Rapid detection and enumeration of spoiler microorganisms from beer mixes with low or 0.0 alcohol content Marta Orive Camprubí, EBC Symposium 2014, Wien
BIOLOGICAL STABILITY: Ecological aspects +++ high cotnent 0 Absence or near Ecological aspects Lager Beer Lager Beer 0.0 Soft drinks Beer mixes Sugars + + +++ +/+++ Other nutrients ++ ++ - +/++ Fruit juices 0 0 0/++ 0/++ Alcohol +++ 0 0 0/+ Hops ++ ++ 0 +/++ ph 4-4.5 4-4.5 2-3.5 3-4.5 CO2 ++ ++ +++ ++/+++ Oxygen 0 0 +++ 0/+ 2
+++ high level 0 Low/Absence or near Product Container Filling/ Pasteurisation Rapid release Safety Spoilage risks Natural image Beer Bottles /Cans Filling / Pasteurisation +++ +++ 0 +++ Beer Kegs Pasteurisation / Filling +++ +++ 0/+ +++ Beer 0 0 Bottles /Cans Filling / Pasteurisation +++ +++ 0 +++ Beer 0 0 Kegs Pasteurisation / Filling +++ +++ 0/+ +++ Soft drinks Bottles /Cans Pasteurisation / Filling / Preservatives 0 +++ 0/+ 0 Soft drinks Kegs /PET Pasteurisation / Filling / Preservatives 0 +++ 0/+ 0 Beer mixes Bottles /Cans Filling / Pasteurisation +++ +++ 0 +++ Beer mixes Kegs Pasteurisation / Filling / Preservatives +++ +++ 0/+ 0 Beer mixes PET Pasteurisation / Filling???? 3
OUR PRODUCTS Beer mixed with soft drinks or juices Low or 0.0 ABV Flash pasteurised In PET bottles With no added preservatives and Quarantined Product A 0.0 ABV beer + lemon juice Product B Lager beer 5.5 ABV + clear lemonade Product C Lager beer 5.4 ABV + hazy lemonade 4
CONTROL PLAN BASED ON TRADITIONAL METHODS Parameter Medium Temperature Days to result Forcing test As is 25 a 30ºC 9 Mesophile total aerobic count PCA 30ºC 3 + 3 = 6 días Yeast count OSA o WLNM o MLBA 27ºC 3 + 6 = 9 días Due to 3 days preincubation + 6 days slow growing REDUCE Dekkera TIME / Brettanomyces TO RESULT sp. IN on MICROBIAL incubation ANALYSES media 5
Available Techniques? ChemScan (solid phase cytometry) PCR FISH Bactrac (impedance) Bactiflow (flow cytometry) Election of Rapid Technique? 6
Evaluation Criteria Shorter time to result to reduce Quarantine? Enumeration of microorganisms Versatile for Membrane Filtration of different products: water, soft drinks, beer, beer mixes,? No inhibition? Validated (similar / better in comparison) against standard? Costs Long term: time, price/analysis / Cost Short term (price instrument, ) Ease of use (sample prep, ) / Ease of implementation (user friendliness, training, hands-on-time, no of samples per day ) Maintenance, repair BAT (Best Milliflex AvailableTechnique)? Quantum 7
MSM protocol for MILLIFLEX QUANTUM Product Pre-incubation 3 days + Membrane Filter as usual + Incubate on Petri dish + staining + result reading Half to 1/3 reduction of traditional time Further identification possible 8
9
Validation and verification 10
Products Product A 0.0 ABV beer + lemon juice Alcohol 0.011 ABV, ph 3.4-4.0 and Isohumolone EBU 9-13 Product B Lager beer 5.5 ABV + clear lemonade Alcohol 0.9 ABV, ph 2.9-3.5 and Isohumolone EBU 2-5 Product C Lager beer 5.4 ABV + hazy lemonade Alcohol 3.2 ABV, ph 3.2-4 and Isohumolone EBU 12-14 11
TRADITIONAL METHODS Parameter Method Temperature Days Yeast count WLNM 27ºC 3 + 6 = 9 days Due to slow growing Dekkera/Brettanomyces sp. on incubation media MICROORGANISMS Zygosacharomyces bailii Dekkera anomala Dekkera bruxellensis 12
Compatibility and determination of minimum days of incubation for approppriate results in MFQ Dekkera bruxellensis in 3 products could be detected in 3 days in MFQ 13
Laboratory validation with spiked samples (ISO 16140) 1-3 products, with and w/o preservatives vs. D.anomala, D.bruxellensis and Zygosaccharomyces bailii. 2- Experimental design - Incubate samples during 3 days - Evaluate 6 bottles of each product spiked with D.anomala and D.bruxellensis (10 CFU/ filterered volume). 6 samples of 1 product with Z.bailii. - Evaluate 2 non-spiked samples of each product - Analise in parallel in MFQ and the reference method 3- Evaluate recovery 4- Calculate relative detection level, relative sensitivity and relative especificity 5.- Inclusivity and exclusivity (Supplier) 14
Reference MFQ Day 0 MF MF Day 3 Incubate 27ºC Incubate 27ºC Staining + MFQ results Re-incubate 27ºC Day 6 Results Results on Petri Dishes 15
Products A with and without Preservatives B with and without Preservatives C with and without Preservatives A with and without Preservatives B with and without Preservatives C with and without Preservatives B with Preservatives Microorganism D. bruxellensis D. bruxellensis D. bruxellensis D. anomala D. anomala D. anomala Z. bailii 16
M F Q COMPARISON BETWEEN MFQ/3rd AND 6th DAY T r a d t i o n a l A with D. bruxellensis B with D. anomala C with Z. bailii 17
Validation results ISO 16140 Recovery >70% Efficacy 100% Relative detection level 4 CFU / 100 ml Relative especificity 100% Relative sensitivity 100% Both methods are equivalent (qualitative comparison) 18
Verification 1.- 6 products were produced in trial fillings to be tested 2.- Experimental design: A number of 15 replicates were analysed per product in both methods. As the results expected from trials might be absence or near 1 CFU/bottle, a number of positive controls were prepared D.anomala and D.bruxellensis and Zygosaccharomyces bailii. In order to make sure that postives with a suitable level of CFU would be available for verification. Preincubation was as in validation 3.- Eficacy 4.- Evaluation of accuracy/recovery and precision/uncertainty were calculated according to ISO 16140. 19
Verification results ISO 16140 Efficacy 90% Relative especificity 93% Relative sensitivity 86% Recovery/Accuracy Precision / Uncertainty >70% and difference in log counts with traditional under 0.015 Similar to traditional Both methods are equivalent (qualitative and quantitative) 20
CONTROL PLANS TRADITIONAL AND NEW Parameter Medium Temperature Days to result Days to result Forcing test As is 25-30ºC 9 6 Mesophile total aerobic count PCA 30ºC 3 + 3 = 6 days 3 + 3 = 6 days Yeast count WLNM 27ºC 3 + 6 = 9 days 3 + 3 = 6 days 21
Evaluation Criteria Shorter time to result? Yes 6 to 3 days incubation time Enumeration of microorganisms Yes Versatile for Membrane Filtration of different products: water, soft drinks, beer, beer mixes,? Yes No inhibition? Absence of inhibition Validated (similar / better in comparison) against standard? Yes Costs Long term: time, price/analysis / Cost Short term (price instrument, ) Easily compensated by shorter time in quarantine Ease of use (sample prep, ) / Ease of implementation (user friendliness, training, hands-on-time, no of samples per day ) Yes, very similar to actual protocols. Maintenance, repair Simple and relatively inexpensive 22
Furthermore Easy to implement due to small changes in laboratory habits All biological stability system uses the same basis: Same microorganism groups detected or not Same specifications Same historical data BAT or MAT (Most Appropriate technique)? Special points to be held in mind: 1. Spoilers isolated from the breweries 2. Pre-incubation of the validation microorganisms must be very well designed as they may vary their metabolism directly affecting the results of a validation (Dekkera) 3. Determine realistic specifications for quality acceptance of a batch 23
Bibliography Walker S. (2010), Bri_Quantum report http://www.briadvantage.com/instrumentcms/pdf/quantumreport.pdf Valton, N.; Caron, A.; Vicente, M. and Chollet, R. (2012) Nicht destruktiver schnellnachvweis mikrobieller Biershädlinge, Brauwelt, Nr 50, 1507-1509 Hutzler., M.; Wellhoener, U.; Tenge, C. and Geiger.E., (2008) Brauwelt International, IV, 206-208. 24
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