Evaluation of electrolyzed water for Clean-in-place of ... Water For NICMA 1-17-2… · EVALUATION OF ELECTROLYZED WATER FOR CLEAN-IN-PLACE OF DAIRY PROCESSING EQUIPMENT Yun Yu and

EVALUATION OF ELECTROLYZED WATER FOR CLEAN-IN-PLACE OF DAIRY PROCESSING EQUIPMENT

Yun Yu and R. F. RobertsPresented at NICMA

1-17-2017

Clean in place - CIP• The cleaning of items of equipment or pipeline

circuits without dismantling and with little orno manual involvement on the part of theoperator.

• CIP cleaning is accomplished by circulating solutions through the system.

(Tamime and Robinson, 1999)

4-step CIP procedure• Pre-rinse

– to remove residual milk (soil)• Alkaline detergent circulation

– to remove bound protein and fat deposit• Intermediate rinse

– to remove all traces of detergent and entrained soil

– cool the system down for next step• Sanitizing

– to kill bacteria, and to retard bacterial growth

Conventional CIP chemicals• Alkaline detergent

– NaOH• Saponification• Providing negative ions to disrupt soil

– Other components: sodium hypochlorite, surfactants

• Acidic Sanitizer– Chlorine

• Attacking bacterial cell wall and membrane– Other sanitizers: iodophors, peroxy acids

Membrane

NaCl Solution Water

Acidic Alkaline

Na++ H- NaOHCl2+H2O HOCl+H++Cl-

Principle of electrolyzed water generation(Modified from www.kangenwater-asia.com) {Kim,2000} {Huang, 2008}

Electrolyzed water (EW)

Electrolyzed water generator

(www.ewatersystems.com)

Conventional CIP Chemicals• Stored in

concentrated solutions

• Risk of burn on skin

• Cost

• Produced on-site

• Safe and easy to handle

• More cost effective– Water– Salt– Electricity

Electrolyzed Water

Acidic EO water

• Electrolyzed oxidizing (EO) water pH: 2.3~2.7, ORP: >1100 mV, Free chlorine: 80-100 ppm

• Application (as disinfectant)– Medical, dental equipment– Food industry– Food service (restaurant, public eating

facilities)

Fabrizio & Cutter, 2003, 2004, & 2005; KIM, Huang, Brackett, & Frank,2001; Kim, Hung, & Brackett, 2000; Rahman et al., 2010; Venkitanarayananet al., 1999, Ozer and Demirci, 2006

Alkaline ER water• Electrolyzed reducing (ER) water

– pH: 10.0~11.5– ORP: -800~ -900 mV

• Application in CIP (along with EOW)– for CIP of milking system on dairy farm

Walker, S.P., A. Demirci, R.E. Graves, S.B. Spencer, and R.F. Roberts. 2005. Response surface modelling for cleaning and disinfecting materials used in milking systems with electrolysed oxidizing water. Int. J. Dairy Technol. 58:65–73.

Walker, S., and A. Demirci. 2005. Cleaning milking systems using electrolyzed oxidizing water. Trans. ASAE. 48:1827–1834.

Dev, S.R.S., A. Demirci, and R. Graves. 2011. Mathematical modeling of CIP of milking systems using electrolyzed oxidizing water. Northeast Agric. Biol. Eng. Conf.

Wang, X., S.R.S. Dev, A. Demirci, R.E. Graves, and V.M. Puri. 2012. Electrolyzed oxidizing water for cleaning-in-place of milking systems on dairy farms – Performance evaluation and assessment. NABEC-CSBE/SCGAB 2012 Jt. Meet. Tech. Conf. Northeast Agric. Biol. Eng. Conf. Can.

Hypothesis:Electrolyzed water can be used as aneffective cleaning and sanitizing agent formilk processing equipment.

Research question:Can electrolyzed water serve as analternative to conventional CIP chemicalsfor CIP of milk processing equipment?

Objectives1. Construct a pilot-sale test system, and validate its performance and cleanability by CIP.

2. Develop CIP procedure using EW for cleaningof refrigerated milk storage tanks.

3. Optimize CIP procedure using EW for a tank used to heat milk.

CONSTRUCTION, CHARACTERIZATION AND VALIDATION OF TEST SYSTEM

Objective 1

Overview of pilot scale test system

Construction of test system

Test Vessel

TE

Jacket media

Jacket media

Agitator

Test Vessel

TE

Jacket media

Jacket media

Water

Watertank

Drain

CIP tank

Coil heat exchanger

Test Vessel

TE

Jacket media

Jacket media

Water

Watertank

CIP tank

Coil heat exchanger

Schematic of pilot CIP process test systemDrain

Construction of test system

Characterization of test system• Determine flow rate of each pump

setting

• Check coverage of 360° static spray ball using riboflavin test

Volumetric flow rate measurements

1.83.9

6.08.3

10.011.8

13.515.3

17.3

0.003.006.009.00

12.0015.0018.00

10 20 30 40 50 60 70 80 90

Mea

n flow

rat

e (L

/min)

VFD pump setting

Mean volumetric flow rates (L/min) at different VFD pump settings. The regression equation is: Flow Rate (L/min) = 0.18 + 0.19 (Pump Setting).

196.0 L/min 8.3 L/min

3.9 L/min

1.8 L/min 10 L/minRiboflavin solution (0.2 gram/L)

Experiment: Riboflavin coverage test

Before rinse 1.8 L/min for 1 min

3.9 L/min for 7 min 6.0 L/min for 17 min 8.3 L/min for 1 min

Residual riboflavin pattern at different flow rates

To validate CIP performance of the test system, efficacy of conventional CIP was evaluated after using the tank to heat milk

Validation

Pre-clean manually

Alkaline wash SanitizingPost-rinse

Soiling

Pre-rinse

Experiment: Validation of test system

Soiling:heat whole milk to 74°C (165°F) under agitation then hold for 15 min

Pre-rinse Post-rinse

Soiling

Pre-clean manually

Pre-clean manually:rinse -> wash -> rinse -> sanitize(water) (HC-10) (water) (XY-12)

Pre-rinse & Post-rinsewith water at room temperature for 3 min

Alkaline wash Sanitizing

Alkaline washwith Principal (3200 ppm)at 63°C for 15 min

Sanitizing:with XY-12 (100 ppm)at room temperaturefor 3 min

Cleanliness assessments

( ) Cleanliness assessments– ATP bioluminescence assay– Residual protein detection

Pre-clean manually

Alkaline wash SanitizingPost-rinse

Soiling

Pre-rinse

1

432

0

ATP Bioluminescence Method

– Quantitative result : relative light units (RLU)

www.charm.comPocket swab NovaLum ATP analyzer

50 cm2

Protein Detection Method

– semi-quantitative result : 1~4 score

www.hygiena.net

Result: Conventional CIP returned the test system to a cleaning condition

SamplingCommercial treatment

ATP Protein (μg)

After soiling Log10 RLU = 5.38 ± 0.27 Fail >100 Fail

After pre-rinse Log10 RLU = 5.60 ± 0.35 Fail >100 Fail

After post-rinse RLU = 0 ± 0 Pass 0~20 Pass

After sanitizing RLU = 0 ± 0 Pass 0~20 Pass

Cleanliness assessments of ATP and protein measurements after different steps of CIP using commercial chemicals following conventional procedure

For swab sampling…• Each 50 cm2 inner surface could be

swabbed only once• Does sampling location effect the

variation of cleanliness measurements?

Acid sanitizing with EOW

Test for swab sampling variability

Schematic of sampling locations

Soiling (heated)

Rinse

( ) ATP bioluminescence assayTo evaluate:– location effect,

swab three areas for each assessment

– repeatability between trials,repeated experiment in three times

– quench effect of EOW on ATPassay,

rinse with EOW before assessment

log10 RLU comparison

Average log10 RLU comparison between trials and CIP steps

AB AB ABA A ABAB B AB

0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

Trial #1 Trial #2 Trial #3

Log1

0 RL

U

After soilingAfter pre-rinseAfter sanitizing

Summary of objective-1• A test system was constructed and characterized

to evaluate CIP of milk processing equipment.• Flow rate of 8.3 L/min was determined to provide

adequate coverage.• Sampling location was not a factor in RLU value

variations.• The repeatability of the test system was

acceptable.• The test system was able to be cleaned using a

conventional CIP procedure after soiling with milk under heating.

CIP CLEANING USING ELECTROLYZED WATER FOR REFRIGERATED MILK STORAGE TANK

Objective 2

To mimic raw milk silo…• Inoculated bacterial cocktail:

– 10 ml Escherichia coli (9.1 log10 CFU/ml)– 10 ml Enterococcus faecalis (9.4 log10 CFU/ml)– 10 ml Pseudomonas fluorescens (9.1 log10 CFU/ml)

into 11.4 L (3 gal.) of pasteurized whole milk

• Bacterial level in inoculated milk, 6.7 log10 CFU/ml

Milk

Preparation of EW• pH and ORP • Chlorine

EO Water ER Water

pHORP (mV)

Total [Cl-]

(ppm)

Free [Cl-]

(ppm) pHORP (mV)

[OH-](ppm)

Minimum 2.19 1157 64.1 60.5 11.58 -897 64.7

Maximum 2.51 1182 126.7 122.0 11.78 -844 102.5

Average 2.38 1169 90.5 85.8 11.66 -872 77.7

St.Dev. 0.06 6.08 12.76 12.27 0.06 13.21 10.21

Chemical properties of EW

Alkaline wash SanitizingPost-rinsePre-rinse

Soiling

Experimental design

Soiling:Inoculated whole milk and hold at 2-4°C for 18 hours


Soiling

Pre-clean manually

Pre-clean manually:rinse -> wash -> rinse -> sanitize(water) (HC-10) (water) (XY-12) `0

Washing temperature and time of four treatments

TreatmentsWash

Chemical Temp. (°C) Time (min)

Pos Ctrl Principal 63 15

EW Long ER water 40 15

EW Short ER water 40 5

Neg Ctrl water 40 15



Sanitizing temperature and time of four treatments

TreatmentsSanitizing

Chemical Temp. (°C) Time (min)

Pos Ctrl XY 12 25 1

EW Long EO water 25 1

EW Short EO water 25 1

Neg Ctrl water 25 1


Soiling

Assess the cleanliness

Soiling:Inoculated whole milk to 2-4°C for 18 hours

( ) Cleanliness assessmentsATP bioluminescence assayResidual protein detectionMicrobial enrichment analyses

1

432

0

Means of RLU values. Tukey’s comparison were conducted between 16 means of log10(RLU) measurements. Means that do not share a letter indicates significant difference (P < 0.05)

RLU resultsa

b

d d

a

b

d d

a

b

c

d

a

bb

b

1

10

100

1000

10000

100000

1000000

After Soiling After Pre-rinse After Post-rinse Sanitizing

RLU

Pos ctrlEW longEW shortNeg ctrl

Protein (ug) After Soiling

After Pre-rinse

After Post-rinse

After Sanitizing

Pos ctrl 60 ~ 100 20 ~ 40 0 ~ 20 0 ~ 20EW long 60 ~ 100 20 ~ 40 0 ~ 20 0 ~ 20

EW short 60 ~ 100 20 ~ 40 0 ~ 20 0 ~ 20Neg ctrl 60 ~ 100 20 ~ 40 20 ~ 40 0 ~ 20

Protein results

Means of protein residue levels after different CIP steps

Microbial enrichment analyses

Micro APC (log10cfu/ml)

Soiling Pre-rinse Post-rinse Sanitizing

Pos ctrl 6.4±0.01 + + - -EW long 6.5±0.08 + + - -EW short 6.6±0.11 + + - -Neg ctrl 6.6±0.09 + + - -

Microbiological enrichment results after different CIP steps

Incubate at 30°C for 48 h

Swab 50 cm2

using sterile alginate swab

+ -

Summary of objective-2• A CIP procedure was developed for cleaning

of refrigerated milk storage tank:Pre-rinse Wash Post-rinse Sanitizing

Agent Water ERW Water EOWTemperature RT 40°C RT 25°CTime 1 min 15 min 3 min 1 min

• So far…– The use of test system for evaluation CIP

procedure was validated.– A refrigerated milk tank was able to be

cleaned using EW.

CIP USING ELECTROLYZED WATER FOR HEATED MILK PROCESSING TANK

Objective 3


Soiling

Experimental design

Soiling:heat whole milk to 74°C (165°F) under agitation then hold for 15 min


Soiling

Pre-clean manually

Pre-clean manually:rinse -> wash -> rinse -> sanitize(water) (HC-10) (water) (XY-12) `


Soiling

Experimental designSoiling:

heat whole milk to 165 °F (74 °C) under agitation then hold for 15 min

Soiling

Alkaline wash Sanitizing

Code -1 0 1WashTemp (°C) 40 55 70WashTime (min) 5 15 25SaniTemp (°C) 25 35 45SaniTime (min) 1 5.5 10

4 factors, 3 levels

Response surface model• Box-Behnken design matrix:

27 trials including 3 center points

Trials Run order WTemp WTime STemp STime

1 2 40°C 5 min 35°C 5.5 min2 10 70°C 5 min 35°C 5.5 min3 26 40°C 25 min 35°C 5.5 min4 6 70°C 25 min 35°C 5.5 min5 18 55°C 15 min 25°C 1 min6 13 55°C 25 min 45°C 5.5 min// // // // // //

25 27 55°C 15 min 35°C 5.5 min26 8 55°C 15 min 35°C 5.5 min27 23 55°C 15 min 35°C 5.5 min


Soiling

Cleanliness assessmentsSoiling:

heat whole milk to 165 °F (74 °C) under agitation then hold for 15 min

( ) Cleanliness assessmentsATP bioluminescence assay (RLU 1~4)Residual protein detection (Protein 1~4)

1 432

RLU3 data analysis and modeling• Factors:

• WashTemp• WashTime

• Percentage RLU reduction (PRR): ((RLU2−RLU3)RLU2

)100%

• Transformed Response:(Natural logarithms transformation of PRR)

𝑙𝑙𝑙𝑙((RLU2−RLU3)RLU2

) (range: ≤0 )

Regression model

ln(RLU2 − RLU3

RLU2)

= −0.6038 + 0.014045 × WashTemp+ 0.0205 × WashTime − 0.0001 × WashTemp 2

− 0.0002 × WashTime 2 − [0.0002 × WashTemp× WashTime]

Factor P-valueWashTemp 0.068

WashTime 0.011

WashTemp2 0.195

WashTime2 0.128

WashTemp × WashTime 0.068

(R2 = 48%)

WashTemp

Was

hTim

e

656055504540

25

20

15

10

5

> – – – – – – – – – – <

-0.039 -0.027-0.027 -0.015

-0.015

-0.135-0.135 -0.123-0.123 -0.111-0.111 -0.099-0.099 -0.087-0.087 -0.075-0.075 -0.063-0.063 -0.051-0.051 -0.039

ln((RLU2-RLU3)/RLU2)

Contour Plot of ln((RLU2-RLU3)/RLU2) vs WashTime, WashTemp

After alkaline wash and post-rinse

CurHighLow0.98160

DNew

d = 0.98160

Targ: 0.0ln((RLU2

y = -0.0092

0.98160DesirabilityComposite

5.025.0

40.069.40

WashTimeWashTemp

[54.6497] [20.5043]

𝑙𝑙𝑙𝑙(𝑅𝑅𝑅𝑅𝑅𝑅𝑅 − 𝑅𝑅𝑅𝑅𝑅𝑅𝑅

𝑅𝑅𝑅𝑅𝑅𝑅𝑅)

Optimization of ER water wash stepOptimum wash temperature = 54.6°C

Optimum wash time = 20.5 min

Predicted RLU reduction = 99.08%

Conventional ctrl:100% RLU reduction

Conclusions• The test system developed was suitable for

evaluating CIP cleaning of milk tanks.

• It was possible to CIP clean a refrigerated milk storage tank using EW.

• It was possible to CIP clean a tank used to heat milk using the optimal condition, but the system was not as effective as when cleaned with conventional chemicals.

Future research• Consider methods to improve the cleaning

efficacy of alkaline ER water – add surfactants– Increase alkalinity– Add other detergent builder

• To improve acidic EO water– Use neutral electrolyzed water with less corrosive

characteristic– Add corrosion inhibitors, such as silicates to acid

EO water to prevent corrosion

Documents

Evaluation of electrolyzed water for Clean-in-place of ... Water For NICMA 1-17-2… · EVALUATION OF ELECTROLYZED WATER FOR CLEAN-IN-PLACE OF DAIRY PROCESSING EQUIPMENT Yun Yu and