CEN/TS 15790:2008

Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae (yeast)

This Technical Specification defines a polymerase chain reaction (PCR) methodology for the identification of S. cerevisiae probiotic yeast strains. Additionally, a method for the extraction of high quality DNA from yeast is suggested.

Futtermittel - PCR-Typisierung der probiotischen Stämme von Saccharomyces cerevisiae (Hefe)

Diese Technische Spezifikation legt eine Polymerase-Kettenreaktion-(PCR)-Methodik für die Identifizierung
von probiotischen Saccharomyces cerevisiae-Hefestämmen fest. Zusätzlich wird ein Verfahren für die
Extraktion hochreiner DNA der Hefe vorgeschlagen.

Aliments des animaux - Typage par réaction de polymérisation en chaîne (PCR) des souches probiotiques de Saccharomyces cerevisiae (levure)

La présente Spécification technique définit une méthode d’amplification en chaîne par polymérase (ACP)
destinée à l’identification des souches de levure probiotiques de Saccharomyces cerevisiae. Elle présente
également une méthode d’extraction d’un ADN de bonne qualité à partir de la levure.

Krma - PCR-tipizacija probiotičnih sevov Saccharomyces cerevisiae (kvasovk)

General Information

• Status: Published
• Publication Date: 09-Dec-2008
• ICS: 07.100.30 - Food microbiology
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 3 - Feed additives and drugs
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 26-May-2011
• Completion Date: 26-May-2011
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Mandate: M/382 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for animal Feedingstuffs
• Ref Project: SIST-TS CEN/TS 15790:2009 - Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae (yeast)

Overview

CEN/TS 15790:2008 is a Technical Specification developed by the European Committee for Standardization (CEN) that defines a standardized method for the polymerase chain reaction (PCR) typing of probiotic strains of Saccharomyces cerevisiae yeast used in animal feeding stuffs. This document provides a reliable molecular approach for the identification and characterization of authorized probiotic yeast strains in animal feed supplements. Additionally, it outlines a procedure for extracting high-quality genomic DNA from yeast, which is fundamental for effective PCR analysis.

The specification aims to ensure consistency and reliability in detecting probiotic S. cerevisiae strains, which are pivotal for improving animal health and feed efficiency. The method is applicable across European member states and supports compliance with regulatory requirements in animal feed production.

Key Topics

• Scope and Purpose
  The standard focuses on the PCR-based identification of S. cerevisiae probiotic strains isolated from animal feeding stuffs or supplements, providing a robust approach to confirm authorized strains and maintain product quality.

• DNA Extraction Method
  Emphasizes extraction of high molecular weight, pure genomic DNA from yeast cells, crucial for reliable PCR amplification. The protocol includes sequential steps such as cell wall removal, nuclei lysis, protein precipitation, and nucleic acid purification.

• PCR Typing Principle
  Utilizes primers targeting δ elements within the yeast genome. The PCR amplification yields specific banding patterns for authorized probiotic strains after agarose gel electrophoresis. This molecular fingerprinting enables differentiation of strains.

• Reagents and Apparatus
  Detailed reagent list includes modified primers, dNTP mix, reaction buffer, magnesium chloride solution, and DNA Taq polymerase. Equipment specified includes thermal cyclers, electrophoresis systems, transilluminators, and imaging devices for result analysis.

• Analytical Procedure
  Steps comprise DNA extraction, PCR reaction mix preparation, thermal cycling under defined conditions, gel electrophoresis using ethidium bromide staining, and UV visualization of DNA bands. The results are compared against reference strain patterns for confirmation.

• Validation and Reliability
  The standard provides validation data from collaborative trials across multiple European laboratories, confirming reproducibility and robustness in identifying probiotic yeast strains via this method.

Applications

• Animal Feed Quality Control
  Ensures verified identification of probiotic S. cerevisiae strains incorporated into animal feeds, promoting uniformity and safety of supplements.

• Probiotic Strain Authentication
  Supports producers and regulatory bodies in authenticating authorized probiotic yeast strains, preventing contamination with unauthorized or potentially ineffective variants.

• Research and Development
  Facilitates microbial strain characterization in feed additive development and probiotic efficacy studies.

• Regulatory Compliance
  Assists laboratories performing standard testing under European Union directives related to animal feed additives and probiotics.

• Feed Industry Laboratory Use
  Ideal for feed manufacturers and testing labs to monitor production batches and validate probiotic strain presence.

Related Standards

• CEN/TC 327 - Animal feeding stuffs methods of sampling and analysis
• Standards covering microbial methods in animal feed to complement genetic typing techniques.
• Molecular biology standards for DNA extraction and PCR in food and feed microbiology.
• Guidelines on probiotic strain identification and validation in animal nutrition.

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By following the CEN/TS 15790:2008 Technical Specification, stakeholders in the animal feed industry can implement a dependable, scientifically validated method to typify probiotic Saccharomyces cerevisiae strains. This ensures probiotic efficacy, feed safety, and regulatory adherence, supporting the overall goal of enhancing animal health through targeted feed supplementation.

Standards Content (Sample)

SLOVENSKI STANDARD
01-april-2009
.UPD3&5WLSL]DFLMDSURELRWLþQLKVHYRY6DFFKDURP\FHVFHUHYLVLDHNYDVRYN
Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae
(yeast)
Futtermittel - PCR-Typisierung der probiotischen Stämme von Saccharomyces
cerevisiae (Hefe)
Aliments des animaux - Typage ACP des souches probiotiques de Saccharomyces
cerevisiae (levure)
Ta slovenski standard je istoveten z: CEN/TS 15790:2008
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 15790
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
December 2008
ICS 07.100.30; 65.120
English Version
Animal feeding stuffs - PCR typing of probiotic strains of
Saccharomyces cerevisiae (yeast)
Aliments des animaux - Typage ACP des souches Futtermittel - PCR-Typisierung der probiotischen Stämme
probiotiques de Saccharomyces cerevisiae (levure) von Saccharomyces cerevisiae (Hefe)
This Technical Specification (CEN/TS) was approved by CEN on 25 August 2008 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15790:2008: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Principle .5
3 Reagents .5
3.1 PCR .5
3.1.1 Primers .5
3.1.2 dNTP mix .5
3.1.3 Buffer .6
3.1.4 Magnesium Chloride solution .6
3.1.5 DNA Taq polymerase .6
3.2 Gel Electrophoresis.6
3.2.1 Agarose, molecular-grade agarose free from DNase and RNase contamination. .6
3.2.2 Molecular weight marker .6
3.2.3 Tris-Borate-EDTA buffer .6
3.2.4 Loading dye .6
3.2.5 Water .6
3.2.6 Ethidium bromide .6
4 Apparatus .6
4.1 PCR .6
4.1.1 PCR Tubes .6
4.1.2 Pipets and sterile tips .7
4.1.3 Thermal cycler .7
4.2 Gel Electrophoreses .7
4.2.1 Horizontal gel electrophoresis system .7
4.2.2 Microwave oven .7
4.2.3 Conical flask .7
4.2.4 Balance .7
4.2.5 Transilluminator .7
4.2.6 Image analysis system or Polaroid camera .7
5 Procedure .7
5.1 PCR reaction .7
5.2 Thermal Cycle .8
5.3 Agarose gel electrophoresis .8
6 Analysis of the results .9
Annex A (informative) Example Gel - PCR of probiotic strains of Saccharomyces cerevisiae . 10
Annex B (informative) Validation data from the European Collaborative trial [4][5] . 11
Bibliography . 12

Foreword
This document (CEN/TS 15790:2008) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs – Methods of sampling and analysis”, the secretariat of which is held by NEN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and United Kingdom.
Introduction
This methodology is based on specific polymerase chain reaction (PCR) amplification of a genetic sequence
for the detection of Saccharomyces cerevisiae isolated from animal feed or animal feed probiotic supplement.
The aim of this method is to identify authorised probiotic yeast strains. Molecular typing methods and
especially PCR amplification based methods used to characterise the yeast strains require high quality high
molecular weight genomic DNA. The method of DNA extraction from the yeast must facilitate these
requirements.
1 Scope
This Technical Specification defines a polymerase chain reaction (PCR) methodology for the identification of S.
cerevisiae probiotic yeast strains. Additionally, a method for the extraction of high quality DNA from yeast is
suggested.
2 Principle
This method is based upon the amplification of δ elements which are present in the yeast genome. Two
primers are used for the PCR reaction, which are a modification of Ness et al. [1]. Distinct patterns are
produced for probiotic S. cerevisiae strains when separated in agarose gels by electrophoresis. Patterns are
visualised under UV light after electrophoresis and ethidium bromide staining of the agarose gel.
The PCR analysis of individual yeast colonies isolated from aga
...