Microbiological safety of fruit juices served in cafes/restaurants, Jimma town, South west, Ethopia

Abstract

BACK GROUND: Traditionally, fruit products have been regarded as microbiologically safer than
other unprocessed foods. However, many outbreaks of human infections have been associated with the
consumption of contaminated fruit juices. The objective of this study was to evaluate the microbiological
safety and quality of fruit juices being served in Jimma town, Southwest Ethiopia.
MATERIALS AND METHODS: The microbiological safety of different fruit juices being served in
cafés/ restaurants in Jimma town were assessed based on the isolates API-20E (Analytical Profile Index
used to test for twenty substrates to differentiate Enterobacteriaceae) profile from February, 2005 to
July, 2006. A total of 90 samples (30 samples each for avocado, papaya and pine-apple), collected from
six randomly selected cafés and/or restaurants in Jimma town, were analyzed. In addition, some physicochemical
parameters of the juices, such as pH and Titratable acidity were determined following standard
procedures.
RESULT: The mean aerobic mesophilic bacteria counts (CFU/ml) of avocado, papaya and pine-apples
were 8.0 x106
, 3.1x107
, and 7.9x106
, respectively. The counts of yeasts were relatively higher in avocado
(4.5x105CFU/ml) and pine-apple (5.0 x106 CFU/ml) as compared to that of papaya (6.2x103CFU/ml).
The pH and Titratable acidity (TA) of all fruit juices were 4.0-5.84 and 0.08-0.223 (g lactic acid/100g
sample), respectively. Pine-apple was more acidic (pH= 4±0.001) than avocado (pH= 5.84 ±0.14) and
papaya (5.23±0.02). The dominant bacterial groups isolated from the fruit juices included two Klebsella ,
three Enterobacter , and three Serratia species.
CONCLUSION: The microbial loads of most of the fruits juices were higher than the specifications set
for fruit juices sold in the Gulf region and other parts of the world. To the authors’ knowledge, there is
no specification set for the permissible level of microbes in fruit juices being served in Ethiopia. As
dominant isolates were colonies of organisms, the poor hygienic practice of the fruit juice handlers and
lack of sound knowledge of using disinfectant during processing, besides the conducive physicochemical
profiles of the fruit juices, might have contributed to the high microbial load. Thus, high level
of workers hygiene should be enforced and the use of disinfectant better practiced to improve the
microbial quality, safety, and shelf-life of the final product.

Full text

Microbiological Safety of Fruit Juices Served Tsige K. et al
98
ORIGINAL ARTICLE
MICROBIOLOGICAL SAFETY OF FRUIT JUICES SERVED IN
CAFES/RESTURANTS, JIMMA TOWN, SOUTHWEST ETHIOPIA
Tsige Ketema1, MSc, Tsegaye Gaddisa1, MSc, Ketema Bacha1,2* PhD
ABSTRACT
BACK GROUND: Traditionally, fruit products have been regarded as microbiologically safer than
other unprocessed foods. However, many outbreaks of human infections have been associated with the
consumption of contaminated fruit juices. The objective of this study was to evaluate the microbiological
safety and quality of fruit juices being served in Jimma town, Southwest Ethiopia.
MATERIALS AND METHODS: The microbiological safety of different fruit juices being served in
cafés/ restaurants in Jimma town were assessed based on the isolates API-20E (Analytical Profile Index
used to test for twenty substrates to differentiate Enterobacteriaceae) profile from February, 2005 to
July, 2006. A total of 90 samples (30 samples each for avocado, papaya and pine-apple), collected from
six randomly selected cafés and/or restaurants in Jimma town, were analyzed. In addition, some physico-
chemical parameters of the juices, such as pH and Titratable acidity were determined following standard
procedures.
RESULT: The mean aerobic mesophilic bacteria counts (CFU/ml) of avocado, papaya and pine-apples
were 8.0 x106, 3.1x107, and 7.9x106, respectively. The counts of yeasts were relatively higher in avocado
(4.5x105CFU/ml) and pine-apple (5.0 x106 CFU/ml) as compared to that of papaya (6.2x103CFU/ml).
The pH and Titratable acidity (TA) of all fruit juices were 4.0-5.84 and 0.08-0.223 (g lactic acid/100g
sample), respectively. Pine-apple was more acidic (pH= 4±0.001) than avocado (pH= 5.84 ±0.14) and
papaya (5.23±0.02). The dominant bacterial groups isolated from the fruit juices included two Klebsella ,
three Enterobacter , and three Serratia species.
CONCLUSION: The microbial loads of most of the fruits juices were higher than the specifications set
for fruit juices sold in the Gulf region and other parts of the world. To the authors’ knowledge, there is
no specification set for the permissible level of microbes in fruit juices being served in Ethiopia. As
dominant isolates were colonies of organisms, the poor hygienic practice of the fruit juice handlers and
lack of sound knowledge of using disinfectant during processing, besides the conducive physico-
chemical profiles of the fruit juices, might have contributed to the high microbial load. Thus, high level
of workers hygiene should be enforced and the use of disinfectant better practiced to improve the
microbial quality, safety, and shelf-life of the final product.
KEY WORDS: Fruit juice, Jimma, Microbial safety, physicochemical parameters
INTRODUCTION
Fruit juices are common beverages in many
countries of the world. In hot climate areas, cafés,
restaurants and road side stalls have local facilities
to extract the juice from fresh fruits and then
serving the juice liberally dozed with ice, to the
thirsty customers (1).
The consumption of fruit juices could have
both positive and negative effect on the part of
consumers. Fruit juices processed under hygienic
condition could play important role in enhancing
consumers health through inhibition of breast
cancer, congestive heart failure (CHF), and urinary
tract infection (2,3). In absence of good
manufacturing practice, however, the nutritional
richness of fruit juices makes the product good
medium for microbial growth, vehicle of foodborne
pathogens and associated complications (1).
Fruit juices contaminated at any point of processing
could be the source of infectious pathogens. Study
conducted on the microbiological safety of some
fruit juices showed Salmonella in apple and
orange juices (4). E. coli O157:H7 infection has
been linked with consumption of apple juices (5).
The prominent pathogens involved in un-
pasteurized juice outbreaks have been identified as
E. coli O157:H7, Samonella spp and
Cryptosporidium (6).
Although scanty on Ethiopian side, some
countries of the world have set standards for the
maximum permissible level of microbes in fruit
juices and related products (1).
1Department of Biology, Jimma University, P.O.Box 378, Jimma, Ethiopia
2Department of Biology, Addis Ababa University, P.O.Box 1176, Addis Ababa, Ethiopia
* Corresponding Author

Ethiop J Health Sci. Vol. 18, No. 3 November 2008
96
The setting of standards with respect to microbial
load and physico-chemical parameters of products
could have paramount important not only for
regulatory bodies, but also to the consumers’
health.
In Ethiopia, particularly in large urban areas,
fruit juices are available in supermarkets in canned
or bottled forms. In addition, fruit juice vending
houses, which have been serving different types of
fruit juices in fresh forms, are proliferating.
However, the scientific information on the safety of
fruit juices prepared and consumed in Ethiopia is
scanty. The objective of this study was, therefore,
to evaluate the physico-chemical quality and
microbiological safety of some fruit juices being
served in Jimma town, southwest Ethiopia.
MATERIAL AND METHODS
A total of 90 fruit juice samples (30 each of
avocado, papaya, and pine-apple) were collected
from six randomly selected cafés/restaurants from
among 30 cafés/restaurants in Jimma from
February 2005 to July 2005. As some of the fruit
juice vending cafés/restaurants were serving either
one, two or three types of the fruit juices, only
those serving the maximum number of fruit juices
were considered and six of them were selected for
sampling following lottery method. The maximum
types of fruit juices encountered in the study area
were juices made from avocado, papaya, and pine-
apple. Samples (250ml each) of these fruit juices
were collected in sterile flask (500ml) separately
and transported to laboratory at Jimma University,
Biology Department, using cold chain. Samples
were processed within maximum of an hour after
its collection and arrival at laboratory.
Questionnaire was used to obtain preliminary
information on the demographic characteristics of
the fruit juice makers, servers, and cares being
taken during processing of the fruit juices. All the
personnel’s involved in the processing and/or
serving of the fruit juices in the six selected
cafés/restaurants were included.
Twenty five milliliters (25ml) of the fruit
juices was separately drawn and blended in 225ml
of sterile physiological saline solution (0.85%
NaCl). The samples were homogenized and
appropriate dilutions were plated in duplicate on
pre-dried surfaces of respective media for microbial
count: aerobic mesophilic bacteria (AMB) were
counted on Plate Count Agar (PCA) after
incubation at 32 oC for 48 hours; Violate Red Bile
Agar (VRBA) was used to count coliforms after
incubation for 48 hours at 32oC. Purplish red
colonies surrounded by reddish zone of precipitated
bile were counted as coliforms. Enterobacteriaceae
were counted on MacConkey agar after incubated
at 32oC for 48 hours. Pink to red purple colonies
with or without haloes of precipitation were
counted as member of Enterobacteriaceae.
Staphylococci were counted on Mannitol Salt agar
(MSA) after incubation at 32oC for 48 hours.
Similarly, Yeasts and molds were counted on
Sabouraud agar plus 0.1g chloramphenicol
incubated at 25-28oC for 2-5 days. Smooth (non-
hairy) colonies without extension at periphery
(margin) were counted as yeasts. Hairy colonies
with extension at periphery were counted as molds.
After enumeration, ten colonies were
randomly picked from countable plates of PCA,
MSA, VRBA, and MacConkey Agar plates and
further purified by repeated plating on PCA. Cell
morphology, Gram reaction, colony
characterization and broth features were determined
following standard microbiological methods: KOH
test, a test for lipopolysaccharide, was made to
distinguish between gram-positive and gram-
negative bacteria (7). Catalase test was performed
by adding few drops of 3% H2O2 on plates of an
overnight culture of the pure isolates (8).
Isolates were purified by repeated plating on
appropriate media. Rod shaped, Gram-negative,
non-spore forming bacteria were considered as
members of Enterobacteriaceae and were
biochemically characterized using API 20E kit
(Biomeriuex, Marcy I’Etoile, France). Biochemical
profiles of the isolates were analyzed to species and
subspecies level using APIWEB® Stand Alone V
1.1.0 software (Biomeriuex, Marcy I’Etoile,
France).
pH was measured using digital pH meter (Nig
333, Naina Solaris LTD, India) after homogenizing
10ml of the fruit juices in 90 ml of distilled water
(9). Standard method was used to measure
Titratable acidity (10). The fruit juice sample (5ml)
was homogenized in distilled water (20ml) and
filtered through whatman No.1 filter paper. Two-
three drops of phenolphthalein were added to 20ml
of the filtrate as indicator and titrated against 0.05M
NaOH to the end point of phenolphthalein.
Titratable acidity was expressed as g lactic
acid/100g of juice and calculated using the formula:
TA = MNaOH x ml NaOH x 0.09 x 100
ml juice sample
Where, TA = titratable acidity; MNaOH = Molarity
of NaOH used; ml NaOH = amount (in ml) of
NaOH used; 0.09 = equivalent weight of lactic acid
To check the reliability of the test, API
System was used. The standard reference strain
used in this study was Escherichia coli ATCC
25922 (Oxoid).
Microbiological and some physico-chemical
data were expressed as average of the total samples
for each fruit juice analyzed. The significance of
differences (P<0.05) of the mean microbial count
among the fruit juices was evaluated with one-way
ANOVA using SPSS for windows version 10.0.

Microbiological Safety of Fruit Juices Served Tsige K. et al
98
RESULTS
All the ninety fruit juice makers interviewed were
females and 54 (60.0%) of them were younger than
30 years. Although 45 (50%) of them had
completed or were attending high school education,
none of the fruit juice makers had any exposure to
professional training related to their current career.
Moreover, the fruits used for juice preparation were
all bought from open markets in Jimma Town with
preference to the ripened fruits. In all studied
cafés/restaurants, there was no experience of using
disinfectant or any other method of disinfection
during processing of juice and relied solely on tap
water for all purposes (Table 1).
Table1. Fruit juice processing conditions in cafés /restaurants in Jimma town, 2005 - 2006.
Characteristics
Number of respondents
Percent
Source of fruits
Open market
Directly from producers
75
15
83.3
16.7
Nature of fruit used:
Ripened
Over ripened
75
15
83.3
16.7
Temporary storage site of fruit
Shelf
Basket
Refrigerator
No special storage
45
20
15
10
50.0
22.2
16.7
11.1
The mean count of AMB was the highest (6.6 log
cfu/ml) in papaya fruit juices. All the three fruit
juices had closer counts of Enterobacteriaceae (5.4
to 6.1 lof cfu/ml), although the count was relatively
higher (6.1 log cfu/ml) in juice made of avocado.
Likewise, the mean counts of staphylococci, yeasts
and molds were the highest in avocado (5.41 log
cfu/ml), pine-apple (5.75 log cfu/ml) and avocado
(5.9 log cfu/ml), respectively, with counts ranging
between 5 to 6 log cfu/ml in all cases. Thus, the
mean microbial counts were above detectable level
in all the fruit juice samples examined (Fig. 1).
Except for pine-apple (pH=4.0), the mean pH
of juices made from avocado and papaya was in a
range that support the growth of most bacteria and
molds. In agreement with its low pH, the highest
titratable acidity was recorded in pine-apple juice
(Table 2).
Table 2 pH and Titratable acidity (TA) of fruit juices served in cafés/ restaurants in Jimma town, 2005 –
2006.
Types of fruit
juices
Sample
size
pH
TA
Avocado
30
5.84±0.14
0.081
Papaya
30
5.23±0.01
0.110
Pine-apple
30
4.00±0.001
0.223
Where, TA= Titratable acidity (g lactic acid/100g fruit juice)
Some members of the Enterobacteriaceae family
were encountered in almost all the three fruit juices
types. Of the ninety fruit juice samples examined,
65 (72.0 %) yielded enteric bacteria, where all
avocado and papaya samples were positive. The
most frequently found enteric bacteria were
Klebsiella oxytoca, K. pneumoniae, Enterobacter
aerogenes, E. cloacae, E .sakazaki, Serratia
liquefaciens, S. odoifaction and S. marcescens
(Table 3).

Ethiop J Health Sci. Vol. 18, No. 3 November 2008
98
Table 3. API-20E profile of Enterobacteriaceae isolate from fruit juices, Jimma town, 2005- 2006.
ONPG= ß-galactosidase production, ADH= arginine dihydrolase, LDC= lysine decarboxylase, ODC= ornithine decarboxylase, URE= urease, TDA= tryptophane deaminase, CIT=
Citrate, H2S= production of hydrogen sulphide, and IND= indole, GLU= glucose, MAN= mannitol, INO= inositol, VP= Vogus prausker (acetoin production), GEL= Gelatin
liquefaction, SOR= sorbitol, RHA= rhaminose, SAC=saccharose, MEL= melibiose, AMY= amylase, and ARA=arabinose
Sources
Biochemical Tests for utilization of different substrates
Isolates possible identity
Percent
identification
ONPG
ADH
LDC
ODC
CIT
H2S
URE
TDA
IND
VP
GEL
GLU
MAN
IND
SOR
RHA
SAC
MEL
AMY
ARA
NO3-
NO2
Avocado
+
-
+
-
+
-
-
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella oxytoca
97.0
Avocado
+
-
-
-
+
-
+
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella pneumonia
98.0
Avocado
+
+
-
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
Enterobacter cloacae
90.0
Avocado
+
-
+
-
+
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella oxytoca
97.4
Avocado
+
-
-
-
+
-
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
Klebsiella pneumoniae
93.8
Avocado
+
-
+
-
+
-
+
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella pneumoniae
97.6
Avocado
+
+
-
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
-
Enterobacter cloacae
98.4
Avocado
+
+
+
-
+
-
+
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella pneumoniae
97.5
Avocado
+
+
-
+
+
-
+
-
-
+
+
+
+
-
+
+
+
+
+
+
+
Enterobacter sakazaki
78.0
Avocado
+
+
+
-
+
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
Klebsiella oxytoca
97.0
Papaya
+
-
+
+
+
-
-
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Enterobacter aerogenes
96.6
Papaya
+
+
+
+
+
-
-
-
+
-
+
+
+
+
+
+
+
+
+
+
+
Serratia odoification
99.9
Papaya
+
-
+
-
+
-
-
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella oxytoca
97.7
Papaya
+
-
+
+
+
-
-
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Enterobacter aerogenes
96.0
Papaya
+
-
+
+
+
-
-
-
+
-
-
+
+
+
+
+
+
+
+
+
+
Klebsiella spp
85.2
Papaya
+
-
+
-
+
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Klebsiella oxytoca
97.4
Papaya
+
-
+
+
+
-
-
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Enterobacter aerogenes
96.0
Papaya
+
+
-
+
+
-
+
-
-
+
-
+
+
+
+
+
+
+
+
+
+
Enterobacter cloacae
96.6
Papaya
+
+
-
+
+
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
Enterobacter sakazaki
90.4
Pine apple
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
Serratia liquefaciens
67.4
Pine apple
+
-
+
+
+
-
-
-
-
+
+
+
+
+
-
+
+
+
+
-
+
Serratia marcescens
85.5

Microbiological Safety of Fruit Juices Served Tsige K. .et al
99
0
1
2
3
4
5
6
7
AMB
Coliform
Entrobacteriacae
Staphylococcus
Yeasts
Molds
count in type of fruit juice
log count (CFU/ml)
Avocado
Papaya
Pine-apple
Figure1. Microbial load of fruit juice served in cafés/restaurants in Jimma town, 2005 – 2006. Where,
AMB=aerobic mesophilic bacteria.
DISCUSSION
Plant foods, especially plant juices, tend to
have redox potential (Eh) values from 300 to
400 millivolt. Having such a high redox
potential is an indication for availability of
sufficient amount of free oxygen accessible to
aerobic microbes. Thus, the survival and
growth of aerobic bacteria and molds in such
products are highly likely, with the same
microbial groups being responsible for
spoilage of the same products (5). High loads
of different microbial groups, including
coliforms and other Enterobacteriaceae were
recorded from the fruit juices examined in
this study. The range of microbial counts
recoded in the fruit juices analyzed in this
study (6.2x103 - 3.1x107CFU/ml) was
relatively higher than the microbial load (102-
105 CFU/ml) reported in some earlier woks
(11). To the authors’ knowledge, there is no
specification set for the permissible level of
microbes in fruit juices being served in
Ethiopia. However, the recommended
specifications for fruit juices served in the
Gulf region suggests that the maximum count
permitted for total colony count of coliforms,
yeast and molds are 1x104, 100, and
1x103CFU/ml, respectively (12). On the basis
of the gulf standards, it is clear that the
colony counts of almost all the microbial
groups in our fruit juices exceeded the
standard by considerable margin. These high
counts ,however, may not necessarily pose
hazard to the health of consumers provided
that there are no potential pathogenic strains
such as strains of E. coli and Salmonella
species within the fruit juices to be
consumed(2).
The pH of fruit juices is usually too low
with good potential of inhibiting the growth
of pathogenic bacteria (5, 13) although some
molds and yeasts could tolerate the acidity.
Thus, the high magnitude of members of
coliforms and other
Enterobacteriaceae in the juices examined in
this study could be due to the high water
activity of ready- to -serve -juices (10).
Products with high water activity possess
good amount of un-bound water molecule
that supports growth and survival of
microorganisms. However, the low acidity
(i.e., higher pH) and viscosity of avocado,
besides its nutrient content, makes it good
medium for growth of microorganisms.
The mean microbial counts of pine
apple juices were significantly different
(P<0.05) from that of both avocado and
papaya. Pine-apple juice had lower microbial
loads than the other two. This could be
attributed mainly to the very low pH observed
(3.94- 4.04). In addition, conditions under
which the juice was processed, stored, and/or
served might have contributed to the
betterment of the product. In fact, its low pH
did not inhibit the growth of acid tolerant
yeasts and allowed their proliferation to level
as high as 6 log CFU/ml. The spoilage of
acidic foods is most often due to
contamination of the foods with aerobic acid
tolerant bacteria as well as yeasts and moulds
(5). Debaryomyces, for example, are among
the frequently reported genera of yeasts
responsible for spoilage of fruit juices (5).
Aerobic bacteria isolated in this study
were species of Klebsiells, Serratia, and
Enterocobacter. Although reports on the
microbiology of fruit juices were scanty,
Serratia and Enterobacter spp, were reported

Ethiop J Health Sci. Vol. 18, No.3 November 2008
100
to dominate the early phases of fermentation
of Nigerian palm wine (5).
In addition to the fruit, the equipment
used for processing of the juices could
contribute to the number of bacterial and
fungal species. Regulating the microbial
safety of facilities to be used for processing
and the use of good quality fruits and surface
disinfection besides cleaning with pure water
will certainly improve the microbiological
quality of these juices. For longer shelf-life
and safety of the juices against fungi and
molds, the use of an approved food additives
could be another best option. Many organic
acids with Generally Regarded as Safe
(GRAS) status have been currently used for
preservation of many foods and juices.
In general, most of the fruit juices being
served in Jimma had higher microbial load
than the specification set for fruit juices in
some parts of the world. As these products
could be the cause of health problems and
potential vehicle of foodborne outbreaks, high
level of workers hygiene should be enforced
and the use of disinfectant better practiced to
improve the microbial quality, safety, and
shelf-life of the final product.
ACKNOWLEDGEMENTS
This study was partly sponsored by Research
and Publication office of Jimma University.
All expenses on API kits and its reagents
were covered personally by Dr. Ketema
Bacha. We would like to thank anonymous
reviewers for their critical and constructive
comment.
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