Andrea BUDDE-NIEKIEL, Stephane CHARTIER, Sandra CASANI, Jing GENG IPA WORLD CONGRESS MAY 31 - June 2, 2016 CHICAGO

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1 Quantification of lactic acid bacteria by flow cytometry in starter cultures, probiotics and fermented milk products - a new ISO/IDF standardized method Andrea BUDDE-NIEKIEL, Stephane CHARTIER, Sandra CASANI, Jing GENG IPA WORLD CONGRESS MAY 31 - June 2, 2016 CHICAGO

2 ANALYTICAL MICROBIOLOGY IN DAIRY

3 ANALYTICAL MICROBIOLOGY IN DAIRY Worldwide preferred and accepted No specific equipment required ISO/IDF Standards available Quantification of culturable cells Long time to result (2 to > 5days) No/less automatisation High variability of results (strong dependency on biological factors)

4 ANALYTICAL MICROBIOLOGY IN DAIRY Quantification of different populations (active, non-active) Rapid method (nearly real time) High throughput, partly automated High precision and accuracy Already introduced in dairy industry

5 General Optimized & validated analytical protocol: Fit for purpose Internationally recognized Ready-to-use in lab Analytical equivalence, i.e. comparison of results between labs world-wide Facilitates global trade Improves competitiveness General accepted (reference) & adopted by regulatory bodies Avoids duplications unique protocol Helps reducing negative impacts on the environment Why standardizing analytical methods? Flow cytometry of LAB Assessment of quality of starter cultures, probiotics and fermented milk products Differentiation between active and total fluorescent units More relevant, appropriate & reliable criteria for certain applications Rapid (nearly real time) High throughput Partly automated; less handling, less errors High precision & accuracy Not existing as an International method standard before

consensus process Develop International Standards Dairy

6 IDF & ISO IDF International Dairy Federation ISO International Organization for Standardization Global consensus process Develop International Standards Dairy sector 170 IDF ISO standards - 60 recognized by the Codex Alimentarius

7 ISO/IDF PROJECT Aim & Scope Obtaining a standard analytical method for the use in quantification of active and total cells: Lactic Acid Bacteria and Probiotics in starter cultures Lactic Acid Bacteria and Probiotics in fermented milk products Not in scope: - differentiation of bacteria - stability tests 7 This is NOT an alternative method (to CFU count methods ) but another method to quantify Lactic acid bacteria & Probiotics

study Enquiry Draft International Standard DIS Approval Final draft International Standard FDIS

8 Project time frame 8 ISO Stage Product Acronym Preliminary Preliminary work item PWI Proposal New proposal for a work item NP Preparatory Working draft WD Committee Committee draft Collaborative study Enquiry Draft International Standard DIS Approval Final draft International Standard FDIS Publication International Standard IS CD Voting Voting Voting February 2012 ~ 3 3/4 years December 2015

9 9 Collaborative Study Aims and Setup - Defining precision for each matrix and sample type - (Demonstrating equivalence between the 3 staining protocols) 15 labs (5 countries) 9 different FCM machines 10 samples (2 different batch) 8 single strain starter cultures 1 blend starter culture 1 Yoghurt Sample 3 staining protocols 2 repetitions per lab per strain per protocol 1800 test samples analyzed for both active and total cells according to ISO and IDF bulletin 453

10 Collaborative Study Project Manager: Sandra Casani, Chr. Hansen A/S, Denmark Project group 10

Collaborative Study Results of the collaboratory study Error bars

23 Conclusions Collaborative Study Good Repeatability values with low rate of outliers & stragglers (<5%) In general, no significant difference between protocols The variability of results is mainly explained by the labs and not by the protocols or the instruments

Collaborative Study 2015 January: ISO standard draft 2015 August : Publication of a

24 Collaborative Study Project Milestones 2011 September: Questionnaire 2012 February: Kick off Meeting 2012 May: Pre-study 2012 October: Pilot Trial 2014 January: Collaborative Study 2015 January: ISO standard draft 2015 August : Publication of a bulletin on Intercollaborative FCM data 2015 December: International Standard publication

25 Collaborative Study Summary and Key learnings Flow cytometry is a rapid method for the quantification of cells Flow cytometry has been standardized by IDF/ISO for its use in starter cultures, probiotics and fermented milk products enabling worldwide comparison of results

26 Collaborative Study Summary and Key learnings Flow cytometry allows assessing the fitness of a population by differentiation between active and total cells Further advantages of flow cytometry include low variability and possibility of high analysis throughput In case of stressed cells the validity of flow cytometry might be limited

of the ISO/IDF Project Group on quantification of Lactic acid bacteria by flow

27 I am Sandra CASANI, Project Manager of the D06 and Deputy Chair of SCAMDM* I am Stephane CHARTIER, Chair of the SCAMDM* and member of the D06 project Members of the ISO/IDF Project Group on quantification of Lactic acid bacteria by flow cytometry (D06) * Standing Committee of Analytical Methods for Dairy Microorganisms

28 Acknowledgments Project group Labs participating at the collaborative study Standardization organizations IPA for inviting me YOU for your kind attention

29 Thank you for your attention

30 Table 3 Recommendations for flow cytometer: configuration and settings Optical configuration Excitation source Laser 488 nm Minimum 20mW Detectors Emission: minimum 2 Fluorescence channels and Scatter channels FL1: Green Channel Protocol A: nm Protocol B: nm Protocol C: nm FL2 or FL3: Orange or Red channel Protocol A: Orange/red > 570nm Protocol B: Red > 630 nm Protocol C: Red > 650 nm Light Scattering: Fluidic configuration Sample flow rate 15 to 120 µl per min a Sample volume analysed 20 µl to 250 µl Event rate Side Scatter Channel (SSC) and Forward Scatter Channel (FSC) Max: events/sec a Overall analysis parameters Triggering parameters used SSC or both FSC and SSC Amplifier and signal conditioning (linearity or logarithmic scale) a Settings for these parameters depend on the instrument. Manufacturer guidelines should be followed. Log scale

Application Note. Yeast Analysis with a life - dead staining kit (Yeast control - viability)

Application Note. Yeast Analysis with a life - dead staining kit (Yeast control - viability) Application Note Yeast Analysis with a life - dead staining kit (Yeast control - viability) (CyFlow Cube 8 and CyFlow Cube 6 with CyView TM version 1.5 CyFlow Space) Introduction The Partec Yeast control