inactivation of listeria monocytogenes by disinfectants ... · inactivation of listeria...

Inactivation of Listeria monocytogenes by Disinfectants andBacteriophages in Suspension and Stainless Steel Carrier Tests

N CHAITIEMWONG W C HAZELEGER AND R R BEUMER

Laboratory of Food Microbiology Wageningen University PO Box 17 6700 AA Wageningen The Netherlands

MS 14-151 Received 31 March 2014Accepted 16 July 2014

ABSTRACT

To simulate food contact surfaces with pits or cracks stainless steel plates with grooves (depths between 02 and 5 mm)

were constructed These plates were artificially contaminated with Listeria monocytogenes in clean conditions with organic

soiling or after 14 days of biofilm formation after which inactivation of the pathogen by Suma Tab D4 (sodium

dichloroisocyanurate 240 and 300 mgliter) Suma Bac D10 (quaternary ammonium compound 740 mgliter) and bacteriophage

suspension (Listex P100) was determined Both chemical disinfectants performed well in suspension tests and in clean carrier

tests according to the European standard with a reduction of more than 5 and 4 log units respectively of Listeria cells after 5 min

of contact time However for the plates with grooves the reduction could not meet the standard requirement although a higher

reduction of L monocytogenes was observed in the shallow grooves compared with the deeper grooves Furthermore presence of

food residues and biofilm reduced the effect of the disinfectants especially in the deep grooves which was dependent on type of

food substrate Bacteriophages showed the best antimicrobial effect compared with the chemical disinfectants (sodium

dichloroisocyanurate and quaternary ammonium compound) in most cases in the shallow grooves but not in the deep grooves

The chlorine based disinfectants were usually less effective than quaternary ammonium compound The results clearly

demonstrate that surfaces with grooves influenced the antimicrobial effect of the chemical disinfectants and bacteriophages

because the pathogen is protected in the deep grooves The use of bacteriophages to inactivate pathogens on surfaces could be

helpful in limited cases however use of large quantities in practice may be costly and phage-resistant strains may develop

Listeria monocytogenes a ubiquitous pathogenic bac-

terium is widely found in the natural environment including

plants soil sewage water and also in human and animal

feces (27 29 67) Due to this widespread occurrence in

nature the pathogen can enter food processing plants via

several vectors such as personnel equipment raw materi-

als pest animals and process waste (67) The pathogen is

able to adhere to and form biofilms on several surface

materials such as stainless steel rubber and plastic which

are used in food processing facilities (11 14 49)Furthermore the bacterium has been frequently isolated

from wet and cold spots (23) as well as from surfaces and

equipment that are difficult to clean (18 33 48)In food processing plants cleaning and disinfection are

the most important steps to eliminate dirt and microorgan-

isms that accumulate on food-contact surfaces equipment

walls and floors Improperly cleaned and disinfected

surfaces can lead to cross-contamination when food

products come into contact with the surfaces or when dirt

and microorganisms are dispersed during processing

Additionally biofilms can be formed on surfaces particu-

larly in pits or cracks that are difficult to clean Once the

biofilm is formed it is problematic to remove the pathogens

by normal cleaning and disinfecting procedures (8 54)because abraded surfaces can provide protection to firmly

attached bacteria Moreover microorganisms inside soil are

protected against disinfectants (2 9)Various disinfectants such as peracetic acid quaternary

ammonium compounds (Qacs) hypochloride sodium

dichloroisocyanurate (SDC) and iodofores have demon-

strated their efficacy against L monocytogenes in both

suspension (12 24 59 69) and surface tests (2 10 41)where the antimicrobial activity of the disinfectants was

more effective in suspension than in surface tests In a

suspension the disinfectants come into contact with free-

floating cells whereas on a surface the disinfectants can

reach the organisms only from one side (28 36 63)Moreover the effectiveness of the disinfectants will be

reduced when the disinfectants are inactivated by or bind to

organic matter present on the surface resulting in less

disinfectant coming into contact with the microorganism

(32 68)Apart from chemical disinfectants bacteriophages

(viruses that infect bacteria) have been used to control

bacteria in all stages of food production from preharvest to

finished products (22 44 47 52 57) Although some

studies on the efficiency of bacteriophages in removing

bacterial biofilm cells have been performed with Esche-richia coli (26) L monocytogenes (38) and Pseudomonas

Author for correspondence Tel z317-484982 E-mail

wilmahazelegerwurnl

2012

Journal of Food Protection Vol 77 No 12 2014 Pages 2012ndash2020doi1043150362-028XJFP-14-151Copyright G International Association for Food Protection

fluorescens (60) still little information is available on the

efficiency of bacteriophages on the inactivation of patho-

gens in biofilms (43 65 66)The effectiveness of disinfectants against bacteria on

smooth surfaces such as stainless steel coupons or chips (17 12 32 34) buna-N rubber (58 64) Teflon (55)polyester fabrics and polyurethane surfaces of conveyor

belt (64) has been well documented however only few

studies have been performed on surfaces with pits or

grooves The purpose of this study was to simulate the

practical situation where a Listeria biofilm is formed in

cracks in the equipment and to investigate the effect of two

commercial disinfectants based on chlorine and Qacs and a

Listeria bacteriophage product against L monocytogenes in

suspension and in carrier tests with flat and rutted (grooved)

surfaces

MATERIALS AND METHODS

Bacterial strains L monocytogenes LF 38 (serotype 12a

isolated from cooked ham) LF 36 (serotype 12b isolated from

cooked sausage) and LF 29 (serotype 4e isolated from cooked

sliced sausage) were obtained from The Netherlands Food and

Consumer Product Safety Authority (Wageningen The Nether-

lands) Stationary-phase cultures of all strains (24 h 30uC) were

stored in 1-ml cryo vials (Greiner Bio-one Frickenhausen

Germany) with 25 (volvol) glycerol (Fluka-Chemica Buchs

Switzerland) in brain heart infusion (BHI) broth (Difco BD

Sparks MD) and 2-mm glass beads (Emergo Landsmeer The

Netherlands) at 220uC For each experiment one glass bead of

each strain was inoculated to a separate tryptone soy agar plate

(Oxoid Basingstoke England) and then incubated at 30uC for

24 h A single colony was picked inoculated into BHI broth and

incubated at 30uC for 24 h Test suspensions were prepared by

mixing cultures of the three L monocytogenes strains where 2 ml

of overnight cultures of each strain (BHI broth 30uC for 24 h)

were combined to give a 6-ml mixed culture of L monocytogeneswhich was serially diluted in peptone saline solution (PSS 85 g

liter NaCl Merck Darmstadt Germany) and 1 gliter Neutralised

Bacteriological Peptone (Oxoid) to obtain appropriate dilutions for

inoculation

Bacteriophages Listeria bacteriophage Listex P100 was

obtained from Micreos Food Safety BV (Wageningen The

Netherlands) as a suspension with a concentration of 1011 PFU

ml Listex P100 was serially diluted with 9 ml of PSS to obtain

109 PFUml The phage suspensions were freshly prepared

before use to prevent deterioration during storage The

concentration of the phages was determined by a double agar

overlay method according to Carey-Smith et al (19) using BHI

agar (BHI plus Agar technical Agar no 3 12 and 4 gliter for

bottom and top layer respectively Oxoid) and Listeria innocua(serovar 5 Micreos Food Safety BV) as the indicator organism

The plates were incubated at 30uC for 24 h and plaques were

counted

Test surfaces Two types of surfaces were used in this study

stainless steel coupons (AISI type 304 standard ODS Bare-

ndrecht The Netherlands) with a total surface (both sides) of

38 cm2 and stainless steel plates (AISI type 304 standard ODS)

measuring 12 by 10 by 1 cm in which two identical sets of grooves

(5- 2- 1- 05- 02-mm depth 1-mm width and 10-cm length)

were cut (Fig 1)

Disinfectants Suma Tab D4 (SDC Diversey Inc Utrecht

The Netherlands) and Suma Bac D10 (Qac Diversey Inc) were

used in this experiment at concentrations of 240 mgliter for SDC

(pH 6) and 740 mgliter for Qac (pH 101 to 103) according to the

manufacturerrsquos recommendations Sterile water was used as a

control in the experiments The disinfectants were freshly prepared

shortly before use on the day of the experiments to prevent

deterioration during storage

Neutralizers For the chemical disinfectants inactivation

liquid was used as a neutralizer (6) containing 1 gliter L-histidine

(Merck) 3 gliter lecithin (VWR International Poole England)

5 gliter sodium thiosulfate pentahydrate (Na2S2O3-5H2O VWR

International) 30 mlliter Tween 80 (Merck) and 10 mlliter

phosphate buffer The phosphate buffer was prepared by dissolving

34 g of potassium dihydrogen phosphate (KH2PO4 Merck) in

500 ml of demi water after adjustment of the pH to 72 with 1 M

sodium hydroxide (NaOH VWR Prolabo Lutterworth England)

the volume was filled with 1 liter of demi water There were 9 ml

of inactivation liquid that were dispensed into test tubes and

sterilized at 121uC for 15 minutes

For Listeria bacteriophages a dead cell suspension of Linnocua (serovar 5) was used as a neutralizer (37) which was

prepared by adding 1 ml of an overnight culture (BHI broth 30uC

24 h) of L innocua to 9 ml of PSS which were subsequently

sterilized for 15 min at 121uC

Preparation of food residues Modified atmospherendashpacked

cooked ham vacuum-packed smoked salmon and chopped

endives were bought from a local supermarket kept in a

refrigerator at 4 to 7uC and used within 24 h To prepare a 10

FIGURE 1 Pictures of the stainless steel plate (12 by 10 by 1 cm)with 2 sets of 5 grooves (5- 2- 1- 05- 02-mm depth 1-mmwidth 10-cm length) used in carrier tests top (a) and side view (b)

J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2013

suspension of the food residue 10 g of each food was aseptically

placed in a stomacher bag with 90 ml PSS and subsequently

homogenized in a stomacher (Stomacher 400 Circulator Seward

England) for 1 min at 260 rpm kept at 4 to 7uC and used within 4 h

One milliliter of ultrahigh-temperature-processed milk was asep-

tically added to 9 ml PSS vortexed and used immediately Prior to

the test all food residues were tested for the presence of Lmonocytogenes according to ISO 11290-11996Amd 12004 (5) to

verify if the food matrix was free of L monocytogenes

Suspension tests (case 1) The tests were performed

according to the European standard EN 10402005 (6) An

overnight culture of L monocytogenes with or without 10 food

residue was added to each disinfectant at recommended concen-

trations at room temperature for 5 min of contact time The tests

were performed in triplicate for each disinfectant on different days

Figure 2 shows a diagram of all tests that were performed in this

study

Carrier tests The tests were performed on smooth surfaces

(case 2) and surfaces with grooves (case 3) Before the experiments

started the stainless steel coupons and plates with grooves were

soaked overnight in anionic-active detergent (Dreft Procter amp

Gamble Netherlands BV Rotterdam The Netherlands) Thereafter

both surfaces were rinsed with hot water (70 to 80uC) and air dried

The coupons were put in a glass container covered with aluminum

foil while the plates were wrapped in paper and then all were

sterilized at 121uC for 15 min

Carrier tests on smooth surfaces (case 2) (4) To inoculate

the stainless steel coupons 10 ml of the test suspensions (109

CFUml resulting in 64 log CFUcm2) were put on one side of

the coupon and allowed to air dry for 1 h in a biological safety

cabinet After inoculation of the stainless steel coupons these were

placed in a 12-well plate (Greiner Bio-One BV Alphen ad Rijn

The Netherlands) containing 1 ml of SDC (240 mgliter) or Qac

(740 mgliter) and left at 1- 2- and 5-min contact time at room

temperature The experiment was repeated three times on different

days for each disinfectant

Carrier tests on surfaces with grooves (case 31) To

inoculate the surface with grooves (case 31) 10 ml of test

suspension (107 CFUml resulting in 6 log CFUcm2) with or

without 10 food suspension were put on the plates and evenly

spread over the surfaces to fully fill the grooves by using a sterile

sponge The plates were then kept at room temperature for 15 min

and 24 h to simulate wet and dry conditions respectively After

inoculation of the plates 240 mgliter SDC or 740 mgliter Qac

were sprayed on the plates (5 ml) by using a sterile spray bottle

(perfume spray bottle Etos Wageningen The Netherlands) and

left to achieve 5- 10- 15- or 20-min contact time at room

temperature The experiment was repeated two times on different


Carrier tests on surfaces with grooves in presence of abiofilm (case 32) To mimic the situation in a food processing

plant with biofilm formation in cracked equipment a biofilm was

grown by daily inoculating a diluted overnight culture of the three

L monocytogenes strains with each food residue (3 ml 106 CFU

ml) in the grooves for 7 consecutive days Thereafter only food

suspension (3 ml) was added daily to the grooves for another 7 days

to allow further development of the biofilm During this period the

plates were stored at 15uC in a closed plastic box to maintain

stable humidity (50 relative humidity measured by a digital

hygrothermometer VWR International BV Amsterdam The

Netherlands) The disinfectant tests were carried out as described

in the previous paragraph for 20-min contact time

For the bacteriophage test 5 ml of a 109 PFUml phage

suspension were sprayed over the biofilm plates Thereafter the

plates were put in sterile plastic bags (Aseptic vacuum bags 400

by 510 mm Hevel Zaandam The Netherlands) to maintain

humidity and left for 30-min contact time

The tests on surfaces with grooves were performed in

duplicate on different days for each disinfectant and for

bacteriophages

Effect of disinfectants on suspended biofilm cells The 2-

week biofilm cells were taken out of each groove by scraping the

grooves with sterile extra-thin toothpicks (Jordan Intec BV

Utrecht The Netherlands) The cells were mixed with 9 ml PSS

and tested with disinfectants as described in the suspension test

Microbiological analysis In the suspension test (case 1) the

reaction was terminated after the required contact time by

FIGURE 2 Diagram shows all tests per-formed in this study The suspension andcarriers tests on smooth surface wereperformed with or without food residues(w or wo) according to the Europeanstandard EN 10402005 Tests on thesurface with grooves were performed withL monocytogenes cells on wet and dryconditions in presence and absence of foodresidues and 2-week food biofilm Thedisinfectants used were SDC (240 mgliter)and Qac (740 mgliter) while sterile waterwas used as a control

2014 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12

transferring 1 ml of the suspensions to 9 ml of neutralizer for 1 min

Of these suspensions 100 ml was then plated on Agar Listeriaaccording to Ottaviani and Agosti (ALOA BioMerieux SA Marcy

lrsquoEtoile France) using a spiral plater (Eddy Jet IUL Instrument

SA Barcelona Spain) For the carrier tests on flat surfaces (case

2) the disinfectant reaction was terminated by transferring the

coupons plus disinfectant to a 9-ml tube containing neutralizer and

45-g glass beads (1 mm Emergo Landsmeer The Netherlands)

As a blank inoculated coupons were transferred to 10 ml of PSS

plus glass beads The cells were removed from the coupons by

vortexing the tubes for 30 s Thereafter 1 ml of the suspension was

pour plated in tryptone soy agar

For the carrier tests on surfaces with grooves (case 31)

samples were taken using cotton swabs and cotton threads after the

required contact time Cotton swabs made with extra-thin toothpicks

and cotton wool (01 g 100 Hygienic cotton Etos) and cotton

threads (27-cm length 075-mm diameter [01 g] Super Durable

Glanskatoen no 8 HWS Markoma BV Ninove Belgium) were wet

sterilized at 121uC for 15 min The sterile cotton swabs were used to

take the samples from the 2- 1- 05- and 02-mm grooves by

swabbing three times through the grooves The sterile cotton threads

were used to take samples from the 5-mm groove by flossing and

scraping the groove for 30 s After sampling the cotton swabs and

cotton threads were put in 9-ml neutralizer vortexed and 100 ml

were plated on ALOA plates using a spiral plater

For the carrier tests with bacteriophages (case 32) the cotton

threads and cotton swabs were soaked for 1 min in a heat killed

suspension of L innocua (109 CFUml) to bind free phage particles

as neutralizer Subsequent sampling and plating were similar as

described above

During the 14 days of growing biofilms the numbers of

Listeria aerobic plate count and lactic acid bacteria were

determined at different time intervals on separate plates using

ALOA agar tryptone soy agar and De Man Rogosa and Sharpe

Agar (Lactobacillus broth Merck and 12 [wtvol] Agar

technical Agar no 3 Oxoid) respectively

Data and statistical analysis The volume of each groove

(cubic millimeters) was calculated and transformed to milliliters

The amount of organisms in a groove was then divided by this

volume to obtain a concentration in log CFU per milliliter The

value was calculated as log CFU per milliliter of the grooves in

order to equally compare the results obtained from the grooves that

have different depths

For statistical analysis the results of duplicate experiments

were combined The significance of differences (P 005) in

numbers of L monocytogenes that were reduced by different types

of disinfectants food residues and types of surface were

determined with analysis of the variance and a general linear

model using PASW Statistics 17 (SPSS Inc Chicago IL)

RESULTS AND DISCUSSION

Suspension tests (case 1) Reduction of more than 5

log units of Listeria planktonic cells and biofilm cells in

suspension was observed after 5-min contact time with

240 mgliter SDC and 740 mgliter Qac in all test

conditions where there was no significant difference (P

005) in reduction of the cells between the two disinfectants

in absence of food residues In contrast the effectiveness of

SDC (1-log reduction in 5 min with 3 ultrahigh-

temperature-processed milk) was less than that of Qac (5-

log reduction in 1 min with 10 ultrahigh-temperature-

processed milk) in presence of food residues as expected

(21 25 46) The effectiveness of SDC and Qac in absence

of food residues was higher than in presence of food

residues (data not shown) However these results comply

with the European standard EN1040 (6) which indicates a

reduction of 5 log units should be obtained after 5-min

contact time for an effective disinfectant

Carrier tests on smooth surfaces (case 2) The results

from the tests on the stainless steel coupons showed more

than a 5-log reduction after 1- 2- and 5-min contact time

with no significant difference (P 005) between SDC and

Qac (data not shown) These results complied with the

European standard EN13697 requirement (4) for a reduction

of 4 log units on clean surfaces indicating an effective

disinfectant in the surface test

Carrier tests on surfaces with grooves (case 3) The

shape of the 1-mm wide grooves with depths of 02 05 1

2 and 5 mm used as test surfaces in this study was beyond

the capability of standard sampling methods such as surface

contact plates and swabbing techniques Therefore several

methods eg the use of filter paper cotton swabs and

flossing threads were evaluated for their effectiveness to

recover Listeria cells from the grooves The results showed

that the flossing technique with cotton thread led to 70

recovery for the 5-mm groove For the other grooves this

method could recover only 20 With cotton swabs

prepared from extra-thin toothpicks and cotton wool a

recovery of 70 could be obtained for the remaining

grooves (data not shown) Therefore flossing and swab

methods were chosen as sampling methods for the 5-mm

groove and the other grooves respectively

In these tests the contact times of disinfectant on carriers

were 5 10 15 and 20 min Since the inactivation obtained for

the different contact times was not significantly different (P 005) which may be due to exhaustion of the disinfectants

(3 51) only the results from the contact time of 5 min are

shown Furthermore reduction of L monocytogenes was

dependent on the groove depth the deeper the groove the

lower the reduction This indicates that the activity of the

disinfectants stopped due to the inability to reach the cells in

the deeper areas Only the results of the extremes the 5- and

02-mm grooves are presented in Figures 3 and 4

All food residues were tested negative for L monocy-togenes In absence of food residues on a wet surface after

exposure to water (control) and the disinfectants a high

reduction of the cells was observed in the 02-mm grooves

whereas a similar trend but less reduction was observed in

the 5-mm grooves On the dry surfaces treatment of the

cells in the 02-mm groove resulted in levels below the

detection limit which can be partly explained by a high

reduction observed already as a result of the drying of the

plates for 24 h For the 5-mm grooves this initial reduction

was much lower and not all listerias were inactivated by the

treatment In most cases there was no significant difference

(P 005) between the control and disinfectants The

results demonstrated that drying of surfaces alone could

already largely reduce the cells on the surfaces especially in

the shallow grooves where deeper grooves provided suitable


FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method

FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit


conditions to prevent complete drying protecting some of

the cells from the disinfectants Drying of surfaces has been

considered as a significant bactericidal method to maintain

low numbers of bacteria in any environment and is

dependent on type of organism temperature relative

humidity and soiling (39 45 53) Furthermore type of

surface and topography have been reported to play an

important role on efficiency of disinfectants (30 40) in

particular surfaces with scrapes or defects where soil can

accumulate and protect bacteria against cleaning and

disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm

grooves Listeria was reduced 2 to 3 log dependent on

types of food residues (Fig 3a) After exposure to the

disinfectants a reduction to below the detection limit was

observed for all food residues with no significant difference

(P 005) between SDC and Qac In the 5-mm grooves

before treatment less reduction (2 log CFUml) was

observed SDC was significantly (P 005) less effective

(2 log) than Qac ($2 log CFUml) for all food residues

except fish In the dry 02-mm grooves already a reduction

of 3 log CFUml was observed 24 h after inoculation

without treatment After exposure to the disinfectants the

numbers were further reduced to below the detection limit

In the 5-mm groove only a small (05 log CFUml ie

vegetable fish and ham residues) or no reduction (milk

residue) was observed immediately before treatment

probably because the cells were protected from the drying

in the deep grooves Treatment with SDC and Qac did not

inactivate all listerias with no significant difference (P

005) between the two disinfectants except in the case of

ham and vegetable residues where Qac performed signif-

icantly better than SDC in the wet grooves and for

vegetables in the dry grooves Previous studies have

reported an inactivation of the antimicrobial activity of

disinfectants by organic matter on surfaces showing 1- to 2-

log reductions on soiled surfaces compared with 3- to 4-

log reductions on clean surfaces (2 34) This could be due

to the chemical compositions such as protein and fat which

can bind with the disinfectants or reduction of penetration

into the bacterial layer (32 68) The chlorine-based

disinfectants have been found to be easily inactivated by

organic materials (25 46) whereas Qac was reported to be

less inactivated by organic matters (21) which was

confirmed in most cases in this study In general the test

on surfaces is dependent on the surface materials used and

viability of the cells dried on the surfaces resulting in

inconsistent results when compared with the test in

suspensions (15) Under practical conditions high amounts

of organic materials may be present resulting in less or no

activity of the disinfectant When the concentration of SDC

was increased from 240 to 300 mgliter a comparable

reduction of the cells to that of Qac was obtained however

there was no effect on reduction in the deep grooves (data

not shown) The results indicated that the disinfectant could

not reach all cells in the bottom of the groove while such

grooves (5-mm depth 1-mm width) might be present in

practice (eg small spaces between connection parts of

slicers) The effectiveness of the disinfectants in the deep

areas could be increased by using high pressure spraying

(32) however this method might have adverse effects since

it could also spread the bacterial cells to other areas and

environments in particular when high numbers of bacterial

cells are accumulated (42)

Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in

02-mm grooves (Fig 4) the number of cells remained

more or less stable with a decrease of the cells in vegetable

and ham biofilms (15 and 18 log CFUml respectively) In

5-mm grooves however growth of biofilm cells was

observed in all food residues A previous study reported that

L monocytogenes has the ability to form biofilms at

numbers of 104 to 107 CFUcm2 (34) and adheres to a

variety of food contact surfaces such as plastic glass

rubber and stainless steel particularly in pits or crevices

that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the

disinfectants than the planktonic cells the biofilm cells were

brought into suspension and then tested The obtained

results a 5-log reduction in all conditions after 5-min

exposure to the disinfectants indicated no difference in

reduction of biofilm cells (case 32) compared with

planktonic cells (case 1) These results complied with a

previous study where reduction of the suspended biofilm

cells was higher than that of biofilm cells attached to

surfaces after exposure to disinfectants (50) It has been

described that a 10 to 100 times higher concentration of

disinfectants may be required to obtain the same reduction

of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation

For the bacteriophages an efficacy test was previously

performed with L monocytogenes strains in presence and

absence of food residues on flat stainless steel surfaces The

results demonstrated that rapid inactivation of Listeria cells

(2 to 3 log CFUcm2) within 30 min of contact time was

obtained (data not shown) which was similar with the

results of Soni and Nannapaneni (65) where a reduction of

35 log CFUcm2 was obtained in the same time with a

phage treatment on a 1-week-old L monocytogenes biofilm

on stainless steel coupons

In the 02-mm grooves (Fig 4a) Qac showed better

reduction (2 to 3 log) than SDC (12 to 27 log) with

inactivation below the detection limit in vegetable biofilms

The phages showed higher reduction (3 log CFUml P

005) than that of water or the chemical disinfectants in three

food biofilms except for the vegetable biofilms where all

cells were inactivated by every disinfectant In the 5-mm

grooves SDC and Qac did not reduce Listeria better than

water alone However the phages reduced approximately

14 log CFUml in ham and fish biofilms and approxi-

mately 08 log CFUml in milk and vegetable biofilms

which was not significantly different (P 005) from that of

the disinfectants except for ham biofilms The results

indicated that only in the 02-mm grooves the phages were

likely to be more effective in the biofilms with food matrix

than the chemical disinfectants This was probably due to

the fact that the disinfectants and phages did not penetrate


the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of

other bacteria (6 to 7 log CFUml) including lactic acid

bacteria (4 log CFUml) in the grooves as well (data not

shown) Phage efficacy depends on susceptibility of the

bacterial cells to the phages and availability of receptor sites

between phages and the bacterial cells (13) which could be

blocked by the food matrix (35) In this study the phage

efficacy was enhanced by keeping the biofilm plates in

plastic bags after application of the phages to maintain a

high humidity that would enhance the diffusion and the

chance of contact between Listeria cells and phage particles

(66)Strikingly the biofilm removal by sterile water

(control) resulted in a similar reduction of the biofilm cells

compared with disinfectants In the present study the tests

were performed to simulate survival of bacteria in presence

or absence of organic matter in pits or cracks on food

contact surfaces and to test the effect of disinfection

therefore cleaning of the surfaces before using disinfectants

was excluded So in this case plates and grooves were

dirtier than soiled surfaces in reality which explains the

observed low efficacy of the disinfectants and the water

washed away the biofilm cells from the grooves Rinsing the

biofilm on surfaces by water has been reported to reduce the

biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that

presence of grooves or spaces on surfaces humidity and

presence of food substrates influenced the antimicrobial

effect of disinfectants and bacteriophages Bacteriophages

showed a better antimicrobial effect than the chemical

disinfectants (ie SDC and Qac) in the shallow grooves but

not in the deep grooves Use of bacteriophages as a

biocontrol could only be a promising method in limited

cases however use of large quantities in practice may be

costly and phage-resistant strains may occur (35 62)

ACKNOWLEDGMENTS

The authors thank Diversey Inc (Utrecht The Netherlands) who

provided the chemical disinfectants for this study We also thank Micreos

Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage

Listex P100 and L innocua (serovar 5) Our great appreciation goes to

Feifei Gao for her technical assistance in the phage experiments and Hilda

Nyati for her work on smooth surfaces

REFERENCES

1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007

Susceptibility of Listeria monocytogenes strains to disinfectants and

chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048

2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T

Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal

efficiencies of commercial disinfectants against Listeria monocyto-

genes on surfaces J Food Saf 20237ndash250

3 Adams M R A D Hartley and L J Cox 1989 Factors affecting

the efficacy of washing procedures used in the production of prepared

salads Food Microbiol 669ndash77

4 Anonymous 2001 European Standard EN 136972001 Chemical

disinfectants and antisepticsmdashqualitative non-porous surface test for

the evaluation of bacteriocidal andor fungicidal activity of chemical

disinfectants used in food industrial domestic and institutional

areasmdashtest method and requirements without mechanical action (phase

2 step 2) Available at httpwwwceneucenSectorsTechnical

CommitteesWorkshopsCENTechnicalCommitteesPagesStandards

aspxparam~6197amptitle~Chemical20disinfectants20and20

antiseptics Accessed February 2014

5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash

horizontal method for the detection and enumeration of Listeria

monocytogenesmdashpart 1 detection method ISO 11290-11996Amd

12004 International Organization for Standardization Geneva

Available at httpswwwisoorgobpuiisostdiso11290-1ed-1

v1enamd1amd1 Accessed January 2014


disinfectants and antiseptics Quantitative suspension test for the

evaluation of basic bactericidal activity of chemical disinfectants

and antisepticsmdashtest method and requirements (phase 1) Available

at httpwwwceneucenSectorsTechnicalCommitteesWorkshops

CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~

Chemical20disinfectants20and20antiseptics Accessed February

2014

7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D

Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013

Effectiveness of phages in the decontamination of Listeria monocy-

togenes adhered to clean stainless steel stainless steel coated with

fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash

1116

8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003

Characterization of persistent and sporadic Listeria monocytogenes

strains by pulsed-field gel electrophoresis (PFGE) and amplified

fragment length polymorphism (AFLP) Syst Appl Microbiol 26

539ndash545

9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen

and L Gram 2003 The microbial ecology of processing equipment

in different fish industriesmdashanalysis of the microflora during

processing and following cleaning and disinfection Int J Food

Microbiol 87239ndash250

10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis

2011 Efficiency of different sanitation methods on Listeria

monocytogenes biofilms formed under various environmental condi-

tions Int J Food Microbiol 145S46ndashS52

11 Beresford M R P W Andrew and G Shama 2001 Listeria

monocytogenes adheres to many materials found in food-processing

environments J Appl Microbiol 901000ndash1005

12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a

variety of disinfectants against Listeria spp Appl Environ Micro-

biol 56377ndash380

13 Bigwood T J A Hudson and C Billington 2009 Influence of host

and bacteriophage concentrations on the inactivation of food-borne

pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash

64

14 Blackman I C and J F Frank 1996 Growth of Listeria

monocytogenes as a biofilm on various food-processing surfaces J

Food Prot 59827ndash831

15 Bloomfield S F M Arthur B Klingeren W van Pullen J T

Holah and R Elton 1994 An evaluation of the repeatability and

reproducibility of a surface test for the activity of disinfectants J

Appl Bacteriol 7686ndash94

16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine

1997 Adhesion of Streptococcus thermophilus to stainless steel

with different surface topography and roughness Biofouling 11

201ndash216

17 Bower C K J McGuire and M A Daeschel 1996 The adhesion

and detachment of bacteria and spores on food contact surfaces

Trends Food Sci Technol 7152ndash157

18 Bryan F L 2002 Where we are in the retail food safety how we got

to where we are and how do we get there J Environ Health 6529ndash

36

19 Carey-Smith G V C Billington A J Cornelius J A Hudson and

J A Heinemann 2006 Isolation and characterization of bacterio-

phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash

186


20 Carpentier B and O Cerf 1993 Biofilms and their consequences

with particular reference to hygiene in the food industry J Appl

Bacteriol 75499ndash511

21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36

61ndash62

22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S

Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100

combined with chemical antimicrobials in reducing Listeria mono-

cytogenes in cooked turkey and roast beef Int J Food Microbiol

167208ndash214

23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C

Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food

processing non-food and domestic environments Food Microbiol 6

49ndash61

24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo

recommended concentrations of commercial sanitizers on inactivation

of Listeria monocytogenes Food Control 26194ndash199

25 De Beer D R Srinivasan and P S Stewart 1994 Direct

measurement of chlorine penetration into biofilms during disinfec-

tion Appl Environ Microbiol 604339ndash4344

26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic

infection of Escherichia coli biofilms by bacteriophage T4 Can J


27 Environmental Protection Agency (EPA) Office of Wastewater

Enforcement and Compliance 2014 Guide to field storage of

biosolids and other organic by-products used in agriculture and for

soil resource management Available at httpwaterepagovscitech

wastetechbiosolidsguidecfm Accessed January 2014

28 Fatemi P and J F Frank 1999 Inactivation of Listeria

monocytogenesPseudomonas biofilms by peracid sanitizer J Food

Prot 62761ndash765

29 Fenlon D R J Wilson and W Donachie 1996 The incidence and

level of Listeria monocytogenes contamination of food sources at

primary production and initial processing J Appl Bacteriol 81641ndash

650

30 Frank J F and R A N Chmielewski 1997 Effectiveness of

sanitation with quaternary ammonium compound or chlorine on

stainless steel and other domestic food-preparation surfaces J Food

Prot 6043ndash47

31 Frank J F and R A Koffi 1990 Surface-adherent growth of

Listeria monocytogenes is associated with increased resistance of

surfactant sanitizers and heat J Food Prot 53550ndash554

32 Gibson H J H Taylor K E Hall and J T Holah 1999

Effectiveness of cleaning techniques used in the food industry in terms

of the removal of bacterial biofilms J Appl Microbiol 8741ndash48

33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey

of Listeria monocytogenes in foods J Food Prot 66559ndash569

34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel

2007 Influence of food soiling matrix on cleaning and disinfection

efficiency on surface attached Listeria monocytogenes Food Control

181165ndash1171

35 Greer G G 2005 Bacteriophage control of foodborne bacteria J


36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A

Sjoberg 1999 Screening of antimicrobial activities of disinfectants

and cleaning agents against foodborne spoilage microbes Eur Food

Res Technol 208289ndash298

37 Hagens S 2014 Personal communication

38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and

removal of L-forms of Listeria monocytogenes with bred bacterio-

phage Int J Food Microbiol 34197ndash207

39 Hirai Y 1991 Survival of bacteria under dry conditions from a

viewpoint of nosocomial infection J Hosp Infect 19191ndash200

40 Holah J T 1992 Industrial monitoring hygiene in food processing

p 645ndash659A In L F Melo T R Bott M Fletcher and B

Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-

demic Publishers Dordrecht

41 Holah J T C Higgs S Robinson D Worthington and H

Spenceley 1990 conductance-based surface disinfection test for food

hygiene Lett Appl Microbiol 11255ndash259

42 Holah J T J H Taylor and J S Holder 1993 The spread of

Listeria by cleaning systems part II Technical Memorandum no 673

Campden and Chorleywood Food Research Association Chipping

Campden UK

43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm

susceptibility to bacteriophage attack the role of phage-borne

polysaccharide depolymerase Microbiology 1443039ndash3047

44 Joerger R D 2003 Alternatives to antibiotics bacteriocins

antimicrobial peptides and bacteriophages Poult Sci 82640ndash647

45 Kusumaningrum H D G Riboldi W C Hazeleger and R R

Beumer 2003 Survival of foodborne pathogens on stainless steel

surfaces and cross-contamination to foods Int J Food Microbiol 85

227ndash236

46 Lee S-H and J Frank 1991 Inactivation of surface adherent

Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash

6

47 Leverentz B W S Conway M J Camp W J Janisiewicz T

Abuladze M Yang R Saftner and A Sulakvelidze 2003

Biocontrol of Listeria monocytogenes on fresh-cut produce by

treatment with lytic bacteriophages and a bacteriocin Appl Environ


48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A

Hall and M P Doyle 2006 Cross-contamination between

processing equipment and deli meats by Listeria monocytogenes J


49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of

persistent Listeria monocytogenes contamination between food-

processing plants associated with a dicing machine J Food Prot

651129ndash1133

50 Luppens S B I M W Reij R W L van der Heijden F M

Rombouts and T Abee 2002 Development of a standard test to

assess the resistance of Staphylococcus aureus biofilm cells to

disinfectants Appl Environ Microbiol 684194ndash4200

51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990

Efficiency of sanitizing agents for destroying Listeria monocytogenes

on contaminated surfaces J Dairy Sci 733428ndash3432

52 Mahony J O McAuliffe R Paul and D van Sinderen 2011

Bacteriophages as biocontrol agents of food pathogens Curr Opin

Biotechnol 22157ndash163

53 McEldowney S and M Fletcher 1988 Effect of temperature and

humidity on survival of bacteria attached to dry solid surfaces Lett

Appl Microbiol 783ndash86

54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-

tion of Listeria monocytogenes from an ice-cream plant by serotyping

and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash

192

55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against

attached bacteria in a milk biofilm J Food Prot 5634ndash41

56 Norwood D E and A Gilmour 2000 The growth and resistance to

sodium hypochlorite of Listeria monocytogenes in a steady-state

multispecies biofilm J Appl Microbiol 88512ndash520

57 Oliveira M I Vinas P Colas M Anguera J Usall and M

Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria

monocytogenes on fresh-cut fruits and fruit juices Food Microbiol

38137ndash142

58 Ronner A B and A C L Wong 1993 Biofilm development and

sanitizer inactivation of Listeria monocytogenes and Salmonella

typhimurium on stainless steel and buna-n rubber J Food Prot 56

750ndash758

59 Sallam S S and C W Donnelly 1992 Destruction injury and

repair of Listeria species exposed to sanitizing compounds J Food

Prot 55771ndash1033

60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J

Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas

fluorescens planktonic cells versus biofilms Biofouling 20133ndash138

61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to

and viability of Listeria monocytogenes on food contact surfaces J


62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction

Int J Food Microbiol 2965ndash14 (Review)


63 Somers E B J L Schoeni and A C L Wong 1994 Effect of

trisodium phosphate on biofilm and planktonic cells of Campylo-

bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes

and Salmonella typhimurium Int J Food Microbiol 22269ndash

276

64 Somers E B and A C L Wong 2004 Efficacy of two cleaning

and sanitizing combination on Listeria monocytogenes biofilm

formed at low temperature on a variety of materials in the presence

of ready-to-eat meat residue J Food Prot 672218ndash2229

65 Soni K A and R Nannapaneni 2010 Removal of Listeria

monocytogenes biofilms with bacteriophages P100 J Food Prot 73

1519ndash1524

66 Sutherland I W K A Hughes L C Skillman and K Tait 2004

The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6

(MiniReview)

67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R

Beuchat M P Doyle and T J Montville (ed) Food microbiology

fundamentals and frontiers 2nd ed ASM Press Washington DC

68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An

updated review of Listeria monocytogenes in the pork meat industry

and its products J Appl Microbiol 1017ndash17

69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy

of quaternary ammonium compound iodophor and chlorine on

Listeria J Food Prot 531029ndash1033


fluorescens (60) still little information is available on the

efficiency of bacteriophages on the inactivation of patho-

gens in biofilms (43 65 66)The effectiveness of disinfectants against bacteria on

smooth surfaces such as stainless steel coupons or chips (17 12 32 34) buna-N rubber (58 64) Teflon (55)polyester fabrics and polyurethane surfaces of conveyor

belt (64) has been well documented however only few

studies have been performed on surfaces with pits or

grooves The purpose of this study was to simulate the

practical situation where a Listeria biofilm is formed in

cracks in the equipment and to investigate the effect of two

commercial disinfectants based on chlorine and Qacs and a

Listeria bacteriophage product against L monocytogenes in

suspension and in carrier tests with flat and rutted (grooved)

surfaces

MATERIALS AND METHODS

Bacterial strains L monocytogenes LF 38 (serotype 12a

isolated from cooked ham) LF 36 (serotype 12b isolated from

cooked sausage) and LF 29 (serotype 4e isolated from cooked

sliced sausage) were obtained from The Netherlands Food and

Consumer Product Safety Authority (Wageningen The Nether-

lands) Stationary-phase cultures of all strains (24 h 30uC) were

stored in 1-ml cryo vials (Greiner Bio-one Frickenhausen

Germany) with 25 (volvol) glycerol (Fluka-Chemica Buchs

Switzerland) in brain heart infusion (BHI) broth (Difco BD

Sparks MD) and 2-mm glass beads (Emergo Landsmeer The

Netherlands) at 220uC For each experiment one glass bead of

each strain was inoculated to a separate tryptone soy agar plate

(Oxoid Basingstoke England) and then incubated at 30uC for

24 h A single colony was picked inoculated into BHI broth and

incubated at 30uC for 24 h Test suspensions were prepared by

mixing cultures of the three L monocytogenes strains where 2 ml

of overnight cultures of each strain (BHI broth 30uC for 24 h)

were combined to give a 6-ml mixed culture of L monocytogeneswhich was serially diluted in peptone saline solution (PSS 85 g

liter NaCl Merck Darmstadt Germany) and 1 gliter Neutralised

Bacteriological Peptone (Oxoid) to obtain appropriate dilutions for

inoculation

Bacteriophages Listeria bacteriophage Listex P100 was

obtained from Micreos Food Safety BV (Wageningen The

Netherlands) as a suspension with a concentration of 1011 PFU

ml Listex P100 was serially diluted with 9 ml of PSS to obtain

109 PFUml The phage suspensions were freshly prepared

before use to prevent deterioration during storage The

concentration of the phages was determined by a double agar

overlay method according to Carey-Smith et al (19) using BHI

agar (BHI plus Agar technical Agar no 3 12 and 4 gliter for

bottom and top layer respectively Oxoid) and Listeria innocua(serovar 5 Micreos Food Safety BV) as the indicator organism

The plates were incubated at 30uC for 24 h and plaques were

counted

Test surfaces Two types of surfaces were used in this study

stainless steel coupons (AISI type 304 standard ODS Bare-

ndrecht The Netherlands) with a total surface (both sides) of

38 cm2 and stainless steel plates (AISI type 304 standard ODS)

measuring 12 by 10 by 1 cm in which two identical sets of grooves

(5- 2- 1- 05- 02-mm depth 1-mm width and 10-cm length)

were cut (Fig 1)

Disinfectants Suma Tab D4 (SDC Diversey Inc Utrecht

The Netherlands) and Suma Bac D10 (Qac Diversey Inc) were

used in this experiment at concentrations of 240 mgliter for SDC

(pH 6) and 740 mgliter for Qac (pH 101 to 103) according to the

manufacturerrsquos recommendations Sterile water was used as a

control in the experiments The disinfectants were freshly prepared

shortly before use on the day of the experiments to prevent

deterioration during storage

Neutralizers For the chemical disinfectants inactivation

liquid was used as a neutralizer (6) containing 1 gliter L-histidine

(Merck) 3 gliter lecithin (VWR International Poole England)

5 gliter sodium thiosulfate pentahydrate (Na2S2O3-5H2O VWR

International) 30 mlliter Tween 80 (Merck) and 10 mlliter

phosphate buffer The phosphate buffer was prepared by dissolving

34 g of potassium dihydrogen phosphate (KH2PO4 Merck) in

500 ml of demi water after adjustment of the pH to 72 with 1 M

sodium hydroxide (NaOH VWR Prolabo Lutterworth England)

the volume was filled with 1 liter of demi water There were 9 ml

of inactivation liquid that were dispensed into test tubes and

sterilized at 121uC for 15 minutes

For Listeria bacteriophages a dead cell suspension of Linnocua (serovar 5) was used as a neutralizer (37) which was

prepared by adding 1 ml of an overnight culture (BHI broth 30uC

24 h) of L innocua to 9 ml of PSS which were subsequently

sterilized for 15 min at 121uC

Preparation of food residues Modified atmospherendashpacked

cooked ham vacuum-packed smoked salmon and chopped

endives were bought from a local supermarket kept in a

refrigerator at 4 to 7uC and used within 24 h To prepare a 10

FIGURE 1 Pictures of the stainless steel plate (12 by 10 by 1 cm)with 2 sets of 5 grooves (5- 2- 1- 05- 02-mm depth 1-mmwidth 10-cm length) used in carrier tests top (a) and side view (b)

















study




















































bacteriophages






































described above














































































































































































































































ACKNOWLEDGMENTS







REFERENCES

































2014






1116





539ndash545















biol 56377ndash380




64











201ndash216






36




186






61ndash62





167208ndash214




49ndash61

















Prot 62761ndash765




650




Prot 6043ndash47












181165ndash1171























Campden UK









227ndash236



6













651129ndash1133

















192









38137ndash142




750ndash758



Prot 55771ndash1033














276







1519ndash1524



(MiniReview)


























study




















































bacteriophages






































described above














































































































































































































































ACKNOWLEDGMENTS







REFERENCES

































2014






1116





539ndash545















biol 56377ndash380




64











201ndash216






36




186






61ndash62





167208ndash214




49ndash61

















Prot 62761ndash765




650




Prot 6043ndash47












181165ndash1171























Campden UK









227ndash236



6













651129ndash1133

















192









38137ndash142




750ndash758



Prot 55771ndash1033














276







1519ndash1524



(MiniReview)







































described above














































































































































































































































ACKNOWLEDGMENTS







REFERENCES

































2014






1116





539ndash545















biol 56377ndash380




64











201ndash216






36




186






61ndash62





167208ndash214




49ndash61

















Prot 62761ndash765




650




Prot 6043ndash47












181165ndash1171























Campden UK









227ndash236



6













651129ndash1133

















192









38137ndash142




750ndash758



Prot 55771ndash1033














276







1519ndash1524



(MiniReview)

































































































































































ACKNOWLEDGMENTS







REFERENCES

































2014






1116





539ndash545















biol 56377ndash380




64











201ndash216






36




186






61ndash62





167208ndash214




49ndash61

















Prot 62761ndash765




650




Prot 6043ndash47












181165ndash1171























Campden UK









227ndash236



6













651129ndash1133

















192









38137ndash142




750ndash758



Prot 55771ndash1033














276







1519ndash1524



(MiniReview)















61ndash62





167208ndash214




49ndash61

















Prot 62761ndash765




650




Prot 6043ndash47












181165ndash1171























Campden UK









227ndash236



6













651129ndash1133

















192









38137ndash142




750ndash758



Prot 55771ndash1033














276







1519ndash1524



(MiniReview)















276







1519ndash1524



(MiniReview)











inactivation of listeria monocytogenes by disinfectants ... · inactivation of listeria...

Documents