The effects of lactic acid bacteria inoculants and formic acid on the formation of biogenic amines in grass silages

Silages were prepared in six laboratory experiments from four direct-cut grassland swards and pure swards of perennial ryegrass and false oat with dry matter contents ranging between 180 and 325?g/kg. Grass was fermented at 22°C and silages were stored at the same temperature for 4 months. Untreated silages (negative control) and silages preserved with 3?g/kg of formic acid (positive control) were compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobacillus buchneri and a mixed preparation Microsil. The inoculants were applied at a dose of 5.10⁶ CFU/g of grass. Seven biogenic amines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Common chemical quality parameters of silages were also determined. Tyramine, cadaverine and putrescine were the amines occurring at the highest concentration. As compared to untreated silages, formic acid was most effective to suppress formation of the main amines. Also the inoculants often decreased amine contents significantly (P?相似文献   

The effects of using lactic acid bacteria inoculants in maize silage on the formation of biogenic amines

Silages from five ripened varieties of silage maize with dry matter contents ranging between 275 and 410 g/kg were prepared in five laboratory experiments. Whole-plant maize was fermented at 22°C and silages were then stored at the same temperature for 4 months. Spontaneously fermented silages were prepared as control variants and compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobacillus buchneri and a mixed preparation Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus. The starter cultures were applied at doses 5·10⁵ and 5·10⁶ CFU/g of chopped maize. Seven biogenic amines and polyamines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Common chemical criteria of silage quality were also determined. All three inoculants, mainly at the higher dose, decreased significantly contents of tyramine, putrescine and cadaverine, three undesirable amines occurring at the highest levels. L. plantarum was the most effective. Contents of histamine and tryptamine were low in all experimental silages. Also relatively low were levels of polyamines spermidine and mainly of spermine.     

Fatty acid composition and biogenic amines in acidified and fermented fish silage: a comparison study

In the presented study, ensiling of discard fish by acidification or fermentation was evaluated. Klunzinger’s ponyfish which is a discard fish was used for the production of fish silage by acidification (3% formic acid for Method FA; 1.5% formic and 1.5% sulphuric acid for Method FASA) and fermentation (Lactobacillus plantarum for Method LP and Streptococcus thermophilus for Method ST). The chemical, microbiological and nutritional properties of the differently preserved fish silages were estimated during a storage period of 60 d at ambient temperature. Compared to the raw material, a slight increase in saturated fatty acids and a slight decrease in polyunsaturated fatty acids were observed in all silages. At the end of the storage period, the aerobic bacteria counts after applying Methods FA, FASA, LP and ST amounted to 2.35, 2.39, 5.77 and 5.43 log cfu/g, respectively. The analysis of thiobarbituric acid revealed that acidification of silages accelerated the lipid oxidation. Nine biogenic amines were found in raw fish and different silages. The initial histamine concentration in raw fish was 0.17 mg/100 g and in all silages it remained at low levels during the storage period. The initial tyramine content was found to be 1.56 mg/100 g in raw fish and increased significantly in all silages. The increase of the tyramine content in fermented silages was considerably higher than in acidified silages (23–48 mg/100 g and 5–10 mg/100 g, respectively). It can be concluded that acidified or fermented fish silage should be considered as potential feed component for animals because of its high nutritional value and appropriate microbiological and chemical quality.     

The effect of applying lactic acid bacteria at ensiling on the aerobic stability of silages

The effect of applying commercial lactic acid bacteria inoculants at ensiling on the aerobic stability of silages was studied under laboratory conditions. The silages used were wheat, hedysarum, corn and various sorghum cultivars at various stages of maturity. Three inoculants were used, two containing Lactobacillus plantarum, Enterococcus faecium and Pediococcus acidilactici (H/M F, Medipharm, USA and Sil-All, Alltech, UK) and one containing Ent. faecium (Lacticil, M74, Medipharm, Sweden). The inoculants were applied at 0.5 times 10⁶ cfu g^-1. Silages with no additives served as controls. After treatment, the chopped forages were ensiled in 1.5 1 anaerobic jars; there were six jars per treatment. After ensiling for 45 d, the silages were tested for aerobic stability in a test in which CO₂ production was measured along with chemical and microbiological parameters.
The inoculated silages that spoiled upon aerobic exposure faster than the controls were those of wheat and of the sorghum cultivar FS5 at the milk stage of maturity. This was evident from intensive CO₂ production and development of yeasts and moulds. Regression analysis indicated that aerobic deterioration of inoculated silages was associated with high levels of residual water-soluble carbohydrates and lactic acid and lack of volatile fatty acids. Aerobic spoilage of inoculated silages was attributed mainly to yeast activity.     

Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

Effects of maturity stage, wilting and acid treatment on crude protein fractions and chemical composition of whole crop pea silages (Pisum sativum L.)

In order to determine if maturity stage, and wilting or acid treatment, change the crude protein (CP) fraction distribution (determined according to the Cornell Net Carbohydrate and Protein System) of whole crop pea silages, a pea with variegated flowers (Pisum sativum ssp. arvense L., cv Timo) was compared to a white-flowered, semi-leafless pea (P. sativum ssp. hortense L, cv Capella). Herbage was harvested at three maturity stages being: pod set, pod swell and full pod, and either acid-treated or wilted. Timo was acid-treated using 4 (acid4), 6 (acid6) or 8 (acid8) L/tonne fresh matter (FM) with a 2:1 mixture of formic and propionic acid, or wilted to a dry matter (DM) content of about 400 g/kg. Capella was treated with acid6 or wilted. Herbage was ensiled for 103 days in 10 kg laboratory silos. Despite differences in wilting conditions, all wilted herbages had similar protein fraction distributions. In the Capella silages the soluble CP content was lower in the later maturity stages, but this was not the case in the Timo silages. The amount of acid added only affected the B1 CP fraction content, which decreased with increasing acid. At pod set and pod swell for Timo, and at pod set for Capella, the direct-harvested herbages were difficult to ensile because of the high buffering capacity and low level of water soluble carbohydrates. Wilting improved ensilability. Acid treatment reduced proteolysis, but crops with DM contents below 150 g/kg must be acid treated with at least 6 L/tonne FM to ensure stable fermentation. Timo silages were more prone to malfermentation, probably caused by lodging, which made Capella the preferred cultivar for producing pea silages harvested at the pod swell stage or later. Proteolysis and the amount of soluble CP in the silage were lower in later maturity stages in the Capella, but not the Timo, cultivar.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

The effect of temperature and Lactobacillus amylovorus and Lact. plantarum, applied at ensiling, on wheat silage

The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on wheat silage stored at 25 and41 °C was studied under laboratory conditions. The inoculants were applied at 10⁶ cfu g⁻¹.Silages with no additives served as controls. Three jars per treatment were sampled on days 2, 8 and 60 after ensiling, for chemical and microbiological analyses. After the ensiling period, the silages were subjected to an aerobic stability test. The control and Lact. plantarum inoculated wheat fermented faster at 25 than at 41 °C, whereas silages inoculated with Lact. amylovorus fermented faster at 41 °C. This was apparent from the rate of pH decrease and from the contents of residual sugars and lactic acid in the final silages. The numbers of lactobacilli in the control and Lact. plantarum silages at 41 °C after 2 and 8 days of ensiling were lower than in the corresponding silages at 25 °C. For the Lact. amylovorus silage the opposite held true. The control silages at both temperatures and the Lact. plantarum silage at 41 °C were the most stable silages under aerobic exposure.     

Characterization of protein fractions and amino acids in ensiled alfalfa treated with different chemical additives

Formic acid, formaldehyde, tannic acid or mixtures of two were studied on their effects on ensiled alfalfa (Medicago sativa L.) amino acids and N fractions by the Cornell Net Carbohydrate and Protein System (CNCPS). The alfalfa forage was a second cut and was wilted to a mean over-dry dry matter (DM) content of 330 g/kg. All silages were prepared as mini-silos using 100 ml polypropylene centrifuge tubes (50 g) on a small laboratory-scale, with the additives added in 20 ml aliquots/kg herbage fresh weight (FW). After 35 d of ensiling, most of forage true protein was converted to fraction A and all of the added additives reduced fraction A content in the ensiled forages (P<0.05). The content of fraction B₁ in all of the additive-treated silages was higher (P<0.05) than that in control silage. Large proportions of true protein in the tannic acid/formaldehyde- and formic acid/formaldehyde-treated silages were fractions B₂ and B₃, respectively. No difference was observed on fraction C content between the control silage and silages treated with additives except for the formaldehyde or tannic acid-treated silages. Amino acids were well preserved in additive-treated silages compared with the control silage. Concentration of total amino acid was higher in formic acid-treated silages than that in the control and the other additive-treated silages (P<0.05). The pattern of changes in individual amino acid in all of the silages indicated that branched chain amino acids and methionine were relatively well preserved during fermentation but the basic and acidic amino acids were not.     

A calorimetric investigation of the nutritive value of formic acid- and formaldehyde—Formic acid-treated grass silages

Experiments were conducted to study the utilisation of a ryegrass mixture preserved chemically by formic acid (silage 1) of mean gross energy (GE) 18.6 MJ/kg dry matter (DM), and formaldehyde—formic acid (silage 2) of mean GE 18.3 MJ/kg DM. The parent grass and resulting silages were analysed chemically and calorimetric balance trials were performed with four sheep fed at maintenance and 1.5 times maintenance.Silages 1 and 2 had similar, high digestibility coefficients for DM (0.732 and 0.718), organic matter (OM) (0.755 and 0.742), and energy (E) (0.730 and 0.710). The digestible energy (DE) contents of silages 1 and 2 were 13.3 and 12.7 MJ/kg DM, respectively, at the lower level of feeding and 13.8 and 12.8 MJ/kg DM, respectively, at the higher level. The metabolisable energy (ME) contents of silages 1 and 2 were 11.6 and 10.8 MJ/kg DM, respectively, at the lower level of feeding and 12.0 and 11.0 MJ/kg DM, respectively, at the higher level.The mean efficiency of utilisation of ME for growth (K_g) for silages 1 and 2 was 0.43 and 0.39, respectively; these results are in broad agreement with those predicted by other workers.     

The effect of Lactobacillus buchneri on the fermentation, aerobic stability and ruminal degradability of maize silage

AIMS: To evaluate the effect of Lactobacillus buchneri, heterofermentative lactic acid bacteria (LAB), on the fermentation, aerobic stability and ruminal degradability of whole-crop maize silages under laboratory conditions. Two homofermentative LAB were tested for the purpose of comparison. METHODS AND RESULTS: Maize was harvested at early dent [290 g kg(-1) dry matter (DM)] and one-half milk line (355 g kg(-1) DM) stages. Both homofermentative LAB were applied at 1 x 10(5) CFU g(-1) of fresh forage. Lactobacillus buchneri was applied at 1 x 10(5), 5 x 10(5) and 1 x 10(6) CFU g(-1) of fresh forage. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-l anaerobic jars. Three jars per treatment were sampled on day 60. After 60 days of storage, silages were subjected to an aerobic stability test lasting for 5 days, in which CO(2) production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. Both homofermentative LAB increased the concentration of lactic acid and the numbers of yeasts, and decreased the concentration of acetic acid and impaired the aerobic stability of silages. In contrast, applying L. buchneri decreased the concentration of lactic acid and increased the concentration of acetic acid of the silages. Under aerobic conditions, silages treated with 5 x 10(5) and 1 x 10(6) CFU g(-1) of L. buchneri, had lower pH, CO(2) production and the numbers of yeasts than the silages treated with 1 x 10(5) CFU g(-1) of L. buchneri (P < 0.05). However, all doses of L. buchneri and both homofermentative LAB did not affect in situ rumen DM, organic matter and neutral detergent fibre degradability of the silages. CONCLUSIONS: Lactobacillus buchneri was very effective in protecting maize silages exposed to air under laboratory conditions. All doses of L. buchneri, especially 5 x 10(5) CFU g(-1) or more, markedly decreased the numbers of yeasts and improved the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of L. buchneri, as a silage inoculant, can improve the aerobic stability of maize silages by inhibition of yeast activity.     

Viability of Cryptosporidium parvum during ensilage of perennial ryegrass

The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg^-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg^-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 × 10⁴ to 1 x 10⁴ g^-1 fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.     

The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages

AIMS: To determine the effect of Propionibacterium acidipropionici, alone or in combination with Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages. METHODS AND RESULTS: The inoculants were applied at 1.0 x 10(6) CFU g(-1). Silages with no additives served as control. Fresh forages were sampled prior to ensiling. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, the silages were subjected to an aerobic stability test. The P. acidipropionici-inoculated silages had significantly higher levels of acetic and propionic acid than the L. plantarum or P. acidipropionici + L. plantarum-inoculated silages (P < 0.05). Therefore, yeast activity was impaired in the P. acidipropionici-inoculated silages. As a result, P. acidipropionici decreased CO(2) production and improved aerobic stability of wheat, sorghum and maize silages. However, the combination of P. acidipropionici + L. plantarum did not improve aerobic stability of the silages. CONCLUSIONS: The P. acidipropionici was very effective in protecting the wheat, sorghum and maize silages exposed to air under laboratory conditions, probably because the acidic environment under ensiling conditions is favourable for this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of P. acidipropionici, as a silage inoculant can improve the aerobic stability of silages by inhibition of yeast activity.     

Silages containing buckwheat and chicory: quality, digestibility and nitrogen utilisation by lactating cows

The suitability of silages containing buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) for the nutrition of dairy cows was determined. Buckwheat and chicory were sown in mixture with ryegrass (Lolium multilorum), and a pure ryegrass culture served as a control forage. Swards were harvested 55 d after sowing and were ensiled after wilting, without additives in small round bales. Finally, buckwheat and chicory made up the dietary dry matter (DM) proportions of 0.46 and 0.34, respectively. Concentrates were restricted to 2 kg/d. Diets were fed to 3 x 6 late-lactating cows for 15 d at ad libitum access. During the collection period (days 10-15) amounts of feed intake and faeces, urine and milk were recorded and samples were taken. Ensilability was good for buckwheat and ryegrass swards, but was so less for the chicory sward, which was rich in total ash. The buckwheat silage was rich in acid detergent fibre (445 g/kg DM) and lignin (75.7 g/kg DM) and contained less crude protein (135 g/kg DM) and ether extract (15.8 g/kg DM) than the other silages. Consistent with that, the apparent digestibility of the organic matter and fibre were lowest when feeding this silage. The potassium concentrations in the chicory and ryegrass silages were high (61 g/ kg) and lower in buckwheat (47 g/kg). No significant treatment effects on intake, body weight, milk yield or milk composition as well as plasma beta-hydroxybutyrate and non-esterified fatty acids occurred. Being lowest in nitrogen (N) content, the buckwheat silage resulted in the lowest urine N losses and the most efficient N utilisation for milk protein synthesis, but this at cost of body N retention. The results show that silages containing buckwheat and chicory may be used as components of the forage part of dairy cows' diets even though they were found to have a lower feeding value than ryegrass silage.     

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover

Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.Disclaimer: Mention of products is for informational purposes only and does not imply a recommendation or warranty by USDA over other products that may also be suitable     

Effects of formic acid,molasses and inoculant additives on corn silage composition,organic matter digestibility and microbial protein synthesis in sheep

This study examined the production of com silages with low or high lactic acid concentrations, provided by the addition of formic acid (0.5%), molasses (5%) or microbial inoculant (homofermentative lactic acid bacteria, 10 g/tonne). After the fermentation period, sheep were fed the silages to determine true and apparent digestibility of the organic matter and microbial protein synthesis. The experiment were carried out with four KıvırcıkxMorkaraman sheep, 1.5 years old, fixed with cannula in their rumen and duodenum.Lactic acid concentrations were significantly higher in silages treated with enzyme or molasses compared to other specific treatments. Acetic acid concentration was highest in silage treated with formic acid, and lowest in silage treated with molasses (P < 0.05). The by-pass of crude protein was highest in silage treated with formic acid.     

The effect of a propionic acid bacterial inoculant applied at ensiling on the aerobic stability of wheat and sorghum silages

The effect of a new strain ofPropionibacterium shermanii (PAB), applied at ensiling, on the aerobic stability of wheat and sorghum silages was studied in several experiments under laboratory conditions. In the one experiment with wheat and in those with sorghum a lactic acid bacteria (LAB) inoculant (Lactobacillus plantarum andPediococcus cerevisiae) was also included. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars which were sampled in triplicate on predetermined dates to follow fermentation dynamics. At the end of the experiments, the silages were subjected to an aerobic stability test. The PAB inoculant improved the aerobic stability only in one experiment with wheat, in which the decrease in pH was very slow; the final pH remained relatively high (4.5). The PAB-treated silages contained 19.5±2.0 g of propionic acid per kg of dry matter. In the experiments with sorghum, the control and PAB-inoculated silages were stable, whereas LAB-inoculated silages deteriorated. The results suggest that PAB can survive in and improve the aerobic stability of only slow-fermenting silages which are prone to aerobic deterioration.     

Prediction of the digestibility of primary growth and regrowth grass silages from chemical composition, pepsin-cellulase solubility and indigestible cell wall content

Prediction of organic matter (OM) digestibility (OMD) of primary growth and regrowth grass silages was studied based on their chemical composition, pepsin-cellulase solubility of OM (OMS) and indigestible neutral detergent fibre (INDF) content. Twenty-five primary and 28 regrowth silages were harvested from mixed timothy (Phleum pratense) meadow fescue (Festuca pratensis) or timothy cocksfoot (Dactylis glomerata) swards by varying the dates of the first and second harvest. In vivo OMD was measured with sheep and indigestible NDF was determined by 12 days ruminal incubation of forage samples in nylon bags using dairy cows fed a forage-based diet. Chemical composition of the silages was significantly correlated with digestibility, but single regression equations led to an unacceptable prediction accuracy of OMD (RMSE>40 g/kg DM). Pepsin-cellulase solubility reliably predicted OMD of primary growth silages (RMSE = 10.8 g/kg DM), but was less accurate for regrowth silages (RMSE = 25.9 g/kg). The prediction accuracy of OMD could be improved by using different equations for the two silage types. Indigestible NDF predicted OMD more accurately than OMS for all silages and especially for the regrowth silages. In contrast to OMS, the relationship between INDF and OMD was similar for both silage types.     

Growth and survival of lactic acid bacteria in lucerne silage

A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were used for the experimental production of lucerne silage. Laboratory silage without inoculants served as a control. Counts of total anaerobes, total lactic acid bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days 14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage microflora, reaching a percentage between 59 and 95 % of total anaerobes. Lactobacilli were found as a predominant group of LAB during the whole study. Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g in the control at the first observation. Their counts decreased to 4.23 and 4.92 log CFU/g in treated silage and the control, respectively, on day 63 of fermentation. Similar decreases were observed in all bacterial groups. The treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and contained more lactic acid compared to control silage. The identity of re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne was evaluated by fingerprinting techniques. The fingerprint profiles of re-isolated bacteria corresponded to the profiles of strains used for the treatment. It could be concluded that supplemented LAB dominated in laboratory silage and overgrew naturally occurring LAB.     

Different lactic acid bacteria and their combinations regulated the fermentation process of ensiled alfalfa: ensiling characteristics,dynamics of bacterial community and their functional shifts

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