Food Control 23 (2012) 282e285

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Food Control
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Short communication

Incidence of microorganisms from fresh orange juice processed by squeezing

machines
I. Sospedra*, J. Rubert, J.M. Soriano, J. Maes
Department of Preventive Medicine and Public Health, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrs Estells s/n, 46100 Burjassot, Spain

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

Article history: This study was carried out to evaluate the microbiological quality of orange juice obtained from
Received 15 March 2011 squeezing machines in foodservice establishments. The samples included fresh squeezed orange juice
Received in revised form and juice which is maintained in metal jugs until consumption. According to the European Commission
22 June 2011
Regulation (No. 2073/2005 and No. 1441/2007) and Spanish microbiological criteria (No. 3484/2000), 12%
Accepted 28 June 2011
and 43% of the total examined lots exceed the adopted limits of mesophilic aerobic counts and Enter-
obacteriaceae, respectively. Possibly, this contamination is caused by incorrect handling of oranges and
Keywords:
juices and also by inadequate cleaning and sanitization of squeezing machine and metal jugs. Further-
Orange juice
Squeezing machine
more, 0.5 and 1% of all the examined lots were positive for the presence of Salmonella spp. and Staph-
Microbiological quality ylococcus aureus, respectively. However all lots were negative for Escherichia coli and Listeria
monocytogenes. These results emphasize the need for applying and maintaining good hygienic practices
in the restaurants.
2011 Elsevier Ltd. All rights reserved.

1. Introduction decades in comparison with previous decades (ranged from 0 to 1

Orange juice is known as a popular juice among many consumers
(Dennison, 1996) for its pleasant taste, fresh avor, and nutritional
value as an important source of bioactive compounds such as
phenolics (e.g. avanone glycosides and hydroxycinnamic acids).
The high content in vitamin C, carotenoids and natural antioxidants
is particularly appreciated (Abeysinghe et al., 2007; Bull et al., 2004),
but technologies used for their processing and subsequent storage
may cause alterations in their contents so they may not provide the
benets expected by the consumer (Martinelli, 1999). In fact, orange
juice, during storage, undergoes a number of deteriorative reactions,
including ascorbic acid degradation, cloud loss, microbial spoilage,
development of off-avor, changes in colour, texture and appear-
ance among others, resulting in quality degradation of the product
(Ayhan, Yeom, Zhang, & Min, 2001; Bezman, Rouseff, & Naim, 2001;
Ewaidah, 1992; Goyle & Ojha, 1998; Roig, Bello, Rivera, & Kennedy,
1999). Orange juice producers have traditionally relied on the
acidity of their products to assure microbiological safety. Never-
theless, several incidents of foodborne disease have been associated
with juices. Raybaudi-Massilia, Mosqueda-Melgar, Soliva-Fortuny,
and Martn-Belloso (2009) reviewed and observed that the number
of documented outbreaks of human infections associated with fruit
juices (ranged from 1 to 5 per year) has increased in the last two
per year). Total colony count, coliforms and pathogenic bacteria, e.g.
Salmonella spp., Escherichia coli and Staphylococcus aureus, have
been isolated from this type of juice (Al-Jedah & Robinson, 2002;
Martnez-Gonzalez, Navarro-Hidalgo, Castillo, Villarruel-Lpez, &
Torres-Vitela, 2006; Pil et al., 2009; Raybaudi-Massilia et al., 2009).
Other bacteria such as Alicyclobacillus spp., Enterobacter spp., Erwi-
nia spp., Propionibacterium cyclohexanicum, Pseudomonas spp., and
lactic acid bacteria have been also detected as spoilage organisms in
cut fruit and juices by several authors (Brackett, 2001; Chang &
Kang, 2004; Paof & Petracek, 1997; Walker & Phillips, 2008).
In 2009, population of Spain drunk on 138 million liters of orange
juice (Spanish Ministre of Agriculture), around 40% being consumed
in the foodservice establishments as fresh squeezed juice but this
product is obtained with squeezing machines which have contact
surfaces difcult to clean (Lakshmanan & Schaffner, 2005).
The aim of this study was to analyze, in foodservice establish-
ments, the microbial quality of orange juice obtained from
squeezing machines which is maintained in glass or metal jugs
until consumption.
2. Materials and methods
2.1. Samples and sampling procedure

* Corresponding author. Tel.: 34 963543056; fax: 34 963544954. To obtain the orange juice from squeezing machines in all
E-mail address: isabel.sospedra@uv.es (I. Sospedra). studied foodservice establishments, the procedure was the

0956-7135/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.06.025
 I. Sospedra et al. / Food Control 23 (2012) 282e285
following; i) Valencia and Navel oranges are introduced through
the feeding tube of the machines, ii) the switch is turned on and iii)
freshly squeezed orange juice is collected in glass or kept in metal
jugs under the machines until the consumption. All squeezing
machines used in these establishments were Auto Orange Juicer
Machine OJ2AAP (Frucosol, Calahorra, Spain). The temperature and
pH of the oranje juice was measured after the sampling with
a Crison 638 Pt digital thermometer (Crison Instruments, Barce-
lona, Spain) and a Crison PH-25 Model portable pH Meter (Crison
Instruments), respectively. A total of 190 lots of this type of juice
were collected aseptically in sterile bottles (VWR International
Eurolab, Barcelona, Spain). For each sample ve replicates were
taken. Immediately after collection, samples were chilled to 4  C
and transported to the laboratory for analysis. The microbiological
analysis was made on the same day. The microbial quality of the
lots was evaluated according to specications of the Commission
Regulation No. 2073/2005 (European Union, 2005) and No. 1441/
2007 (European Union, 2007) and Spanish Regulation No. 3484/
2000 (Anonymous, 2001. pp. 1435e1441). According to these
legislations and ICMSF (1986), two-class attributes plan is preferred
when the microorganism of concern is not permitted in food (Lis-
teria monocytogenes and Salmonella spp.). In this plan, if the all
samples are  m (microbiological limit) or  c (the maximum
allowable number of samples that yielded unsatisfactory test) of
samples are > m, lots are accepted. However, if > c of samples are
within >m, lots are rejected. If the number of microorganisms must
be counted (APC, Enterobacteriaceae, E. coli, S. aureus), a three-class
attributes plan is adopted. For a three-class attributes plan, two
microbiological limits, m and M (limit beyond which the level of
contamination is hazardous or unacceptable), are set and if all
samples are  m or  c of samples are > m, lots are accepted.
However, if a number of samples > c are within >m and <M or any
sample is >M, lots are rejected.
2.2. Analysis
Freshly squeezed orange juice (25 ml) were diluted with 225 ml
buffered peptone water (BPW) (Oxoid, Unipath, Hampshire, UK)
and homogenized in a Stomacher (Classic, IUL, Barcelona, Spain).
The homogenates were used for the plating and incubation
procedures. Four 10-fold dilutions were made with each sample,
1 ml of each dilution was surface plated by duplicate onto standard
Plate Count Agar (PCA) (Oxoid) and incubated at 30  C for 72 h,
according to the International Organization for Standardization
(ISO, 2003) to determine the aerobic plate counts (APC).
Enterobacteriaceae were determined according to the ISO
(2004a, b), using duplicate poured plates of Violet Red Bile
Glucose agar (VRBG) (Oxoid), over layered with a further 10e15 ml
of VRBG agar. The plates were incubated at 37  C for 24 h and
typical colonies were counted on all plates having not more than
150 typical colonies.
To isolate E. coli, the previous BPW tubes were inoculated onto
CHROMagar ECC (CHROMagar Microbiology, Paris, France). After
Table 1
Microbial quality, according to the European and Spanish legislations, for total aerobic mesophilic bacteria, Enterobacteriaceae and S. aureus in fresh squeezed orange juice
collected from studied foodservice establishments.
Microbial quality (%)
Total aerobic mesophilic bacteria
Source (no. of lots) Acceptable Marginal Unacceptable
Glassa (n 163) 95 3 2
Metal jugb (n 27) 47 40 13
a
Fresh orange juice processed by squeezing machines served directly in a glass.
b
Fresh orange juice processed by squeezing machines maintained in metal jug during one day.
283
incubation at 37  C for 48 h, colonies were conrmed using Rapid
ONE System (REMEL Inc. Santa Fe, USA) as described before
(Soriano, Rico, Molt, & Maes, 2002). For enumeration of S. aureus,
a 0.1 ml of the inoculated BPW was surface plated on Baird-Parker
agar containing egg-yolk tellurite emulsion (Oxoid), and incubated
at 37  C for 24 h (ISO, 1999). Typical colonies (i.e., black, convex and
with or without halo on BP agar) were counted and examined
microscopically. Colonies were also tested for catalase reaction and
conrmed with agglutination Staphytect Plus test (Oxoid).
Isolation and identication of Salmonella spp. was done according
to the ISO (2004) and performed using the homogenate in BPW.
Briey, this microorganism was analyzed using 1 and 0.1 ml of the
BPW into Tetrathionate broth with Novobiocin (TTn) (Oxoid) and into
Rappaport-Vassiliadis (RV) broth (Oxoid), respectively. The enrich-
ment broths were incubated for 24 h at 37  C (for TTn broth) and 42  C
(for RV broth). The positive cultures were streaked onto CHROMagar
Salmonella (CHROMagar Microbiology) and the conrmation was
done by the Rapid ONE System (REMEL Inc. Santa Fe, USA).
L. monocytogenes was detected according to the International
Organization for Standardization (1996). Samples (25 ml) were
weighed into sterile stomacher bags, diluted and homogenized
with 225 ml of Fraser broth (Oxoid). After homogenizing and pre-
culturing at 37  C for 48 h, the positive broth was streaked onto
Listeria Palcam agar (Oxoid) and incubated at 37  C for 48 h.
Characteristic colonies were Gram stained, tested for motility,
oxidase and catalase followed by identication with the API Listeria
system (BioMrieux, Mancy lEtoile, France).
3. Results and discussion
In our study, the fresh orange juice was processed by squeezing
machines in restaurants. It was never refrigerated, because the
juices were served in a glass or maintained in a metal jug until the
consumption. The average of temperature and pH of all studied
orange juice samples were 17.1  C and 3.5, respectively. These
machines are easily cleaned but they are time-consuming. In fact,
we have observed in sampling that some of these machines have
scraps of oranges in internal tubing. For this reason and according
to Lakshmanan and Schaffner (2005), the presence of dirty places in
these machines is reected in the formation of biolms.
Our analysis showed that 2 and 22% of the studied lots for fresh
orange juice sampled from the glass exceeded the European and
Spanish maximum level of mesophilic aerobic counts and Enter-
obacteriaceae, respectively (Table 1), whereas a high percentage, 13,
81 and 1% of the lots of orange juice taken from a metal jug, exceeded
these levels for mesophilic aerobic counts, Enterobacteriaceae and
S. aureus, respectively (Table 1). The study of Martn-Diana, Rico,
Barat, and Barry-Ryan (2009) also showed a signicant increase of
aerobic counts over storage time. According to Sospedra, Rubert,
Soler, Soriano, and Maes (2009), the samples obtained from
metal jugs had a high level of microbial contamination due to
contamination from the environment and/or inadequate hygienic
handling and unsanitary conditions ICMSF (1998). Lakshmanan and
Enterobacteriaceae Staphylococcus aureus
Acceptable Marginal Unacceptable Acceptable Marginal Unacceptable
64 14 22 100 0 0
13 6 81 99 0 1
284 I. Sospedra et al. / Food Control 23 (2012) 282e285
Schaffner (2005) studied the total plate counts on beverage
dispensers in use in University dining halls as well as on an identical
but new beverage dispenser situated in their laboratory. In the rst
equipment the microbial counts were highest immediately after
a beverage had been dispensed and then declined gradually over
time. However, microbial counts in the laboratory dispenser were
initially low, but increased over time. Furthermore, they observed
a relationship between high microbial counts obtained by swabbing
the inside of the dispenser tips and the presence of biolms. These
authors reected that the use of a higher concentration (100-ppm
chlorine solution) of sanitizer may reduce microbial counts on
beverage dispenser tips. In our study 12% and 43% of the total
examined lots exceed the adopted limits of mesophilic aerobic
counts and Enterobacteriaceae, respectively. Pil et al. (2009)
detected S. aureus in two out of twenty orange juice samples in
a range from 2 to 6.5  103 CFU/ml and all samples (n 20) were
positive for total coliforms and thermotolerant coliforms from <2.2
to >16.0 MPN/ml and from <2.2 to 16.0 MPN/ml, respectively. Iha
et al. (2000) evaluated the physicochemical and hygienic-sanitary
quality of the fresh and pasteurized orange juices consumed in
Ribeiro Preto and Araraquara, located in the State of So Paulo
(Brazil). One hundred and thirty fresh and thirty-three pasteurized
orange juice samples provided by different plants located in the
Araraquara and Ribeirao Preto regions were analyzed. The results
showed that for fresh juice 63 (48.5%) samples were in disagreement
with physicochemical standards established by the legislation, 8
(6.1%) with relation to microscopic and 65 (50.0%) with relation to
microbiological analysis. With regard to the pasteurized juice, 3
(9.1%) and 5 (15.1%) samples did not agree with the legislation
according to physicochemical analysis and microscopic examina-
tion, respectively. Therefore, no pasteurized juice sample was in
disagreement with the microbiologic examination. Training and
awareness of school food handlers is a fundamental condition for
avoiding contamination and ensuring the quality and safety of the
food produced (Pistore & Gelinskib, 2006).
In our study, Salmonella spp. was detected in one lot of orange
juice sampled from metal jug.
Jain et al. (2009) concluded that unpasteurized orange juice was
responsible for foodborne salmonellosis in 152 people in six states
in the USA. Vojdani, Beuchat, and Tauxe (2008) reviewed fruit
juice-associated salmonellosis of illness reported, from 1995 to
2005, from Centres for Disease Control (CDC). Eight of the
outbreaks reported were linked to orange juice and ve of them
were caused by Salmonella serovars. Salmonellosis outbreak
occurred during the summer of 1995 among individuals who
consumed nonpasteurized orange juice. Furthermore, Salmonella
cells in juice were associated with the levels of faecal coliforms in
the population and also E. coli was found above the limits of
detection (>110/ml) based on the most probable number (MPN).
However, in our study, neither E. coli nor L. monocytogenes were
detected from any of the samples.
4. Conclusions
The presence of the microorganisms isolated (aerobic mesophilic
bacteria, Enterobacteriaceae and Salmonella spp.) from the samples,
indicates that the conditions of preparation, handling, and storage are
not hygienic. Most outbreaks of foodborne infections are generally
caused by foods that have been mishandled or mistreated during
preparation or storage. To decrease microbial growth in orange juice,
correct handling practices, efcient cleaning, sanitization of cont-
ainers and implementation of prerequisite programs and HACCP plan
are needed. These procedures must be done to improve the quality of
orange juice and consequently to protect consumers health.
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