Biofilms and microbial testing in the dairy industry'

1
Biofilms and microbial testing in the dairy
industry.

• S. Flint Guelph University
• 2nd April 2007

2
Outline

• Introduction to Fonterra
• Biofilm research group
• Features of dairy biofilms
• Current strategies to control biofilms
• Alternative strategies
• Current research
• Alternative test methods

3
Introduction to Fonterra
4
Long History, New Beginning
1927 Amalgamated Dairies established in London
to market New Zealand butter and cheese
2000 Milk supply by two major companies New
Zealand Dairy Group Kiwi Cooperative Dairies
1963 New Zealand Dairy Board established
1800s Dairying in NZ began
2001 Fonterra formed
2006 Total business aligned under one brand
1882 SS Dunedin sailed to London with first
refrigerated shipment of butter in the world
1930 Many towns have dairy factories in New
Zealand
1970-1990 Dairy factories merged
5
Fonterra Today

• Co-operative owned by 11,600 supplier
  shareholders
• Total Sales of 12.7 billion
• Process nearly 2 billion kilograms of milk solids
  annually
• Export 95 of production to more than 100 markets
  around the world
• Employ 18,600 people worldwide

6
Innovation in Fonterra
CUSTOMERS
FonterraIngredients
FonterraSpecialty Products
FonterraBrands
FonterraFood Service
Ingredients Innovation
Brands Innovation
Group Innovation
Manufacturing Innovation
Fonterra Group Manufacturing
7
Palmerston North
Population 73,000
City Square
8
Globally Recognised Facilities People
9
One of Worlds Largest Registered Dairy Pilot
Plants
10
A Cluster of Science Organisations
11
Biofilm research groups
12
Biofilm Research Groups

• Steve Flint - Fonterra

• Phil Bremer - University of Otago

Dunedin
John Brooks Massey University
13
Biofilm Research at Fonterra current strategy

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

14
Biofilm research at Fonterra
Laboratory reactor system
15
Biofilm research at Fonterra
Robbins device
16
Biofilm research at Fonterra
Bactrac impedance system
17
Features of dairy biofilms
18
Features of Dairy Biofilms

• Process biofilms
• Generally single species
• Limited thickness and density ( 106 cells/cm2)
• Rapid growing
• Environmental biofilms
• Mixed species
• Thick layer

19
Process Biofilms

• Streptococcus thermophilus
• Pasteuriser plate heat exchangers (PHEs)
• Recycle loops in scraped surface plants
• Thermophilic bacilli
• Preheating and evaporation sections of milk
  powder plants
• Pasteuriser PHEs
• Hot separators
• Cream heaters in anhydrous milk fat (AMF) plants
• Hot ultrafiltration plants

20
Process Biofilms S. thermophilus

• Thermoduric
• Optimum growth temperature of 40-45ºC
• Grow on the cooling side of pasteurisers
• Causes off flavours excess open texture in
  cheese

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Process biofilms Thermophilic bacilli

• True thermophiles
• Geobacillus spp.
• Anoxybacillus flavithermus
• Facultative thermophiles
• Bacillus spp.
• Optimum growth temperature of 40 65 ºC
• Difficult to eliminate
• Fast growth rate (approximately 15 - 20 min
  generation time)
• Heat and chemical resistant spores
• Wide temperature growth range

22
Environmental Biofilms

• Membranes in reverse osmosis (RO) and
  ultrafiltration (UF) plants
• Mixed species
• Bacillus sp.
• Enterobacteriaceae, Pseudomonas
• Lactococcus
• Blastoshizomyces captitatum
• Limits operating time
• Contamination of product

23
Strategies to control biofilms
24
Current strategies to control dairy biofilms

• Frequent cleaning
• Use of sanitisers
• Cold processing
• Ultrafiltration
• Centrifugal milk separation
• Reducing surface area over the optimal
  temperature zone for biofilm growth
• Direct steam injection (DSI)
• Steam infusion.
• Dual PHEs

25
Current cleaning procedures

• Water rinse
• NaOH (1.5) 75ºC
• Water rinse
• Nitric acid (0.5) 70ºC
• Water rinse
• Sanitiser flush - 200 ppm free available chlorine
  (FAC)
• Water rinse

26
Alternative strategies

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

27
Alternative strategies

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

28
Cleaning procedures activated water

• Cleaning sanitising solution
• Salt solution converted to
• Catholyte (pH 11.0
• NaOH 0.1)
• Anolyte (pH 2.5
• FAC 500 ppm)

29
Activated water potential applications

• Replacing NaOH with Catholyte for cleaning
• Replacing hypochlorite sanitisers with Anolyte to
  control bacteria
• Improve control of bacteriophage with Anolyte

30
Activated water - catholyte for biofilm removal

• Method based on microtitre plate assay (Pitts
  et al. 2003)
• Microtitre plate incubated with culture to allow
  biofilm to form
• Serial dilutions of catholyte into microtitre
  plate
• Biofilm stained with crystal violet then rinsed
  with water ethanol
• Biofilm remaining detected by measuring the OD
  _at_540nm

Bacteria
Catholyte concentration
31
Effect of catholyte on biofilm removal
32
Activated water - catholyte for biofilm removal

• Effective at concentrations of gt10 (equivalent
  to 0.01 NaOH)
• Some variation between different bacteria

33
Activated water - anolyte as a sanitiser

• Method
• Serial 2-fold dilutions of anolyte into
  microtitre plate
• 106 cells of bacteria added
• Incubation 1 h at room temperature
• Survivors detected by Alma Blue (Redox indicator)

Bacteria
Anolyte concentration
34
Activated water - anolyte as a sanitiser

• Efficacy less than sodium hypochlorite based on
  FAC levels
• Minimal effect of organics
• Biofilm inactivation needs higher concentration
  than planktonic cells

35
Activated water - anolyte efficacy against
bacteriophage

• Method
• Phages exposed to activated water at room
  temperature
• Surviving phage numbers were determined after
  exposure times of one minute and five minutes
• Sodium hypochlorite was used as the control
  sanitiser
• Up to 6 log reduction
• Superior to sodium hypochlorite at an equivalent
  FAC level

36
Alternative strategies

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

37
Altering surfaces

• Coating stainless steel with a protein
• Using impregnated stainless steel
• Molecular brush
• Anti-stick solutions

38
Coating surfaces with a protein

• Coating stainless steel with different proteins

39
Coating surfaces with lysozyme
Rinse manufacturing plant with lysozyme
Lysozyme removes biofilm
40
Coating surfaces with lysozyme

• 1 Lysozyme effective for 18 h
• Re-use of lysozyme solution at least 10 times
• Storage life of the solution at 10ºC is at least
  50 days
• Not currently used because of risk of lysozyme in
  product

41
Impregnated stainless steel

• University of Surrey provided samples
• Tested attachment of thermo-resistant
  streptococci to ion impregnated samples and
  compared with 316 stainless steel
• CrN 4 log reduction
• CrC 2 log reduction

42
Surface modifications to stainless steel

• Kenifine Coatings - proprietary Nickel plated
  surfaces to produce sanitary stainless steel
  surfaces
• 1 log reduction in attachment
• Electroless Ni-P based composite coatings
  prepared by University of Auckland
• Preliminary trials no difference in attachment

43
Molecular brush

• Developed by Wageningen University

Complex coacervate micelles unfold on surface
Brush layer
Substrate
Complex coacervate layer
Figure adapted from a presentation by de Kiezer
(2006)
44
Molecular brush
Control
Treated
No difference
Pseudomonas fluorescens
2 log reduction
Streptococcus thermophilus
45
Anti-stick solutions

• Commercial anti-stick preparations (Automate
  Process 731B from Ecolab) containing
• Sodium dichloroisocyanurate
• Sodium metasilicate
• Traditionally used in butter manufacturing plants
• Preliminary trials - treated stainless steel
  samples resulted in 2 log reduction of
  Geobacillus sp. attachment

46
Alternative strategies

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

47
Disrupting technologies temperature spike

• Control of Streptococcus thermophilus in
  pasteurisers

48
Disrupting technologies temperature spike

• The preheat section of the pasteuriser was
  increased from 34ºC to 55C for 10 min every hour
  for 20 h.

(s)
49
Disrupting technologies temperature spike

• Temperature spike controlled S. thermophilus
  growth

50
Current research
51
Biofilm Research at Fonterra current strategy

• Alternative Cleaning systems (CIP chemicals)
• Bioactive Surfaces (substrate focus)
• Disruptive Technologies (process/environment
  manipulation)
• Technical advancement (microbial interactions)

52
Current research (Jon Palmer)

• Identification of cell surface adhesins on A.
  flavithermus (Jon Palmer)
• Successful isolation of a mutant with a 10-fold
  decrease in attachment
• Detection of two proteins not present in the
  mutant

53
Current research (Brent Seale)

• Understand factors controlling thermophilic spore
  attachment
• Spores attach quickly to surfaces.
• Attach in similar numbers to all surfaces
• Increasing ionic strength increases attachment
• Developing anti-adhesive stainless steel surfaces
• Examine proprietary metallic surfaces and polymer
  coatings

54
Current research (Xumei Tang)

• Reverse Osmosis / Ultrafiltration biofilm
  investigation
• Isolate, identify and characterise microflora
  from membrane samples
• Develop a laboratory model which can mimic
  factors (e.g. pH, temperature, pressure,
  concentration) found in RO/UF membrane plants
• Investigate critical factors for biofilm
  colonisation
• Identify strategies to reduce biofilm formation

55
Future work?

• Altering surfaces
• immobilising lysozyme onto UF/RO membranes
• Disruptive technology
• pulsed flow
• Bacteriophage in biofilms

56
Acknowledgements

• John Brooks
• Phil Bremer
• Frank van de Ven
• Sara Scott
• Kylie Walker
• Bryan Waters

• Anita Clarke
• Jon Palmer
• Brent Seale
• Xuemei Tang

57
Method Development
58
Rapid Micro Methods - Categories

• Methods for typing/characterisation/detection
• Well researched
• Methods for enumeration
• Not so well researched

59
Faster, Smarter (cheaper)

• Fonterra spends x per year on testing milk
  powder.
• Faster Smarter a series of initiatives to
  reduce costs for manufacturing.
• Faster test results means a reduction in test
  numbers.
• Greater control over processing means less need
  for exhaustive testing on final product.
• Rapid trace back when things go wrong.

60
Industry wish list for rapid bacterial
enumeration assays

• Lower detection limit 1-100 cells/ml
• Specificity selective
• Assay time 30 minutes max
• Protocol Simple
• Measurement Direct
• Format Automated
• Cell viability Yes
• Cost Cheap
• Precision /- 0.2 Log

61
Potential rapid enumeration methods - studies at
Fonterra (thermophile focus)

• Impedance /metabolite based assays
• ATP assays
• Quantitative PCR
• BioGeorge in-line monitor
• Dip-stick assays
• Flow Cytometry
• Biosensors

62
Potential rapid enumeration methods - studies at
Fonterra (thermophile focus)

• Impedance /metabolite based assays
• ATP assays
• Quantitative PCR
• BioGeorge in-line monitor
• Dip-stick assays
• Flow Cytometry
• Biosensors

63
Flow cytometry most promising

• Flow cytometry provides a unique opportunity to
  achieve this.
• Results available in under 2 hours.
• Plate counts 2 5 days.
• Flow cytometry is well established in other
  industries.
• Cosmetics
• Raw milk
• Fruit juices
• Brewing and winemaking

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Flow Cytometry - BactiFlow

• Detection Limit 100 - 1000 cells/ml
• Specificity Non selective
• Assay time 1-3 h
• Protocol Simple
• Measurement Direct
• Format Semi Automatic
• Cell viability Yes
• Cost 100,000
• Precision lt /- 0.5 log

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Flow Cytometry - outline.

• Direct fluorescent labelling of viable
  micro-organisms.

Cell Membrane
Substrate
66
Flow Cytometry - outline.

• Fluorescent labelling

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Flow Cytometry - outline.

• Sample hydro-dynamicallyfocused.
• Laser excites fluorescently labelled cells.
• Fluorescent emission signal split up into red
  and green wavelengths.
• Fluorescence information collected by sensitive
  photo-multipliers and converted into data.

Labelled Sample
Green Detector
488 nm laser
Red Detector
Waste
68
Flow Cytometry - outline.
Sample placed into holder.
Data analyzed by computer.
Easy to interpret results displayed on screen.
69
Flow Cytometers.

• D-Count (AES Chemunex, France)
• Fully automated
• High throughput (50 samples/hour)
• Bactiflow (AES Chemunex, France)
• Manual sample preparation
• Lower throughput (15 samples/hour)
• RBD 3000 (AATI, USA)
• Fully automated
• Low throughput (6 samples/hour)
• Higher sensitivity

70
Fonterra and flow cytometry.

• Bactiflow instruments in routine usage
• Fonterra Clandeboye (2005)
• Fonterra Whareroa (2004)
• Fonterra Te Rapa (2005)
• Fonterra Innovation (PN) (2004)
• D-Count instrument in routine usage
• Fonterra Whareroa (2001)
• RBD 3000 instrument (research)
• Fonterra Innovation (PN) (2006)

71
Methods

• Total Viable Count (Protocol B)
• Total Viable Count for milkpowder (Protocol A)
• Thermophile Count (Protocol T)
• Mould Count (Protocol Y)
• Mesophile Count (Protocol C)

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Protocol B (TVC Basic)Outline

• Fastest, simplest test for bacterial enumeration.
• Add labelling reagents (buffer, counterstain,
  fluorochrome)
• Incubate for 20 minutes at 30C.
• Add reducing agent to quench unbound
  fluorochrome.
• Read each sample in the instrument (1 minute per
  sample)

73
Protocol C (TVC Basic) Limitations

• Does not distinguish between thermophilic/mesophil
  ic bacteria.
• Milk powders have very high background
  fluorescence.

74
Protocol A (TVC Advanced)Outline

• Adapted from Protocol B (TVC Basic)
• Pre-treatment at 37C/10 minutes with A31
  proprietary chemical enzyme based.
• Centrifiltration Pre-treated sample passed
  through 25µm nylon mesh filter, and centrifuged
  at 1000g for 8 minutes.
• Successfully reduces background fluorescence for
  most milkpowders.

75
Protocol A (TVC Advanced)Performance
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Protocol A (TVC Advanced)Limitations

• Does not distinguish between thermophilic/mesophil
  ic bacteria.

77
Protocol T (Thermophile Protocol)Outline

• Adapted from Protocol A by Fonterra Innovation
  (Flint et al. 2005)
• Changed pre-treatment temperature to 62.8C from
  37C.
• Changed labelling temperature to 40C from 30C.

78
Protocol T (Thermophile protocol)Performance
79
Protocol Y (Mould Protocol)Outline

• Adapted by Fonterra innovation from Chemunex
  standard method to count yeast and moulds in
  cultured dairy products.
• Changed sample size from 1ml to 10mL in order to
  achieve greater sensitivity. (Fonterra limits
  around 50 cfu/g for mould)

80
Protocol Y (Mould Protocol)Performance
81
A cool reception

• Traditional plate count method has been in use
  for decades at the 3 main laboratories.
• A potentially very beneficial opportunity exists
  in using Flow Cytometry one test instead of two.
• Reduced costs
• Even faster grading
• Potential changes to the fundamental way testing
  is carried out has encountered mixed responses
  from the industry.
• Scepticism.
• Resistance to change.

82
A cool reception

• Bactiflow technology requires competent
  laboratory trained staff.
• Original plan for 10 Bactiflows throughout
  Fonterra manufacturing sites, 3 currently.
• On site validation trial problems poor
  correlations initially.
• Poor sample selection sensitivity of test is
  poor below 103 counts/g.
• Inconsistent testing of both reference and flow
  cytometry methods.
• Resource and space issues.

83
More challenges.

• Flow cytometers are expensive to buy and to run.
• Management want business as usual immediately.
• Only one supplier of equipment and reagents.
• Manufacturer based in France.
• Plate count method is imperfect.

84
Future

• Instruments at factories without laboratories.
• More products to be tested. E.g. whey, lactose,
  butter, cheese etc
• More specialised testing. E.g. enterobacteriaceae,
  spore testing, specific mesophile test

85
Rapid Bacterial Detection (RBD) 3000
86
Sample trays
Un-attended analysis of 42 samples
Priority tray
87
Different labelling options

• Biomass enumeration
• Total Viable Organism (TVO) count
• Dead cell enumeration
• Antibody detection
• rRNA detection

88
Advantages

• Automated labelling system similar to the
  D-count
• More labelling options available
• Different protocols on one tray
• Addition of trays at any time
• Priority tray

89
Disadvantages

• Not proven for milk product assays
• Requires air pressure system

90
Acknowledgements

• Kylie Walker
• Sara Scott
• Bryan Waters
• Michelle Dwyer
• Desmond McGill

91
Final Slide This slide appears at the start of
each presentation. This slide is optional.
92
Thank you