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Journal of Food Safety ISSN 1745-4565

MICROBIAL LEVELS IN APPLE MUST AND THEIR ASSOCIATION

WITH FRUIT SELECTION, WASHING AND SANITIZATION
TATIANE APARECIDA GOMES1, MAURO RODRIGUES SALVADOR FILHO1,
ACÁCIO ANTONIO FERREIRA ZIELINSKI1, GIOVANA DE ARRUDA MOURA PIETROWSKI2 and
ALESSANDRO NOGUEIRA1,3
1
Food Science and Technology Master Programme, State University of Ponta Grossa, Av. Carlos Cavalcanti 4748, Uvaranas Campus, CEP 84.030-
900, Ponta Grossa, PR, Brazil
2
Department of Food, Federal Technological University of Paraná, Ponta Grossa, PR, Brazil

3
Corresponding author. ABSTRACT
TEL: 55 42 3220 3775;
FAX: 55 42 3220 3072; This study evaluated the effect of the operations of selection, cleaning and sanitiz-
EMAIL: alessandronog@yahoo.com.br ing with sodium hypochlorite, on the populations of yeasts and lactic acid bacteria
present in apple cider must, elaborated from commercial and industrial fruit. The
Accepted for Publication February 9, 2014
microbial load differed between the cultivars and classes of fruit. The total popula-
doi: 10.1111/jfs.12107
tions of yeasts and lactic acid bacteria were higher in the industrial fruit. The
operation of selection avoided cross-contamination and the presence of organic
compounds that might react with the chlorine. Washing the apples decreased the
populations of these microorganisms by one logarithmic cycle. Hierarchical
cluster analysis indicated that sanitization was effective at a concentration of
100 mg/L for 5 min. The sanitizer was not effective in totally eliminating the
microorganisms in populations greater than 104 CFU/mL. Selection and washing
help reduce natural microbiota, making sanitization more effective, and positively
affecting food safety and the quality of unpasteurized cider.

PRACTICAL APPLICATIONS
This article presents the results, and a thorough discussion, of the use of pre-
fermentation operations that can be used to reduce the microbial load of apple
must, which is to be turned into cider. Among all the various techniques and con-
trols, the three that were most noteworthy were analyzed (selection of fruits,
washing and sanitizing with chlorine, followed by rinsing). These operations
are critical control points, and if they are planned and monitored then can reduce
or eliminate microorganisms (pathogens or not), eliminate or reduce the need
for sulfite, facilitate the cleaning and disinfection of the environment and equip-
ment, increase food safety, and ensure the production of high-quality unpasteur-
ized cider.

organoleptic characteristics and physiological or morpho-

INTRODUCTION
Cider is a carbonated or uncarbonated beverage that is
obtained by the alcoholic fermentation of apple must and it
is produced in more than 20 countries around the world.
Apples used for cider processing consist of dessert (sweet
and aromatic) and industrial varieties (bitter, sour and
acid), either blended or unblended. Industrial fruit has
no commercial value for fresh consumption because of
logical defects and infection by microorganisms (Nogueira
and Wosiacki 2012).
In the manufacture of cider, the selection and exclusion
of fruit with superficial rot, injuries or different stages of
ripeness is precarious and often nonexistent. The exclusion
of damaged fruits results in a substantial decrease in the
total microbiota of fresh must (Parish 1997). The removal
of damaged apples before washing is a means to prevent

Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc. 141
SAFE PRACTICES IN APPLE MUST MANUFACTURE
further increases in the microbial load of the water that is
used (washing, rinsing and hydraulic transport water) and
to reduce the contamination of other apples in the tank
(Snowdon et al. 2006). Fruits with injuries or cuts can be
quickly contaminated by microorganisms (Janisiewicz et al.
1999; Kenney and Beuchat 2002).
The lack of efficient methods to remove wild and
unwanted microorganisms in apples leads to the necessity
for the application of a combination of mechanical
methods (chemical washes, high-pressure sprays and brush-
ing) or new methods (ozone, ultraviolet light and pulsed
electric field) and sanitization treatments that may be effec-
tive in achieving the necessary reductions (Kenney and
Beuchat 2002). However, the sanitization process is often
not performed, on the grounds that the operation of
extracting the apple must is continuous, which generates
more costs, because, as well as the equipment and sanitizer
(often chlorinated compounds), this will require efficient
rinsing because waste can harm the alcoholic fermentation
and/or sensory quality of the cider.
In countries that utilize industrial apple varieties, the
fruit is mainly collected from the ground (indicating the
point of harvest). The production of fruit can occur in asso-
ciation with action from livestock (sheep, goats and cattle)
or manure can be used as fertilizer, making the soil an envi-
ronment with a high concentration of pathogens; because
of this factor, the fruit should not be harvested from the
ground. These practices are common in the U.S.A. and in
European countries, where there have been reports of
numerous outbreaks of illness (17 cases between 1972 and
2012) associated with unpasteurized apple juice and cider
caused by Escherichia coli 0157:H7, Salmonella and
Cryptosporidium parvus (Millard et al. 1994; Parish 1997;
Mihajlovic et al. 2013), in addition to the high presence of
coliforms. E. coli is the main contaminating microorganism
because it causes infection at low concentrations and sur-
vives high acidity (pH ≤ 4.6) for up to 4 weeks (Zhao et al.
1993; Keene et al. 1994; Miller and Kaspar 1994; Mihajlovic
et al. 2013). Other forms of contamination by pathogens in
this product, including irrigation water, lack of hygiene
practices in collection and disinfection of transport boxes,
as well as the period and conditions of storage, have all been
documented (Buchanan et al. 1999; Uljas and Ingham 2000;
Garcia et al. 2006).
In the U.S.A., cider (hard cider) is not subject to the
Food and Drug Administration Juice Rule requirements
of Hazard Analysis and Critical Control Points. The fermen-
tation process itself is an effective means of reducing patho-
gen load and patulin contamination. However, many
microorganisms (yeast and bacteria), can still affect cider
quality and stability, so Standard Sanitary Operating Proce-
dures (SSOPs) and product stabilization should still be
addressed in the production plan (Uljas and Ingham 2000).
142
T. A. GOMES ET AL.
Cider can be prepared by spontaneous fermentation or
by inoculum using commercial yeasts and bacteria. There
is a diversity of wild species of Saccharomyces and
nonconventional yeasts on the surface of apples that come
from the fruit itself and the processing equipment (Michel
et al. 1988; Laplace et al. 1998; Coton et al. 2006; Valles et al.
2007; Bedriñana et al. 2010). These microorganisms develop
a complex fermentation process, which can result in prod-
ucts with variation in standards of identity and sensory
quality, and if they are present in populations above
105 CFU/mL they can even compromise the quality of ciders
prepared with commercial yeast inoculums.
Likewise, lactic acid bacteria (LAB) are always present,
and their population can reach 109 CFU/mL. In acid apple
musts, these bacteria can promote the decarboxylation of
malic acid into lactic acid and reduce the acidic taste, which
can be positive for the quality of the cider. However, in
musts with low acidity, this is not necessary, and depending
on the species of LAB, other metabolites may be synthesized
in excess, such as diacetyl and geraniol, which de-
characterize the flavor of cider (Jarvis and Lea 2000;
Reguant and Bordons 2003).
To solve this problem, the addition of sulfite (SO2) in the
triturated material (before pressing), or in the apple must
and after fermentation, becomes usual and necessary. In
Brazil, the legislation allows the addition of up to 350 mg/L
of sulfite, well above the doses used in some European
countries, where the limit is 180 mg/L (Nogueira and
Wosiacki 2012). This compound has bacteriostatic, anti-
fungal and antioxidant properties (Jarvis and Lea 2000).
Besides these advantages, yeasts, mainly of the Saccharomy-
ces genus, exhibit resistance to sulfite, which avoids compe-
tition from other microorganisms, allowing them to
multiply and dominate the fermentation medium (Lea and
Drilleau 2003). Although sulfites do not cause a true allergic
reaction, sulfite-sensitive people may experience similar
reactions to people with food allergies. Asthma sufferers are
most at risk to sulfite sensitivity and other forms of sulfite
reactions (Vally et al. 2009).
Many producers have used post-harvest chlorination for
sanitizing fruits, because of its rapid action, easy application
and complete dissociation in water (Hong and Gross 1998).
Chlorine rinses are frequently used, with concentrations
varying from 50 to 200 mg/L, and with typical contact times
of from less than 5–20 min (Artés et al. 2009). This com-
pound is a strong oxidizing agent with broad-spectrum
antimicrobial activity (Joshi et al. 2013). Some of the disad-
vantages associated with its use are that high concentrations
(up to 250 mg/L) can cause discoloration and modify
the aroma of fruits; it also has low efficiency at high pH
(Oliveira et al. 2003). When NaClO is added to water, it
increases pH and generates hypochlorous acid (HOCl),
which is the active antimicrobial species. The acid
Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc.
T. A. GOMES ET AL.
dissociates readily to hypochlorite ions (OCl−) at high pH,
or chlorine gas (Cl2) at low pH, thus the pH must be kept in
the range of 6.5−7.5 for HOCl to be stable and efficient, and
to reduce the risk of corrosion of metallic processing equip-
ment (Artés et al. 2009; Joshi et al. 2013; Ramos et al. 2013).
Chlorination can be a strategy to decrease the popula-
tions of yeasts and LAB that may compromise or alter the
quality of the beverage (Lang et al. 1999; Lea and Drilleau
2003; Guerrero-Beltrán et al. 2005) in addition to increasing
the food safety of unpasteurized ciders, which depend only
on their acidity, ethanol content and the addition of chemi-
cal preservatives (Downing 1989).
Therefore, the selection, washing, sanitizing and monitor-
ing of the quality of apples are critical control points, and
they are extremely important for companies producing
unpasteurized cider. This study aimed to evaluate the effect
of the selection, washing and chlorination of fruits on
the wild yeast and lactic bacteria populations present in
apple must.
MATERIALS AND METHODS
Chemicals
Sodium hypochlorite (Vitta Pury) was purchased from
Meneghetti Industry (São Paulo, Brazil). Agar was acquired
from HiMedia (Mumbai, India). Lysine was acquired from
Biotec (Pinhais, Brazil). Potato dextrose agar (PDA) and
Man, Rogosa and Sharp Agar (MRS) were purchased from
Acumedia (Lansing, MI).
Selection and Washing of Fruits
Samples of 52 kg of Gala and Fuji apples, from the 2010–
2011 harvest, were classified as industrial (apples with
physiological and morphological defects, scars and rot) and
commercial (without the defects previously described). The
fruits were manually washed in potable water.
Sanitization of Apples with
Sodium Hypochlorite
Different concentrations of sodium hypochlorite (2.5%)
and different immersion times (0, 5, 15 and 20 min) were
used for the experiments. For each treatment (1 kg of fruit),
different concentrations of sodium hypochlorite were
diluted in 3 L of distilled water with pH 6.81 (84.8% of
chlorine in HOCl and 15.2% of chlorine in OCl−). The solu-
tions and sanitization processes were performed at tempera-
tures between 3 and 4C. The sanitizing conditions were:
apples without the sanitizing agent (control), 50, 100, 150
and 200 mg/L, at times of 5, 15 and 20 min, respectively.
The fruits were subsequently rinsed in clean water (3 L) to
Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc.
SAFE PRACTICES IN APPLE MUST MANUFACTURE
remove excess sanitizer and sent for the processing of the
must.
Apple Must Processing
The apples (Gala and Fuji) were separated into commercial
and industrial categories and divided into four groups. For
each sanitizing condition, 1 kg of fruit was used, and
samples of freshly extracted juice from unwashed apples,
apples washed with potable water and apples subjected to
sanitization with sodium hypochlorite (2.5% active chlo-
rine), according to the conditions presented in item 2.3,
were analyzed. After sanitizing, the juice processing was per-
formed using a domestic extractor (Juicer extractor model
NCF01 cod. 1260-01; Mondial M.K. Eletrodomesticos Ldta.,
Conceição do Jacuípe, Bahia, Brazil).
Microbiological Analysis
The populations of total yeasts (SCH), oxidative yeasts
(OXY) and LAB were monitored because of their significant
presence in apples, their greater resistance to sulfite (rather
than pathogenic) microorganisms and because it is known
that they change the standard of quality of unpasteurized
ciders (Snowdon et al. 2006; Pizarro et al. 2009).
OXY. Dilutions were made from freshly extracted juices
according to Tortora et al. (2010). The AgarLys culture
medium was prepared using agar and lysine, diluted in dis-
tilled water, according to the manufacturer’s specifications.
The sterilization of the medium and glassware were per-
formed in an autoclave (Phoenix VA 13811) for 15 min
(121C, 1.01 kgf/cm). The analysis procedure was performed
as described by Nogueira et al. (2007), using about 100 μL
of the sample, with scattering and using a Drigalski spatula.
The plates were then incubated at 26C for 24 h, with sub-
sequent counting of specific colonies. The results were
expressed as CFU/mL.
SCH. The culture medium was PDA, which was hydrated
and sterilized according to the manufacturer’s specifica-
tions. About 10 mL of culture medium and 50 μL of 10%
tartaric acid (Biotec, Pinhais, Brazil), were poured into
90 mm Petri plates and subsequently, the solid medium
received 100 μL of sample following the same analysis of
OXY as described in the previous section. After incubation
at 26C for 48 h, the colonies were manually counted and the
result was expressed in CFU/mL (Nogueira et al. 2007).
LAB. The determination of LAB was performed on selec-
tive medium, using MRS, dissolved in distilled water, fol-
lowed by sterilization. The Pour Plate procedure was used
by inoculating 1 mL of sterile sample on the plate and then
143
SAFE PRACTICES IN APPLE MUST MANUFACTURE
pouring approximately 15 mL of MRS medium (about
45C), homogenized with circular movements, and awaiting
complete solidification, with the subsequent addition of an
overlayer. The plates were incubated at 28C for 8 days, with
subsequent counting of specific colonies. The results were
expressed as CFU/mL (Nogueira et al. 2007).
Hierarchical Cluster Analysis
Data were presented as means. Hierarchical cluster analysis
(HCA) was performed to evaluate the similarity between
the microbiological data obtained from the Gala and Fuji
apples using an array of three variables (LAB, OXY and Sac-
charomyces genus [SCH] ) of each fruit sample that received
different treatments. The data were autoscaled, and the
similarity of the samples was calculated, based on the
Euclidean distance. Ward’s method was used to form and
suggest groups of similar samples. The clustering imposed
a hierarchy on this similarity, making it possible to have
a two-dimensional view of the whole set of samples used in
the study (Braga et al. 2013). In order to compare the results
within the three recovered groupings, Levene’s test was con-
ducted to check the homogeneity of variance. Kruskal–
Wallis, analysis of variance and multiple comparison tests
were applied to verify differences between the clusters. A
P-value below 0.05 was considered significant. All statistical
analyses were performed using Statistica software v. 7
(Statsoft, Tulsa, OK).
RESULTS AND DISCUSSION
Figure 1 shows the effect of classifying the apples into com-
mercial and industrial fruits for the evaluated microorgan-
isms. The Gala apples had a higher microbial load
compared with the Fuji cultivar. This may have been mainly
FIG. 1. EFFECT OF APPLE CLASSIFICATION (COMMERCIAL AND
INDUSTRIAL) ON WILD YEAST AND LACTIC ACID BACTERIA POPULA-
TIONS IN APPLE MUST
SCH, total yeasts; OXY, oxidative yeasts, LAB: lactic acid bacteria.
144
T. A. GOMES ET AL.
related to factors such as climate, degree of ripeness, injuries
and the incidence of superficial rot (Parish 1997). However,
it was found that not all the populations of microorganisms
were higher in the industrial fruit. The natural yeasts of the
Saccharomyces genus showed a high population for the
industrial fruit of both cultivars (105–106 CFU/mL). In fresh
apple musts, the yeasts population varies between 103 and
104 CFU/mL (Le Quéré and Drilleau 1998; Dierings et al.
2013). The populations of OXY were very similar between
the industrial and commercial apples, but showed large
variations among cultivars (104–107 CFU/mL). Dierings
et al. (2013) found a similar variation between the popula-
tion of OXYs in apple musts (103–106 CFU/mL). The LAB
count was similar in the musts of the Gala cultivar, but
showed large variations in the commercial and industrial
musts of the Fuji cultivar, with values of 103–105 CFU/mL,
respectively. The LAB were the microorganisms that showed
most variation in the fresh apple musts, reaching values
greater than 200 % (Dierings et al. 2013). The vegetative
form of the filamentous fungi does not survive chlorine,
sulfite or fermentation. However, enzymes produced by
these fungi remain, and they can compromise the sensory
quality of cider, turning volatile phenolic compounds into
unpleasant aromas in the final product (Snowdon et al.
2006; Nogueira and Wosiacki 2012) requiring the removal
of fruit with phytopathological defects caused by filamen-
tous fungi.
The operation of washing and rinsing of industrial and
commercial fruits in running potable water decreased the
population of all the evaluated microorganisms by one
logarithmic cycle, regardless of the initial count (Fig. 2).
Lang et al. (1999) and Buchanan et al. (1999) observed that
an increase in coliform and E. coli count can occur in reused
washing water, which results in a larger population of these
bacteria, and possibly other microorganisms in the must.
Thus, the washing water should always be potable water that
has not been reused. The brushing of fruit is recommended
when they are very dirty or when they have been collected
from the soil. In France, the harvest of apples begins when
50% of the fruits are on the ground, indicating the degree of
ripeness that is ideal for processing (Lea and Drilleau 2003).
According to Uljas and Ingham (2000), of the 93 cider pro-
ducers in the region of Wisconsin (U.S.A.), 93% washed
their apples, 87% reported brushing their apples, and only
16% sanitized their fruits. These results are similar to
a survey of the cider producers in Virginia (U.S.A.), where
93% washed their fruits, 64% used brushing and 37% used
disinfectants (Wright et al. 2000).
These results (Figs. 1 and 2) demonstrate that operations
that are performed prior to the extraction of apple must,
such as the selection of apples (mainly with drawing those
with rot and injuries) and washing with clean, nonreused
water, significantly decreases the concentration of organic
Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc.
T. A. GOMES ET AL.
FIG. 2. EFFECT OF WASHING APPLES ON
WILD YEAST AND LACTIC ACID BACTERIA
POPULATIONS IN APPLE MUST
SCH, total yeasts; OXY, oxidative yeasts; LAB,
lactic acid bacteria; cg, commercial Gala; ig,
industrial Gala; cf, commercial Fuji; if, indus-
trial Fuji.
compounds that can react with chlorine, either inactivating
it or forming chlorinated by-products, with potentially
adverse health effects (Ramos et al. 2013). There are various
techniques and products for the disinfection of apples that
are described in the literature (Evrendilek et al. 1999; Zook
et al. 1999; Rogers and Ryser 2004; Ramos et al. 2013), but
which are not being used in practice due in part, to the cost,
availability of technology and lack of knowledge of produc-
ers. Thus, chlorine continues to be the most commonly used
sanitizer because of its efficacy, cost-effectiveness ratio and
simplicity of use (Joshi et al. 2013; Ramos et al. 2013).
Because of this, the sanitization of apples with chlorine is
the sanitizing operation that can be most feasibly used,
mainly by small cider producers.
Tables 1 and 2 show the effect of the chlorine sanitization
on the microorganisms in apples, which was classified into
two groups: commercial and industrial. The results show
that the effect of sanitizing on the elimination of SCH, OXY
and LAB was not effective. A study by Kenney and Beuchat
(2002), which investigated the possibility of treating apples
with sanitizers, verified that treatment with sodium hypo-
chlorite at 200 mg/L did not significantly reduce the
number of yeasts and molds on the surface of the apples.
Chlorine kills only what it directly comes into contact with.
Water films that form on very small contours on plant sur-
faces may prevent the chlorinated water from directly con-
tacting the target microorganisms. In the present study,
surfactants were not used in the process to reduce the
surface tension of the water, which may have influenced the
efficiency of chlorination (Suslow 1997).
However, it was possible to observe a reduction in micro-
organisms at concentrations above 50 mg/L (Tables 1 and
2). The initial populations of SCH between 102 and 103 were
completely removed at concentrations of 100, 150 and
200 mg/L; in populations above 104 CFU/mL the same
treatments were ineffective (Tables 1 and 2). The effects on
Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc.
SAFE PRACTICES IN APPLE MUST MANUFACTURE
OXY and LAB were dependent on the microbial load, the
sensitivity of the microorganisms to sodium hypochlorite,
the concentration of the sanitizer, and the reaction time
(Tables 1 and 2).
The similarity among the samples was evaluated using
HCA and three clusters were recovered (Fig. 3).
The samples in cluster 1 (Table 3) represent the higher
counts for all the evaluated microorganisms (SCH, OXY,
LAB). This cluster was formed mainly by samples that had
not been washed and sanitized (Fig. 3). Cluster 3 showed
intermediate count values for samples that were mainly sub-
mitted to lower hypochlorite concentrations (50 mg/L).
Cluster 2 presented the lowest counts for all the microor-
ganisms. This cluster was formed by all treated samples and
it is conclusive that a treatment using 100 mg/L for 5 min
can be effective in eliminating the majority of fermentative
and OXYs and LAB. This treatment, with shorter time and
followed by efficient rinsing, is sufficient to decrease the
population of microorganisms to levels that do not affect
the development of commercial yeasts and reduce the risk
of remaining residue, which might alter the sensory charac-
teristics of the beverage, such as the formation of “off”
flavors. It is ideal for an industrial process (Suslow 1997;
Ramos et al. 2013) HCA was successfully used to classify the
treated samples with respect to the concentration and time
of sanitization to be used for reduction/removal of SHC,
OXY and LAB.
These unit operations (selection, washing/rinsing and
sanitization) can reduce or avoid the use of the preservative
sulfite, which is responsible for symptoms of allergic reac-
tions in people sensitive to this chemical compound (Vally
et al. 2009), especially in countries that permit concentra-
tions above 300 mg/L, which can affect the sensory quality
of the cider (Nogueira and Wosiacki 2012).
The microorganisms evaluated in this study had a higher
resistance to the sanitizer than strains that are pathogenic to
145
146
TABLE 1. EFFECT OF DIFFERENT CONCENTRATIONS AND TIMES HYPOCHLORITE IMMERSION IN THE PROCESS SANITIZING MICROBIOTA SURFACE OF GALA APPLES

Sodium hipochlorite concentration (mg/L) and immersion times (min)

50 100 150 200

Gala apples MO 0′* 5′* 15′* 20′* 5′* 15′* 20′* 5′* 15′* 20′* 5′* 15′* 20′*

Commercial SCH 6.80 × 103 2.80 × 103 0.00 2.50 × 104 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
OXY 2.50 × 105 6.40 × 105 1.20 × 104 3.31 × 106 1.80 × 104 4.20 × 104 1.80 × 104 4.10 × 103 0.00 7.60 × 103 0.00 0.00 2.61 × 104
LAB 7.70 × 103 3.00 × 105 3.40 × 102 1.37 × 103 0.00 6.10 × 102 9.10 × 103 0.00 2.60 × 102 3.40 × 103 1.20 × 102 6.10 × 103 0.00
Industrial SCH 9.00 × 105 9.80 × 103 3.00 × 103 0.00 0.00 7.90 × 103 5.50 × 103 3.00 × 103 1.59 × 104 0.00 1.61 × 104 0.00 0.00
OXY 4.50 × 105 7.60 × 103 1.01 × 105 0.00 4.60 × 104 2.10 × 105 2.50 × 105 1.11 × 104 2.48 × 105 1.08 × 105 3.10 × 105 3.80 × 103 6.40 × 103
LAB 1.14 × 104 6.10 × 102 4.10 × 104 6.30 × 104 2.40 × 103 0.00 7.20 × 102 5.20 × 102 4.00 × 103 0.00 2.90 × 103 1.51 × 103 9.70 × 102
SAFE PRACTICES IN APPLE MUST MANUFACTURE

Note: * Time (minutes) immersion in sodium hypochlorite solution at 4–5C.

Counts were expressed in CFU/mL.
MO, microorganism; SCH, total yeast; OXY, oxidative yeasts; LAB, lactic acid bacteria.

TABLE 2. EFFECT OF DIFFERENT CONCENTRATIONS AND TIMES HYPOCHLORITE IMMERSION IN THE PROCESS SANITIZING MICROBIOTA SURFACE OF FUJI APPLES

Sodium hipochlorite concentration (mg/L) and immersion times (min)

50 100 150 200

Fuji apples MO 0′* 5′* 15′* 20′* 5′* 15′* 20′* 5′* 15′* 20′* 5′* 15′* 20′*
2 3 3
Commercial SCH 3.00 × 10 4.60 × 10 2.50 × 10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
OXY 2.30 × 103 3.20 × 103 0.00 0.00 0.00 1.40 × 104 3.90 × 103 0.00 0.00 0.00 2.50 × 104 2.80 × 103 0
LAB 1.70 × 102 8.60 × 102 6.80 × 102 3.90 × 102 0.00 6.60 × 102 5.00 × 102 0.00 0.00 0.00 0.00 0.00 0.00
Industrial SCH 2.50 × 104 0.00 0.00 1.34 × 104 0.00 5.20 × 104 0.00 4.00 × 102 4.10 × 104 0.00 0.00 0.00 0.00
OXY 6.90 × 104 2.70 × 104 3.40 × 104 1.76 × 105 2.50 × 104 3.60 × 104 2.50 × 105 1.42 × 104 2.50 × 105 1.46 × 104 4.50 × 104 4.60 × 103 2.50 × 105
LAB 5.00 × 102 3.10 × 103 2.60 × 102 1.08 × 105 6.20 × 103 3.70 × 103 1.53 × 104 1.50 × 102 1.03 × 104 6.20 × 102 6.50 × 102 1.41 × 103 2.50 × 104

Note: * Time (min) immersion in sodium hypochlorite solution at 4–5C.

Counts were expressed in CFU/mL.
MO, microorganism; SCH, total yeast; OXY, oxidative yeasts; LAB, lactic acid bacteria.
T. A. GOMES ET AL.

Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc.

T. A. GOMES ET AL. SAFE PRACTICES IN APPLE MUST MANUFACTURE

FIG. 3. DENDROGRAM OF HIERARCHICAL CLUSTER ANALYSIS (HCA) OF SANITIZATION USING DIFFERENT CONCENTRATIONS AND DIFFERENT
IMMERSION TIMES FOR GALA AND FUJI APPLES
IG, industrial Gala; CG, commercial Gala; IF, industrial Fuji; CF, commercial Fuji; (I), control; (II), 50 mg/L–0 min; (III), 50 mg/L–5 min; (IV), 50 mg/L–
15 min; (V), 50 mg/L–20 min; (VI), 100 mg/L–5 min; (VII), 100 mg/L–15 min; (VIII), 100 mg/L–20 min; (IX), 150 mg/L–5 min; (X), 150 mg/L–15 min;
(XI), 150 mg/L–20 min; (XII), 200 mg/L–5 min; (XIII), 200 mg/L–15 min; (XIV), 200 mg/L–20 min.

humans. Consequently, their reduction or elimination may
represent an indicator of the effectiveness of this compound
against pathogens.
Therefore, adopting SSOPs that incorporate critical pre-
fermentation control points, such as harvesting, storage,
sorting, washing, sanitizing, rinsing of fruit, and the
cleaning/disinfection of plant and equipment, are proce-
dures that guarantee a lower microbial load in apple must,
eliminating or reducing the need to add sulfite and also
decreasing the amount of microorganisms, which facilitates
the cleaning and disinfection of plant and equipment and
produces a beverage with greater food safety and at the
same time maintains the sensory quality of the product.
ACKNOWLEDGMENTS
The authors are deeply grateful to the National Council of
Technological and Scientific Development (CNPq), Arau-
caria Foundation (FA), and Coordination for the Improve-
ment of Personnel in Higher Level (CAPES) for financial
support and scholarships.

TABLE 3. MEAN COUNT OF

Microorganisms Cluster 1 Cluster 2 Cluster 3 P-value* P-value**
MICROORGANISMS ACCORDING TO GROUPS
PROPOSED SCH 1.81 × 10 a 6
4.21 × 10 b 3
1.57 × 10 ab5
<0.0001 0.0016
OXY 3.15 × 106a 5.27 × 104b 7.14 × 105ab <0.0001 0.0015
LAB 2.21 × 105a 1.94 × 103c 4.16 × 104b <0.0001 <0.0001

Notes: P-value*: probability value obtained by Levene’s Test.

P-value** – probability value obtained by Kruskal–Wallis’ Test – analysis of variance. Different
letters represent averages with values significantly different (P < 0.05) and the same letter means
no significant difference (P > 0.05).

Journal of Food Safety 34 (2014) 141–149 © 2014 Wiley Periodicals, Inc. 147
SAFE PRACTICES IN APPLE MUST MANUFACTURE
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