1736

Journal of Food Protection, Vol. 82, No. 10, 2019, Pages 1736–1743
https://doi.org/10.4315/0362-028X.JFP-19-071
Copyright Ó, International Association for Food Protection

Research Paper

Fate of Spoilage and Pathogenic Microorganisms in Acidified

Cold-Filled Hot Pepper Sauces
ARIELA LOBO,1† CAROLINA ZÚÑIGA,1† RANDY W. WOROBO,2 OLGA I. PADILLA-ZAKOUR,2 AND JESSIE USAGA2,3*

1Escuela de Tecnología de Alimentos, Universidad de Costa Rica (UCR), Ciudad Universitaria Rodrigo Facio, CP 11501-2060, San José, Costa Rica;
2Department of Food Science, Cornell University, 665 West North Street, Geneva, New York 14456, USA; and 3Centro Nacional de Ciencia y Tecnología de
Alimentos (CITA), Universidad de Costa Rica (UCR), Ciudad Universitaria Rodrigo Facio, CP 11501-2060, San José, Costa Rica

MS 19-071: Received 12 February 2019/Accepted 15 June 2019/Published Online 19 September 2019

ABSTRACT
Consumption of spicy foods and hot sauces is currently a popular trend worldwide. Shelf-stable acidified sauces are
commonly hot-filled to ensure commercial sterility, but cold-fill-hold processes might also be suitable if microbial safety and
stability are ensured. For this study, model acidified hot pepper sauces were developed and characterized. The effects of sauce
pH and of two different organic acids on the survival of Pichia manshurica and Lactobacillus curvatus isolated from
contaminated commercial hot sauces and on pathogenic Escherichia coli O157:H7, Salmonella enterica, and Listeria
monocytogenes were assessed. Full factorial designs with three levels for pH (3.2, 3.5, and 3.9) and two for organic acid (citric
and acetic) were used to determine the effects of these factors and their interactions on the survival of the microorganisms.
Commercially sterile sauces were independently inoculated and kept at ambient temperature. Microbial counts were determined
at different sampling times, depending on the treatment evaluated. Sauces acidified to pH 3.2 with citric or acetic acid were
inoculated with cocktails of five strains or serotypes of the three pertinent pathogens, and inactivation curves were determined.
Trials were performed in triplicate. A greater than 5-log reduction of P. manshurica and L. curvatus was achieved in less than 6 h
in sauces adjusted to pH 3.2 with acetic acid. Greater than 5-log reductions of pathogenic bacteria were achieved 0.5 h after
inoculation in sauces acidified to pH 3.2 with acetic acid. In contrast, at least 48 h was required to guarantee the same
inactivation for the most tolerant pathogen when citric acid was used. Thus, a cold-fill-hold process may be a suitable alternative
for acidified hot pepper sauces. Based on survival of the microorganisms evaluated in this study, microbial safety and stability
can be achieved by adjusting the pH to 3.2 or less by the addition of acetic acid.

HIGHLIGHTS

pH and acidifier influence safety and stability of cold-filled acidified sauces.

Safe and stable cold-filled sauces were obtained at pH 3.2 with acetic acid.

Results help establish science-based conditions for cold-filled products.

Key words: Cold-filled acidified foods; Hot pepper sauce; Lactobacillus curvatus; Pichia manshurica

Hot peppers and other spices have traditionally been origin, and the use of different peppers including habanero,
used to color, flavor, and preserve foods because of their jalapeño, chipotle, ghost, green chile, cayenne, espelette,
typical color, pungency, taste, aroma, and composition (4, arbol, and others (23). Thus, it is not surprising that the food
9). Recent epidemiological studies have demonstrated industry (including artisanal producers and private labels)
potential health benefits associated with the consumption has developed and launched an important number of
of spicy foods (4, 19). A recent population-based prospec- cooking and table hot sauces in recent years.
tive cohort study showed that consumption of hot red chili Acidified hot pepper sauces are traditionally sold at
pepper was associated with reduced mortality (9). Spicy hot room temperature due to their commercial sterility in
foods represent one of the most significant flavor trends in unopened containers. According to the U.S. Food and Drug
the United States in the last few years (23). This trend has Administration (FDA) Code of Federal Regulations (CFR
prompted the development of a wide variety of spicy 21 Part 114), ‘‘acidified foods means low-acid foods to
products, including hot sauces. Sauce manufacturers are which acid(s) or acid food(s) are added; these foods include,
highlighting the piquancy of their products, country of but are not limited to, beans, cucumbers, cabbage,
artichokes, cauliflower, puddings, peppers, tropical fruits,
* Author for correspondence. Tel: þ506 2511-3465; Fax: þ506 2253-
and fish, singly or in any combination. They have a water
3762; E-mail: [email protected]. activity greater than 0.85 and have a finished equilibrium
† These authors contributed equally to this work. pH of 4.6 or below’’ (26). According to regulations, these
J. Food Prot., Vol. 82, No. 10 SPOILAGE AND PATHOGENIC MICROORGANISMS IN COLD-FILLED HOT SAUCES 1737

TABLE 1. Formulations of the hot sauces acidified to three pH values using citric and acetic acid
Formulation (%)

pH 3.2 pH 3.5 pH 3.9

Ingredient Citric acid Acetic acid Citric acid Acetic acid Citric acid Acetic acid

Habanero puree 71.93 69.64 72.14 71.21 72.36 72.14

Water 24.80 24.02 24.88 24.58 24.95 24.88
Salt 2.48 2.40 2.49 2.45 2.49 2.49
Acidulant 0.79 3.94 0.49 1.76 0.20 0.49

products ‘‘shall be thermally processed to an extent that is yeasts are responsible for about three-fourths of the cases in
sufficient to destroy the vegetative cells of microorganisms these products (18, 20). Saccharomyces, Pichia, and
of public health significance and those of nonhealth Candida are the most frequently isolated yeasts in low-pH
significance capable of reproducing in the food under the products (13, 20). It is well known that preservatives may
conditions in which the food is stored, distributed, retailed be used to inhibit reproduction of microorganisms of
and held by the user’’ (26). All processors of acidified foods nonhealth significance in lieu of thermal processing (26).
are mandated to submit a scheduled process for each of their However, this alternative may not be an appealing
products to the FDA before commercialization. This may be marketing option given the major consumer-led movement
particularly challenging for small companies and entrepre- to limit the consumption of foods containing preservatives
neurs, considering that shelf-stable acidified foods must be (10).
thermally processed to ensure microbial safety and stability The pH of acidified foods is typically adjusted to
(7, 26). On the other hand, FDA allows the use of a cold- between 3.2 to 3.9, using acetic acid as the acidulant of
fill-hold process for products that have inherent qualities, preference (20). However, the characteristic strong aroma
such as pH, that destroy vegetative cells of microorganisms and flavor of this organic acid has led some companies to
of concern at ambient temperatures. In this case the attempt the use of alternative acidifiers with a milder profile
containers do not require a thermal treatment. However, a (20), such as citric, malic, and lactic acids. A cold-fill-hold
challenge study must be provided to demonstrate that the process can be an extremely attractive alternative for small
product, as formulated, will result in the destruction of processors because of the reduced production times and
pathogens and spoilage microorganisms of concern (25). To energy savings associated with this approach in comparison
complete a scheduled process, the processes applied to the with hot-filled or in-bottle pasteurized products. Likewise,
product must be supported by scientifically based findings considering that some acidified foods may have inferior
that substantiate the conditions of the selected treatment. quality if subjected to a thermal treatment and that some
Currently, this is a major concern for the industry, given the plastic containers will deform if a product is hot-packed, the
limited availability of published research that can be used to definition of appropriate conditions for cold-fill-hold
define critical limits for cold-filled acidified products. processes for acidified products is of particular industrial
Moreover, few laboratories are equipped to appropriately interest. Nevertheless, such development attempts should be
perform microbial challenge studies with pathogens and undertaken with due diligence to verify that the replacement
spoilage microorganisms, and few currently offer the of acetic acid with another acidulant does not compromise
service to the public. In addition, the cost of microbial the microbiological stability or safety of the modified
challenge studies may be prohibitive for most small-scale products (20, 27) and that the cold-fill process would indeed
processors. represent a suitable packing alternative for a particular
The exceptional resistance to microbiological spoilage formulation without compromising its microbial stability
of acidified specialty products, including hot pepper sauces, and safety during shelf life.
is attributable to low pH values (below 4.0), high Therefore, the objective of this study was to determine
concentrations of acetic acid (mainly added as vinegar), the suitability of a cold-fill-hold process by evaluating the
and limited headspace and oxygen availability in the effect of pH and acidulant type on the fate of selected
container (20). Food safety incidents, as well as economic spoilage and pathogenic microorganisms of concern in
losses, due to microbiological issues in acidified foods are acidified hot pepper sauces.
very few. However, the absence or neglect of formulation
and production controls may cause major spoilage incidents MATERIALS AND METHODS
(20). The low pH and high acetic acid concentration in most
Hot sauce formulation and preparation. Six standard
acidified products prevent the growth of almost all acidified hot sauce formulations were developed using the
vegetative and spore-forming bacteria, including pathogens ingredients and proportions shown in Table 1. A base formulation
of concern in low-acid products such as Clostridium containing 72.5% habanero hot pepper puree, 25.0% water, and
botulinum, but not the growth of several species of lactic 2.5% salt was consistently used in all formulations. Citric or acetic
acid bacteria (LAB) and some fungi. According to the acid (Insumos Químicos y Servicios de Costa Rica, San José ,
literature, approximately one-fourth of the spoilage cases of Costa Rica), two of the most commonly used organic acids in
acidified specialty foods are caused by LAB (18, 20) and industry, were added to adjust the pH to three target values within
1738 LOBO ET AL. J. Food Prot., Vol. 82, No. 10

the range cited in the literature for effective control of microbial Salmonella enterica, and Listeria monocytogenes were prepared
growth (3.2, 3.5, and 3.9) (20). Fresh habanero hot peppers were as described by Usaga and Worobo (24). An isolated colony of
purchased locally, in Alajuela, Costa Rica; they were manually each culture was transferred into 5 mL of Trypticase soy broth
washed and sanitized by immersion in a 150-ppm chlorine (Difco, BD) and incubated for 18 6 2 h at 35 6 28C in an Innova
solution for 5 min. Peppers were mashed and homogenized using 2300 rotary platform shaker (New Brunswick Scientific Co.,
an industrial blender (model 37BL19, Waring, Torrington, CT) Edison, NJ) at 175 rpm. A cocktail composed of five strains or
and were blended with the other ingredients in a Groen steam serotypes of each pathogen was prepared by mixing individual
kettle (Dover Corporation, Downers Grove, IL). The pH of the stationary-phase cultures.
sauces was adjusted to the target values using the selected
acidifiers. Sauces were heated and hot-filled at a minimum Survival of spoilage microorganisms in hot sauces. To
temperature of 838C in food-grade glass woozy bottles (Vicesa, determine the survival of P. manshurica in the acidified hot sauces,
Cartago, Costa Rica) that had plastic lids with tamper-evident a full factorial design was used with three levels for pH (3.2, 3.5,
hermetic seals to maintain commercial sterility and, therefore, and 3.9) and two levels for organic acidulant (citric and acetic
avoid the presence of unwanted background microbiota that might acid). An isolated colony of P. manshurica was transferred into
negatively interfere with the results. During hot filling, the 100 mL of acidified potato-dextrose broth (Difco, BD) and
plastisol in the interior of the lids softens and molds itself around incubated for 24 6 2 h at 35 6 28C in a Lab Companion SI-600
the glass to guarantee a hermetic seal. Following the closure step, shaking incubator (Jeio Tech, Daejeon, Korea) at 150 rpm. A
passive cooling of the product creates a vacuum inside the bottles; volume of 49 mL of each sauce formulation was independently
container vacuum may be used as a hermetic seal indicator. All inoculated with 1 mL of the stationary-phase culture to obtain an
samples were kept at ambient temperature until use. Three initial population of 106 CFU/mL. To prevent potential contam-
independent batches for each sauce formulation were prepared. ination during sampling, aliquots of inoculated sauces were
Each batch was manufactured on a different occasion, but all three aseptically transferred to sterile test tubes. Test tubes were
batches were prepared from the same lot of hot peppers. To verify immediately capped and kept at ambient temperature (23 6
the absence of background microbiota in the sauces that could 28C) until sampling to simulate the storage conditions of a sauce
interfere with microbiological analyses, three replicate samples of after a cold-fill process. Sauces were sampled and analyzed for
each formulation were plated on standard plate count agar and on yeast counts by spread plating 1 mL of product, or the respective
acidified (pH 3.5) potato-dextrose agar (Difco, BD, Sparks, MD) serial dilution, on acidified (pH 3.5) potato-dextrose agar (Difco,
and were incubated at 35 6 28C for 24 h and 30 6 28C for 48 h, BD) and incubation at 30 6 28C for 48 h until enumeration (21,
respectively. Total aerobic plate count and mold and yeast counts 22). The sauce with pH 3.2 acidified with acetic acid was sampled
were determined. every 30 min for up to 6 h. The other sauces were sampled every
12 h for 6 days. Sampling times were selected based on
Physicochemical characterization. Hot pepper sauces were preliminary trials. Each sample was subjected to serial dilutions
characterized in terms of moisture, ash, fat, protein, carbohydrate, in 0.1% sterile peptone water and was plated in duplicate.
sodium, and capsaicin contents. Soluble solids (expressed as To evaluate the survival of L. curvatus, the same experimen-
8Brix), pH, and titratable acidity (expressed as citric or acetic acid, tal design was followed. An isolated colony of L. curvatus was
depending on the acidifier used) were also quantified for each transferred into 100 mL of de Man Rogosa Sharpe agar (MRS)
sauce. Moisture, ash, protein, and sodium contents were broth (Difco, BD) and incubated for 24 6 2 h at 35 6 28C in a
determined using standard AOAC International methods (2). Fat Lab Companion SI-600 shaking incubator (Jeio Tech, Daejeon,
content was determined as previously described (8), and Korea) at 150 rpm. A volume of 59 mL of each hot sauce
carbohydrate content was determined by calculation. Capsaicin formulation was inoculated with 1 mL of stationary-phase culture
was determined according to methods previously described by to obtain the target initial population of 106 CFU/mL. The
Collins et al. (11) and Al Othman et al. (1). Soluble solids were inoculated product was portioned in sterile test tubes and kept at
determined using a NAR-1T Abbe Refractometer (Atago Co., 23 6 28C until sampled. Samples (1 mL) were spread plated in
Tokyo, Japan). Sauce pH was measured using an Oakton 2700 duplicate on MRS agar (Difco, BD) after appropriate serial
Benchtop pH meter (Eutech Instruments, Singapore). Titratable dilutions in 0.1% sterile peptone water and were incubated at 35
acidity was determined using an 836 Titrando automatic titrator 6 28C for 72 6 2 h in anaerobic jars until enumeration. Sauces
(Metrohm AG, Herisau, Switzerland). acidified to pH 3.2 with citric acid were sampled every 20 min for
2 h, and sauces acidified to pH 3.2 with acetic acid were sampled
Description and preparation of cultures. Two spoilage every 15 min for 1 h. Sauces acidified to pH 3.5 with citric and
microorganisms, a lactic acid bacterium and a yeast, were isolated acetic acid were sampled at 0, 1, 2, 3, 7, and 24 h, and 0, 1, 2, 3, 4,
from a commercial hot pepper sauce with evident microbial 5, and 24 h, respectively. Sauces with pH 3.9, acidified with citric
growth. The lactic acid bacterium, Lactobacillus curvatus, was and acetic acid, were sampled every 12 and 24 h, respectively, for
identified based on the 16S gene sequence (GenBank accession 5 days. Sampling times were selected based on preliminary trials.
no.: L. curvatus KX816336), and the yeast (Pichia manshurica)
was identified using the 26S and 18S gene sequence (GenBank Survival of pathogenic microorganisms in hot sauces. Two
accession no.: P. manshurica KX816337 and KX816338, hot sauces, both with a pH of 3.2 with either acetic or citric acid as
respectively). Cultures were maintained in 20% (v/v) glycerol, a the acidulant, were selected for a challenge study with pathogenic
cryopreservative that serves as a carrier, and were stored at 808C bacteria. A full factorial design with three levels for pathogen (E.
until use. Pathogenic vegetative bacteria were obtained from the coli O157:H7, S. enterica, and L. monocytogenes) and two levels
Food Microbiology Laboratory at Cornell AgriTech (Geneva, for organic acidulant (citric and acetic acid) was used. A volume of
NY). All selected pathogenic cultures had been implicated in 15 mL of shelf stable hot pepper sauce (pH 3.2, acidified with
foodborne disease outbreaks in the United States, most of which acetic or citric acid) was independently inoculated with 1 mL of
were caused by contaminated fruits or vegetable-derived products. each of the three pathogen cocktails to obtain an initial target
Cocktails of five strains or serotypes of E. coli O157:H7, population of about 108 CFU/mL. Initial counts were determined
J. Food Prot., Vol. 82, No. 10 SPOILAGE AND PATHOGENIC MICROORGANISMS IN COLD-FILLED HOT SAUCES 1739

TABLE 2. Physicochemical characterization of the acidified hot pepper sauces used to evaluate the survival of spoilage and pathogenic
microorganismsa
pH 3.2 pH 3.5 pH 3.9

Parameter (units) Citric acid Acetic acid Citric acid Acetic acid Citric acid Acetic acid

Moisture (g/100 g) 91.58 6 0.05 92.40 6 0.30 91.70 6 0.20 92.20 6 0.70 91.70 6 0.60 92.20 6 0.70
Ash (g/100 g) 3.08 6 0.03 2.90 6 0.20 3.10 6 0.10 3.00 6 0.30 3.20 6 0.30 3.00 6 0.20
Fat (g/100 g) 0.30 6 0.10 0.21 6 0.03 0.10 6 0.10 0.20 6 0.10 0.30 6 0.20 0.14 6 0.05
Protein (g/100 g) 0.50 6 0.02 0.46 6 0.03 0.47 6 0.01 0.48 6 0.03 0.50 6 0.10 0.49 6 0.04
Carbohydrates (g/100 g) 4.6 6 0.1 4.0 6 0.2 4.7 6 0.2 4.1 6 0.5 4.3 6 0.2 4.2 6 0.5
Sodium (mg/100 g) 967 6 184 773 6 89 885 6 79 906 6 120 730 6 55 759 6 96
Capsaicin (mg/100 g) 5.4 6 0.7 5.2 6 0.7 4.6 6 0.6 6.0 6 1.0 5.9 6 0.9 5.7 6 0.8
Soluble solids (8Brix) 9.20 6 0.40 9.90 6 0.40 9.20 6 0.30 9.40 6 0.50 9.39 6 0.01 9.00 6 0.40
Titratable acidity (g/100 g) 1.10 6 0.60 4.40 6 0.30 0.73 6 0.06 2.03 6 0.07 0.46 6 0.01 0.74 6 0.02
pH 3.17 6 0.05 3.20 6 0.02 3.46 6 0.01 3.50 6 0.02 3.86 6 0.03 3.90 6 0.01
a
Values are means 6 standard deviations, n ¼ 3.

immediately after inoculation using the pour plate technique with However, in sauces acidified with acetic acid, the change in
Trypticase soy agar (Difco, BD). Inoculated hot sauces were yeast population was dependent on the pH of the sauce. At
stored at 23 6 28C until sampling. Sampling intervals for each pH 3.9, the increase in the population of P. manshurica was
treatment (pathogen and organic acidulant) were determined in similar to the increase in sauces acidified with citric acid
preliminary trials. Each sample was subjected to serial dilutions in
(regardless of adjusted pH). In sauces acidified to lower pH
0.1% sterile peptone water and plated in duplicate. Petri dishes
values (3.2 and 3.5) using acetic acid, the yeast population
were incubated for 22 6 2 h at 35 6 28C before enumeration.
For each experiment, three independent biological replicates
decreased over time. Two-way ANOVA (r2 ¼ 0.9971), used
were prepared for each treatment. The headspace in every vessel to evaluate the effects of sauce pH, organic acidulant, and
containing inoculated hot sauce was greater than 40% to ensure their interaction on the final yeast population, showed a
the presence of oxygen and, therefore, provide worst-case scenario significant interaction between factors (P , 0.001). Tukey’s
conditions for the potential growth of the tested microorganisms. HSD showed no differences in mean values of final yeast
population among the three sauces acidified with citric acid
Statistical analyses. Two-way analyses of variance (AN- and the sauce acidified to pH 3.9 with acetic acid. These
OVA) followed by Tukey’s honestly significant difference (HSD) treatments differed from sauces acidified to pH 3.2 and 3.5
tests for means comparisons were performed. To predict the time with acetic acid, for which a higher than 5-log reduction of
required to reach targeted microbial populations for spoilage P. manshurica was achieved after a cold-fill-hold process.
microorganisms, a logistic model with four parameters was used
Using equation 1 to model these two population curves,
(equation 1), where a corresponds to the inactivation rate, b to
inflection point, c to lower asymptote, and d to upper asymptote.
followed by inverse prediction, showed that, for sauces at
pH 3.2 (r2 ¼ 0.9837), a hold time of 1.7 6 0.1 h at 23 6
dc 28C would be required to achieve a 5-log reduction of P.
Population ¼ c þ ð1Þ
1 þ eaðtimebÞ manshurica, whereas for sauces at pH 3.5 (r2 ¼ 0.9768), 108
Although quantification limit of the counting method 6 9 h would be required. These results suggest that with a
corresponds to 1.4 log, the model takes into account every formulation adjusted to pH of 3.2 with acetic acid, a cold-
sampling point, regardless of results. All statistical analyses were fill-hold process might represent a suitable processing
performed with JMP 11 (SAS Institute Inc., Cary, NC), and alternative that ensures commercial sterility when P.
differences were considered significant at P , 0.05. manshurica is the only spoilage microorganism of concern.
Worth noting, further research using different strains of this
RESULTS AND DISCUSSION yeast and other spoilage organisms, linked to acidified food
The physicochemical properties of the acidified hot products, would be highly suggested to support the finding
pepper sauces are summarized in Table 2. Properties were reported in this study. For other formulations, a hot-fill-hold
similar among formulations, except for pH and total process would be recommended to ensure the microbiolog-
titratable acidity, which differed due to the experimental ical stability of the product over the course of the shelf life.
design. Otherwise, the addition of preservatives may be required to
avoid spoilage incidents, but further experiments would be
Survival of spoilage microorganisms in hot sauces. necessary for confirmation. According to the literature,
In hot sauces acidified with citric acid and inoculated with acetic acid has an expected enhanced antimicrobial effect in
P. manshurica in a simulated cold-fill-hold process, the comparison with other acidulants, including citric acid (20),
yeast population increased to a maximum of about 8.5 log which may be attributed to its pKa value (17). The enhanced
after 3 days of storage at ambient temperature (23 6 28C) at antimicrobial activity of acetic acid is due to the presence of
all pH levels evaluated (Fig. 1). The populations remained both dissociated and nondissociated structures in solution
constant for the remainder of the test period (6 days). (16). At low pH values (lower than pKa), the number of
1740 LOBO ET AL. J. Food Prot., Vol. 82, No. 10

FIGURE 1. Survival curves for P. manshurica determined in a FIGURE 2. Survival curves for L. curvatus determined in a cold-
cold-filled acidified hot pepper sauce, at pH values (A) 3.2, (B) filled acidified hot pepper sauce, at pH values (A) 3.2, (B) 3.5, and
3.5, and (C) 3.9, using two organic acids (citric and acetic), and (C) 3.9, using two organic acids (citric and acetic), and stored at
stored at ambient temperature (23 6 28 C). Error bars represent ambient temperature (23 6 28 C). Error bars represent standard
standard deviations (n ¼ 3). Dotted line represents quantification deviations (n ¼ 3). Dotted line represents quantification limit of
limit of 1.4 log. 1.4 log.

nondissociated molecules is higher; thus, the acid may go Figure 2 shows the survival curves for L. curvatus at 23
through cell membranes and alter the internal microbial pH 6 28C after inoculation in hot pepper sauces in a simulated
and inhibit the cells (14). This would explain the observed cold-fill. Populations decreased in sauces with pH adjusted
results. However, as demonstrated, the effectiveness of the to 3.2 or 3.5 with either acidulant, whereas populations
initially decreased and then increased in sauces adjusted to
acidulant against P. manshurica was dependent on the pH of
pH 3.9. Analysis of the effects of main factors (sauce pH
the product. Therefore, both the acidulant and the product and organic acidulant) and their interaction on the final
equilibrium pH should be considered when acidified food bacterial population using two-way ANOVA (r2 ¼ 0.9643)
products are formulated, particularly when a cold-fill-hold showed a significant interaction between factors (P ,
process is used. 0.0398). Tukey’s HSD showed no differences between
J. Food Prot., Vol. 82, No. 10 SPOILAGE AND PATHOGENIC MICROORGANISMS IN COLD-FILLED HOT SAUCES 1741

FIGURE 3. Survival curves for cocktails of E. coli O157:H7, S. enterica, and L. monocytogenes determined in a cold-filled acidified hot
pepper sauce (pH 3.2) acidified with (A) acetic acid and (B) citric acid and stored at ambient temperature (23 6 28 C). Error bars
represent standard deviations (n ¼ 3). Dotted line represents quantification limit of 1.4 log.

mean values for final populations in treatments adjusted to 3.2 and 3.9 effectively prevents the growth of microorgan-
pH 3.2 and 3.5 with citric and acetic acid. This group isms and that acetic acid provides a major additional
differed from sauces acidified to pH 3.9. For all sauces with microbial barrier. Although under some conditions this was
pH 3.2 and 3.5, a full factorial design with two levels for proven correct, our results suggest that this recommendation
sauce pH and two levels for organic acidulant was used. should not be generalized for the cited pH range because the
Data were analyzed with a two-way ANOVA, with the time effect of the acidulant depends on the pH of the product and
to achieve a 5-log reduction of the population of L. curvatus the microorganisms present in the food.
as the response variable. To predict this time, the population
curves were modeled using equation 1. Inverse prediction Survival of pathogenic microorganisms in hot
showed that hold times required to achieve the desired log sauces. For the challenge study, the first sauce (pH 3.2,
reduction at 23 6 28C were 0.4 6 0.1 h (r2 ¼ 0.9771) and acidified with acetic acid) was selected given that it
2.0 6 0.3 h (r2 ¼ 0.9129) for sauces at pH 3.2, acidified achieved a greater than 5-log reduction of both tested
with acetic and citric acid, respectively. Values were 5 6 2 spoilage microorganisms in less than 24 h after inoculation.
h (r2 ¼ 0.9384) and 6.6 6 0.6 h (r2 ¼ 0.9477) for sauces The second sauce, same pH but acidified with citric acid,
acidified to pH 3.5 with acetic and citric acid, respectively. was chosen as an alternative formulation. Although P.
The two-way ANOVA (r2 ¼ 0.8473) showed that the manshurica was not controlled at pH 3.2 in citric acid, this
interaction was not significant (P ¼ 0.9939). After removal sauce was included in the study because of the 5-log
of the interaction term, the main factors of organic acidulant reduction of L. curvatus in less than 24 h observed in the
(P ¼ 0.0428) and sauce pH (P , 0.0001) were deemed previous study, and also because this formulation may be
significant. As expected, sauces with lower pH (3.2) currently used by the industry. Survival curves for
acidified with acetic acid required shorter hold times to pathogenic E. coli O157:H7, S. enterica, and L. monocy-
achieve the 5-log reduction in the population of L. curvatus. togenes at ambient temperature (23 6 28C) after simulated
Overall, for products with pH adjusted to 3.2 and 3.5 using sauce contamination followed by a cold-fill-hold process are
either organic acidulant, less than 12 h would be required to shown in Figure 3. For statistical purposes, the survival
achieve higher than 5-log reductions of L. curvatus. This response corresponded to the sampling point (hours) at
suggests that when this spoilage bacterium is the microbial which a higher than 5-log reduction in the population of the
concern, a cold-fill-hold process may be a suitable approach inoculated pathogen occurred in each of the three
that ensures the microbiological stability of the sauce. independent replicates. Two-way ANOVA showed a
However, because control of both P. manshurica and L. significant interaction between main effects (P , 0.0001).
curvatus was achieved only in sauces acidified to pH 3.2 A post hoc mean comparison (Tukey’s HSD) showed no
with acetic acid, a cold-fill-hold process using this differences among survival responses of the three pathogens
formulation would be recommended to control both in sauces acidified with acetic acid. In less than 0.5 h, a
spoilage microorganisms. Note that only one strain of the larger than 5-log reduction in population was observed for
LAB was evaluated in this study, and thus, additional the three pathogens. Survival response varied significantly
experimental trials (including different strains and other for the pathogens inoculated in the sauce acidified with
spoilage microorganisms of concern in acidified products) citric acid (48 h for L. monocytogenes, 14 h for S. enterica,
would be recommended to support our conclusions. and 8 h for E. coli). These results are similar to those
The presence of yeasts and heterofermentative LAB are obtained for the LAB and yeast, which were significantly
undesirable in acidified foods such as hot sauces because of less tolerant in samples containing acetic acid at a pH level
production of off-flavors (20) and gas during storage. of 3.2. In agreement with our findings, Breidt et al. (6)
Sperber (20) previously mentioned that a pH range between indicated that the 5-log reduction times for pathogenic
1742 LOBO ET AL. J. Food Prot., Vol. 82, No. 10

bacteria in acidified foods containing acetic and benzoic acidified hot sauce. Likewise, further studies considering
acids at pH 3.5 and 3.8 revealed that acid killing was other spoilage microorganisms and a formal shelf-life study
dependent on the concentration and type of acid. In a for a particular formulation are highly suggested when a
previous study (5) and in contrast with our results, E. coli cold-fill-hold process is selected as a processing alternative.
O157:H7 was found to be the most acid-resistant microor-
ganism in an acidified vegetable product (cucumbers) with a ACKNOWLEDGMENTS
pH of 3.3 or below and using acetic acid. In this case, to This study was funded by University of Costa Rica (UCR), project
achieve a 5-log reduction, at least 1.4 days at 258C were 735-B5-246, and Cornell University College of Agriculture and Life
required. Differences encountered may be associated with Sciences. Authors thank M. Espinoza and V. Mora (UCR), as well as J.
Churey (Cornell University), for their technical assistance in the Food
the use of different strains and environmental and analytical
Microbiology Laboratories.
conditions, in addition to intrinsic differences in the
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