Concept of Bacterial Silage inoculant & organic acid: Role of Inoculants in Silage Fermentation

A commonly asked question about silage management is, “What are the differences between using a bacterial silage inoculant versus a direct acidification product such as organic acids, like propionic acid?” This question is most easily addressed once we establish a foundational understanding of the similarities and differences between the two types of products.

Silage additives and inoculants are typically incorporated into silage crops to help balance deficiencies in bacteria population and support an efficient fermentation process.

Silage additives can be used to remedy deficiencies such as lack of sufficient population of bacteria to support adequate fermentation and low levels of fermentable carbohydrates. Most of the silage additives are applied as forages and are chopped or added during the loading phase. Silage inoculants tend to be expensive, but safe and noncorrosive. The standard silage inoculant, lactic acid bacteria (LAB), usually reduces fermentation losses but often increases losses during feeding. In most cases, if spoiling during feeding becomes a problem in a specific farm, the use of LAB may increase overall shrink losses and would not be recommended. However, if spoilage has not been a problem, then the use of LAB should be considered because it reduces fermentation losses.

Bacterial silage inoculants have been investigated since the early 1900’s. The very first silage inoculants were often not viable due to limitations in the microbial technology at the time. The theory behind the use of silage inoculants was that by adding live, viable bacteria we could drive fermentation toward a desirable endpoint. The original theory still holds true today.

This is accomplished by using specifically-selected bacteria that outcompete the epiphytic bacteria in terms of replication and production of fermentation acids. This efficiency provides for a rapid fermentation which helps reduce energy losses associated with fermentation and dry matter loss.

Lactobacillus plantarum was the dominant silage inoculant species until about 40 years ago when the use of additional species of bacteria was introduced. The different species provided unique characteristics such as production of large amounts of lactic acid early in the fermentation process, or the ability to continue producing lactic acid at lower pH levels. In the mid-1990’s research began on Lactobacillus buchneri, which is heterofermentative, meaning it can produce more than one product during fermentation.

Simply stated, in addition to producing lactic acid, it produces acetic acid which helps limit yeast and mold growth and thereby enhances aerobic stability of the silage.

Fairly recently Lactococcus lactis O-224 has been included in SILOSOLVE® FC to replace some of the more-established strains. This strain has some very unique properties as it is a superior oxygen scavenger that has enabled early opening of silos in as few as 7 days. Without question, bacterial silage inoculants are generally regarded as safe (GRAS), easy to handle and non-corrosive to equipment.

When reviewing some of the classical data from Bolsen et al. (1992), we see improved DM recovery by 1.3 percentage points, 1.8% more efficient gains and 3.6 lbs. more gain per ton of crop ensiled when a single strain silage inoculant was compared to an untreated control. More recent research has shown improvements of more than 3.5% in DM recovery (Figure 1). A few studies have reported improved efficiency of energy-corrected milk production through the use of science-based, research-proven bacterial inoculants.

The research that has been conducted on bacterial silage inoculants by Chr. Hansen and others overwhelmingly has shown the value of using bacterial silage inoculants. By comparison, research is somewhat lacking for organic acid products. Silage review articles give little thought to the use of this technology. While it can work if used correctly based on moisture, crop type and stage of maturity, it is more cumbersome than inoculants which are proven effective and can be applied at the same rate for all crops.

Organic acid products such as propionic acid are direct acidifiers as they decrease the pH of the silage mass directly. This is simply due to adding an acid to the silage mass. Historically, larger amounts were used that resulted in a restricted fermentation. The addition of organic acids can inhibit yeast and mold through antimycotic activity. This simply means that yeast and molds typically do not survive in the presence of organic acids; thereby increasing aerobic stability.

The application rate of acids tends to be variable and is dependent on the moisture of the crop ensiled. Likewise, crops with higher buffering capacity, such as alfalfa, require higher rates of application. If propionic acid is applied in its base form it can be very corrosive to equipment. As salts of acids have become more readily available, this has become less of a concern. Research using these products is fairly limited and is only applicable to similar crops at similar moistures.

A comparison of the favorable and unfavorable attributes of bacterial silage inoculants compared to organic acids is shown in Table 1.

Silage technology has progressed significantly in the last 40 years, evolving from single species (not strain) bacterial inoculants to modern multi-strain inoculants where each strain is selected for a specific purpose. These modern products outcompete the native microflora and facilitate the achievement of ideal fermentation endpoints.

While organic acids have had a significant value to silage making in the past, this technology is not able to progress as it is based on acidifying the silage mass, not fermentation. Bacterial inoculants have been proven to improve DM recovery, and enhance aerobic stability, both of which are financially important to operations feeding ensiled feeds.

Inoculants are extensively studied both during development and after a product is introduced. This continued research furthers the understanding of the silage making process and helps determine what future improvements can be made. This is important for both producers and the livestock they feed as further improvements in silage fermentation can have an impact on animal performance, feed quantity and quality, and the bottom line of the operation. A science- based, research-proven bacterial silage inoculant provides all of the features and benefits of organic acids along with several additional advantages.

TYPES OF SILAGE INOCULANTS
A wide variety of silage inoculants are available on the market. These can be broadly grouped into 3 different categories.
• Bacteria
• Preservatives or organic acids
• Enzymes

BACTERIA
Most silage inoculants are lactic acid bacteria (LAB). Some products contain only homofermentative strains or heterofermentative strains while others are a combination of both types of LAB.

Homofermentative bacteria such as Lactobacillus plantarum, Pediococcus, Enterococcus and Lactococcus enhance the production of lactic acid, which lead to a faster drop in pH value and improved fermentation, thus reducing DM losses, protein breakdown and growth of undesirable microorganisms.

Heterofermentative bacteria such as Lactobacillus brevis, L. kefiri and L. buchneri convert forage sugars to lactic and acetic acid. The production of acetic acid will improve aerobic stability of the silage by preventing proliferation of undesirable yeast and mold keeping silage highly nutrient and hygienic.

In grass silage, the main challenge is acidification—in which case an adequate amount of homofermentative lactic acid bacteria (LAB) should be applied. A combination of homo- and heterofermentative lactic acid bacteria guarantees not only optimal fermentation but also enhanced aerobic stability.

Application of inoculant bacteria
Silage inoculants are generally applied as the forage is being picked-up or baled, using a specific applicator. While the forage will already have a range of naturally occurring bacteria on them including lactic acid bacteria species, the microbial community present may not drive optimum fermentation and may even have high levels of detrimental bacteria.

The aim with an inoculant is to supply a sufficient amount of selected strains with known effects on fermentation to help ensure that fermentation proceeds rapidly and in the right direction.

The rate of 100 000 (1 x 105) colony forming units (cfu) per gram of fresh forage will provide enough microorganisms to dominate fermentation. If a silage inoculant has a lower level than this, or does not even specify a cfu count, then there may be insufficient bacteria to really influence silage fermentation in positive way.

Be aware that not all inoculant bacteria are equal. Even within the same species, there is wide variation in what effect the bacteria will have on fermentation. Products and the published evidence of efficacy should specify the actual strain numbers to provide assurance to customers.

Quality of the packaging and storage conditions are also important. These should prevent exposure to oxygen, moisture and heat that could reduce the viability of bacteria. Follow manufacturer instructions on storage and use and ensure that application is even and comprehensive over the whole forage.

PRESERVATIVES
The use of organic acids such as propionic and formic acids are aimed at lowering the silage pH to make it less favorable for undesirable bacteria such as Clostridia. Other organic acids and their salts including potassium sorbate and sodium benzoate target the growth of yeasts and mold fungi either in fermentation or during feed out.

There needs to be sufficient amount of the additive to provide a concentration in the bulk forage that will actually be sufficient effect on the growth of those undesirable organisms. That rate is typically at 5 to 10 kg of active ingredient per ton of forage to preserve the silage or around 1 to 2.5 kg/ton to restrict yeast amount at feed out. Compare those values to what is actually contained in a product that claim a preserving affect.

At the lower inclusion rate (less than 5 kg/ton of active ingredient), organic acids do not provide full preservation. To ensure adequate fermentation it is advisable to use silage inoculants. Bear in mind that organic acids and silage inoculants cannot be mixed together.

ENZYMES
The aim of adding enzymes to silage is usually to aid the breakdown of plant cell walls (e.g. use of celluloses and hemi-celluloses). The main benefit of this appears to be an increase in the amount of sugars available for LAB bacteria to convert to lactic acid for more rapid acidification.

While there is some evidence of favorable outcomes of this on silage quality and animal production this is less reliable than general silage inoculant approach. In some cases, there are claims of increasing forage digestibility for livestock but evidence for this is less clear. There are also some enzymes aimed at improving starch availability for either bacteria or livestock but research on this is still at an early stage.

Role of Inoculants in Silage Fermentation

Silage

Any plant material that has been fermented or “pickled” in a silo/bale is referred to as silage. A silo, on the other hand, is any storage structure used to keep green, moist fodder. The main purpose of silage preparation is to preserve green fodder during the time of adequate availability, for usage during the lean season and to maximise the retention of natural nutrients in the fodder crop during the preservation process.

Ensilage

Ensilage, often known as ensiling, is a method of conserving green fodder for use as animal feed later. Ensilage’s principles are well-known. The primary goal is to create anaerobic environment in which natural fermentation can occur. This is accomplished by compacting and consolidating the material, as well as sealing the silo to prevent air re-entry. Respiratory enzymes quickly remove air that has been trapped in the herbage. Conversely, aerobic microbial activity happens when oxygen is in touch with herbage for a prolonged period of time with development yeast and mould. As a result, the fodder degrades into a worthless, unpalatable and often harmful product. Improved compaction and fermentation of silage come from finer chopping of plant material. As a result, the palatability and intake of silage improves. The second goal is to prevent the multiplication of undesirable microbes such as Clostridia and Enterobacteria. Clostridia can be found in the form of spores on crops and in the soil. Clostridia multiply in anaerobic surroundings, producing butyric acid and breaking down amino acids, resulting in a foul-tasting silage as well as a reduced nutritional value. Enterobacteria are also capable of degrading amino acids. Lactic acid production can arrest growth of clostridia and enterobacteria. Lactic acid bacteria are naturally found on harvested crops and ferment available carbohydrates such as glucose and fructose to produce lactic acid. The lactic acid produced enhances hydrogen ion concentrations and undissociated acids to a level that inhibits unwanted organisms. The moisture content and temperature of the silage influence the pH at which clostridia and enterobacteria cannot grow. Lower the pH, wetter is the substance. A pH little below 5.0 inhibits the development of most acid-tolerant clostridia provided the dry matter content should be more than 15%. Clostridia are extremely water-sensitive organisms that require damp circumstances to thrive. Clostridia growth can be slowed by wilting of the fodder before ensiling to reduce moisture content. Lactic acid bacteria can dominate the fermentation of high dry matter crops due to their strong tolerance to low moisture conditions.

The first few days of ensiling are crucial which decides the success or failure of fermentation. Unwanted microorganisms, primarily enterobacteria, clostridia, and yeasts will be able to compete for nutrition if the pH is not dropped soon enough. This will make it more difficult to obtain a steady silage. Lactic acid bacteria will rapidly acidify the environment under favourable conditions to the point where spoilage organisms will be unable to thrive, resulting in a stable silage.

Silage Additives

For many years, researchers have investigated for additives to improve the nutritional value of silages and to eliminate some of the undesirable events associated with the ensiling process. Silage additives are added to the forage or crop during the ensiling process to improve the ensiling process, reduce nutritional losses, reduce aerobic deterioration at feed out, improve hygienic quality of the silage, limit secondary fermentation, improve aerobic stability, increase the nutritive value of the silage, and as a result increase animal production and give the livestock raiser a good ROI – Return on Investment.

Types of Silage Additives/Inoculants:

1) Fermentation stimulants:

a) Inoculant microbes e.g., Lactic acid bacteria (LAB)

b) Enzymes Cellulases, Hemicellulases, Amylases

c) Fermentable carbohydrates & Sugar sources such as Molasses, sucrose, glucose, citrus pulp, pineapple pulp, sugar beet pulp

2) Fermentation inhibitors:

a) Organic acid and their salts Acids and organic acid salts such as Mineral acids (e.g., formic acid, acetic acid, lactic acid, acrylic acid, calcium formate, propionic acid, propionates)

b) Other chemical inhibitors such as Formaldehyde, sodium nitrite, sodium metabisulphite

3) Aerobic spoilage inhibitors:

Propionic acid, propionates, acetic acid, caproic acid

4) Nutrients

Urea, ammonia, grain, minerals

5) Absorbents

Grain, straw, bentonite, sugar beet pulp, polyacrylamide

Biological Additives

Microbial inoculants and enzyme preparations are natural ingredients that are safe to handle, non-corrosive to machinery, do not pollute the environment and their usage has increased rapidly in recent years. Bacterial inoculants have perhaps gained more attention from researchers and livestock producers than any other aspect of silage management.

Microbial Inoculants

Organisms

Inoculants are applied to silage to control the epiphytic bacteria population on plants that produce DM losses due to poor sugar fermentation. The two main types of microbial inoculants are 1) homofermentative and 2) heterofermentative.

Lactobacillus plantarum, Pediococcus, and Lactococcus species are homofermentative inoculants. They induce a fast fermentation that produces mostly lactic acid and quickly lowers the pH to 4, inhibiting further degradation of the crop’s sugar and protein.

Lactobacillus buchneri and Lactobacillus brevis are microorganisms belong to heterofermentative inoculants. They create a mixture of lactic and acetic acid, which causes fermentation to be slower than with homofermentative inoculants. They’re required to prevent yeast and mould from initiating the aerobic process.

• Enzyme Additives

The method of treating forages with enzymes increases digestibility through several processes, including direct hydrolysis, improved palatability, changes in intestinal viscosity, and changes in digestive site. Cellulase and Hemicellulase are enzymes that break down cell walls into sugars. The sugars generated by the enzymes facilitate the development of silage microorganisms, while fibre degrading enzymes can also improve fodder digestibility. Low-lignin feedstuffs, such as cereal silages and immature, cool-season grasses, are the best candidates for these enzymes. Fibre-digesting enzymes have been found to be successful in lowering the fibre content of grass and alfalfa crops ensiled at 60 to 70 percent moisture, with grasses having the highest beneficial effect.

Enzymes require optimal conditions of pH and temperature for their action. For instance, most cellulase enzymes require a pH of 4.5 and a temperature of 50°C. Enzyme activity is influenced by surface area, binding sites, moisture level, and plant proteases.

	Feed Ingredients and By-Products as Additives	Effect in fermentation
1.	Grain	While adding grain to corn silage isn’t beneficial, doing so with hay crop silage offers advantages. Adding grain to hay crop silage boosts its energy level. For better results, grain should be broken or rolled before ensiling.
2.	Molasses	Cane molasses (75 percent DM) has been frequently utilised to give quick fermentable carbohydrate for the ensilage of tropical herbages, with up to 10% w/w inclusion levels. Because of its viscosity, it is difficult to apply and should be diluted with a little amount of warm water to prevent seepage.
3.	Starch Sources	Its usage depends on the amount of starch that is accessible as a substrate for lactic acid bacteria.
4.	Citrus Pulp	Fresh citrus peels have been added to the ensilage of Napier grass at concentrations up to 50%, enhancing fermentation quality as evaluated by low pH, low butyric acid content, and appropriate lactic acid generation.

 

	Acids	Effect in fermentation
1.	Formic Acid and/or Formaldehyde Treatments	Commercial formic acid (85 %) has long been used to ensilage unwilted grasses but owing to its dangers in handling and application as well as its caustic nature it is replaced with biological additives.
2.	Propionic Acid	Propionic acid has the most anti-mycotic effect among the short-chain fatty acids. It is not as strong as formic or mineral acids but is safe for use in silage. Propionic acid has been proven to reduce moulds and yeasts that cause silage to deteriorate aerobically.
3.	Sodium Diacetate	Acetic acid and sodium salt combination has comparable effects to propionic acid and is very good in preventing top spoiling.

 

	Nutrient Additives	Effect in fermentation
1.	Ammonia and Urea	With variable degrees of efficacy, ammonia has been used to treat corn silage, tiny cereal grain silage, and high moisture corn, but not alfalfa silage.
2.	Minerals	At the time of ensiling, minerals often have little effect on fermentation. Adding minerals at the time of ensiling will improve the nutritional profile of the silage.

Conclusion:

Improved ensiling process requires Homofermentative as well as Heterofermentative bacterial inoculation. In which Heterofermentative Lactobacillus buchneri improve aerobic stability of silages by reducing the growth of yeasts. Homofermentative Lactobacillus plantarum, Pediococcus acidilactici and Enterococcus faecium induce a fast fermentation that produces mostly lactic acid and maintain pH between 3.5-4.2. Enterococcus faecium is effective in the reduction of E. coli and enterobacteriae in the silage. Cellulase and Hemicellulase are enzymes that break down cell walls into sugars that make more availability of carbohydrates for ensiling process. The net effect is that silages inoculation with Bacterial inoculants are more resistant to aerobic condition at feed out (exposure to air) and enzyme inoculants improve digestibility of the silage as compared to untreated silages.

Compiled  & Shared by- This paper is a compilation of group work provided by the Team, LITD (Livestock Institute of Training & Development)

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