Pro Sil M100 Silage Additive Incorporating a Unique

Pro. Sil 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 1

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 2

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 • Pro. Sil M-100 contains Lactobacillus fermentum which produces acetic acid, which inhibits the activities of yeasts 3

Unique Combination Inoculant • There are 2 main types of lactic acid bacteria: homofermentative and heterofermentative • Pro. Sil 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 4

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 p. H fall, resulting in: - improved silage fermentation - lower losses - improved nutritional value of silage - improved livestock performance 5

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 6

Pro. Sil M-100 Acetic Acid Pathways • From hexose sugars: 3 fructose → 1 lactic acid + 1 acetic acid + 2 mannitol + 1 CO 2 1 glucose + 2 fructose → 1 lactic acid + 1 acetic acid + 2 mannitol + 1 CO 2 • 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 CO 2 * Proposed molar levels 7

It Begins with Good Strain Selection (1) • The 2 homofermentative LAB in Pro. Sil 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 p. H 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 8

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 9

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 Pro. Sil M-100 to achieve both improved stability and improved silage fermentation and feeding quality 10

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 11

Fermentation of the most important (5 and 6 Carbon) Sugars P. acidilactici L. plantarum L. fermentum Arabinose Ribose Galactose Xylose Glucose Galactose Fructose Glucose Mannose Fructose Mannose 12

More Acetic Acid = Improved Aerobic Stability in Maize Silage Parameter DM % p. H Lactic acid % DM Acetic acid % DM Average temperature after opening °C Control 32. 5 3. 6 4. 2 1. 3 25. 5 Pro. Sil M-100 Significance 32. 2 3. 6 ns 4. 3 ns 1. 9 P<0. 001 24. 7 P<0. 01 Pro. Sil 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 13

More Acetic Acid = Improved Aerobic Stability in Whole-Crop Wheat Silage Parameter DM % p. H Lactic acid % DM Acetic acid % DM Average temperature after opening °C Control 38. 6 4. 2 3. 5 1. 4 25. 6 Pro. Sil M-100 Significance 38. 5 4. 1 ns 3. 8 ns 2. 3 P<0. 001 21. 5 P<0. 001 Pro. Sil M-100 significantly increased (P<0. 001) acetic acid in wholecrop wheat silage, leading to a significantly lower (P<0. 001) average temperature after opening 14

Pro. Sil M-100 Reduces Temperature after Opening Trial 3 15

Change in temperature with time of maize silages exposed to air 30 Untreated Pro. Sil M-100 20 Homoferm. Ambient 10 Temperature deg C 1 13 25 37 49 61 73 85 97 109 121 133 145 Time hours Maize dry matter 31. 7% 16

Pro. Sil M-100 Reduces Temperature after Opening Trial 4 17

Effect of different treatments on heating of maize silage 24 20 Untreated 18 Pro. Sil M-100 16 ambient 14 12 118 109 100 91 82 73 64 55 46 37 28 19 10 10 1 Temperature deg C 22 Time Hours Maize dry matter 29. 3% 18

Hours to +2°C Rise above Ambient Trial Control Significance 21 Pro. Sil M-100 3 4 30 105 P<0. 001 Stability significantly extended (P<0. 001) by over 3 days after opening in both trials 19

Reduced Yeast in Silage Yeasts CFU / g Control 2, 100, 000 Pro. Sil M-100 17, 000 CFU = colony forming units • Substantial reduction in the number of yeasts at opening • Reduction in yeasts leads to greater stability 20

Specifications • Active ingredients: Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus acidilactici. • Carrier: Highly soluble dextrose • Packed in 100 g 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). 21

Usage instructions • One 100 g sachet of Pro. Sil 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. • Pro. Sil 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 22

Pro. Sil 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 23