ProSil M-100 Silage Additive Incorporating a Unique Combination of Lactic Acid Bacteria for Improving Stability of Whole-Crop Cereal, Maize and Crimped Grain Silage after Opening

Features & Benefits Contains 3 unique lactic acid bacteria (LAB) isolated from farm silages: Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus acidilactici. Improves the stability of whole-crop cereal, maize and crimped grain silages after opening Increases acetic acid to improve stability after opening Reduces the number of yeasts which cause stability problems in silage Improves silage fermentation Successfully used on farms Easy to use Safe EU notified strains

Maize Post-opening problems Maize is a great forage for silage due to it’s high dry matter and energy contents The well known problem with maize silage is that it is very susceptible to heating after opening This heating is mainly caused by yeasts ProSil M-100 contains Lactobacillus fermentum which produces acetic acid, which inhibits the activities of yeasts

Unique Combination Inoculant There are 2 main types of lactic acid bacteria: homofermentative and heterofermentative ProSil M-100 contains a combination of: Homofermentative LAB: Lactobacillus plantarum and Pediococcus acidilactici, which improve silage fermentation, and Heterofermentative LAB: Lactobacillus fermentum, which improves the stability of silage

Homofermentative Mode of Action Homofermentative LAB are very efficient at fermenting hexose sugars in forage to lactic acid: 1 glucose or 1 fructose → 2 lactic acid Fermentation of the rarer pentose sugars: 1 arabinose or 1 xylose → 1 lactic acid + 1 acetic acid This efficient utilisation of sugar results in a fast pH fall, resulting in: - improved silage fermentation - lower losses - improved nutritional value of silage - improved livestock performance

Heterofermentative Mode of Action The mode of action of the heterofermentative Lactobacillus fermentum is similar to that of L. buchneri Acetic acid is formed from the fermentation of hexose and pentose sugars and from the fermentation of lactic acid Pathways for fermentation of lactic acid to acetic acid in silage are not fully understood and the pathway shown in slide 7 is one proposed without an external electron acceptor and under anaerobic conditions by Oude Elferink et al. 2001. Other possible pathways also exist

ProSil M-100 Acetic Acid Pathways From hexose sugars: 3 fructose → 1 lactic acid + 1 acetic acid + 2 mannitol + 1 CO2 1 glucose + 2 fructose → 1 lactic acid + 1 acetic acid + 2 mannitol + 1 CO2 From pentose sugars: 1 arabinose or 1 xylose → 1 lactic acid + 1 acetic acid From lactic acid: 2* lactic acid → 1 acetic acid + 1 1,2-propanediol + (trace) ethanol + 1 CO2 * Proposed molar levels

It Begins with Good Strain Selection (1) The 2 homofermentative LAB in ProSil M-100 were each isolated from well preserved silage, so they originate from silage and grow in that habitat. Good selection criteria are essential to ensure that the best lactic acid bacteria are selected for inoculants. These selection criteria included: Strain safety Fast growth rate The ability to ferment silage quickly / fast pH fall Strong production of lactic acid, with check on when the lactic acid was produced Non-proteolytic to preserve more protein in silage No production of extracellular polysaccharides

It Begins with Good Strain Selection (2) The heterofermentative Lactobacillus fermentum was selected from over 600 strains of LAB which were isolated from stable high dry matter silages on dairy farms. It originates from silage and grows in that habitat. The selection criteria for L. fermentum included: Strain safety The ability to ferment lactic acid to acetic acid when presented with lactic acid as the only source of carbon and energy Non-proteolytic to preserve more protein in silage No production of extracellular polysaccharides

A Combination That Works Homofermentative LAB are faster growing than heterofermentatives (see slide 11) and would dominate silage fermentation if the wrong ratio of each was used. Extensive testing on dairy farms has identified the optimum ratio of homofermentatives and heterofermentatives to include in ProSil M-100 to achieve both improved stability and improved silage fermentation and feeding quality

Doubling time and Temperature Range Double in number every: Pediococcus acidilactici 35 minutes Lactobacillus plantarum 33 minutes Lactobacillus fermentum 53 minutes Temperature range for growth Pediococcus acidilactici 20 - 50°C Lactobacillus plantarum 15 - 45°C Lactobacillus fermentum 20 - 45°C This covers the range of temperatures found in silage

Fermentation of the most important (5 and 6 Carbon) Sugars P. acidilactici Arabinose Ribose Galactose Glucose Fructose Mannose L. plantarum Xylose L . fermentum

More Acetic Acid = Improved Aerobic Stability in Maize Silage Parameter Control ProSil M-100 Significance DM % 32.5 32.2 pH 3.6 ns Lactic acid % DM 4.2 4.3 Acetic acid % DM 1.3 1.9 P<0.001 Average temperature after opening °C 25.5 24.7 P<0.01 ProSil M-100 significantly increased (P<0.001) acetic acid in maize silage, leading to a significantly lower (P<0.01) average temperature after opening Lower temperatures mean that more energy is retained in the silage, which increases feeding value

More Acetic Acid = Improved Aerobic Stability in Whole-Crop Wheat Silage Parameter Control ProSil M-100 Significance DM % 38.6 38.5 pH 4.2 4.1 ns Lactic acid % DM 3.5 3.8 Acetic acid % DM 1.4 2.3 P<0.001 Average temperature after opening °C 25.6 21.5 ProSil M-100 significantly increased (P<0.001) acetic acid in whole-crop wheat silage, leading to a significantly lower (P<0.001) average temperature after opening

ProSil M-100 Reduces Temperature after Opening Trial 3

Temperature deg C Change in temperature with time of maize silages 10 20 30 1 13 25 37 49 61 73 85 97 109 121 133 145 Temperature deg C Time hours Change in temperature with time of maize silages exposed to air Untreated ProSil M-100 Homoferm. Ambient Maize dry matter 31.7%

ProSil M-100 Reduces Temperature after Opening Trial 4

Effect of different treatments on heating of maize silage 10 12 14 16 18 20 22 24 1 19 28 37 46 55 64 73 82 91 100 109 118 Time Hours Temperature deg C Untreated ProSil M-100 ambient Maize dry matter 29.3%

Hours to +2°C Rise above Ambient Trial Control ProSil M-100 Significance 3 21 100 P<0.001 4 30 105 Stability significantly extended (P<0.001) by over 3 days after opening in both trials

Reduced Yeast in Silage Control ProSil M-100 Yeasts CFU / g 2,100,000 17,000 CFU = colony forming units Substantial reduction in the number of yeasts at opening Reduction in yeasts leads to greater stability

Specifications Active ingredients: Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus acidilactici. Carrier: Highly soluble dextrose Packed in 100g amounts in alufoil sachets to treat 100 tonnes of whole-crop cereal, maize or crimped grains All strains have been notified to the European Commission and to the European Food Safety Authority Storage stability: 18 months from the date of manufacture when stored at ambient temperature (<20°C).

Usage instructions One 100g sachet of ProSil M-100 treats 100 tonnes of whole-crop cereal, maize or crimped grains Mix the contents thoroughly in a small amount of clean cold tap water Dilute the solution as necessary in clean cold tap water to suit the applicator and speed of ensiling. ProSil M-100 can be applied at any application rate between 100 ml and 2 litres per tonne of forage, provided always that 1 g of product is applied per tonne of forage

ProSil M-100 Summary Contains unique lactic acid bacteria isolated from good quality silages and from stable silages The formulation is based on what works on dairy farms Lengthens the stability of whole-crop cereal and maize silage after opening Increases the content of acetic acid Fewer yeasts recorded in maize silage Improves silage quality EU notified strains Easy to use