In developing countriesincluding India,livestock sector is one of the fastest growing segment of the agricultural economy. The supply of the green forage throughout the year is an economic priority to the farmers, in orderto maintain the production from the ruminant stock. But forage production is mostlyseasonal in many parts of the world, with surplus availability in harvesting season and shortage of fodder during dry season. Silage making is an option to preserve the green fodder to make the greens available throughout the year.Ensiling is based on natural lactic acid fermentation under anaerobic conditions. The most important foddercrops for ensiling are corn, sorghum, barleyand various othergrasses. Beside these crops various moist “by-products” of the food industry, such as apple pomace, beet pulp and brewer’s mash can alsobe used for silage preparation. Ensiling process has many steps which should be timed and controlled carefully to ensure successful ensiling.
Ensiling process and role of different bacterial groups during ensiling:
The ensiling process completes in the following steps: harvesting the crop (30-35%DM), chopping, loading into a silo, compacting and sealing to exclude air, storing and feed out phase (unloading for animal feeding). Biochemical and microbiological incidents can ariseduring the different stages of ensiling which may affect the silage quality. At first step of ensilingisthe enzymatic activity of intact plant cells whenresidual respiration occurs. The intact cellsuse glucose and fructose as carbohydrate source and consume oxygen entrapped in the silage.Early consumption of carbohydrates is detrimental for the subsequent anaerobic lactic acid fermentation. In initial phase of ensiling epiphytic aerobic flora such as Enterobacteria, yeasts and molds develop until oxygen has been entirely consumed or acidification is sufficient to stop their metabolism. At ensiling, the facultative anaerobic bacteria carry out a heterolactic fermentation which slightly decreases the pH of the silage.
As the conditions become anaerobic in silagepit, fermentation phase starts.This phase continue for several days or weeks, during which different groups of facultative aerobic or anaerobic microorganisms naturally found in plants compete for available nutrients. With gradual acidification,acid tolerant bacterial development start; convert water soluble carbohydrates into lacticacid. In well-processed silage, LAB dominate the fermentation, rapidly producing the low pH conditions that help to preserve the silage. In case of accidental soil incorporation in ensiled material, a long aerobic phase or slow acidification, the microbial communities in the silage will be dominated by Clostridia, yeasts, molds and accidentally incorporated pathogenic microorganisms such as Listeria sp.
As long as the pH is sufficiently low and anaerobiosis is maintained, storage phase lasts and few changes occur. Numbers of LAB and other viable microorganisms decline over time, except for some specialized species such as L. buchneri with continues to be active at low population densities. Some acid-tolerant microorganisms can survive this storage period in an almost inactive state (e.g. acid-tolerant yeasts) or as spores (e.g. butyric acid bacteria). Homofermentative lactobacilli such as L. plantarum and Lactobacillus curvatus tend to predominate in well preserved silage until the final stage of fermentation, when they are invariably replaced by heterofermentative species such as L. brevis and L. buchneri.
The fourth phase is the unloading or feedout phase. On opening of silos air penetrates into the silage depending on the density and porosity of the plant material and the rate of silage removal. This causes the growth of undesirable aerobic microorganisms initially present in the silage, such as yeasts and molds and an increase in pH.
Undesirable microorganisms and their metabolites
Several undesirable microorganisms can grow during ensiling process which can affect silage quality and thus affect animal performance or both animal and human health. These microorganisms are known as spoilage microorganisms; responsible for silage degradation mainly induce economic losses.
Yeasts and molds: Yeasts are considered to be the most important groupamong the undesirable microorganisms of silage because they are involved in aerobic spoilage either during the aerobic phase at the beginning of ensiling or during the unloading phase. The organic acid metabolism pathways (succinic, citric and lactic acids) of yeasts restarts on exposure of silage to air; inducing a pH increase and allowing the growth of less acid-tolerant microorganisms. Yeasts present in silage convert WSC into CO2 and alcohols; impair silage quality and lead to a decrease in feed intake. The alcohol production also has negative effect on milk taste.
Moulds are eukaryotic micro-organisms and develop in part of silage where oxygen is present. Many mould species produce the large filamentous structures and coloured spores in silage..Penicillium (70%), Fusarium (47%) and Aspergillus (34%) are the most frequent mycotoxin-producing fungi isolated from corn silage. There metabolites remain in the silage even after the fungus has disappeared. More than 20 mycotoxins can be produced by Fusarium sp., mainly dioxynivalenol(DON), zearalenone (ZEN) and fumonisin (FB).
Chronic exposure to mycotoxins produce non-specific symptoms such as immune system impairment, increased infections and metabolic and hormonal imbalances. Ruminants are better protected than other animals against many mycotoxins but fumonisin B1 is only poorly metabolized in the rumen.In high producing dairy animals fed on silage-based diets with high levels of concentrates; the consequent acidificationof the rumen environment may increase the animal’s sensitivity to mycotoxins.
Butyric acid bacteria (BAB): Soil accidentally included with the plant material during silo filling is the source of Butyric acid bacterial contamination of silage. Endospore-forming bacteria of the genera Clostridium, especially C. tyrobutyricum and C. butyricum and Bacillus are main BAB found in silage.At a relatively low ph,BABconvert lactic acid into butyric acid, hydrogen and carbon dioxide. A typical “clostridial silage” is characterized by a high butyric acid content of more than 5 g/kg DM, a high pH (over pH 5 in low DM silages), and a high ammonia and amine content. Excess butyric acid from feeding butyric silages results in higher levels of plasma Ketones. Daily doses of over 50 – 100g of butyric acid can cause ketosis.Feeding butyric silage has long term negative effects on production, fertility and health, thus economy of the farm business.
Bacterial spores can survive the passage through the gastrointestinal tract in dairy cows. Bacillus cereusis an important spoilage microorganism in pasteurized milk and milk products and can lead to food poisoning.The occurrence of Clostridium in milk can lead to off-flavors and excessive gas formation in semi-hard or hard ripened cheeses.
Listeria: The presence of L. monocytogenes in silage, feces or both increases the risk of its presence in milk and hence of its transmission to humans. pH of silage over 4.5 increases the risk of presence of Listeria sp. In ruminants it could cause encephalitis, absorption or septicemia, even death.
E. coli (STEC):E. coli and higher prevalence of E. coli O157 or E. coli O157:H7 has been found in herds fed corn silage.At ensiling, insufficient anaerobiosis could delay the establishment of lactic acid fermentation, slowing the pH decrease andincreasing the survival of pathogenic E. coli, which is a food born pathogen.
Biogenic amines (BA): Putrescine, cadaverine and tyramine are main biogenic amines found in silage; derived from arginine, lysine and tyrosine, respectively. These are produced due to amino acid decarboxylation by enzymes of several lactic acid bacteria. Species of many genera present in silagesuch as Clostridia, Bacillus, Klebsiella, Escherichia and Pseudomonas can also cause biogenic amine production. Several factors such as temperature, oxygen availability and rapidity of pH decrease during the initial stage of fermentation results inthe formation of BA. Feeding of silage having BA acts as causative factors in ketonemia. BA presence in silage decrease the palatability and reduce DryMatter Intake (DMI) and cattle performance.
Strategies to limit silage degradation by undesirable microorganisms:
Spoiled silage has to be thrown out because till now no any measure has been develop to improve the quality of spoiled silage. Main factors which affect the quality during silage processing are the use of poor quality or immature plant material, insufficiently rapid filling (causes delay in establishment of anaerobiosis leading to weak silage acidification), and contamination by pathogenic or spoilage microorganisms. Preventive measures which should be taken care for quality silage production are discussed below.
Promoting acidification:Silage preservation is mainly based on acidification,whichdepends on anaerobiosis (promoting LAB fermentation), buffering capacity and DM of the crop. Soil incorporation in silage increases its buffering capacity, encourages the growth of aerobic microorganisms, whichreduce the quantity of hexoses and pentoses available for further LAB fermentation. This all leads to delay in silageacidification during which lactic acid is converted tobutyric acid, followed by pH increase and further spoilage due tosecondary fermentation by Clostridia. Mineral acids such as sulfuric and chlorhydric acids has been used to promote silage acidification and to limit the pathogenic microorganism growth.
Establishment of anaerobiosis during ensiling: For good silage production anaerobic conditions should be established as soon as possible in the silo. This can be achieved by rapid filling of silo and compaction of the silo to exclude the trapped air. Compaction of silage is easy with small particle size of crops. So chopping length plays an important role in good silage production, whilevery small particle size mayimpair the rumen function. For grass silage optimum chopping length is 4-6 cm. The corn silage prepared for dairy cow feeding should contain less than 1% of large particles (>2 cm), 8–12% of medium particles from 1 to 2 cm and less than 50% of very short particles (<6 mm). Harvesting of crop at appropriate dry matter is helpful to reduce effluent losses, which may impair anaerobiosis.
Prevention of air ingression during storage of silage:Silo should be sealed properly to avoid air ingression during storage, which can lead silage spoilage. A barrier made up of plastic sheets is helpful to prevent air ingressionand giveprotection against damage by birds, rodents and UV rays. To reduce spoilage of silage due to exposure during feed-out phase,silage requirement and silo pit dimensions should be calculated prior to silage making.
Improving the aerobic stability of silage: To improve aerobic stability of silage chemical additives like formic acid, silage additives containing partially neutralized acids in salt form (nitrites, sulfates) in association with formol-based preservatives can be used.Bacterial additives like Lactobacillus buchneri and Lactobacillus plantarumPediococcusacidilactici, Pediococcus cerevisiae and Propionibacterium acidipropionici can alsobe used during ensiling to improve the silage quality. Bacterial inoculants also limit pathogen development andproduce substances which may have antimicrobial potential (H2O2, ethanol, diacetyl, exopolysaccharides) and antibacterial peptides such as bacteriocins.
Prevention of pathogen introduction during harvesting and ensiling of crop:Entry of soil in the silage is the source of butyric acid bacterial/ pathogen contamination. Crops should be harvested 4 weeks after manure application takingcare to prevent contamination by soil.
Preservation of surplus fodder as silagemakes it possible to have the green fodder availability throughout the year. Lactic acid fermentation is important for good silage production, but some undesirable bacteria can make entry during ensiling, which candegrade the quality of silage and cause economic lossesto the farmer. Safety control measures and additives may behelpful used in producing quality silage.