1468

Journal of Food Protection, Vol. 74, No. 9, 2011, Pages 1468–1474

doi:10.4315/0362-028X.JFP-11-110
Copyright G, International Association for Food Protection

Efficacy of a Novel Sanitizer Composed of Lactic Acid and

Peroxyacetic Acid against Single Strains of Nonpathogenic
Escherichia coli K-12, Listeria innocua, and Lactobacillus
plantarum in Aqueous Solution and on Surfaces of Romaine
Lettuce and Spinach
KAI-LAI GRACE HO,* DIEGO A. LUZURIAGA, KENNETH M. RODDE, SHANNON TANG, AND CUONG PHAN

Research and Development Department, Chiquita Brands International, J. R. Lugg Research Center, 607 Brunken Avenue, Salinas, California 93901, USA

MS 11-110: Received 9 March 2011/Accepted 19 May 2011

ABSTRACT
A novel sanitizer composed of lactic acid and peroxyacetic acid (LA-PAA) was developed as an alternative to chlorinated
water (CW) for fresh produce processing. Single strains of Lactobacillus plantarum, nonpathogenic Escherichia coli K-12, and
Listeria innocua were used to demonstrate the microbial efficacy of LA-PAA. LA-PAA achieved a .7.8-log reduction of L.
innocua and L. plantarum suspended in water at 4uC for 20 s, and LA, PAA, and CW achieved reductions of 0.4, 4.8, and 2.7 log,
respectively. LA-PAA, when compared with LA, PAA, and CW, enhanced the reduction of L. innocua attached to romaine leaves
by .2.2 log, and improved the removal of E. coli attached to spinach leaves by .2.4 log. The exponential improvement in the
microbial efficacy of LA-PAA showed synergism between LA and PAA. LA-PAA microbial efficacy was inversely proportional
to pH value and directly correlated with residence time and concentration. Despite an improvement in microbial reduction
through the addition of surfactant to LA-PAA, the usage of surfactant in washing fresh produce was impeded by excessive
foaming during actual processing. Effects of organic matter on the performance of LA-PAA were minimal. External sensory
evaluations showed that LA-PAA had no negative effects on the quality of lettuce and tender leaves. Temperature-abuse studies
demonstrated that LA-PAA reduced decay by ,50% when compared with CW. Overall, these results support the premise that
LA-PAA has significant potential to be an alternative to CW for fresh produce processing.

Fresh-cut produce is an increasingly popular food
category because vegetables and fruits are important
components of a healthy diet (27); consumers seek
freshness, nutrition, variety, and convenience. From 1994
to 2007, U.S. supermarket salad sales increased from $3.3 to
$15.5 billion (6). Currently, chlorinated water (CW) is used
to maintain the microbial quality and safety of wash water
for fresh-cut produce. The microbial efficacy of CW is
affected by pH and reduced by soil and organic matters (9,
13, 16), and this affects its ability to reduce microbial
contamination during washing of fresh-cut produce. Hence,
there is a need to develop alternate sanitizers to replace CW
in produce wash water systems.
Two common types of sanitizers used in the food
industry include natural organic acids (acetic, citric, lactic,
malic, and propionic) and oxidation-reduction potential
sanitizers (sodium hypochlorite, chlorine dioxide, hydrogen
peroxide, peroxyacetic acid, and ozone) (3, 18, 21). Studies
using more than one sanitizer in sequence from the same
category (ozone and sodium hypochlorite) (8, 20) or the
addition of surfactants like sodium lauryl sulfate (SLS) to
* Author for correspondence. Tel: 831-772-6091; Fax: 831-759-4782;
E-mail: gho@chiquita.com.
organic acids (11, 18, 32), have demonstrated the increase of
the microbial efficacy of such sanitizers to overcome high
organic loads and low temperatures in fresh-cut produce wash
water. A novel sanitizer, lactic acid plus peroxyacetic acid
(LA-PAA), trademarked as Fresh Rinse, was invented (12) to
enhance microbial efficacy for fresh produce processing.
The bactericidal property of LA is due to the undisso-
ciated form of the LA being able to pass through the cell
membrane, to dissociate back to its ionic form, and to reduce
intracellular pH, thereby disrupting the transmembrane proton
motive force of the cell (7, 10, 21, 22, 25). In addition, LA
permeabilizes the water-soluble porin protein molecules that
are located on the bacterial outer membrane (1). PAA inhibits
microorganisms by oxidizing their outer cell membranes (7,
31). The oxidation-reduction of PAA (1.81 eV), is 15 and
33% higher than chlorine dioxide (1.57 eV) and sodium
hypochlorite (1.36 eV), respectively, and 12.5% less than that
of ozone (2.07 eV) (19). The antimicrobial activity of PAA is
therefore greater than chlorine dioxide and sodium hypo-
chlorite but less than that of ozone.
The purpose of this study was to compare the microbial
efficacy of LA-PAA to CW, LA, and PAA, under laboratory
and pilot plant scale-up conditions. In addition, studies have
J. Food Prot., Vol. 74, No. 9 NOVEL SANITIZER AGAINST NONPATHOGENIC BACTERIA IN SOLUTION AND ON LEAVES
shown that organic acids negatively affect the organoleptic
properties of leafy greens (2, 17, 23, 29); hence, the effects
of LA-PAA on the organoleptic properties of leafy products
was assessed by temperature-abuse storage studies and
sensory evaluations.
MATERIALS AND METHODS
Bacterial strains. Freeze-dried cultures of streptomycin-
resistant Escherichia coli K-12 (ATCC 25253), Listeria innocua
(ATCC 33090), and Lactobacillus plantarum (ATCC 14917), were
obtained from the American Type Culture Collection (Manassas,
VA). E. coli K-12 and L. innocua stock cultures were transferred to
10-ml tubes of tryptic soy broth (Difco, BD, Sparks, MD) and
incubated at 37uC for 24 h. L. plantarum stock culture was
transferred to 10-ml tubes of Lactobacilli de Man Rogosa Sharpe
broth (MRS; Difco, BD) and incubated at 37uC for 48 h. Overnight
enrichments of E. coli K-12, L. innocua, and L. plantarum were
streaked on streptomycin (100 mg/ml) tryptic soy agar, modified
Oxford agar, and MRS agar (Difco, BD) prepoured plates,
respectively, to confirm purity.
Preparation of inocula for laboratory tests. Overnight
enrichments of E. coli K-12, L. innocua, and L. plantarum were
subcultured with their corresponding growth medium and
incubated at 37uC for 24, 36, and 48 h, respectively, to form
stationary-phase cells. Stationary-phase cultures were cooled (1 h
at 4uC), centrifuged (4uC, 10,000 rpm for 15 min; Sorvall Legend
RT, Thermo Electron Corporation, Osterode, Germany), and
resuspended in an equal volume of 0.1% peptone water (Difco,
BD) and 5% horse serum (Sigma Chemical Co., St. Louis, MO) for
suspended and attached cells challenge tests, respectively (15). The
microbial population of the E. coli K-12, L. innocua, and L.
plantarum resuspended inocula was enumerated with streptomycin
(100 mg/ml) tryptic soy agar, modified Oxford agar, and MRS agar
(Difco, BD) prepoured plates, respectively.
Preparation of inocula for pilot plant scale-up test.
Overnight enrichment of L. plantarum at ,108 CFU/ml was
spread plated onto the surface of 600 MRS prepoured agar plates at
1 ml per plate. The inoculated MRS agar plates were incubated at
37uC for 48 h and then wetted with 2 ml of sterile MRS broth. The
L. plantarum colonies were harvested to form 1 liter of inoculum
stock, ,109 to 1010 CFU/ml. The microbial population of the
inoculum was enumerated at the beginning and end of the test by
using MRS prepoured agar plates.
Preparation and measurement of sanitizers. Four saniti-
zers (CW, LA, PAA, and LA-PAA solution) were prepared in
triplicate with 4uC municipal tap water (MTW). CW was prepared
by mixing cooled MTW with 15% (wt/wt) sodium hypochlorite
solution and 50% (wt/wt) citric acid solution (both solutions from
Basic Chemical Solutions, Redwood City, CA) for pH adjustment
to 7.0 ¡ 0.2 (a common practice in the industry to maintain free
chlorine in its bactericidal form, hypochlorous acid, for effective
microbial reduction without liberation of chlorine gas (26)). Food-
grade 88% LA (Purac Biochem, Gorinchem, The Netherlands) and
35% PAA (Pfaltz and Bauer, Inc., Waterbury CT) were used to
prepare LA, PAA, and LA-PAA solutions. Solutions were kept
refrigerated until testing.
Free chlorine was measured by a DR/820 colorimeter (Hach,
Loveland, CO). LA was determined by titration with a TITRONIC
basic titrator (Schott Instruments, Mainz, Germany), with sodium
hydroxide solution (0.05 N; Fisher Scientific, Fairlawn, NJ) and
1469
1% phenolphthalein (Ricca Chemical Co., Arlington, TX).
PAA was determined by a colorimetric method that used
N,N-diethyl-p-phenylenediamine sulfate and the DR/820 colorimeter.
Suspended cells challenge study. The suspended cells
challenge study was performed at 4uC. LA, PAA, and LA-PAA
solutions were tested at various concentrations by using MTW and
CW as controls. One milliliter of the single-strain inoculum was
added to 9 ml of a treatment solution, vortexed for 20, 30, or 40 s,
and immediately transferred to 9 ml of Butterfield’s phosphate
buffer containing 10 ml of sodium thiosulfate (Fisher Scientific) to
neutralize the sanitizer. The neutralized samples were serial diluted
and enumerated by spread plating with corresponding growth
medium of each strain at 37uC for 24 h (E. coli K-12 ) or 48 h (L.
innocua and L. plantarum).
Suspended cells challenge test scale-up at the pilot plant.
The scale-up study treatments were MTW, CW, and LA-PAA
solutions at 4uC. A NonLine double-tank produce wash system
was filled with 300 gal (1,135 liters) of circulating MTW at 1.7uC.
One liter of L. plantarum stock was spiked into the wash system at
nine different locations simultaneously (this was time zero for the
test). Four 1-ml spiked water samples were retrieved at different
locations of the wash system after 20 or 30 s and immediately
transferred into 9 ml of Butterfield’s phosphate buffer containing
10 ml of sodium thiosulfate to neutralize the sanitizer. The
neutralized samples were serial diluted and enumerated by spread
plating with MRS agar plates at 37uC for 48 h.
Attached cells challenge study. Fresh romaine lettuce (1.5
by 2.5 in. [3.81 cm by 6.35 cm]) and spinach were provided by
Fresh Express, Inc., (Salinas, CA) and kept at 4uC. Two leaves of
the test produce were spot inoculated (one on the upper epidermis
and the other on the lower epidermis) with 50 ml of ,109 CFU/ml
E. coli K-12 or L. innocua (10 to 15 droplets) and air dried in a
class II, type A2 biological safety cabinet (NuAire, Plymouth, MN)
for 45 to 60 min at 20 to 25uC and 35 to 40% relative humidity.
The spot-inoculation method was used, as it imitates contamination
on produce from a point source (4, 5).
Study treatments included MTW, CW, LA, PAA, and LA-
PAA solutions. To simulate commercial conditions, organic matter
in the form of chopped romaine lettuce was added to the treatment
wash solutions at 1:6.5 by weight. The two spiked leaves were
submerged into 3 liters of 4uC test solution, with continuous
stirring for 20 or 30 s, macerated by mallet hammer, and then
immediately stomached by a 400 circulator stomacher (Seward,
Ltd., London, UK) at 260 rpm. The samples were enumerated by
spread plating at 37uC for 24 h (E. coli K-12 ) or 48 h (L. innocua
and L. plantarum) (28).
Evaluation of factors affecting microbial efficacy. Effects
of pH were determined by a challenge test of E. coli K-12
suspended cells at pH 3.0, 3.3, 3.6, and 3.9. Solutions were
adjusted to their corresponding pH by 0.1 N sodium hydroxide
solution. Effect of residence time was evaluated by exposing L.
innocua to tested solution at 20, 25, 30, and 35 s of residence time,
following procedures laid out by Ho (12). Influence of surfactants
on the microbial efficacy of LA-PAA was assessed by L. innocua
and E. coli K-12 attached cells challenge tests with spinach and
romaine leaves, using SLS at 220 to 250 ppm. Effects of organic
matter were determined by washing chopped romaine leaves that
were continuously fed into a fixed volume (,1,200 gal [,4,500
liters]) of sanitizer (CW and LA-PAA solution) that was circulating
in a NonLine wash system at 3uC. No additional sanitizer was
1470 HO ET AL. J. Food Prot., Vol. 74, No. 9

TABLE 1. Log reduction of Listeria innocua (ATCC 33090) and Lactobacillus plantarum (ATCC 14917) in suspension treated with LA-
PAA, LA, PAA, and CW a
Peroxyacetic acid (ppm):

Lactic acid (ppm) 0 63.0 67.5 72.0

L. innocua
0 NAb 1.70 ¡ 0.13 B
c
2.27 ¡ 0.12 B 3.02 ¡ 0.31 B
1,800 0.08 ¡ 0.05 A 4.19 ¡ 0.32 C 5.68 ¡ 0.11 D .7.80 F
2,250 0.19 ¡ 0.08 A 4.61 ¡ 0.74 C 6.00 ¡ 0.05 D .7.80 F
2,700 0.05 ¡ 0.03 A 5.19 ¡ 0.06 CD 6.76 ¡ 0.25 E .7.80 F
CW (15.4 ppm, pH 7.0) 0.09 ¡ 0.05 A

L. plantarum
0 NA 3.93 ¡ 0.06 C 4.25 ¡ 0.15 C 4.79 ¡ 0.87 C
1,800 0.13 ¡ 0.11 A 5.21 ¡ 0.08 D 5.13 ¡ 0.10 D 5.24 ¡ 0.26 D
2,250 0.19 ¡ 0.02 A 5.11 ¡ 0.48 D 5.51 ¡ 0.50 DE 5.86 ¡ 0.10 DE
2,700 0.40 ¡ 0.17 A 5.09 ¡ 0.27 D 6.46 ¡ 0.04 E .7.79 F
CW (15.4 ppm, pH 7.0) 2.68 ¡ 0.20 B

a
Test performed at 4uC and 20 s of residence time.
b
NA, not applicable.
c
Log reduction values with different letters indicate statistical significance at P , 0.05.

added to the wash water in order to measure the loss in active
ingredients caused by organic matter. Collected wash water
samples were analyzed for the active ingredients concentrations
and total aerobic plate counts, while the romaine leaves samples
were enumerated for total aerobic plate counts only.
Sanitizer affect on product quality. Temperature-abuse
storage studies at 7uC on spinach and tender leaves mix bagged
under commercial conditions at a pilot plant were used to
determine the effects of CW and LA-PAA on product quality.
Six bags of each treatment were selected randomly from boxes
situated at different locations of a pallet. Percent decay (wt/wt) of
each bag sample was determined by randomly retrieving 80 g
(,50% of the bag sample weight) of leaves from the bag. The
retrieved leaves were individually inspected for signs of decay, and
the weight of the decay leaves was weighed and recorded. The
percent decay (wt/wt) of each bag sample was calculated by
dividing the weight (in grams) of the decay leaves with the total
weight (in grams) of the sample. The average of six bag samples
provided the average percent decay (wt/wt) of each treatment on a
certain sampling day for a particular test.
External sensory evaluations employing a nine-point hedonic
scale on appearance, color, aroma, taste, texture, and overall liking
were used to assess the organoleptic properties of CW- and LA-
PAA–washed leafy green products including chopped iceberg,
chopped romaine, and tender leaves mix with spinach. Each
replication of the sensory evaluation test was conducted by 100
panelists. Evaluated products were produced at a commercial
facility.
Statistical analysis. Statistical analysis was carried out with
analysis of variance (ANOVA). The least significant difference test
was used to determine significant differences (P , 0.05) among
mean values. Microbial data were log transformed before
performing ANOVA. Each study was repeated three separate
times independently.
RESULTS
Suspended cells challenge study. The log reductions
by LA (1,800 to 2,700 ppm) on L. innocua and L.
plantarum at 4uC were similar (0.1 to 0.4 log). The
reductions by 63.0, 67.5, and 72.0 ppm of PAA on L.
innocua were 1.70, 2.27, and 3.02 log, respectively, while
those on L. plantarum were 3.93, 4.25, and 4.79 log,
respectively (Table 1).
The log reductions on L. innocua and L. plantarum by
LA-PAA (1,800 ppm of LA and 63.0 ppm of PAA) were
4.19 and 5.21 log, respectively. A reduction of .7.80 log
(,1 CFU/ml) resulted when the LA-PAA level was
increased to 2,700 ppm of LA and 72 ppm of PAA. The
reduction of L. innocua was enhanced from 4.19 to .7.80
log as the PAA in the LA-PAA solution was increased from
63.0 to 72.0 ppm, with LA constant at 1,800 ppm. At
72 ppm of PAA, the reduction of L. plantarum improved
from 5.24 to .7.79 log, as the LA was elevated to 1,800
and 2,700 ppm, respectively (Table 1).
Under all replications, the log reduction of CW with
free chlorine of 15.4 ppm and at pH 7.0 was 0.09 and 2.68
log on L. innocua and L. plantarum, respectively. The
reductions were equal to or better than the tested LA levels,
but significantly less than that of PAA alone and LA-PAA
solution.
Suspended cells challenge test scale-up at the pilot
plant. Minimal decrease in L. plantarum (0.02 to 0.06 log)
population at the beginning of test showed that homoge-
neous mixing was achieved in the 300-gal (1,136-liter) wash
solution. CW with free chlorine of 9.9 and 16.5 ppm at 20 s
delivered 1.6- and 2.3-log reductions, respectively (Fig. 1)
and the reduction increased by 0.85 and 0.25 log,
respectively, at 30 s.
Treatment of L. plantarum by LA-PAA at 20 and 30 s
resulted in 5.1- and .7.2-log reductions, respectively. The
increase in exposure time improved significantly the log
reduction of L. plantarum by .2.4 log (Fig. 1). The
microbial efficacy of LA-PAA at 20 and 30 s was ,3.0 and
.4.7 log better than CW, respectively. The suspended cells
J. Food Prot., Vol. 74, No. 9 NOVEL SANITIZER AGAINST NONPATHOGENIC BACTERIA IN SOLUTION AND ON LEAVES 1471

FIGURE 1. Pilot plant scale-up results for log reduction of
Lactobacillus plantarum (ATCC 14917) suspended in 300 gal
(1,136 liters) of MTW, CW, and LA-PAA solution for 20 and 30 s.
Log reductions values with different letters indicate statistical
significance at P , 0.05.
challenge test at the pilot plant with L. plantarum confirmed
the results obtained from the bench top (laboratory scale)
test.
Attached cells challenge study. At 4,500 and
6,000 ppm, LA decreased L. innocua attached to chopped
romaine leaves by 0.69 and 1.32 log, respectively, and E.
coli K-12 attached to spinach leaves by 0.43 log and 0.83
log, respectively. LA-PAA (4,500 to 6,000 ppm LA and
80 ppm PAA) achieved .3.58 log on L. innocua attached to
romaine leaves. The inactivation achieved by the LA and
PAA solutions alone, however, were both only 1.3 log
CFU/ml. Reductions .4.88 log on E. coli K-12 attached to
spinach leaves were delivered by LA-PAA (6,000 ppm of
LA and 80 ppm of PAA), whereas reductions of only 0.83
and 2.47 log were obtained from the LA and PAA solutions,
respectively. The log reductions by CW (55 ppm of free
chlorine at pH 6.8) on the attached cells of L. innocua and
FIGURE 2. Effects of pH on the microbial efficacy of LA-PAA on
suspended cells of Escherichia coli K-12 (ATCC 25253). LA-PAA
concentration: 1,555 ppm of LA and 55 ppm of PAA.
E. coli K-12 were only 0.87 and 1.36 log, respectively
(Table 2).
Factors affecting microbial efficacy of LA-PAA. The
log reduction of LA-PAA on E. coli K-12 decreased from
4.07 to 2.83 log as the pH of the solution increased from
pH 3.0 to 3.9 (Fig. 2). LA-PAA microbial efficacy on L.
innocua at 20-, 25-, 30-, and 35-s residence times was 4.7,
5.4, 6.2, and 7.3 log, respectively, better than CW (Fig. 3).
The addition of SLS at 250 ppm to the PAA alone, LA
alone, and LA-PAA solution enhanced the reduction on
suspended L. innocua by 2, 3, and 1 log, respectively
(Fig. 4A). There were no significant differences between
log reductions of L. innocua and E. coli K-12 attached to
romaine and spinach leaves by LA-PAA, with or without
SLS (Fig. 4B).
Residual free chlorine dropped sharply to 10% after one
bin (,600 lb [272.3 kg]) of chopped romaine leaves was
washed under the enclosed wash system, with no replen-
ishment of sodium hypochlorite (Fig. 5). The percentage of

TABLE 2. Log reduction of Listeria innocua (ATCC 33090) attached to chopped romaine leaves and Escherichia coli K-12 (ATCC
25253) attached to spinach leaves treated with LA-PAA, LA, PAA, CW, and MTW a
Peroxyacetic acid (ppm):

Lactic acid (ppm) 0 70.0 80.0

L. innocua
0 NAb 1.10 ¡ 0.04 C
c
1.38 ¡ 0.01 D
4,500 0.69 ¡ 0.16 B 1.75 ¡ 0.16 E .3.58 F
6,000 1.32 ¡ 0.07 CD 2.00 ¡ 0.05 E .3.58 F
CW (55 ppm, pH 6.8) 0.87 ¡ 0.00 B
MTW 0.14 ¡ 0.03 A

E. coli K-12
0 NA 1.34 ¡ 0.01 D 2.47 ¡ 0.01 F
4,500 0.43 ¡ 0.07 B 1.94 ¡ 0.13 E 3.72 ¡ 0.11 G
6,000 0.83 ¡ 0.13 C 2.40 ¡ 0.17 F .4.88 H
CW (55 ppm, pH 6.8) 1.36 ¡ 0.09 D
MTW 0.01 ¡ 0.03 A

a
Tests performed at 4.4 to 7.2uC and 30 s of exposure time.
b
NA, not applicable.
c
Log reduction values with different letters indicate statistical significance at P , 0.05.
1472 HO ET AL.
FIGURE 3. Effects of residence time (20, 25, 30, and 35 s) on the
log reduction of Listeria innocua (ATCC 33090) suspended in
MTW, CW, and LA-PAA. Log reduction values with different
letters indicate statistical significance at P , 0.05. CW con-
centration: 10 ¡ 2 ppm of free chlorine at pH 7.0 ¡ 0.2. LA-PAA
concentration: 2,000 ppm of LA and 70 ppm of PAA.
residual LA-PAA ingredients remained at 100% after
washing three bins (,1,800 lb [818.2 kg]) and ,75% after
six bins (,3,600 lb [1,632.4 kg]) of chopped romaine
leaves. The log reductions by LA-PAA on the indigenous
total aerobic plate counts attached to the romaine leaves
were 1.7 to 2.5 log, while that of CW was ,0.4 log (Fig. 6).
FIGURE 4. Effects of surfactant on log reduction of (A) Listeria
innocua (ATCC 33090) in suspension by PAA, LA, and LA-PAA,
and (B) Listeria innocua (ATCC 33090) and Escherichia coli K-12
(ATCC 25353) attached to romaine and spinach leaves by LA-
PAA. Log reduction values with different letters indicate statistical
significance at P , 0.05. LA-PAA concentration: 4,500 ppm of LA
and 70 ppm of PAA.
J. Food Prot., Vol. 74, No. 9
FIGURE 5. Percentage of active ingredients left in CW and LA-
PAA during washing of chopped romaine lettuce in a 1,200-gal
(4,500-liter) NonLine wash system with no sanitizer replenishment.
No residual cells (,1 CFU/ml) were enumerated from all
the collected LA-PAA solution samples throughout the
entire test, whereas the total aerobic plate counts in the CW
samples were between 4.5 and .6.0 log CFU/ml.
Sanitizer effect on product quality. The log reduc-
tions on the indigenous total aerobic plate counts on spinach
and tender leaves mix by CW and LA-PAA were 0.55 log,
and 1.5 to 1.7 log, respectively (Fig. 7). After 15 days of
storage under temperature-abuse conditions (7uC), the
percent decay (wt/wt) of LA-PAA–washed spinach was
54% less than that of CW (Fig. 8). The percent decay
(wt/wt) of LA-PAA–washed tender leaves mix after 14 days
of temperature-abuse storage at (7uC) was 54% less than
that of CW (Fig. 8).
The leafy green products external sensory evaluations
results showed that for all three replicates, there were no
significant differences between the LA-PAA and CW
average sensory scores on appearance, color, aroma, taste,
texture, and overall liking for chopped iceberg, chopped
romaine, and tender leaves mix with spinach.
FIGURE 6. Log reduction of indigenous total aerobic plate
counts attached to chopped romaine leaves by washing with CW
and LA-PAA with a NonLine 1,200-gal (4,500-liter) wash system
with no sanitizer replenishment.
J. Food Prot., Vol. 74, No. 9 NOVEL SANITIZER AGAINST NONPATHOGENIC BACTERIA IN SOLUTION AND ON LEAVES
FIGURE 7. Initial log reduction of indigenous total aerobic plate
counts on spinach and tender leaves mix by washing with CW and
LA-PAA with a 1,200-gal (4,500-liter) NonLine wash system. Log
reduction values with different letters indicate statistical signifi-
cance at P , 0.05. CW concentration: 10 ¡ 2 ppm of free chlorine
at pH 7.0 ¡ 0.2. LA-PAA concentration: 2,000 ppm of LA and
70 ppm of PAA.
DISCUSSION
In this study, all trials (laboratory, pilot plant, and
commercial facilities) were conducted at 4uC with 20 to 30 s
of residence time, using organic matter containing water.
These testing parameters are normal commercial processing
conditions for leafy greens; thus, the results are valid and
applicable for commercial scale-up. Previous works on other
sanitizers were performed at higher temperatures (20 to 25uC)
and longer dwell times (1 to 10 min) (11, 17, 20, 24, 30, 32),
which did not simulate commercial processing conditions.
LA-PAA demonstrated significant enhancement in
reducing microorganisms that were suspended in wash
water and attached to surfaces of the leaves, when compared
with CW, LA, and PAA. The surprising advancement in
antimicrobial activity of LA-PAA demonstrated the pres-
ence of synergistic effects between the antimicrobial
properties of LA and PAA. All pilot plant microbial tests
were performed with commercial-scale systems, and the
results mirrored that obtained from bench-top, indicating
that LA-PAA is scalable for commercial applications.
The chopped-romaine pilot plant study demonstrated
both the resilience of LA-PAA to organic matter in the wash
water (a serious problem for maintaining the free chlorine
concentration in CW) and the capability of LA-PAA in
keeping fresh produce wash water microbial population low
(,1 CFU/ml in wash water enumeration), even in the
presence of high organic load. This indirectly supports the
premise that LA-PAA is highly probable in preventing
cross-contamination in wash water, a study that will be
reported in our sequel article.
Similar to other studies that showed significant
improvements in microbial efficacy with surfactants (11,
18, 32), the inactivation of L. innocua suspended in solution
by LA, PAA, and LA-PAA was increased by 3.0, 1.8, and
1.4 log, respectively. In the case of L. innocua and E. coli
K-12 that were attached to romaine and spinach leaves, the
average improvement in the antimicrobial activities of LA-
PAA with the addition of SLS was from 0.26 to 0.90 log.
1473
FIGURE 8. Percentage of decay (wt/wt) of CW- and LA-PAA–
washed spinach and tender leaves mix after storage at 7.2uC for 15
and 14 days, respectively. Log reduction values with different
letters indicate statistical significance at P , 0.05. CW con-
centration: 10 ¡ 2 ppm of free chlorine at pH 7.0 ¡ 0.2. LA-PAA
concentration: 2,000 ppm of LA and 70 ppm of PAA.
However, there was no statistically significant difference (P
, 0.05) between the microbial efficacy of LA-PAA with or
without SLS on attached cells. In addition, excessive
foaming was observed during scale-up studies because of
the properties of the wash system. These are reasons why
we do not recommend the addition of surfactant in the LA-
PAA formulation for fresh produce processing.
Unlike other studies that showed sogginess of leaves
resulting from PAA washed produce (2, 17, 23, 29), sensory
evaluations from three studies with 100 panelists each,
showed that LA-PAA presented no negative effects on the
organoleptic properties of chopped iceberg, chopped
romaine, and tender leaves mix with spinach. In addition,
temperature-abuse storage studies demonstrated that LA-
PAA reduced spinach and tender leaves mix decay
percentage (wt/wt) by ,50%. This reduction in decay was
attributed in part to the higher reduction in indigenous
microorganisms attached to the leaves surfaces by LA-PAA.
Our validation of LA-PAA as an effective new sanitizer
was comprehensive, as recommended by others for
development of such novel technologies (14). It involved
studies conducted at bench-top, pilot plant, and commercial
facilities, with sensory evaluation performed on treated
produce after temperature-abuse storage. LA-PAA is
scalable from bench-top to commercial scale, and it
possesses no negative influence on produce quality.
In this study, L. innocua and E. coli K-12 were used to
demonstrate the microbial efficacy of LA-PAA, as these two
strains have served commonly as the nonpathogenic
surrogates in produce cases that involved L. monocytogenes
and E. coli O157:H7, respectively (5). Results of studies
using pathogens cocktails will be published subsequently to
substantiate the inactivation of LA-PAA on produce-
pertinent pathogens, as many pathogens are known to be
significantly more resistant to inactivation intervention
treatments than other nonpathogenic variants within the
same species or genera. Nonetheless, the overall results
reported in this article indicate that LA-PAA has great
potential to be an excellent alternative for CW in fresh
produce processing.
1474 HO ET AL.
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