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
ANALYTICAL MICROBIOLOGY IN DAIRY
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)
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
Why standardizing analytical methods?
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
General 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
Flow cytometry of LAB
IDF & ISO
International Dairy Federation
International Organization for Standardization
Develop International Standards Dairy sector
170 IDF | ISO standards - 60 recognized by the Codex Alimentarius
Global consensus process
IDF ISO
7
ISO/IDF PROJECT
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
This is NOT an alternative method (to CFU count methods ) but another method to quantify Lactic acid bacteria & Probiotics
Aim & Scope
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 CD
Collaborative study
Enquiry Draft International Standard DIS
Approval Final draft International Standard FDIS
Publication International Standard IS
~ 3 3/4 years
December
2015
February
2012
Project time frame
Voting
Voting
Voting
9
Collaborative Study
• 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
- Defining precision for each matrix and sample type - (Demonstrating equivalence between the 3 staining protocols)
1800 test samples analyzed for both active and total cells according to ISO 5725-2 and IDF bulletin 453
Aims and Setup
10
Project Manager: Sandra Casani, Chr. Hansen A/S, Denmark
Project group
Collaborative Study
11
Strains tested
Collaborative Study
© ISO and IDF 2015. All rights reserved
12
Protocols
Collaborative Study
© ISO and IDF 2015. All rights reserved
13
Definitions
Collaborative Study
© ISO and IDF 2015. All rights reserved
Collaborative Study
Protocol A
© ISO and IDF 2015. All rights reserved
Protocol A
Collaborative Study
© ISO and IDF 2015. All rights reserved
Protocol B
Collaborative Study
© ISO and IDF 2015. All rights reserved
Protocol B
Collaborative Study
© ISO and IDF 2015. All rights reserved
Protocol C
Collaborative Study
© ISO and IDF 2015. All rights reserved
Protocol C
Collaborative Study
© ISO and IDF 2015. All rights reserved
Results of the collaboratory study
Error bars indicate one standard deviation
Collaborative Study
© ISO and IDF 2015. All rights reserved
Probiotics
AFU/g
Results for log10 AFU/g
© ISO and IDF 2015. All rights reserved
Probiotics
Results for log10 TFU/g
TFU/g
© ISO and IDF 2015. All rights reserved
Conclusions • 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
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
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
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
Collaborative Study
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
* Standing Committee of Analytical Methods for Dairy Microorganisms
Members of the ISO/IDF Project Group on quantification of Lactic acid bacteria by flow cytometry (D06)
Project group
Labs participating at the collaborative study
Standardization organizations
IPA – for inviting me
YOU – for your kind attention
Acknowledgments
Thank you for your attention
Optical configuration Excitation source Laser 488 nm Minimum 20mW
Detectors Emission: minimum 2 Fluorescence channels
and Scatter channels
FL1: Green Channel
Protocol A: 500–570nm
Protocol B: 500–540nm
Protocol C: 515–545nm
FL2 or FL3: Orange or Red channel
Protocol A: Orange/red > 570nm
Protocol B: Red > 630 nm
Protocol C: Red > 650 nm
Light Scattering:
Side Scatter Channel (SSC) and Forward Scatter
Channel (FSC)
Fluidic configuration Sample flow rate 15 to 120 µl per min a
Sample volume analysed 20 µl to 250 µl
Event rate Max: 20 000 events/sec a
Overall analysis parameters Triggering parameters used SSC or both FSC and SSC
Amplifier and signal conditioning (linearity
or logarithmic scale)
Log scale
a Settings for these parameters depend on the instrument. Manufacturer guidelines should be followed.
Table 3 — Recommendations for flow cytometer: configuration and settings
