AGRE-0041 Development of Biological Silage Additives

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Agricultural Residues : Biological Conversion : Biotechnology : ECLAIR Cluster VII - Proteins : Sugar

Silage Additives

SUMMARY

This summary is based on the final report to the European Commission concerning work funded under the ECLAIR programme. The objective was to improve the characteristics of bacteria and enzymes used as inoculants in the preparation of silage from grass, as well as various agricultural residues. There are a number of aims in producing any new product which is to compete in the highly competitive area of silage inoculants (for instance over 110 products are currently registered in the UK under the UKASTA, ADAS-DANI-SAC scheme. Taking this scheme as an example, the requirements cover short term nutrition studies and long term animal performance, as well as the impact on the silage itself. Inoculants are expected to improve fermentation, this means faster reduction in pH through faster sugar utilisation, better spectrum of organic acids (enhanced lactic acid), and nitrogen retention. Environmental aspects are also important with a reduction in effluent production, whilst from the economic viewpoint the use of the additive must significantly reduce the amount of dry matter, energy and nitrogen lost from the silo. Screening of a large number of lactic acid bacteria resulted in selection of , as prototypes: Lactobacillus plantarum DCU101, L plantarum B41 and Pediococcus acidilactici G24. A number of hydrolytic enzymes were also tested as to their ability to release fermentable carbohydrate from the insoluble storage or cell wall carbohydrates. The final choice of Lp DCU101 and Pa G24 were used to produce silage from grass (fed to Charolais crossbred heifers) and alfalfa (fed to sheep for metabolism studies and lactating Fresian cows for milk production). Enzymes were not found to confer any benefit as they increased effluent, but had no significant effect on lactic acid fermentation. The research included the use of genetic probing methods (RFLP, RAPD and riboprobing) and endogenous plasmid probing to study population kinetics. The results of these studies showed that the introduced strains were able to multiply in competition with the natural flora, resulting in an initial faster rate of drop in pH. Attempts were made to enhance the rate of lactic acid production by transformation introducing multiple copies of the enzyme lactic dehydrogenase. However, no significant improvement was observed. No benefit was observed by disrupting the pathway to D-lactic acid. Animal trials showed a slight beneficial effect on milk production and a small positive effect was observed on carcass weight of finishing bulls. The product is being test marketed.

INTRODUCTION

Production of silage (ensiling) has traditionally been used to preserve forage for use as animal feed. The fresh crop is chopped and tightly packed and then undergoes fermentation by lactic acid generating bacteria under anaerobic producing acidic conditions which prevent the growth of undesirable microorganisms and allow the forage to be kept for months without deterioration. Ensiling can produce higher annual yields, of higher quality feed, with less dependence on sunny conditions, than traditional hay-making. It is also suited to preservation of higher yielding crops such as forage maize and permits several harvests per annum of crops such as rye-grass. However, adverse condition of the starting material, such as low dry matter content, poor natural bacterial flora (range of species) and insufficient bacteria or fermentable sugars, produce a low-quality feed which may lack palatability if other acids, particularly butyric acid, are produced.

BACKGROUND

Modern agricultural practice aims to produce a good-quality silage by use of improved harvesting techniques, tight packing and covering of silos and appropriate desiling methods. However, lack of fermentation substrates or suitable natural bacteria can only be compensated for by use of additives. One approach is to use organic acids which chemically increase the acidity of the silage. However, mixing may be a problem and the use of chemical additives may create more hazards to the user and threats to the environment. Hence, more recently a trend towards the use of the safer, more environmentally-friendly biological additives has been observed. Many such products are on the market. These differ in the nature of the components, which may contain enzymes, bacteria and/or bacteriophages. Current commercially available products include species such as Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Propionibacterium jensenii, Lactococcus lactis lactis, Butyrivibro fibrisolvens, Streptococcus faecium, supplemented in some cases with bacteriophages or in some cases more closely defined as clostridiaphages; while enzymes include cellulase, hemicellulase, pentosanases, amylase, xylanase, glucanase, galactomannase or glucose oxidase. Other products also include nutrients, either sugars or mineral elements (micronutrients), or natural acidulants such as cider vinegar. Chemical products may include formalin, formaldehyde and acids such as formic acid, propionic acid, acetic acid, sorbic acid, diglyceride surfactants, phosphoric acid, sulphuric acid, sodium nitrite, hexamethylene tetramine and even various low MW alcohols.

Against this background the farmer is faced with a difficult choice, which becomes an act of faith - indeed, since silage making is an annual event one farmer would not be able to try, on a year-by-year basis, all the products on the market. This project aimed to select 'better' organisms and then quantify their effect, as well as investigating the benefits (if any) of enzymes and the possibilities of improving the activity of chosen species of bacteria by genetic manipulation.

APPROACH AND RESULTS

Various enzymes (such as listed above) with the ability to release fermentable sugars from the fresh crop were investigated. However, the use of these had a detrimental effect on the resulting silage and increased liquid effluents.

A collection of lactic acid bacteria was established from ensiled crops and agricultural wastes. Screening for desirable characteristics (rapid growth, producing lactic acid resulting in a faster drop in pH than in natural controls) resulted in two strains of Lactobacillus plantarum and one of Pediococcus acidilactici being retained as prototypes.

The effect of these bacteria on lactic acid fermentation and silage pH was monitored in laboratory silos, producing results as shown below.

Attempts were made to enhance the rate of lactic acid production of these bacteria using recombinant gene technology. However, no positive advantages were recorded.

Prototypes made of these selected strains, single or in combination, have been industrially produced and freezedried similarly to additives currently on the market.

Prototype additives have been tested in farmscale silages made of alfalfa and grass were found to be beneficial for silage conservation

When fed to dairy cows and finishing bulls some evidence of increased nutritional value was obtained. However, the effect was reduced due to the good weather conditions which favoured silage production, even without additives.

Graph of time course of pH decline and growth of Lactobacillus plantarum in grass silage.

COMMERCIALISATION

A mixed inoculant made of L. plantarum DCU101 plus Pediococcus acidilactici G24 will be launched in Ireland and Great Britain as test markets.

PARTICIPANTS

Universita Cattolica del Sacro Cuore (I), Dublin City Universty (IRE), TEAGASC (IRE), Consorzio Agrital Ricerche (I), TEXEL SA (F).