Food Control 50 (2015) 645e651

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Influences of malic acid and nisin supplementations on the decimal

reduction times of Escherichia coli O157:H7 in mildly-heated young
coconut liquid endosperm
Alonzo A. Gabriel, Emil Emmanuel C. Estilo*
Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, Alonso Hall, Ma. Regidor St., Diliman,
Quezon City 1101, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: This study was conducted to determine the effects of malic acid (800e1500 ppm) and nisin (0e150 ppm)
Received 31 March 2014 on the heat resistance of Escherichia coli O157:H7 in young coconut liquid endosperm. The Central
Received in revised form Composite Rotatable Design was applied to determine the various combinations of the two supplements
8 September 2014
to be added to young coconut liquid endosperm. The lowest D55 value was observed in the combination
Accepted 4 October 2014
Available online 14 October 2014
of 1500 ppm malic acid and 75 ppm nisin at 14.89 ± 4.17 min, while highest was 27.04 ± 4.98 min in the
combination of 1150 ppm malic acid and 75 ppm nisin. The D55 values established from the combinations
tested did not significantly vary, indicating that within the ranges of supplementation tested, significant
Keywords:
Escherichia coli O157:H7
influences of both additives and their interaction on the heat resistance of the pathogen did not exist. It
Heat inactivation was however emphasized that all supplementation combinations resulted in D55 values significantly
Malic acid lower than that in the unsupplemented medium, reducing the heat resistance of the cells by as much as
Nisin 3-folds. Therefore, thermal processes to be established in the supplemented liquid endosperm shall be
Young coconut liquid endosperm less severe, and may be applied to preserve sensory attributes.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction market opportunities to be developed both as a refreshing beverage

The Philippines is one of the world's largest producer of co-
conuts and coconut products, playing an essential role in the in-
ternational flow and supply of copra, coconut oil, copra meal and
cake, and desiccated coconut (PCA, 2009). Because of its multiple
uses and versatility, food products such as coconut-derived bever-
ages have found its way into the market. More popularly known as
buko juice, young coconut liquid endosperm has long been a pop-
ular drink in the tropics, especially in Southeast Asia, as it is known
for its energy-giving characteristic and health benefits (Tietze,
Echano & Stepanovs, 2006). Moreover, consumer preferences
have shifted to fruit, vegetables and their derived beverage prod-
ucts (Kaur & Kapoor, 2001) due to an increased awareness to
address health and wellness needs (Sloan, 2010). Young coconut
liquid endosperm is consumed for its nutritional and therapeutic
properties attributed to its composition of sugars, vitamins, min-
erals, amino acids, and phytohormones (Yong, Ge, Ng, & Tan, 2009).
Its consumption is also known to help in alleviating muscle sore-
ness and other stress- and acidity-related conditions, giving it
* Corresponding author.
E-mail address: emil.estilo@gmail.com (E.E.C. Estilo).
and as a sports drink (Tietze et al., 2006). One notable improvement
in the market form of the beverage is the development of bottled
variant, making transport and distribution much easier (Rolle,
2007).
Due to extensive demands for products such as fruit beverages
that are minimally processed and with optimal fresh qualities, the
industry has been continually involved in incidences of foodborne
illness outbreaks, usually due to enteric pathogens such as
Escherichia coli O157:H7 (McClure & Hall, 2000). A previously
conducted study assessing the overall quality of young coconut
liquid endosperm shows that these commercially available bever-
ages do not conform to the allowable microbial density levels
required by the Philippine Food and Drug Administration (FDA
Philippines, 2013), possibly due to the very manual preparation of
the beverages that increases the probability of introducing con-
taminations (Paguirigan, Molina, Lorenzana, Valencia, & Masa,
2000). Indeed, it has been reported that microbial contaminations
of as much as 6.0 log cfu mL
1 may be introduced to the liquid
endosperm when collected in the traditional manual methods
(Reddy, Das, & Das, 2005). Moreover, foodborne outbreaks of E. coli
O157:H7 have been reported with a variety of foods including un-
pasteurized fruit juices (Chauret, 2011).

http://dx.doi.org/10.1016/j.foodcont.2014.10.007
0956-7135/© 2014 Elsevier Ltd. All rights reserved.
646 A.A. Gabriel, E.E.C. Estilo / Food Control 50 (2015) 645e651
One of the methods that may be applied to such products to
inactivate microbial contaminations is the application of heat
(Alabastro, 1987; Lado & Yousef, 2002). Heat pasteurization aims to
increase shelf life during refrigeration and ensure human health
and safety concerns by controlling vegetative pathogens and
reducing microbial load (Gould, 1995; Heldman & Hartel, 1997). The
efficacy of heating in improving product safety and shelf-life sta-
bility of fruit juices through reducing both pathogenic and spoilage
microorganisms is recognized by the United States Food and Drug
Administration (Donahue, Canitez, & Bushway, 2004). Heat appli-
cation is however, known to affect and alter the heat-labile and
sensitive components of food products, which results to a
decreased sensorial acceptability as well as nutritional losses
(Gabriel, 2012). In order to mitigate this unacceptable effect, other
forms of thermal processing aides are being studied in order to
reduce the severity of a heat process schedule, but still result in the
desired level of safety and process lethality. Such concept is the
basis of hurdle technology that combines multiple, mild physical,
chemical process techniques and other preservation factors that
may additively or synergistically inactivate microbial contami-
nants, thereby resulting in safe, stable, nutritious, tasty and
economical foods (Leistner & Gorris, 1995; Ohlsson & Bengtsson,
2002).
Some organic acids and bacteriocins are classified as antimi-
crobials. Malic acid and nisin belong to this classification, and
numerous studies have already investigated and applied their
use as food preservatives. They control microbial growth by
lowering the pH levels and disrupting cellular membrane func-
tionality (Heredia, Wesley, & García, 2009; Theron & Lues, 2009)
as well as inactivating essential cellular enzymes and disinte-
gration of genetic material (Davidson, Sofos, & Branen, 2005).
The application of biopreservatives, in combination with tradi-
tional preservative techniques and Good Manufacturing Practice
(GMP) may effectively control the growth of foodborne patho-
gens along with the microorganisms causing spoilage (Davidson
et al., 2005; Riley, 2005). Furthermore, supplementation of these
additives together with physical processing techniques like
heating might result in more acceptable thermal process
schedules, possessing the desired lethalities without negatively
affecting product qualities.
This study aimed to determine the influences of malic acid and
nisin supplementation on the heat resistance characteristics of
E. coli O157:H7 in mildly heated (55  C) young coconut liquid
endosperm. Specifically, the study aimed to (1) determine the
maximum acceptable supplementation concentrations of malic
acid to young coconut liquid endosperm through multi-level sen-
sory evaluation, (2) determine the effects of the combinations of
malic acid and nisin on the decimal reduction times (D55 values) of
a cocktail of E. coli O157:H7 in mildly heated (55  C) young coconut
liquid endosperm. The results of this study may be used in the
establishment of alternative, less severe thermal process schedules
that shall similarly ensure young coconut liquid endosperm safety
against the tested pathogen.
2. Materials and methods
2.1. Nisin stock solution preparation
The procedure in preparing the nisin stock solution
(5000 IU g
1) was adopted from the Compendium of Food Additive
Specifications (FAO, 2008). Standard nisin (Nisin, Sigma Chemical
Co., Missouri, USA), containing 2.5% active nisin and having a bio-
logical activity of 106 IU g
1, was used to prepare the nisin stock
solution. A nisin stock solution of 200 mL was prepared by diluting
1 g of standard nisin into 200 mL of 0.02 N HCl (pH 1.6), boiled for
5 min and filter-sterilized using a microfiltration setup (Advantec,
Toyo Roshi Kaisha, Ltd., Japan) through a 0.45 mm membrane filter
paper (Advantec, Toyo Roshi Kaisha, Ltd., Japan). The stock solution
was stored in a sterile volumetric flask and refrigerated until
further use.
2.2. Young coconut liquid endosperm suspending medium
preparation
The young coconut (6 months) liquid endosperm used as sus-
pending medium for thermal inactivation were obtained and pro-
cured from a local coconut vendor in Quezon City, Philippines 1101.
The liquid endosperm was obtained by making successive cuts on
the mesocarp until a portion of the endocarp is severed to create an
opening for the liquid endosperm to be aspirated out of the drupes
and subsequently transferred into sterile glass bottles.
Upon arrival in the laboratory, the samples were initially passed
through a filter paper (Whatman, GE Healthcare, UK) and subjected
to physicochemical analyses. (pH, total soluble solids (TSS), titrable
acidity (TA)) before supplementing with malic acid and nisin. TSS
(Bx) and pH level of the liquid endosperm were respectively
measured using a handheld refractometer (Atago 2522 HHR-2N,
Atago Co. Ltd., Japan) and a pH meter (Cyberscan 500, Eutech In-
struments, Singapore) calibrated with pH 7.00 and pH 4.00 stan-
dard solutions. Determination of the inherent malic acid
concentration was also carried out by measuring the TA using
standardized 0.05 N NaOH. Food-grade powdered malic acid and a
nisin stock solution (5000 IU g
1) were used for adjustment. The
Pearson's Square formula (Fellows, 2000) was used to calculate the
exact amounts of malic acid and nisin supplements.
Filter-sterilization of the supplemented liquid endosperm was
done using a microfiltration setup (Advantec) was done using
0.45 mm membrane filter papers (Advantec). Physicochemical
properties such as pH, TSS, and TA were similarly characterized
subsequently. The filter-sterilized 200-mL supplemented liquid
endosperm samples were stored in sterile glass bottles and
refrigerated at 4  C for no more than 3 d, until used in the thermal
inactivation studies.
2.3. Central composite rotatable design for two factors
A two-factor central composite rotatable design (CCRD) was
used in determining the different combinations of malic acid and
nisin supplements on young coconut liquid endosperm. The 10
combinations investigated included 4 factorial combinations (high
e low combinations), 4 axial combinations (very high/low e in-
termediate combinations) and 2 center combinations (inter-
mediateeintermediate combinations). The maximum
supplementation level of malic acid and nisin were designated as
the þa values. The maximum malic acid supplementation level was
based on consumer acceptance established in a preliminary multi-
level consumer acceptance sensory evaluation test. As for nisin, the
maximum supplementation level was based on the provisions of
the Codex Alimentarius (FAO and WHO, 2011).
A multi-level sensory evaluation process, which included a
preliminary evaluation, a focus group discussion (FGD) with 10
semi-trained panelists, and a consumer acceptance test (n ¼ 30
panelists, 7-point standard hedonic scale) (data not presented), was
carried out in order to determine the maximum tolerable malic acid
concentration that can be supplemented to young coconut liquid
endosperm. The limits of malic acid supplementation established in
this phase were used in the determination of the combinations of
malic acid and nisin in the subsequent phases of the study. The
software Design Expert 8.0 (Stat-Ease, Inc., 2010, Minnesota, USA)
was used in order to calculate the concentrations of the 10
 A.A. Gabriel, E.E.C. Estilo / Food Control 50 (2015) 645e651 647

treatments. Table 1 presents the coded and uncoded variable Georgia, USA). The supernatant was decanted and resuspension
combinations of malic acid and nisin tested in the study. was done by vortex-mixing in 1 mL filter-sterilized young coconut
liquid endosperm. The cells were acclimatized in the liquid endo-
2.4. Test organisms and working cultures sperm for not longer than 15 min. Cells only subjected to this
activation, enrichment and acclimatization steps were used in the
All test organisms used in the study were gifts from Dr. Hiroyuki thermal activation studies.
Nakano of the Laboratory of Food Microbiology and Hygiene,
Graduate School of Biosphere Science, Hiroshima University, 2.6. Inoculation and thermal inactivation of E. coli O157:H7
Higashi Hiroshima Campus, Japan. The test organisms included five
strains of E. coli O157:H7. The tested serovars include the HCIPH For the thermal inactivation studies, 9.9 mL filter-sterilized
96055 strain, which was obtained from the Hiroshima City Institute young coconut liquid endosperm supplemented with malic acid
of Public Health, Hiroshima, Japan, and the serovars MY-29, DT-66, and nisin in test tubes (24 mm) were pipetted into 24-mm test
MN-28, and CR-3, which were obtained from the National Food tubes, after which 0.1 mL aliquots of the acclimatized cell suspen-
Research Institute, Ibaraki, Japan. sions with approximately 6.00 log cfu mL
1 were introduced to
Working cultures were prepared by aseptically transferring two each tube. The inoculated tubes were immersed and heated on a
stabs each from the refrigerated stock culture into individual 1-mL water bath (PolyScience, Illinois, USA) maintained at a temperature
sterile nutrient broths (NB, Eiken Chemical Co., Ltd., Japan) and of 56  C. The medium temperature was measured by inserting a
incubated at 37  C for 18 h. Individual loopfuls from the activated thermometer through the cold point of a control tube. Contents of
NB culture strains were aseptically transferred into their corre- the inoculated tubes were heated and observed to have come up
sponding nutrient agar (NA, Eiken Chemical Co., Ltd., Japan) slants time to 55  C of 15 min. Preliminary works showed that the inoc-
before subsequent incubation at 37  C for 18 h. The working slants ulated populations were not significantly affected by the come up
were continually kept in refrigerated storage at 4  C until further time (data not presented). Heat exposure timing commenced as
use in the experiments. Fresh working slants were prepared weekly soon as the cell suspensions reach 55  C. The tubes were constantly
following the aforementioned protocols. and manually agitated throughout the heating period that lasted
for as long as 120 min. After heat treatment, the tubes were
2.5. Test organism propagation and composite inoculum immediately immersed into and kept in an ice bath until survivor
preparation enumerations. All thermal inactivation studies were conducted in
two independent runs, with three replications per run.
Prior to thermal inactivation studies, cells were obtained from
the refrigerated working slants by individually inoculating two 2.7. Survivor enumerations and calculation of D55 values
loopfuls of strain each into sterile 1-mL NB tubes and incubating at
37  C for 18 h. Enrichment was done by obtaining a loopful of cells Surviving cells were enumerated from each of the thermally
from each of the incubated NB into their corresponding 10-mL NB treated tubes by conducting 10-fold serial dilutions using sterile
broths and incubating at 37  C for another 18 h. For the preparation 0.1% peptone water (PW, HiMedia, India) and spread-plating (0.1-
of pathogen cocktails, 2-mL aliquots were obtained from each of the mL aliquots) onto pre-solidified NA plates. The plates were incu-
enriched strains and pooled into a sterile test tube prior to vortex- bated at 37  C for 18 h. Colonies were enumerated and cell pop-
mixing for 1 min. Aliquots of 1 mL from the cocktail of E. coli ulations were reported as log cfu mL
1. Survival curves were
O157:H7 cultures were obtained and transferred into micro generated by plotting the enumerated population against heating
centrifuge tubes. Cells were harvested by spinning at 7000 rpm for time using a freeware program DMFit Version 3.0 (Institute of Food
15 min using a portable centrifuge (ZIP Spin, LW Scientific, Inc., Research, Reading Laboratory, UK). Based on the models estab-
lished by Baranyi and Roberts (1994), the freeware was able to
calculate the death rate (KD) kinetic parameter of the test organisms
Table 1
in each of the tested suspending medium. Heat resistance of E. coli
Malic acid and nisin combinations1 with corresponding D55 values2 of E. coli
O157:H7 in mildly-heated young coconut liquid endosperm.
O157:H7 was reported in terms of decimal reduction times at 55  C
(D55), which was equivalent to the negative inverse of the calcu-
CCRD Combination3 Coded variables Uncoded variables D55 (min)
lated KD values. In a log-linear inactivation curve, the D55 values are
points values values
graphically equivalent to the negative inverse of the slopes of the
Malic Nisin Malic acid Nisin regressed survivor curves in each combination (Jay, 2000) and
acid (ppm) (ppm)
correspond to the number of min of heat exposure at 55  C that will
Factorial 1
1
1 903 22 23.22 ± 2.55ab yield a reduction in the population of E. coli O157:H7 in young
2 +1
1 1397 22 25.26 ± 10.42ab
coconut liquid endosperm by 90% or 1 log cycle. In this study, D55
3
1 +1 903 128 21.06 ± 2.54ab
4 +1 +1 1397 128 26.44 ± 9.81ab values were only calculated from inactivation curves that traversed
Axial 5 0
a 1150 0 19.46 ± 11.88ab more than 1 log cycle and with R2 values not less than 0.9.
6 0 +a 1150 150 23.92 ± 13.37ab
7
a 0 800 75 25.96 ± 6.90ab 2.8. Statistical analysis
8 +a 0 1500 75 14.89 ± 4.17a
Center4 9 and 10 0 0 1150 75 24.83 ± 7.20ab
Control e e e 800 0 45.87 ± 2.10c Data obtained from all independently replicated experiments
a,b,c were subjected to single-factor Analysis of Variance (ANOVA) using
Values on the same column followed by the same letter are not significantly
different. the General Linear Model (GLM) procedure of the software IBM
1
Malic acid e nisin combinations were generated using the CCRD (malic acid:
a SPSS Statistics 19 (SPSS, Inc., 2010, New York, USA). Duncan's
¼ 800 ppm, +a ¼ 1500 ppm; nisin:
a ¼ 0 ppm, +a ¼ 150 ppm) for two-factored Multiple Range Test (DMRT) was used as post-hoc analysis when a
experiments using the software Design Expert 8.0 (Stat-Ease, Inc., 2010). significant difference existed among means (p < 0.05). The effects of
2
D55 values are presented as averages of 6 trials (n ¼ 6) obtained from 2 inde-
pendent experimental runs except for the Control (n ¼ 3).
the malic acid and nisin on the thermal inactivation of the cells
3
Order of experimental run based on Design Expert 8.0 randomization. were characterized through Response Surface Methodology (RSM)
4
Duplicated center points as determined by CCRD. using the software Design Expert 8.0 (Stat-Ease, Inc., 2010).
648 A.A. Gabriel, E.E.C. Estilo / Food Control 50 (2015) 645e651
3. Results and discussion
3.1. Heat resistance of E. coli O157:H7 in young coconut liquid
endosperm
This study utilized E. coli O157:H7 cells as challenge organism
for thermal inactivation in young coconut liquid endosperm (pH:
4.53 ± 0.14, TSS: 5.00 ± 0.02 Bx, TA: 0.11 ± 0.02%). In this phase,
the maximum supplementation levels of malic acid, established in
the previously conducted multi-level sensory evaluations (data not
presented), and nisin from the Codex Alimentarius (FAO & WHO,
2011) were used to generate malic acidenisin combinations using
CCRD for two factors. Table 1 presents the corresponding D55 values
for each malic acidenisin combination and Fig. 1 illustrates repre-
sentative survivor curves for each combination.
The data presented in Table 1 showed that the calculated D55
values in all combinations of malic acid and nisin ranged from 14.89
to 27.04 min. The lowest D55 value was observed in cells suspended
in combination VII at 1500 ppm malic acid and 75 ppm nisin. On the
other hand, the highest D55 value was observed in combination III
at 1150 ppm malic acid and 75 ppm nisin. It was also observed that
the D55 values in the other combinations tested were not signifi-
cantly different (p > 0.05) from each other, indicating that within
the ranges of supplementation tested in this study, the influences of
the additives on the heat resistance of the test pathogen were
similar.
Results of the response surface analysis that fitted the calculated
D55 values in a quadratic model to characterize the influences of the
malic acid and nisin on the heat resistance of the E. coli O157:H7
showed that the D55 values obtained from all combinations did not
significantly fit into the full quadratic model (data not presented).
Such outcome may have been due to the non-significant differences
in the heat resistance of the test pathogen within the operable
region, or across the tested combinations of malic acid and nisin
explored in this study. The model, as well as all its terms, was not
significant (p ¼ 0.95, R2 ¼ 0.19). According to the annotations
appended to the output of the Design Expert, the model p-value
implied that there is a 95.18% chance that non-significance could
occur due to noise. The predicted R2 value was calculated to
be
4.51. This negative value implies that the overall mean
(22.92 min) is a better predictor for response than the full quadratic
model. Neither reduction of the model nor transformation of the
responses resulted in a significant fit.
Regression for the quadratic model coefficients by the same
freeware resulted in the following equation. To characterize the
influences of malic acid and nisin on the heat resistance of the cells
within the tested operable region, the generated equation was used
to plot perturbation and surface plots (Figs. 2e3).
D55 ¼ 1:452313 þ 0:044882ðMalic acidÞ
0:010441ðNisinÞ
þ 6:349562  10
5 ðMalic acid  NisinÞ
2:339651
 10
5 ðMalic acidÞ2
0:000350ðNisinÞ2
The perturbation plot for a variable was generated by fixing the
value of the other variable at the intermediate setting (1150 ppm
malic acid, 75 ppm nisin). In Fig. 2, discernible parabolic plots were
observed to illustrate the relationships between both supplements
and D55 values of E. coli O157:H7, both indicating opposing mech-
anisms between a vertex point. At lower malic acid supplementa-
tion levels, the additive seemed to have slightly enhanced the heat
resistance of the cells. However, beyond the malic acid supple-
mentation level equivalent to about 0.2 (1100 ppm), further in-
creases in malic acid supplementation resulted in more notable
decrease in heat resistance of the test organisms. Similarly,
opposing trends in the D55 values of E. coli O157:H7 were observed
for nisin; the comparable decrease in heat resistance can be
observed in supplementation values below
0.3 (75 ppm). How-
ever, unlike the trend observed for the effects of malic acid, a more
notable increase in the heat resistance prior to reaching the vertex
point concentration of nisin was observed, followed by a slight
decrease in the resistance beyond it.
The young coconut liquid endosperm used in this study had an
inherent malic acid concentration of 800 ppm determined through
the measurement of titrable organic acids. The unsupplemented
liquid endosperm had a pH of 4.72 ± 0.01. Increasing the supple-
mentation level of malic acid to the liquid endosperm resulted in
the steady reduction of pH to 4.31 (1500 ppm). Such change in the
physicochemical characteristic of the liquid endosperm must have
similarly caused the reduction in the D55 value of E. coli O157:H7
from 45.87 min (control: 800 ppm malic acid, 0 ppm nisin) to
19.46 min (combination IX: 1150 ppm malic acid, 0 ppm nisin).
Acidity is recognized as one of the most important factors influ-
encing the heat resistance of bacteria. In fact, the classification of
foods as high- (pH < 4.5) or low-acid (pH > 4.5) commodities has
been the basis for the determination of the severity of heat pro-
cessing (Alabastro, 1987). The study of Juneja and Novak (2003)
similarly investigated the heat resistance of E. coli as affected by
acidulants, and demonstrated that the D55 values were lower in
samples at the pH of 4.5 than those at 5.5. Reichart (1994) explained
that generally, microorganisms usually have maximum heat re-
sistances at pH levels near neutrality. Thus, a decrease in pH by the
supplementation of acidulants like organic acids to the food sus-
pending medium usually results in the reduction of D55 values.
Theron and Lues (2009) also stated that aside from the reduction of
pH due to the liberation of hydrogen ions from the ionization of
organic acid molecules into the cell environment, the undissociated
molecules have also been reported to be able to cross the cell
membrane and diffuse into the cytoplasm where they dissociate
and release hydrogen ions. The cells then must then exert energy to
pump the excess hydrogen ions out of the cell to maintain
homeostasis.
Between the two supplements used, nisin seemed to have lesser
heat sensitizing effect on the test pathogens. Although supple-
mentation of young coconut liquid endosperm with as little as
75 ppm in the absence of additional malic acid resulted in a D55
value of 25.96 min that is significantly lower than that of the
control, further increase in malic acid concentration appeared to
have non-significant effects on the efficacy of the bacteriocin in
inducing heat sensitivity to the cells. In combination pairs 1
(903 ppm malic acid, 22 ppm nisin) e 2 (1397 ppm malic acid,
22 ppm nisin), and 3 (903 ppm malic acid, 128 ppm nisin) e 4
(1397 ppm malic acid, 128 ppm nisin) where nisin supplementation
levels were constant, increase in malic acid supplementation levels
were observed to result in the increase in the heat resistance of the
cells, suggesting a possible cancellation of the efficacy of the
bacteriocin in inducing heat resistance to cells with increasing
malic acid supplementation. The changes in the heat resistance of
the test pathogens as malic acid and nisin supplementation levels
simultaneously vary are presented in Fig. 3.
Delves-Broughton (2005) explained that nisin shows little or no
activity against Gram negative bacteria. Several studies have been
able to demonstrate this effect (Chung, Dickinson, & Crouse, 1989;
Dheraprasart, Rengpipat, Supaphol, & Tattiyakul, 2009; Pol et al.,
2001). Furthermore, Boziaris and Adams (2001) similarly demon-
strated that nisin was only able to reduce the population of Gram-
negative cells that have been previously exposed to sublethal injury
after exposure to 55  C; and that the bacteriocin had little or no
effect on uninjured cells. Hence simultaneous exposures of cells to
heat and malic acid must have obscured the efficacy of nisin against
 A.A. Gabriel, E.E.C. Estilo / Food Control 50 (2015) 645e651 649

Fig. 1. Representative D55 curves for test malic acid and nisin combinations: (a) 1150 ppm malic acid:150 ppm nisin, (b) 1397 ppm malic acid:22 ppm nisin, (c) 1150 ppm malic
acid:75 ppm nisin, (d) 903 ppm malic acid:22 ppm nisin, (e) 1397 ppm malic acid:128 ppm nisin, (f) 1150 ppm malic acid:75 ppm nisin, (g) 1500 ppm malic acid:75 ppm nisin, (h)
903 ppm malic acid:128 ppm nisin, (i) 1150 ppm malic acid:0 ppm nisin (j), 800 ppm malic acid:75 ppm nisin.
650 A.A. Gabriel, E.E.C. Estilo / Food Control 50 (2015) 645e651
Fig. 2. Perturbation plot showing the individual influences of malic acid (C) and nisin
(B) on the D55 of E. coli O157:H7 in young coconut liquid endosperm (malic
acid:
1 ¼ 800 ppm, 0 ¼ 1150 ppm, þ1 ¼ 1500 ppm; nisin:
1 ¼ 0 ppm,
0 ¼ 75 ppm, þ1 ¼ 150 ppm). Each plot was generated at fixed malic acidenisin
level ¼ 0.
the tested pathogens. As shown in this study, individual antimi-
crobials cannot be simply combined without preliminary charac-
terizations of possible interactions such as synergism or
antagonism between hurdles. It has been demonstrated by a
number of studies that combining antimicrobials against foodborne
microorganisms might result in various scenarios of synergy, ad-
ditive efficacy, or antagonism (Casey & Condon, 2002; Lee, 2004;
Lee & Kang, 2009; Lee, Rhee, Dougherty, & Kang, 2010).
It should however be noted that, despite the small range in the
calculated D55 values among the combinations tested, the heat
resistances of the cells in all the test combinations were still
significantly smaller (p < 0.05) than those in the control, unsup-
plemented medium (Table 1) at 45.9 min. In fact, results showed
that the heat resistance of the cells in the supplemented liquid
endosperm were 1.7- to 3.0-folds lower than the control. Therefore,
Fig. 3. Response surface plot for the influences of malic acid and nisin on the D55
values of E. coli O157:H7 in young coconut liquid endosperm.
heat process schedules that will be based on any of the combina-
tions tested shall be able to achieve a desirable lethality at shorter
processing times. For example, in order for a processor to apply a
heat process schedule that is capable of reducing the population of
E. coli O157:H7 by the recommended (Alabastro, 1987) 5 logarith-
mic cycles in unsupplemented liquid endosperm, the commodity
must be heated at 55  C for 229.5 min. In all malic acid- and nisin-
supplemented liquid endosperm tested in this study, the same
lethality can be achieved for only 74.15e135.2 min. These results
may therefore have significant implications in the preservation of
the sensory quality of heat-treated liquid endosperm.
In this study, the supplementation level of nisin was limited by
the maximum allowable limits stipulated by the Codex Ali-
mentarius; and that of malic acid was limited by consumer
acceptance in undiluted and unsweetened young coconut liquid
endosperm. Malic acid supplementation may further be increased
without affecting consumer acceptance if heat resistance of path-
ogens shall be established in sweetened and diluted samples. Slight
increase in process temperature may similarly result in the estab-
lishment of more effective process schedules.
Acknowledgments
This research was supported by the Office of the Vice Chancellor
for Research and Development of the University of the Philippines
Diliman under the PhD Incentive Grant (PhDIA 111123). The authors
would also like to extend their gratitude to Aleli Torralba, Bryan
Atas, Eric Gelido, Hannah Madriaga, Ned Cuartel and Sarah Villostas
for their help in conducting the experiments.
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