fermentation when using cool-season annual mixtures

5 Nutritive value and fermentation characteristics of cool-season annual baleage

Baleage production is gaining popularity in the Southeast due to the ability for more timely harvests and more consistent forage quality compared with hay. The objective of this study was to determine the feasibility of cool-season mixtures for baleage production. Wheat (Triticum aestivum) with T-Raptor (Brassica rapa × napus; WT) or crimson clover (Trifolium incarnatum; WC) were planted at the E.V. Smith Research Center in Shorter, AL, as a 2 × 2 factorial design (n = 3). Forage was treated or un-treated with silage inoculant to determine its efficacy in promoting proper fermentation. Samples were taken at 7 different time intervals to determine fermentation characteristics and quality. There was no difference (P ≥ 0.15) in ADF concentration among treatments, but day 120 after ensiling was greater (P ≤ 0.002) than all other sampling dates. There was no difference (P ≥ 0.11) in NDF between forages, silage inoculants, or among days and averaged 50.75 %. The CP of the WC treatment was greater (P ≤ 0.0001) than WT by 3.6 units. Inoculated forage was greater (P ≤ 0.0001) than non-inoculated by 1.06 units. Crude protein was greater (P = 0.005) on day 120 than day 0. Inoculated WT had greater (P < 0.0001) total acid content than non-inoculated (8.35 vs. 6.19 %, respectively). Wheat+clover had 1.5 units more total acid than WT. Inoculated forage had greater (P ≤ 0.0006) total acid present than non-inoculated forage. Butyric acid concentration increased (P ≤ 0.0001) with increasing days after ensiling. Butyric acid tended to be greater (P = 0.05) in WC than WT, likely due to increased pH in clover treatments as a result of legume buffering capacity. The results indicate that silage inoculants offer minimal benefits to baleage fermentation when using cool-season annual mixtures

The potential of silage lactic acid bacteria-derived nano-selenium as a dietary supplement in sheep

Context Selenium (Se) is a trace element essential for cellular function in animals as a component of the enzymes glutathione peroxidase and iodothyronine-5-deiodinase. In many parts of Europe, Se is often deficient in livestock diets due to the low Se status of soil. Supplementation of diets with selenised yeast (predominately as seleno-methionine) or inorganic sodium selenite is common practice in most livestock systems, including ruminants. Lactic acid bacteria have been shown to convert inorganic Se into predominantly elemental nano-Se, which has been used recently in human pro-biotics as a less toxic form of Se. Therefore, silage lactic acid bacteria may provide a supplementation route of bioavailable nano-Se for ruminants. Aim Here, we report on the effect of feeding inoculated silage enriched with a supra-nutritional level of nano-Se (Selage) versus control inoculated silage (Silage) on the Se status of finishing lambs and their products, followed by a second study where blood parameters were investigated in ewes. Methods In the first study, 40 Charollais × Suffolk lambs (42 ± 1.7 kg) were paired according to weight and sex, then allocated to the two treatments for 8 or 10 weeks. Uptake of Se into wool was temporally assessed, as well as excretion of Se into faeces. Selenium concentrations in blood and muscle, carcass characteristics and meat quality are reported postmortem. In the second study, individually penned Suffolk × Mule ewes (n = 12; 76 ± 4.5 kg) were offered the same diets as in the first study. Blood parameters were assessed at the start and after 6 weeks, with intake and excretion into faeces and urine assessed temporally throughout the study. Key results In the first study, dry-matter (DM) intake was similar in both treatment groups, at 0.8 ± 0.03 kg/day, but Se concentrations of the diets were significantly different, resulting in intakes of ~0.14 and 1.60 mg/day on the Silage and Selage diets, respectively. This was reflected in higher Se concentrations in faeces (0.4 vs 2.0 mg/kg DM; P &lt; 0.001), wool (0.11 vs 0.25 mg/kg DM; P &lt; 0.001), blood (0.19 vs 0.46 mg/L; P &lt; 0.001) and muscle (0.31 vs 0.41 mg/kg: P &lt; 0.01) on the Selage than on the Silage diet. Colour (chroma) shelf life of the meat was significantly higher on the Selage treatment (8.05 vs 9.2 days; P &lt; 0.05). In the second trial, for ewes fed Selage, blood seleno-methionine increased from 0.21 to 0.25 mg/L and seleno-cysteine from 0.25 to 0.35 mg/L after 6 weeks on the treatment, whereas there was no change in ewes fed Silage. Glutathione peroxidase increased, whereas haematocrit, haemoglobin and platelet count were decreased across time during the study, but there was no difference between the treatments. Conclusions Nano-Se provided by the Selage treatment was shown to be available to sheep and improve shelf life, with no adverse haematological effects. Implications There is potential to use silage inoculants to provide bioavailable Se to ruminants. Further research is required to determine the most appropriate dose for animal performance and product quality.