Effect of Cleaning In Place (CIP) on the germination of Bacillus cereus spores

isolated during Extended Shelf Life Milk processing

Chané Kruger Supervisor: Prof Elna M. Buys

Department of Consumer and Food Sciences

University of Pretoria

51st SASDT Symposium,

Pretoria, South Africa, 24-25 April 2018,

Extended Shelf Life (ESL) Milk

Bulk raw milk

Standardisation Homogenisation

Bactofugation Cold filling and

aseptic packaging

Pasteurisation 15s @ 73oC

Packaged Extended Shelf Life Milk

Predominant bacteria in the nozzles of aseptic ESL milk filling machines for isolates (n=80) sampled after CIP over four weeks

Khoza (2015)

Group 1

Group 2

Derived from Mugadza & Buys, International Dairy Journal, 2017

http://circleofdocs.com/wp-content/uploads/2016/08/RAw-Milk.jpg

http://www.dairy-bottle-packaging-machines.com/our-dairy-filling-technology.html

70oC

SUPERKLENZ Liquid (0,5%) wash for 10

min

Clean cold water rinse

70oC

CIP ACID (1%) wash for 30

min

Clean cold water rinse

70oC

SUPER-SAN (1%) for 10

min

Standard CIP

Process

Study Objective

To determine the effect of simulated CIP on

the germination and attachment of Bacillus

cereus spores isolated during Extended Shelf

Life (ESL) milk processing with the aim of

improving the shelf life and safety of ESL

milk.

• Flow Cytometry

• Growth kinetics

• Biofilm assay

• SEM (stainless steel coupons)

• TEM

FCM Principles

Damaged, unstained

Intact, unstained

Intact, stained

Damaged, stained

FCM-derived contour plots of the green fluorescence versus

side scatter intensities of SYTO 9-stained spores of B.

cereus spores following CIP treatment

BC10

Unstained 0,16%

Intact 0,40%

Damaged 98,7%

BC17

Unstained 0,08%

Intact 0,11%

Damaged 98,8%

BC24

Unstained 0,06%

Intact 0,39%

Damaged 98,8%

Lag phase of untreated and CIP treated B. cereus spore strains (BC10, BC17 & BC24)

Lag phase longer in CIP treated spores BC10 and BC17 survived CIP treatment Strain effect significantly influenced lag phase (p≤0,05)

Strain + CIP treatment was significant with regards to variation

Germination and growth of untreated B. cereus spore strains (BC10, BC17 & BC24) following CIP treatment

Growth rate was significantly higher in BC10 & BC24 CIP treated spores (p≤0,05)

Strain effect was responsible for variation in growth rate Strain effect + CIP treatment was significant with regards to variation

Biofilm formation of untreated and CIP treated B. cereus spore strains (BC10, BC17 & BC24) over 24h

Biofilm formation of untreated vs. CIP treated spores significantly different (p≤0,05)

Significant difference between strains with regards to biofilm formation

Untreated spores (SEM)

BC17 Untreated BC10 Untreated BC24 Untreated

Attachment Attachment

Attachment

Biofilm formation

&EPS

Untreated spores (TEM)

EX - Intact Exosporium OC - Outer coat IC - Inner coat CX-PCW - Cortex and primordial cell wall IM - Inner membrane and C - Core

BC10 Untreated BC17 Untreated BC24 Untreated

200 nm

EX

OC

IC

CX-PCW

IM

C EX

C

OC

IC

IM

CX-PCW

200 nm

Intact endospore

Strain BC10 CIP treated

Attachment

Attachment

Damage

SEM SEM

Inner coat

Outer coat

Leaked cell components

Cortex & primordial cell wall complex

200 nm TEM

Strain BC17 CIP treated

Attachment

Attached Bacillus

cereus spores

Damage SEM SEM

Inner coat

Leaked cell components

Cortex & primordial cell wall complex

Outer coat

Exosporium

Core

Inner membrane

500 nm SEM

Strain BC24 CIP treated

Attachment Attachment

Damage

Outer coat Inner membrane

SEM SEM

Inner & outer coat

Leaked cell components

Cortex & primordial cell wall complex

Exosporium

Inner membrane

Core

500 nm SEM

Strain effect?

Derived from Mugadza & Buys, International Dairy Journal, 2017

Conclusions CIP damaged at least 98% of all spores in all three B. cereus

strains, and 0,1% of spores remained intact following CIP

treatment.

Spores were still capable of germination, regrowth and biofilm

formation following CIP treatment.

Spores were capable of attaching to stainless steel surface after

CIP treatment.

Acknowledgements