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Methods for the official control of probiotics used as feed additives
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Methods for the official control of probiotics used as feed additives

Final report SMT4-CT98-2235. Volume III

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Council Directive 70/524/EEC concerning additives in feeds regulates the use of probiotic organisms. At the outset of the project, according to the Official Journal of the European Communities (11.9.96 No C263/3) some eleven genera of bacteria, three yeast and one mould were approved for temporary use within specified member states. This was in line with the provisions of Council Directive 93/113/EC on the use and marketing of enzymes, microorganisms and their preparations in animal nutrition. At the present time some nineteen barterial strains plus Saccharomyces cerevisiae (yeast) are authorised but no mould is permitted for probiotic use for animal feed. Additional microorganisms are on provisional lists. The project aim was to develop and validate methods for enumeration and identiflcation of authorised probiotics at strain level in feedingstuffs. Specifically the project aimed to cover identiflcation and enumeration of authorised Bacillus spp., Lactobacillus spp., Bifidobacterium spp., Pediococcus spp., Enterococcus spp. and yeasts in a range of different animal feeds. Each partner in the project was given responsibility for specified species of organisms. Each was charged with assessing available isolation/identification and enumeration methods, and developing and improving these where necessary to ensure robust procedures were available. Partners were further required to develop molecular biology methods for identification at strain level. Validation of the methods using feed samples was earned out in tive separate exsreises involving from 17 to 21 participants in each instance for three enumeration trials and nine participants for the two FOR trials. Molecular biology methods for typing focussed on PFGE and PCR as the preferred methods Ringtesting was undertaken by partners (and a limited number of specialised reference laboratories) by analysing blind samples containing authorised strains. Methodologies for the isolation/identification and enumeration of yeast, Enterococcus spp., Lactobacillus spp., Pediococcus spp., Bifidobacterium φρ. and Bacillus spp. have been developed by project partners. The enumeration methods gave good performance in inter-laboratory validation exercises and after critical assessment of the results, the project partners recommended procedures for possible adoption as Official Methods. These methods in ISO format are provided as a Compendium in Volume 3 of this Final Report with the precision data. For strain typing of the isolated organisms PCR and PFGE methods were developed by project partners. In the case of Bacillus spp. PFGE equipment was not available to the Project Partner and a PCR method was therefore developed, although indications from the work of other Partners was that PFGE might in fact be preferable. In all cases for the authorised probiotic strains, the patterns by PCR and PFGE were produced which could in future be used as the basis for strain identification. These form the basis of a reference library which is provided in Volume 3. In all procedures strain typing methods were standardised by running commercially available reference strains for comparative purposes. Although validation was attempted by sending samples to independent laboratories the responses were poor for the PFGE methods due to a limited number of reference laboratories with this expertise. Independent validation has therefore been achieved for yeast and bacilli PCR methods whereby the method for yeast was recommended. Molecular biology methods for strain typing are provided in ISO format in Volume 3 with the results from the validation.
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ISBN 92-894-6252-3
Katalogo numeris KI-NC-20873-EN-C
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ISSN ISBN 92-894-6252-3 DOI Katalogo numeris KI-NC-20873-EN-C
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